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The synthetic study of inner-core oligosaccharides of lipopoly-
and lipooligosaccharides produced by gram-negative bacteria
Construction of 45-branched 3-deoxy-D-manno-oct-2-ulosonic
acid structure
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
RUIQIN YI
The United Graduate School of Agricultural Sciences
Tottori University
2015
1
Index
List of Figures 2
List of Schemes 3
List of Tables 4
Abbreviation 5
Chapter 1 General introduction 7
Chapter 2 The observation of the limitation of (2-4)-linked Kdo glycosylation 15
Chapter 3 The new route to synthesize 45-branched inner-core trisaccharides 21
Chapter 4 The covergent synthesis of 45-branched inner-core OSs 28
Chapter 5 Conclusion 38
Chapter 6 Experimental Section 41
References 78
Summary 82
Acknowledge 86
List of publications 87
2
List of Figures
Chapter 1
Figure 11 The structure of LPS and LOS
Figure 12 The inner-core OS structure of general gram-negative bacteria
Francisella tularensis and Pseudomonas cichorii
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
Figure14Paulsenrsquos method and our plan to synthesize 45-branched inner-core
trisaccharides
Chapter 2
Figure 21 The mechanism of the glycosidation of - and -fluoride
Chapter 3
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
Chapter 4
Figure 41 General inner-core structure of LPSLOS
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
3
List of Schemes
Chapter 2
Scheme 21 Preparation of Kdo donors
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Chapter 3
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Scheme 32 The transformation of the azide group to the acetamide group in 14
Scheme 33 Full deprotection of 45-branched Kdo trisccharides 11 13 16
Chapter 4
Scheme 41 Synthesis of Hep building blocks
Scheme 42 Synthesis of Lac(1-4)Hep unit
Scheme 43 Synthesis of Hep(1-3)Hep unit
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Scheme 45 Full deprotection of 45-branched Kdo tetra- and pentasaccharides
4
List of Tables
Chapter 2
Table 21 Glycosidation of Kdo donors 2ndash4 with the 45-diol acceptor 5
Chapter 3
Table 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Chapter 4
Table 41 Glycosylation to synthesize 45-branched inner core OSs
5
Abbreviation
Ac2O Acetic anhydride
AcOH Acetic acid
BF3middotOEt2 Boron trifluoride diethyl etherate
Bn2SnO Dibutyltin oxide
Dppb 14-Bis(diphenylphosphino)butane
Et2O Diethyl ether
GalNAc N-acetyl galactosamine
GalN3 2-Azido-2-deoxy-galatosamine
Glc Glucose
Hep L-Glycero-D-manno-heptopyranose
HgBr2 Mercury(II) bromide
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum correlation
H2SO4 Sulfuric acid
K2CO3 Potassium carbonate
Kdo 3-Deoxy-D-manno-2-octulosonic acid
Lac Lactose
LOS Lipooligosaccharide
LPS Lipopolysaccharide
Man Mannose
MeNO2 Nitromethane
6
MeOH Methanol
MS 4A Molecular sieve 4A
MS AW 300 Molecular sieve acid wash 300
NaOH Sodium hydroxide
N meningitides Neisseria meningitides
NMR Nuclear magnetic resonance
OS Oligosaccharide
P Phosphite
Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(OH)2C Palladium hydroxide on carbon
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
Ph3P Triphenyl phosphite
TBDMSCl t-Butyldimethylsilyl chloride
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TMSOTf Trimethylsilyl trifluoromethane sulfonate
TLC Thin-layer chlormatography
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
1
Index
List of Figures 2
List of Schemes 3
List of Tables 4
Abbreviation 5
Chapter 1 General introduction 7
Chapter 2 The observation of the limitation of (2-4)-linked Kdo glycosylation 15
Chapter 3 The new route to synthesize 45-branched inner-core trisaccharides 21
Chapter 4 The covergent synthesis of 45-branched inner-core OSs 28
Chapter 5 Conclusion 38
Chapter 6 Experimental Section 41
References 78
Summary 82
Acknowledge 86
List of publications 87
2
List of Figures
Chapter 1
Figure 11 The structure of LPS and LOS
Figure 12 The inner-core OS structure of general gram-negative bacteria
Francisella tularensis and Pseudomonas cichorii
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
Figure14Paulsenrsquos method and our plan to synthesize 45-branched inner-core
trisaccharides
Chapter 2
Figure 21 The mechanism of the glycosidation of - and -fluoride
Chapter 3
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
Chapter 4
Figure 41 General inner-core structure of LPSLOS
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
3
List of Schemes
Chapter 2
Scheme 21 Preparation of Kdo donors
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Chapter 3
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Scheme 32 The transformation of the azide group to the acetamide group in 14
Scheme 33 Full deprotection of 45-branched Kdo trisccharides 11 13 16
Chapter 4
Scheme 41 Synthesis of Hep building blocks
Scheme 42 Synthesis of Lac(1-4)Hep unit
Scheme 43 Synthesis of Hep(1-3)Hep unit
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Scheme 45 Full deprotection of 45-branched Kdo tetra- and pentasaccharides
4
List of Tables
Chapter 2
Table 21 Glycosidation of Kdo donors 2ndash4 with the 45-diol acceptor 5
Chapter 3
Table 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Chapter 4
Table 41 Glycosylation to synthesize 45-branched inner core OSs
5
Abbreviation
Ac2O Acetic anhydride
AcOH Acetic acid
BF3middotOEt2 Boron trifluoride diethyl etherate
Bn2SnO Dibutyltin oxide
Dppb 14-Bis(diphenylphosphino)butane
Et2O Diethyl ether
GalNAc N-acetyl galactosamine
GalN3 2-Azido-2-deoxy-galatosamine
Glc Glucose
Hep L-Glycero-D-manno-heptopyranose
HgBr2 Mercury(II) bromide
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum correlation
H2SO4 Sulfuric acid
K2CO3 Potassium carbonate
Kdo 3-Deoxy-D-manno-2-octulosonic acid
Lac Lactose
LOS Lipooligosaccharide
LPS Lipopolysaccharide
Man Mannose
MeNO2 Nitromethane
6
MeOH Methanol
MS 4A Molecular sieve 4A
MS AW 300 Molecular sieve acid wash 300
NaOH Sodium hydroxide
N meningitides Neisseria meningitides
NMR Nuclear magnetic resonance
OS Oligosaccharide
P Phosphite
Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(OH)2C Palladium hydroxide on carbon
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
Ph3P Triphenyl phosphite
TBDMSCl t-Butyldimethylsilyl chloride
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TMSOTf Trimethylsilyl trifluoromethane sulfonate
TLC Thin-layer chlormatography
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
2
List of Figures
Chapter 1
Figure 11 The structure of LPS and LOS
Figure 12 The inner-core OS structure of general gram-negative bacteria
Francisella tularensis and Pseudomonas cichorii
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
Figure14Paulsenrsquos method and our plan to synthesize 45-branched inner-core
trisaccharides
Chapter 2
Figure 21 The mechanism of the glycosidation of - and -fluoride
Chapter 3
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
Chapter 4
Figure 41 General inner-core structure of LPSLOS
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
3
List of Schemes
Chapter 2
Scheme 21 Preparation of Kdo donors
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Chapter 3
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Scheme 32 The transformation of the azide group to the acetamide group in 14
Scheme 33 Full deprotection of 45-branched Kdo trisccharides 11 13 16
Chapter 4
Scheme 41 Synthesis of Hep building blocks
Scheme 42 Synthesis of Lac(1-4)Hep unit
Scheme 43 Synthesis of Hep(1-3)Hep unit
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Scheme 45 Full deprotection of 45-branched Kdo tetra- and pentasaccharides
4
List of Tables
Chapter 2
Table 21 Glycosidation of Kdo donors 2ndash4 with the 45-diol acceptor 5
Chapter 3
Table 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Chapter 4
Table 41 Glycosylation to synthesize 45-branched inner core OSs
5
Abbreviation
Ac2O Acetic anhydride
AcOH Acetic acid
BF3middotOEt2 Boron trifluoride diethyl etherate
Bn2SnO Dibutyltin oxide
Dppb 14-Bis(diphenylphosphino)butane
Et2O Diethyl ether
GalNAc N-acetyl galactosamine
GalN3 2-Azido-2-deoxy-galatosamine
Glc Glucose
Hep L-Glycero-D-manno-heptopyranose
HgBr2 Mercury(II) bromide
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum correlation
H2SO4 Sulfuric acid
K2CO3 Potassium carbonate
Kdo 3-Deoxy-D-manno-2-octulosonic acid
Lac Lactose
LOS Lipooligosaccharide
LPS Lipopolysaccharide
Man Mannose
MeNO2 Nitromethane
6
MeOH Methanol
MS 4A Molecular sieve 4A
MS AW 300 Molecular sieve acid wash 300
NaOH Sodium hydroxide
N meningitides Neisseria meningitides
NMR Nuclear magnetic resonance
OS Oligosaccharide
P Phosphite
Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(OH)2C Palladium hydroxide on carbon
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
Ph3P Triphenyl phosphite
TBDMSCl t-Butyldimethylsilyl chloride
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TMSOTf Trimethylsilyl trifluoromethane sulfonate
TLC Thin-layer chlormatography
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
3
List of Schemes
Chapter 2
Scheme 21 Preparation of Kdo donors
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Chapter 3
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Scheme 32 The transformation of the azide group to the acetamide group in 14
Scheme 33 Full deprotection of 45-branched Kdo trisccharides 11 13 16
Chapter 4
Scheme 41 Synthesis of Hep building blocks
Scheme 42 Synthesis of Lac(1-4)Hep unit
Scheme 43 Synthesis of Hep(1-3)Hep unit
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Scheme 45 Full deprotection of 45-branched Kdo tetra- and pentasaccharides
4
List of Tables
Chapter 2
Table 21 Glycosidation of Kdo donors 2ndash4 with the 45-diol acceptor 5
Chapter 3
Table 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Chapter 4
Table 41 Glycosylation to synthesize 45-branched inner core OSs
5
Abbreviation
Ac2O Acetic anhydride
AcOH Acetic acid
BF3middotOEt2 Boron trifluoride diethyl etherate
Bn2SnO Dibutyltin oxide
Dppb 14-Bis(diphenylphosphino)butane
Et2O Diethyl ether
GalNAc N-acetyl galactosamine
GalN3 2-Azido-2-deoxy-galatosamine
Glc Glucose
Hep L-Glycero-D-manno-heptopyranose
HgBr2 Mercury(II) bromide
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum correlation
H2SO4 Sulfuric acid
K2CO3 Potassium carbonate
Kdo 3-Deoxy-D-manno-2-octulosonic acid
Lac Lactose
LOS Lipooligosaccharide
LPS Lipopolysaccharide
Man Mannose
MeNO2 Nitromethane
6
MeOH Methanol
MS 4A Molecular sieve 4A
MS AW 300 Molecular sieve acid wash 300
NaOH Sodium hydroxide
N meningitides Neisseria meningitides
NMR Nuclear magnetic resonance
OS Oligosaccharide
P Phosphite
Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(OH)2C Palladium hydroxide on carbon
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
Ph3P Triphenyl phosphite
TBDMSCl t-Butyldimethylsilyl chloride
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TMSOTf Trimethylsilyl trifluoromethane sulfonate
TLC Thin-layer chlormatography
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
4
List of Tables
Chapter 2
Table 21 Glycosidation of Kdo donors 2ndash4 with the 45-diol acceptor 5
Chapter 3
Table 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Chapter 4
Table 41 Glycosylation to synthesize 45-branched inner core OSs
5
Abbreviation
Ac2O Acetic anhydride
AcOH Acetic acid
BF3middotOEt2 Boron trifluoride diethyl etherate
Bn2SnO Dibutyltin oxide
Dppb 14-Bis(diphenylphosphino)butane
Et2O Diethyl ether
GalNAc N-acetyl galactosamine
GalN3 2-Azido-2-deoxy-galatosamine
Glc Glucose
Hep L-Glycero-D-manno-heptopyranose
HgBr2 Mercury(II) bromide
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum correlation
H2SO4 Sulfuric acid
K2CO3 Potassium carbonate
Kdo 3-Deoxy-D-manno-2-octulosonic acid
Lac Lactose
LOS Lipooligosaccharide
LPS Lipopolysaccharide
Man Mannose
MeNO2 Nitromethane
6
MeOH Methanol
MS 4A Molecular sieve 4A
MS AW 300 Molecular sieve acid wash 300
NaOH Sodium hydroxide
N meningitides Neisseria meningitides
NMR Nuclear magnetic resonance
OS Oligosaccharide
P Phosphite
Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(OH)2C Palladium hydroxide on carbon
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
Ph3P Triphenyl phosphite
TBDMSCl t-Butyldimethylsilyl chloride
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TMSOTf Trimethylsilyl trifluoromethane sulfonate
TLC Thin-layer chlormatography
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
5
Abbreviation
Ac2O Acetic anhydride
AcOH Acetic acid
BF3middotOEt2 Boron trifluoride diethyl etherate
Bn2SnO Dibutyltin oxide
Dppb 14-Bis(diphenylphosphino)butane
Et2O Diethyl ether
GalNAc N-acetyl galactosamine
GalN3 2-Azido-2-deoxy-galatosamine
Glc Glucose
Hep L-Glycero-D-manno-heptopyranose
HgBr2 Mercury(II) bromide
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum correlation
H2SO4 Sulfuric acid
K2CO3 Potassium carbonate
Kdo 3-Deoxy-D-manno-2-octulosonic acid
Lac Lactose
LOS Lipooligosaccharide
LPS Lipopolysaccharide
Man Mannose
MeNO2 Nitromethane
6
MeOH Methanol
MS 4A Molecular sieve 4A
MS AW 300 Molecular sieve acid wash 300
NaOH Sodium hydroxide
N meningitides Neisseria meningitides
NMR Nuclear magnetic resonance
OS Oligosaccharide
P Phosphite
Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(OH)2C Palladium hydroxide on carbon
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
Ph3P Triphenyl phosphite
TBDMSCl t-Butyldimethylsilyl chloride
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TMSOTf Trimethylsilyl trifluoromethane sulfonate
TLC Thin-layer chlormatography
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
6
MeOH Methanol
MS 4A Molecular sieve 4A
MS AW 300 Molecular sieve acid wash 300
NaOH Sodium hydroxide
N meningitides Neisseria meningitides
NMR Nuclear magnetic resonance
OS Oligosaccharide
P Phosphite
Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(OH)2C Palladium hydroxide on carbon
PMB p-Methoxybenzyl
PMBCl p-Methoxybenzyl chloride
Ph3P Triphenyl phosphite
TBDMSCl t-Butyldimethylsilyl chloride
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TMSOTf Trimethylsilyl trifluoromethane sulfonate
TLC Thin-layer chlormatography
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
7
Chapter 1
General introduction
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
8
11 Neisseria meningitidis
Neisseria meningitidis is a gram negative bacterium that colonizes and infects
only human and has never been isolated from other animals1 N meningitidis is the
main cause of bacterial meningitis in children and young adults2 Children younger
than 5 years are at greatest risk followed by teenagers of high school age The World
Health Organization estimates that there are 12 million cases of meningococcal
meningitis worldwide and 135000 related deaths annually3 Especially the
Sub-Sahara African has been plagued by large epidemics of meningococcal
meningitis for over a century4 Attack rates of 100-800 cases per 100000 are
encountered in this area The meningococcal disease often progresses very rapidly and
is difficult to diagnose and treat
Without treatment meningococcal meningitis is almost fatal Persons with N
meningitidis infection should be hospitalized immediately for treatment with
antibiotics5 However antibiotic treatment has many important limitations including
drug side effects6 and the potential for emergence of resistant organisms Moreover
permanent sequelae such as hearing impairment mental retardation or limb loss are
common in survivors7 Therefore the prevention of meningococcal meningitis is
important
To prevent the meningococcal disease especially to stop an outbreak of
meningococcal disease a dose of meningitis vaccine is recommended The discovery
that serum bactericidal antibodies to meningococcal capsular polysaccharides protect
against meningococcal disease is the basis for development of meningococcal
vaccines8 The capsular polysaccharides of N meningitidis are important virulence
factors that inhibit host cell protection mechanisms According to the immunology
specificity of capsular polysaccharides N meningitidis is classified into 13 clinically
significant serogroups Six most important serogroups A B C Y W-135 and X are
associated with disease in human Serogroup A has been the most prevalent in Africa
and Asia but is rare practically absent in North America9 In the United States and
Europe serogroup B is the predominant cause of disease and mortality followed by
serogroup C
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
9
Several effective vaccines using bacterial capsular polysaccharides as target to
generate bactericidal antibodies against serogroup A C Y and W-135 have been
developed and used10 However vaccine against serotype B disease is difficult to
produce The capsular polysaccharide on the serotype B bacterium is composed of
polysialic acid repeating units that are same with the structures found on human
neuronal cells11 Antibodies generated against serogroup B capsular polysaccharide
are cross-reactive with the polysialic acid moieties expressed on human neural cell
adhesion molecules As a result serogroup B capsular polysaccharides are poorly
immunogenic due to self-tolerance mechanisms To dissolve this problem efforts to
develop serogroup B vaccine have largely focused on other membrane antigens of N
meningitides One attempted solution is focused on the lipooligosaccharides (LOSs)
of N meningitides
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
component of the outer membrane of gram-negative bacteria12 LPSLOS contributes
essentially to the integrity and stability of the outer membrane and is also the first
line of defense for bacteria against a range of environmental factors including
detergents and antimicrobial agents13 As a potent virulence factor LPSLOS also
serves as a surface pathogen-associated antigen for recognition by the host immune
system14 Therefore LPSLOS has attracted much interest for the development of
diagnostic tools therapeutic reagents and vaccine candidates
12 Lipopolysaccharide and lipooligosaccharide
As shown in Figure 1112c an LPS consists of three domains a lipophilic
moiety termed lipid A a core oligosaccharide (core OS) and a hydrophilic glycan
called O-specific polysaccharide (O-antigen) whereas LOS which is limited to 10
saccharide units lacks an O-antigen polysaccharide The lipid A moiety which has a
-(1-6)-linked D-glucosamine disaccharide backbone is essential for bacterial
viability and carries the endotoxic properties of the LPSsLOSs15 The O-antigen
polysaccharide is the most variable portion of the LPS and provides serological
specificity which is response for bacterial serotyping16 In addition the core
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
10
oligosaccharide (OS) provides useful information for vaccine development that a core
oligosaccharide (OS) derived from LOS of pathogenic bacteria strains was reported to
be recognized by the human antibody17 So core OS is a focused target for vaccine
development
Figure 11 The structure of LPS and LOS
13 Branched inner core OS
The core OS can be further separated into two regions one proximal to lipid A
(inner-core OS) and the other is distal from lipid A but proximal to the O-antigen
(outer-core OS)12 The inner-core OS is highly conserved in bacterial species This
inner-core OS consists of mostly the unusual higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) Kdo is the unique compound of the inner core OS and rarely found in other
glycans Recent studies employing LOS of Nesseria gonorrhoeae strain 15253 as
affinity ligand indicated that human antibodies recognized several epitopes including
the Kdo region but also the branched heptosyl epitopes17 Therefore the inner-core
OS containing these epitopes draws more attention for vaccine research
Furthermore the general inner-core OS of LPSLOS is composed of a
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
11
45-branched Kdo structure In Figure 12 for example the inner-core OS of
LPSsLOSs from many gram-negative bacteria such as Neisserial18 Salmonella19
and haemophilus20 and so on contains a 45-branched
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide as the common structure
Moreover the Hep I moiety could be substituted by other saccharides (such as Man
GalNAc) in some other bacteria such as Francisella tularensis21 and Pseudomonas
cichorii22
Figure 12 The inner-core OS structure of general gram-negative bacteria Francisella tularensis
and Pseudomonas cichorii
14 The extraction of inner-core OS
Inner-core OS fragments can be extracted from the bacteria strains For the
fragmental extraction the target bacteria strains are isolated identified and grown in
tryptic soy broth medium The LPSLOS fragments are released from whole
bacterial cells by some mild treatments such as strong saline washes23 the use of
chelating agents24 or aqueous organic solvents25 The most general and widely used
procedure is the treatment of the whole bacterial cells with hot aqueous phenol
followed by cooling to produce a two-phase system26 LPSLOS in crude
aqueous-phenol extracts are collected and purified The presence of LPSLOS in the
resulting extract is confirmed by the SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis27
To obtain released inner-core OS fragments the collected LPSLOS is further
hydrolyzed under mild acid conditions28 After further purification by gel permeation
chromatography and high performance liquid chromatography (HPLC) a set of inner
core OS fragments with the different lengths could be collected These fragments are
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
12
usually used for structure study
However isolation of these oligosaccharides from highly pathogenic bacteria
such as N meningitides is undesirable The purification method of extraction and
nonselectivity hydrolysis of LPSLOS also limits the output and purity of these
oligosaccharides Moreover the vaccine development needs the well defined
oligosaccharides to conjugate with carrier proteins for immunizations It is difficult
to conjugate the extracted oligosaccharides to carrier proteins without destroying
vital immunological domains29
Fortunately chemical synthesis can address these issues Chemical synthesis
offers a much more attractive approach to produce multigram amount of highly pure
oligosaccharides and makes it possible to incorporate an artificial linker for
controlled conjugation to proteins
15 Chemical synthesis of branched inner core OS
Chemical synthetic approaches towards components of the inner-core OS
region have to deal with the elaboration of efficient protocols to prepare multigram
amounts of the higher carbon aldoses 3-deoxy-D-manno-2-octulosonic acid (Kdo) and
L-glycero-D-manno-heptopyranose (Hep) followed by transformation into suitable
glycosyl donor and acceptor derivatives In the past several decades the synthetic
efforts have covered the basic structural units (Kdo30 Hep31) and truncated forms of
inner-core OS32 However the chemical synthesis of inner-core OS is still challenging
due to its highly branched nature which complicates the installation of the various
glycosidic linkages
16 Recent research of Kdo synthesis in our laboratory
Recently we reported a useful Kdo intermediate methyl
(78-di-O-benzoyl-45-O-isopropylidene-D-manno-2-octulopyranosid)onate for the
preparation of 2-4 and 2-8 linked Kdo disaccharides33 This Kdo intermediate was
prepared from the D-mannose via 8 steps in 53 yield (Figure 13) For the synthesis
of 2-4 linked Kdo disaccharide the Kdo intermediate could be converted to the
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)
13
corresponding glycosyl fluoride donor and the 45-diol acceptor with ease And the
glycosylation gave the Kdo(2-4)Kdo disaccharide in a good yield (72 =51)
Figure 13 The synthesis of Kdo(2-4)Kdo disaccharide
17 Aim of the study
Although many chemical syntheses of linear inner-core OS structures have been
described34 there are few reports for the synthesis of branched inner-core OS Only
Paulsen group reported a synthesis of the 45-branched Hep(1-5)Kdo(2-4)Kdo
trisaccharide (Figure 14)35 In their synthesis the 4-OH group of Kdo I was firstly
protected by a p-methoxybenzyl group and sequentially a Hep donor was coupled
with Kdo I to form a Hep(1-5)Kdo disaccharide Then the p-methoxybenzyl group
of Kdo I was deprotected and a Kdo donor was installed to the formed
Hep(1-5)Kdo disaccharide to gave the desired Hep(1-5)[Kdo(2-4)]Kdo
trisaccharide However the yield of this approach appears to be low (4 steps only
22)
In the study of this thesis it is aimed to develop a new chemical synthetic
approach for the 45-branched inner-core OSs and to extend the utility of our previous
synthetic Kdo(2-4)Kdo disaccharide In this research the 45-branched Kdo
structures would be synthesized by glycosylation of the 5-OH group of the 2-4 linked
Kdo disaccharide This thesis contains three parts
1) The observation of the limitation of (2-4)-linked Kdo glycosylation
2) The new route to synthesize 45-branched inner-core trisaccharides
14
3) The convergent synthesis of 45-branched inner-core OSs
Figure 14 Paulsenrsquos method and our plan to synthesize 45-branched Kdo trisaccharide
In Chapter 2 to optimize the reaction condition of Kdo(2-4)Kdo the
glycosidation using several types of Kdo donors and Lewis acid are discussed
These Kdo donors are prepared from a Kdo intermediate which is designed in our
previous research
In Chapter 3 we focus on the discussion of the glycosylation using the
synthetic Kdo(2-4)Kdo in Chapter 2 as the acceptor to synthesize 45-branched
inner-core trisaccharides The reaction conditions of this new route to prepare a
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide are discussed and the result is compared
with Paulsenrsquos method Moreover the first synthesis of Man(1-5)[Kdo(2-4)]Kdo
and GalNAc(1-5)[Kdo(2-4)]Kdo by this new approach is also discussed
In Chapter 4 to further observe the utility of the newly synthetic route
discussed in Chapter 3 a convergent synthesis route using the same Kdo(2-4)Kdo
disaccharide as the acceptor to produce more complex inner core OS is discussed To
test the glycosylation conditions and necessary protecting strategy a lactose donor is
chosen as a model compound Based on the model glycosylation the corresponding
Hep units constructed from the Hep building blocks are coupled with the Kdo moiety
to obtain the desired branched inner-core OS
15
Chapter 2
The observation of the limitation of
(2-4)-linked Kdo glycosylation
16
21 Introduction
The inner-core OS of gram-negative bacteria consists of at least one of the
higher carbon sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) Kdo is rarely found
in other glycans and thus can be considered as a mark for the presence of LPSLOS12
The incorporation of Kdo appears to be a vital step in LPS biosynthesis and in
growth of the gram-negative bacteria Furthermore the inner core OS of many
LPSsLOSs is composed of a 45-branched Kdo structure which contains a
Kdo(2-4)Kdo disaccharide at the reducing end According to the newly hypothetical
route to synthesize the 45-branched Kdo structure the 2-4 linked Kdo disaccharide
would be prepared at first
Although many chemical syntheses of Kdo(2-4)Kdo disaccharide have been
reported36 there is still no generally accepted high-yielding procedure for the
stereoselective preparation of Kdo(2-4)Kdo disaccharide In recent for the synthesis
of Kdo(2-4)Kdo disaccharide our laboratory reported a useful Kdo intermediate
from the D-mannose in 8 steps33 This Kdo intermediate could be easily converted to
the corresponding glycosyl fluoride donor and 45-diol acceptor And the
glycosidation of the fluoride donor with the 45-diol acceptor gave the Kdo(2-4)Kdo
in 72 yield (=51) However the limitation of this glycosidation was not
investigated Therefore in this Chapter to optimize the glycosidation conditions of the
synthesis of Kdo(2-4)Kdo several types of Kdo donors and Lewis acid are examined
22 Result and discussion
221 Preparation of Kdo donors
Three types of Kdo donors 2 3 4 were synthesized from common Kdo
intermediate 1 in Scheme 21 Treatment of compound 1 with NN-diethylaminosulfur
trifluoride at 0 degC gave the fluoride 2 as a mixture of anomers in a good yield (81)33
The anomeric ratio of 2 was 31 and the major isomer was easily isolated by
crystallization from ethyl acetate and hexane The anomeric configuration of the
major product was presumed to be based on a compare with the NMR data of
corresponding Kdo derivatives reported by Imoto37 The N-phenyl
17
trifluoroacetimidate 3 was prepared in quantitative yield from compound 1 with
222-trifluoro-N-phenylacetimidoyl chloride in the presence of potassium
carbonate38 However the reaction needed 1 week to reach completion The reaction
for preparing the Kdo trichloroacetimidate donor also demanded a week This showed
that the reactivity of the anomeric hydroxyl group of the Kdo derivatives was lower
than that of aldose because of steric hindrance and low nucleophilicity Two isomers
could be separated and the major product was presumed to be Dibenzyl phosphite 4
was synthesized in moderate yield (56) from compound 1 with 1H-tetrazole
dibenzyl NN-diisopropylphosphoramidite (DDP)39 Also two isomers were separated
and the major product was presumed to be
Scheme 21 Conditions (a) DAST 0 degC 05 h 81= 31 (b) N-phenyl trifluoroacetimidoyl
chloride K2CO3 CH2Cl2 rt 7 days quant = 32 (c) 1H-tetrazole DDP CH2Cl2 0 degCrarr rt 3
h 56 = 231
222 The glycosylation of the 45-diol acceptor with Kdo donors
Glycosylation of the 45-diol acceptor 533 which was also prepared from the
Kdo intermediate 1 (Figure 14 in Chapter 1) with these Kdo donors 2 3 4 was
examined (Scheme 22) The results are summarized in Table 21 In entry 1 the
glycosidation of Kdo fluoride 2in the presence of BF3middotOEt2 gave the Kdo(2-4)Kdo
6 in 72 yield (=51) When the activator was changed to a combined promoter
Cp2HfCl2-AgOTf40 fluoride 2 was readily converted to glycal 7 (90 entry 2)
18
Scheme 22 The glycosylation of the 45-diol acceptor with Kdo donors
Table 21 Glycosylation of Kdo donors 2-4 with the 45-diol acceptor 5
Entry Donor Lewis acid
(equiv)
Temp Time
(h)
Yield
()
7()a 5 ()
1 2 BF3middotOEt2 (60) -20 degC 15 72 51 - -
2 2 Cp2Hf(OTf)2(22) -20 degC 15 16 351 90
3 2 BF3middotOEt2 (60) -20 degC to rt 10 41 251 73
4 2 TMSOTf (01) -20 degC to rt 10 - - 35b
5 3 TMSOTf (01) -78 degC 20 48 241
6 3 TMSOTf (01) -78 degC 20 61 261
7 4 TMSOTf (01) -20 degC 25 35 381 61 50
8 4 BF3middotOEt2 (10) -20 degC 20 30 211 83 63
a The yield was based on the donor
b Twenty four percent of the donor was recovered
Using -fluoride 2 with BF3middotOEt2 as an activator gave a moderate yield (41
entry 3) whereas using TMSOTf as a promoter gave a poor yield (entry 4) The
longer reaction time and higher reaction temperature also suggest that the reactivity of
-fluoride was lower than that of -fluoride The lower reactivity of -fluoride might
be due to the effect of 45-O-isopropylidene group As shown in Figure 21 in the
glycosidation of fluoride 2 the C-F bond is firstly cleaved by BF3middotOEt2 to give an
oxocarbenium ion intermediate41 With the steric hindrance of isopropylidene group
on the top face the acceptor prefers to attack the oxocarbenium ion intermediate from
the bottom side to give -selective glycoside However in the glycosidation of the
fluoride 2 it seems to be difficult to directly form the oxocarbenium ion
intermediate Due to the large electronegativity of the fluorine atom C-F bond is very
19
short and stable In addition the covalent radius of the fluorine atom is also very small
Therefore with the steric hindrance of 45-O-isopropylidene group the boron atom
(Lewis acid) seems to be difficult to attack the fluorine atom to cleave the C-F bond
from the top face The fluoride 2 might transfer to 2 to give the glycoside
Figure 21 The mechanism of the glycosidation of -and -fluoride
The use of N-phenyl trifluoroacetimidate 3 3 (entries 5 and 6) with
TMSOTf as an activator gave a good yield although the stereoselectivity was modest
The use of glycosyl phosphite (entries 7 and 8) with BF3middotOEt2 or TMSOTf gave a
poor yield and low selectivity These donors gave glycal 7 as the major product
Comparing the glycosylation results above Kdo fluoride 2 (entry 1) with BF3middotOEt2
as the activator provided the best yield and -selectivity Thus the reaction of
glycosyl fluoride 2 in the presence of BF3middotOEt2 was the most effective for this
reaction
23 Conclusion
The change of leaving groups in Kdo donors was not effective on the
improvement in the yield for the glycosidation of Kdo(2-4)Kdo dimer As shown in
Figure 21 the glycosidation of the Kdo donors with a 45-O-isopropylidene
protecting group tends to proceed in a SN-1 reaction41 The leaving groups would be
cleaved to form an oxocarbenium ion intermediate The glycosidation results indicate
that the cleaved leaving groups contribute nothing to avoiding the formation of the
glycal from the oxocarbenium ion intermediate The formation of the glycal would
decrease the yield of the glycosidation Moreover the stereoselectivity was also not
20
influenced by the type of leaving group Due to the effect of the 45-O-isopropylidene
group (Figure 21) all donors produced the -glycoside as the main product These
results are consistent with the results reported by Yoshizaki et al that glycosidation
with a 45-O-isopropylidinene-protected Kdo fluoride donor has a high
-selectivity42
21
Chapter 3
The new route to synthesize 45-branched
inner-core trisaccharides
22
31 Introduction
In Chapter 2 the effect of leaving groups and Lewis acid on the yield and
stereoselectivity for the glycosylation of 2-4 linked Kdo dimer has been discussed
The result suggests that the reaction of -fluoride in the presence of BF3middotOEt2 is
the most effective In this Chapter we focus on the construction of 45-branched
Kdo structures using the Kdo2-4) Kdo dimer which was prepared in Chapter 2
as common acceptor
Although many chemical syntheses of linear core LPSLOS have been
reported34 only Paulsen et al described the synthesis of the 45-branched Kdo
structure35 As shown in Figure 14 of Chapter 1 they installed the
L-glycero-D-manno-heptopyranosyl donor (Hep) on the 5-OH of the Kdo acceptor to
form a Hep(1-5)Kdo disaccharide and then linked a Kdo donor to the 4-OH of the
Kdo moiety to form Hep(1-5)[Kdo(2-4)]Kdo trisaccharide However some defects
limited the application of this approach Pre-protection and deprotection of 4-OH of
the Kdo moiety complicated the reaction Moreover due to the steric hindrance of
heptose in 5-position and fail of the stereo control the second glycosidation of
Hep(1-5)Kdo disaccharide with Kdo gave a low yield (37)
To solve these problems a new synthetic strategy different from Paulsenrsquos
method was proposed We prepared this 45-branched Kdo trisaccharides by
glycosylation of the 5-OH group of the 2-4 linked Kdo disaccharide which was
constructed in Chapter 2 Three types of 45-branched Kdo trisaccharides were
synthesized through this new route
32 Result and discussion
321 The glycosylation to synthesize 45-branched Kdo trisaccharides
Glycosylation of Kdo(2-4)Kdo acceptor 6 with L-glycero-D-manno-heptosyl
mannosyl and 2-azido-2-deoxy-galactosyl imidates 8ndash10 was examined (Scheme 31)
and the results are presented in Table 31
23
Scheme 31 Glycosylation of the dimeric acceptor 6 with donors 8ndash10
Table 31 Glycosylation of the dimeric acceptor 6with donors 8ndash10
Entry Donor Temp
(degC)
TMSOTf
(equiv)
Time (h) Product () 12 () 6
()
1 8 () 0 004 4 10 35 44
2 8 () rt 004 2 28 28 19
3 8 () rt 006 2 87 3 9
4 9 () 0 004 2 91 - -
5 10
(=13)
0 004 2 56 - 40
3211 The glycosidation of Hep donor with Kdo(2-4)Kdo
The reaction of heptosyl trichloroacetimidate 843 with acceptor 6 in the
presence of 004 equiv of TMSOTf at 0 degC gave the corresponding 45-branched
trisaccharide Hep(1-5)[Kdo(2-4)]Kdo (11) in only 10 yield and orthoester 12
was the major product (35) To reduce the formation of 12 the reaction temperature
was raised to room temperature (entry 2) Correspondingly the yield of orthoester 12
reduced to 28 and that of the target Hep(1-5)[Kdo(2-4)]Kdo (11) increased to
28 To suppress the formation of orthoester 12 further more TMSOTf should be
24
used because the orthoester could be transferred to 2-O-acyl glycosides in acid44
Increasing the amount of TMSOTf (entry 3) afforded trisaccharide 11 in good yield
(87) and only a small amount of orthoester 12 was detected (3)
The 45-branched structure of 11 was determined by 2D NMR analysis (COSY
HMQC and HMBC) From the COSY and HMBC spectra we were able to identify
the cyclic proton and carbon atoms of each residue of 11 The newly formed 1-5
linkage was identified by HMBC analysis Figure 31 shows part of the HMBC
spectrum The cross-relay peaks in the HMBC spectrum (Kdo H-5IHep C-1III Hep
H-1IIIKdo C-5I) confirmed that heptosyl donor 8 is linked to the 5-position of
acceptor 6 Moreover the anomeric configuration was determined from the 1JC-1H-1
value The coupling constant between H-1III and C-1III (1JH-1III
C-1III= 178 Hz) of the
Hep residue suggested that the newly formed glycosidic bond was an -linkage45
Thus the trisaccharide Hep(1-5)[Kdo(2-4)]Kdo was successfully synthesized
from the Kdo disaccharide with a heptosyl donor
Figure 31 Partial HMBC spectra of compound 11 in CDCl3 at 25 degC
In conjunction with the synthesis of Kdo disaccharide 6 in Chapter 2 the
25
preparation of Hep(1-5)[Kdo(2-4)Kdo] by our new route was supposed to be more
effective than Paulsenrsquos method Due to a new reaction sequence the absence of
4-OH protection shortened the reaction steps (from four steps to two steps) Without
the disturbance of Hep at the 5-position the Kdo(2-4)Kdo linkage could be smoothly
formed in good yield (72) Correspondingly the total yield of
Hep(1-5)[Kdo(2-4)]Kdo synthesis was improved to 63 (Paulsen 22)
3212 The glycosidationof Man donor with Kdo(2-4)Kdo
Following the synthesis of Hep(1-5)[Kdo(2-4)]Kdo (11) other 45-branched
Kdo trisaccharides were also synthesized by the same route with 6 as an acceptor
By coupling 6 with mannosyl trichloroacetimidate 946 branched trisaccharide 13
was also obtained in good yield (91) (entry 4) The effect of the participating group
(Ac) at the C-2 position meant that only -isomer which was identified by the
1JH-1III
C-1III value of the Man residue (175 Hz) was isolated45 In contrast for the
heptose derivative mannosylation proceeded smoothly at 0 degC with 004 equiv
TMSOTf and no orthoester was detected These results suggest that mannosyl donor
9 was more active than heptosyl donor 8
3213 The glycosidation of GalN3 donor with Kdo(2-4)Kdo
Glycosylation of Kdo dimer 6 with GalN3 trichloroacetimidate 1047 was
accomplished to give GalN3 containing branched trisaccharide 14 as a single isomer in
moderate yield (56) (entry 5) The coupling constant between H-1III and H-2III (JH-1
H-2= 34 Hz) of GalN3 residue indicated that an -glycosidic linkage was formed45
The position of azide group meant this glycosylation exploited the anomeric effect to
give the -isomer In addition the presence of acetyl groups at the 3- and 4-position
was also favorable for -isomer formation48 As observing from the Thin layer
chromatography (TLC) only -anomeric donor was consumed during the
glycosidation After the reaction -anomeric donor was almost recovered These
suggested that for GalN3 donor the -isomer is more active than the -isomer
Therefore these glycosylation results indicate that it is available to synthesize
26
the 45-branched Kdo trisaccharides by our new approach using Kdo(2-4)Kdo 6 as
common acceptor Moreover due to the steric hindrance in the Kdo(2-4)Kdo
acceptor the Man type donors (Hep 8 and Man 9) appear to be easier to couple with
the Kdo acceptor than then GalN3 donor
Next the transformation of the azide group to the original acetamide group in
trisaccharide 14 was carried out The azide group could not be converted directly to
the acetamide group by thioacetic acid (data not shown)49 Hence a stepwise
conversion was used (Scheme 32) Firstly the azide group of GalN3 14 was reduced
to the amine group under Staudinger conditions50 Then the amine group of 15 was
acetylated with anhydrous acetic acid in the presence of NN-dimethylaminopyridine
(DMAP) Finally GalNAc trisaccharide 16 was obtained in moderate yield (64)
Scheme 32 Conditions (a) Ph3P THFH2O= 191 rt 16 h (b) Ac2O DMAP pyridine rt 17 h
two steps 64
322 Full deprotection of 45-branched Kdo trisaccharides
In Final the deprotection of all synthetic Kdo trisaccharide 11 13 and 16 was
carried out Acid hydrolysis of the isopropylidene group of Kdo trisaccharide (11 13
and 16) with aqueous trifluoroacetic acid and subsequent hydrolysis in 01 M sodium
hydroxide to remove the ester groups afforded fully deprotected 45-branched Kdo
trisaccharides 17ndash19 as a disodium salt in good yield (Hep 17 87 Man 18 93
GalNAc 19 quantitative) (Scheme 33)
27
Scheme 33 Conditions (a) 80 TFA CH2Cl2 rt (b) 01 M NaOH MeOH two steps Hep 17
(87) Man 18 (93) GalNAc 19 (quant)
33 Conclusion
A new synthetic strategy using Kdo (2-4)Kdo 6 as an acceptor was developed
for the synthesis of 45-branched Kdo trisaccharides Glycosylation at the 4-OH
position of the Kdo acceptor followed by a second glycosylation at 5-OH position
produced the heptosyl Kdo dimer Hep(1-5)[Kdo(2-4)]Kdo (11) We also achieved
the first synthesis of the 45-branched partial inner-core trisaccharides
Man(1-5)[Kdo(2-4)]Kdo (13) from Francisella tularensis21 and
GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas cichorii22 in good yield and
high -selectivity This new route might provide another choice for the synthesis of
the inner-core oligosaccharides of LPSLOS with the Paulsenrsquos method
28
Chapter 4
The convergent synthesis of 45-branched
inner-core OSs
29
41 Introduction
The highly conserved inner-core OS consists of mostly the higher carbon sugars
3-deoxy-D-manno-2-octulosonic acid (Kdo) and L-glycero-D-manno-heptopyranose
(Hep) For example in Figure 41 the inner core OS of LPSsLOSs from many
gram-negative bacteria contains a 45-branched Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide as the common structure51 An R (R = Lac Glc P) residue is usually
substituted at the 4-O position of Hep I52
Figure 41 General inner-core structure of LPSLOS
In Chapter 3 a new synthetic method to prepare 45-branched inner-core
trisaccharides by coupling monosaccharides (Hep Man GalN3) with a common Kdo
acceptor 6 was introduced To extend the utility of this approach in this Chapter we
prepared more complex 45-banched inner core OS structures by using the same Kdo
disaccharide 6 as the acceptor A lactose donor was initially chosen as a model
compound to optimize the glycosylation conditions Based on the model glycosylation
the corresponding Hep units constructed from the Hep building blocks were coupled
with the Kdo moiety to obtain the desired branched inner-core OS
42 Result and discussion
421 Synthesis of Hep units
To install the Kdo moiety the Hep units Gal(1-4)Glc(1-4)Hep trisaccharide
and Hep(1-3)Hep disaccharide were prepared All the Hep building blocks (21 22
25) required for the Hep units were obtained from known methyl
67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside 2053 (Scheme 41)
30
4211 Synthesis of Hep building blocks
Treatment of 34-diol 20 with t-butyldimethylsilyl chloride (TBDMSCl) and
1H-imidazole in NN-dimethylformamide (DMF) at room temperature gave
3-O-TBDMS ether 21 in 94 yield The acetylation of 21 in acetic anhydride
(Ac2O)pyridine and subsequent de-O-silylation of TBDMS group in aqueous
trifluoroacetic acid gave 3-OH product 22 in 88 yield L-Glycero-D-manno-heptosyl
trichloroacetimidate 25 was also prepared from 34-diol 20 in a 69 yield over four
steps as follows sequential acetylation of 34-diol 20 acetolysis of 23 selective
anomeric deacetylation and treatment of hemiacetal 24 with trichloroacetonitrile in
the presence of potassium carbonate
Scheme 41 Conditions (a) TBDMSCl 1H-imidazole DMF rt 4 h 92 (b) (i) Ac2O DMAP
pyridine 0 degCrarr rt 17 h 95 (ii) 90 TFA aq rt 1 h 93 (c) Ac2O DMAP pyridine 0 degCrarr
rt 2 h 94 (d) (i) H2SO4 Ac2O AcOH rt 2 h 95 (ii) hydrazine acetate 0 degCrarr rt DMF 2 h
80 (e) Cl3CCN K2CO3 CH2Cl2 rt 22 h 96
4212 Synthesis of Lac(1-4)Hep unit
Glycosylation of 4-OH Hep building block 21 with hepta-O-acetyl--lactosyl
trichloroacetimidate 2654 using TMSOTf as the catalyst in CH2Cl2 proceeded
smoothly to afford (1-4)-linked Gal(1-4)Glc(1-4)Hep trisaccharide 2755 as a Hep
unit in 79 yield (Scheme 42) The glucosyl-(1-4)-heptose linkage in trisaccharide
27 was assigned as based on the coupling constant between H-1 and H-2 of the
glucose residue (3JH1H2= 80 Hz) Cleavage of the TBDMS group in
31
Gal(1-4)Glc(1-4)Hep trisaccharide 27 with aqueous trifluoroacetic acid produced
the free 3-OH 28 in 97 yield To characterize the effect of the protecting group of
donor moiety on the glycosidation two types of Gal(1-4)Glc(1-4)Hep donors (31
and 35) with the different protecting groups at C-2 of the Hep residue were prepared
from 28 respectively as follows Immediate acetylation of 28 with acetic anhydride
in pyridine gave 29 in 73 yield Acetolysis of 29 in H2SO4Ac2OAcOH and
subsequent selective anomeric deacetylation afforded hemiacetal 30
Gal(1-4)Glc(1-4)Hep hemiacetal 30 was transformed in quantitative yield to the
corresponding trichloroacetimidate 31 In addition to obtain a per-O-acetylated
Gal(1-4)Glc(1-4)Hep donor the benzyl group at C-2 of the Hep residue in 28 was
removed by hydrogenolysis (10 PdC in EtOAc) to give 23-diol 3255 in 97 yield
Acetylation of 32 with acetic anhydride in pyridine followed by acetolysis produced
33 in 67 yield Selective anomeric deacetylation of 33 with hydrazine acetate in
DMF at 0 degC gave hemiacetal 34 in 90 yield Treatment of 34 with
trichloroacetonitrile in the presence of K2CO3 gave per-O-acetylated
Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 in 92 yield
Gal(1-4)Glc(1-4)Hep trichloroacetimidates 31 and 35 were expected to undergo
[3+2] coupling with the Kdo moiety
32
Scheme 42 Conditions (a) TMSOTf CH2Cl2 MS AW 300 molecular sieves 0 degC 3 h 79 (b)
TFAH2O 91 rt 5 min 97 (c) Ac2O DMAP pyridine rt 2 h 73 (d) (i) H2SO4 Ac2O
AcOH rt 3 h 64 (ii) hydrazine acetate DMF 0 degC 8 h 77 (e) Cl3CCN K2CO3 CH2Cl2 rt
13 h quant (f) 10 PdC H2 ethyl acetate rt 35 h 97 (g) (i) Ac2O pyridine rt 24 h (ii)
H2SO4 Ac2O AcOH rt 15 h two steps 67 (h) hydrazine acetate DMF 0 degC 8 h 90 (i)
Cl3CCN K2CO3 CH2Cl2 rt 24 h 92
4212 Synthesis of Hep(1-3)Hep unit
The (1-3)-linked heptobiose unit 36 was prepared in 50 yield by glycosidation
of imidate 25 with Hep building block 22 by using TMSOTf as a promoter in CH2Cl2
(Scheme 43) The coupling constant between C-1 and H-1 (1JCH= 174 Hz) of
reducing heptose residue suggested the (1-3) linkage was an -linkage No -isomer
was detected Acetolysis of the methyl ether in 36 followed by selective cleavage of
the anomeric acetyl group with hydrazine acetate in DMF at 0 degC produced
disaccharide hemiacetal 37 in 78 yield over two steps Treatment of 37 with
trichloroacetonitrile in the presence of K2CO3 gave Hep(1-3)Hep trichloroacetimidate
38 which was expected to undergo [2+2] coupling with the Kdo moiety
Scheme 43 Conditions (a) TMSOTf CH2Cl2 4Aring molecular sieves -78 degCrarr rt 2 h 50 (b) (i)
H2SO4 Ac2O AcOH rt 2 h (ii) hydrazine acetate DMF 0 degC 7 h two steps 78 (c) Cl3CCN
K2CO3 CH2Cl2 rt 21 h 80 =61
422 Convergent synthesis of 45-branched inner-core OS structures
Next we focused on the glycosidation of the Hep units with the Kdo moiety
(Scheme 44 and Table 41) A model glycosylation using a lactose derivative as a
33
donor was performed to test this convergent approach
Scheme 44 The convergent synthesis of 45-branched inner-core OSs
Table 41 Glycosylation to synthesize 45-branched inner-core OSs
34
4221 Model glycosylation of lactose donor with Kdo disaccharide
Because the glycosylation of per-O-acetylated Hep imidate 8 could provide
Hep(1-5)[Kdo(2-4)]Kdo trisaccharide 11 in good yield (Chapter 3 Table 31)
per-O-acetylated lactosyl imidate 39 was used for coupling with Kdo(2-4)Kdo
acceptor 6 (Scheme 44 and Table 41) However no Lac-Kdo tetrasaccharide was
formed The reactivity of the per-O-acetylated lactose donor was too low to form the
linkage Therefore a more reactive donor hepta-O-benzyl--lactosyl
trichloroacetimidate 4056 was examined To increase the -selectivity in the
lactosylation the reaction was carried out in CH2Cl2Et2O57 and gave branched
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 in only 20 yield as only a
single isomer The coupling constant between H-1 and H-2 of the glucose residue
(3JH1H2= 34 Hz) indicated that the (1-5) linkage was an -linkage The high
stereoselectivity was due to the anomeric effect58 and the solvent effect59 The
introduction of benzyl ethers meant that imidate 40 was more effective in providing
desired tetrasaccharide 41 despite the high steric hindrance
4222 The convergent glycosylation of Lac(1-4)Hep unit with Kdo disaccharide
Following the model glycosylation the [3+2] coupling of the Lac(1-4)Hep
unit with the Kdo moiety was examined According to the glycosidation results of
both Hep donor 8 and 25 giving products in high -selectivity in CH2Cl2 CH2Cl2 was
used as a solvent for the following heptosylation The synthesis of
Lac(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide by coupling of
per-O-acetylated Gal(1-4)Glc(1-4)Hep trichloroacetimidate 35 with Kdo acceptor
6 failed No branched pentasaccharide was found and mainly imidate 35 was
recovered even though the reaction time was extended to 15 h In addition the
decomposition of the acceptor 6 to lactone 42 was observed The donor was changed
to Gal(1-4)Glc(1-4)Hep imidate 31 which contained a benzyl group at C-2 of the
Hep residue and desired Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo
pentasaccharide 43 was obtained in a 26 yield as only the -anomer The anomeric
configuration of the Hep residue in pentasaccharide 43 was confirmed by the coupling
35
constants between C-1 and H-1 of the heptose residue (1JCH= 174 Hz) This was
consistent with the results of the model glycosylation which indicated that the
reactivity of the donor is important for this convergent approach The introduction of
an benzyl ether at C-2 of the Hep residue increased the reactivity of the
Gal(1-4)Glc(1-4)Hep donor to provide the branched pentasaccharide To increase
the pentasaccharide yield further the strategy of benzylating the 3-OH of the heptose
residue was considered Therefore in our convergent approach the sterically crowded
heptose unit can be added to the Kdo moiety to produce the desired 45-branched core
OS structures This approach was also expected to provide the common inner-core OS
structure containing the heptobiose unit
4223 The convergent glycosylation of Hep(1-3)Hep unit with Kdo dimer
Dibenzyl Hep(1-3)Hep trichloroacetimidate 38 was coupled with
Kdo(2-4)Kdo acceptor 6 by using 006 equiv of TMSOTf as the activator As
expected the introduction of the dibenzyl group substantially increased the reactivity
of the Hep(1-3)Hep unit to provide Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 in moderate yield (57) as the -anomer The configuration of
tetrasaccharide 44 was confirmed by the coupling constants between C-1 and H-1 of
the corresponding heptoses (1JC H= 172 174 Hz)
Furthermore all branched structures we synthesized were characterized by
analyzing the corresponding 2D NMR spectra (COSY HMQC and HMBC) For
example the existence of the (1-5) linkage in Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
tetrasaccharide 44 was supported by the HMBC analysis The cross-relay peaks Kdo
H-5IHep C-1III Hep H-1IIIKdo C-5I in the HMBC spectrum (Figure 42) confirmed
that the Hep unit is linked to the 5-position of the Kdo moiety
These results suggest that it is possible to obtain complex 45-branched inner
core OSs of LPSLOS using common Kdo dimmer 6 as an acceptor via a convergent
approach The Lac-Hep imidate 31 with a benzyl group at C-2 of the reducing residue
other than the Lac-Hep peracetate 35 giving the desired product of glycoside
indicates that the effective improvement of the reactivity is supported by the benzyl
36
group Meanwhile the glycosidation of perbenzylated Lac imidate 40 giving less
product might indicate that the perbenzylated donor should be too active to obtain the
glycoside in good yield These results suggest that the introduction of appropriate
number of benzyl protecting groups appears to be important for the yield of this
convergent glycosylation
Figure 42 Partial HMBC spectrum of tetrasaccharide 44 in CDCl3 at 25 degC
423 Full deprotection of 45-branched Kdo tetra- and pentasaccharide
Finally the deprotection of all synthetic Kdo trisaccharide 41 43 and 44 was
carried out As shown in Scheme 45 deprotection of
Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide 44 was performed over three
steps Pd(OH)2C-promoted hydrolysis of the benzyl groups acid hydrolysis of the
isopropylidene group with aqueous trifluoroacetic acid and hydrolysis of the ester
group in 01 M NaOH produced the target 45-branched tetrasaccharide 45 in 90
yield as the disodium salt
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide 43 and
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo tetrasaccharide 41 were subjected to similar
deprotection to afford the corresponding deprotected compounds 46 (53) and 47
37
(60)
Scheme 45 Conditions (a) Pd(OH)2C H2 MeOH rt (b) 80 TFA aq CH2Cl2 rt (c) 01 M
NaOH MeOH three steps 41 (90) 42 (53) 43 (60)
43 Conclusion
The convergent synthetic strategy using Kdo(2-4)Kdo as a common acceptor
was used to prepare more complex 45-branched inner core OS structures Model
glycosylation using a lactose derivative as a test compound suggested that the
reactivity of the donor was important for this convergent synthesis and this was
supported by the subsequent glycosylation Based on the convergent approach the
first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo These results suggested
that it is a available to synthesize complex 45-branced inner-core OS structures by
our new approach and Kdo(2-4)Kdo 6 is a useful intermediate for these synthesis
38
Chapter 5
Conclusion
39
In this study a new synthetic approach using a common Kdo(2-4)Kdo
disaccharide as acceptor to synthesize the 45-branched inner-core OS structures was
described Using this new approach several types of 45-branced Kdo trisaccharides
tetrasaccharides and pentasaccharides were successfully synthesized in good yield
and high -selectivity
In the Chapter 2 to optimize the condition of glycosylation several types of
glycosyl donors with different leaving group and stereoselectivity were prepared from
common Kdo intermediate and were glycosylated with 45-diol acceptor The results
showed that all donors produced the -glycoside as the main product and the
stereoselectivity was not influenced by the type of leaving group Moreover the
-fluoride donor with BF3middotOEt2 as the activator provided the best yield and
-selectivity of product
In the Chapter 3 using the constructed Kdo(2-4)Kdo disaccharide as the
common acceptor three types of 45-branched Kdo trisaccharides were successfully
synthesized in good yield and high -selectivity The glycosylation condition of
Hep(1-5)[Kdo(2-4)]Kdo was discussed and the result seemed to be better than that
of the Paulsenrsquos method in the yield and stereoselectivity The first synthesis of the
45-branched partial inner-core trisaccharides Man(1-5)[Kdo(2-4)]Kdo (13) from
Francisella tularensis and GalN3(1-5)[Kdo(2-4)]Kdo (14) from Pseudomonas
cichorii were also achieved These results suggest that it is available to synthesize the
45-branched Kdo structures by the new reaction sequence of glycosylation at the
4-OH position of the Kdo acceptor followed by a second glycosylation at 5-OH
position This new route should be more effective for the synthesis of the inner-core
oligosaccharides of LPSLOS than Paulsenrsquos method
In the Chapter 4 to extend the utility of the new synthetic strategy more
complex 45-branched inner-core OS structures were synthesized With the same
Kdo(2-4)Kdo disaccharide as acceptor three types of 45-branched Kdo tetra- and
pentasaccharides were synthesized in high -selectivity Model glycosylation using a
lactose derivative as a test compound suggested that the reactivity of the donor was
40
important for this convergent synthesis and this was supported by the subsequent
glycosylation The first synthesis of 45-branched inner-core OSs namely
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo pentasaccharide and a common
inner-core Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo tetrasaccharide was accomplished
by coupling the corresponding Hep units with Kdo(2-4)Kdo
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched inner-core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide It would provide
another synthetic choice to obtain 45-branced inner-core OSs with Paulsenrsquos method
41
Chapter 6
Experimental section
42
61 General procedures
Optical rotation was measured with a Horiba SEPA500 polarimeter in CHCl3
and melting point (uncorrected) was measured with a Yanagimoto micro melting
point apparatus All NMR spectra were recorded at 25 degC in CDCl3 or D2O on a 600
MHz NMR spectrometer (Avance II Bruker) All NMR chemical shifts () were
recorded in parts per million (ppm) and coupling constants (J) were reported in hertz
(Hz) Mass spectrometry (MS) was performed by positive- and negative-mode
electrospray ionization on a Waters LCT Premier spectrometer For high-precision
measurements the spectra were obtained by scanning the voltage over a narrow mass
range at a resolution of 10000 MALDI-TOF spectra were recorded on a Bruker
Daltonics instrument using 35-dihydroxybenzoic acid as the matrix Elemental
analysis was carried out on a performed on Vario ELCUBE and Vario EL III
Elementar Infrared spectra were determined on a JASCO FTIR-4100 Spectrometer
Analytical TLC was performed on Merck silica gel 60 F254 glass plates The TLC
plates were visualized with UV light and by staining with Hannessian solution (ceric
sulfate and ammonium molybdate in aqueous sulfuric acid) and then heating at
200 degC for 3 min Column chromatography was performed on silica gel 60 (flash
column 0040ndash0063 mm open column 0063ndash0200 mm)
62 Methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2-octulopy
-ranosyl N-phenyl trifluoroacetimidate)onate (3)
Compound 1 (2500 mg 05 mmol) was dissolved in dry dichloromethane (50
mL) under argon N-phenyl trifluoroacetimidoyl chloride60 (7160 L 50 mmol) and
potassium carbonate (1130 mg 50 mmol) was added into the reaction After stirring
for 1 week the mixture was filtered through Celite The solution was concentrated
and purified by silica gel column chromatography (ethyl acetatetoluene 15) to give
3 in quantitative yield (3430 mg =32) -isomer []25D = +756 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 124 (s 3H Me) 150 (s 3H Me) 233 (1 H
J3a3e=156 Hz J3a4=34 Hz H-3a) 278 (dd 1H J3a 3e=156 Hz J3e4=40 Hz H-3e)
378 (s 3H OMe) 437 (dd 1H J5 6=20 Hz J67=86 Hz H-6) 441 (dd 1H J45=76
43
Hz J56=20 Hz H-5) 462 (ddd 1H J3a4=34 Hz J3e4=40 Hz J45=76 Hz H-4)
472 (dd 1H J78a=40 Hz J8a8b=124 Hz H-8a) 500 (dd 1H J78b=24 Hz
J8a8b=124 Hz H-8b) 575 (ddd 1H J67=86 Hz J78a=40 Hz J78b=24 Hz H-7)
674-675 (m 2H NPh-Ar) 705 (m 1H NPh-Ar) 724ndash725 (ddd 2H NPh-Ar)
737-744 (m 4H Ar) 751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150
MHz CDCl3) 248 256 (CH3) 330 (C-3) 528 (OCH3) 626 (C-8) 695 (C-4)
701 (C-7) 702 (C-6) 708 (C-5) 990 (C-2) 1101 (Cisop) 1165 (CF3) 1191
(NPh-Ar) 1243 (NPh-Ar) 1284 1285 1287 1294 1296 1297 1298 1331
1333 (14 C NPh-Ar) 1432 (C=N) 1651 1662 (C=O) 1681 (C-1) IR 1736 1727
1229 1215 1202 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941873 -isomer []25D = +66 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
129 (s 3H Me) 154 (s 3H Me) 234 (dd 1H J3a3e=162 Hz J3a4=34 Hz H-3a)
278 (dd 1H J3a 3e=162 Hz J3e4=30 Hz H-3e) 366 (s 3H OMe) 426 (dd 1H J5
6=18 Hz J67=80 Hz H-6) 444 (dd 1H J45=82 Hz J56=18 Hz H-5) 468 (ddd
1H J3a4=34 Hz J3e4=30 Hz J45=82 Hz H-4) 470 (dd 1H J78a=52 Hz
J8a8b=124 Hz H-8a) 497 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8b) 568-571
(ddd 1H J67=86 Hz J78a=52 Hz J78b=24 Hz H-7) 674ndash675 (dd 2H NPh-Ar)
709 (m 1H NPh-Ar) 727ndash728 (ddd 2H NPh-Ar) 739ndash744 (m 4H Ar)
751ndash757 (m 2H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 256
258 (CH3) 297 (C-3) 528 (OCH3) 629 (C-8) 692 (C-4) 705 (C-7) 709 (C-5)
715 (C-6) 993 (C-2) 1100 (Cisop) 1146 (CF3) 1193 (NPh-Ar) 1243 (NPh-Ar)
1284 1285 1286 1288 1296 1297 1298 12983 1300 1340 1332 (14 C
NPh-Cmeta and Ar) 1432 (C=N) 1652 1661 (C=O) 1679 (C-1) IR 1736 1725
1230 1214 1205 cm-1 ESI-HRMS for C34H32F3NO10 6941876 [M+Na]+ Found
6941855
63 Methyl (dibenzyl-78-di-O-benzoyl-45-O-isopropylidene-3-deoxy-D-manno-2
-octulopyranosyl phosphite)onate (4)
1H-tetrazole (1120 mg 16 mmol) was added to a solution of 1 (2000 mg 04
mmol) in dry dichloromethane (130 mL) under argon Then the reaction mixture was
cooled to 0 ˚C and treated with dibenzyl NN-diisopropylphosphoramidite (DDP 322
44
L096 mmol) After stirring for 3 h the solution was quenched with triethylamine
(Et3N) and concentrated The residue was purified by silica gel column
chromatography (ethyl acetate hexane 23+1 Et3N) to give a mixture of anomers 4
in 56 yield (1670 mg =231) -isomer []25D = +338 (c 11 CHCl3)
1H-NMR (600 MHz CDCl3) 120 143 (s 2H CH3) 206 (dd 1H J3a3e=150 Hz
J3a4=34 Hz H-3a) 282 (dd 1H J3a3e=150 Hz J3e4=52 Hz H-3e) 370 (s 3H
OMe) 425 (dd 1H J45=70 Hz J56=20 Hz H-5) 434 (dd 1H J56=20 Hz J67=74
Hz H-6) 447 (ddd 1H J3a4=34 Hz J3e4=52 Hz J45=70 Hz H-4) 466 (dd 1H
J78a=60 Hz J8a8b=124 Hz H-8a) 477 (dd 1H J =124 and 78 Hz OCH2Ph) 481
(d 2H J=78 Hz OCH2Ph) 483 (dd 1H J=122 and 84 Hz OCH2Ph) 500 (dd 1H
J78b=24 Hz J8a8b=124 Hz H-8b) 574 (ddd 1H J67=74 Hz J78a=60 Hz J78b=24
Hz H-7) 718ndash754 (m 16H Ar) 797ndash803 (m 4H Ar) 13C NMR (150 MHz
CDCl3) 250 260 (CH3) 337 (C-3) 527 (OCH3) 633 (C-8) 642 6420 646
647 (2C OCH2) 698 (C-4) 706 (C-7) 707 (C-6) 714 (C-5) 973 9733 (C-2)
1097 (Cisop) 1276 1277 12772 1281 1283 12837 1284 1287 1298 1299
1301 1329 1331 1379 1380 1380 (Ar) 1653 1662 (C=O) 1687 (C-1) IR
1749 1713 1282 1252 1213 973 cm-1 ESI-HRMS for C40H41O12P 7672233
[M+Na]+ Found 7672222 -isomer []25D = +234 (c 10 CHCl3)
1H-NMR (600
MHz CDCl3) 121 142 (s 2H CH3) 240 (dd 1H J3a3e=152 Hz J3a4=30 Hz
H-3a) 294 (dd 1H J3a3e=152 Hz J3e4=56 Hz H-3e) 373 (s 3H OMe) 435 (dd
1H J45=76 Hz J56=20 Hz H-5) 452 (ddd IH J3a4=30 Hz J3e4=56 Hz J45=76
Hz H-4) 465 (dd 1H J56=20 Hz J67=74 Hz H-6) 466 (dd 1H J78a=70 Hz
J8a8b=124 Hz H-8a) 495ndash502 (m 4H OCH2) 502 (dd 1H J78b=24 Hz
J8a8b=124 Hz) 578 (ddd 1H J67=74 Hz J78a=70 Hz J78b=24 Hz H-7) 725ndash753
(m 16H Ar) 798ndash803 (m 4H Ar) 13C NMR (150 MHz CDCl3) 246 254
(CH3) 322 (C-3) 530 (OCH3) 635 (C-8) 695 (C-4) 6955 696 6962 697 (2C
OCH2) 703 (C-7) 713 (C-5) 721 (C-6) 1000 (C-2) 1098 (Cisop) 1281 1282
1283 1284 12842 12846 12848 1285 1297 12975 1298 1301 1328 1331
1356 (Ar) 1653 1662 (C=O) 1672 (C-1) IR 1748 1717 1253 1213 954 cm-1
ESI-HRMS for C40H41O12P 7672233 [M+Na]+ Found 7672223
45
64 (23467-Penta-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (11)
A mixture of 6 (320 mg 326 mol) imidate 8 (550 mg 987 mol) and
MS-AW 300 (400 mg) was suspended in dichloromethane (10 mL) The reaction
mixture was stirred for 1 h under argon and then 001 M TMSOTf (1960 L 196
mol) in dichloromethane was added dropwise to the reaction mixture After stirring
for 2 h the reaction was neutralized by the addition of a few drops of triethylamine
and aqueous saturated sodium hydrogen carbonate The reaction mixture was diluted
with dichloromethane and was filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtrated and concentrated The residue was purified by BioRad S-X3
(tolueneethyl acetate 11) to give compound 11 (39 mg 87) as colorless syrup Mp
882 ˚C []25D = +197 (c 15 CHCl3)
1H-NMR (600 MHz CDCl3) 120 (s 3H
Me) 138 (s 3H Me) 193 202 203 214 (s 3H x 5 Ac) 205 (dd 1H J3a3e=126
Hz J3a4=120 Hz H-3aI) 212 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 227 (dd
1H J3a3e=126 Hz J3e4=44 Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz
H-3eII) 343 (s 3H OMeI) 347 (s 3H OMeII) 373 (brs 1H J45=20 Hz J56=18
Hz H-5I) 393 (dddd 1H J=16 16 48 130 Hz OCH2-) 397 (dddd 1H J=14
16 54 130 Hz OCH2-) 412 (dd 1H J56=18 Hz J67=100 Hz H-6I) 422 (dd
1H J45=100 Hz J56=80 Hz H-5III) 423 (dd 1H J67a=24 Hz J7a7b=120 Hz
H-7aIII) 426 (dd 1H J67b=40 Hz J7a7b=120 Hz H-7b
III) 428 (dd 1H J56=20 Hz
J67=74 Hz H-6II) 437 (dd 1H J45=80 Hz J56=20 Hz H-5II) 452 (ddd 1H
J3a4=22 Hz J3e4=36 Hz J45=80 Hz H-4II) 460 (dd 1H J78a=50 Hz J8a8b=126
Hz H-8aII) 465 (ddd 1H J3a4=120 Hz J3e4=44 Hz J45=20 Hz H-4I) 469 (dd 1H
J7 8a=36 Hz J8a 8b=126 Hz H-8aI) 488 (dd 1H J7 8b=26 Hz J8a 8b=126 Hz H-8b
I)
493 (dddd 1H J=14 16 30 106 Hz =CH2) 501 (dddd 1H J=16 16 32 172
Hz =CH2) 504 (d 1H J12=20 Hz H-1III) 524 (dd 1H J12=20 Hz J23=32 Hz
H-2III) 528 (dd 1H J78b=24 Hz J8a8b=126 Hz H-8bII) 532 (dd 1H J34=102 Hz
46
J45=100 Hz H-4III) 537 (ddd 1H J56=80 Hz J67a=24 Hz J67b=40 Hz H-6III)
551 (dd 1H J23=32 Hz J34=102 Hz H-3III) 562-568 (m 1H -CH=) 565 (ddd
1H J67=74 Hz J78a=50 Hz J78b=24 Hz H-7I) 569 (ddd 1H J67=100 Hz
J78a=36 Hz J78b=26 Hz H-7I) 737ndash745 (m 8H ArI ArII) 751ndash756 (m 4H ArI
ArII) 793ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2074 208
and 211 (Ac-CH3) 247 and 251 (Isop-Me) 319 (C-3II) 344 (C-3I) 5225 (OMeI)
5228 (OMeII) 624 (C-8I) 625 (C-8II) 637 (C-7III) 647 (OCH2-) 652 (C-4III) 674
(C-6III) 676 (C-4I) 687 (C-3III) 692 (C-7I) 692 (C-5III) 700 (C-4II) 703 (C-2III)
704 (C-6II) 705 (C-6I) 707 (C-7II) 723 (C-5I) 740 (C-5II) 970 (C-2II) 987 (C-2I)
990 (C-1III) 1097 (Cisop) 1162 (=CH2) 1283 1284 1285 1293 1296 12985
1299 1302 1328 1331 (ArI and ArII) 1334 (-CH=) 1651 1652 1658 1674
(Bz C=O) 1674 (C-1I) 1692 (C-1II) 1696 1697 1698 1704 and 1707 (Ac
C=O) IR 1745 1725 1278 1248 1218 cm-1 Anal Calcd for C69H76O30 C 5982 H
553 Found C 5965 H 567 Ortho ester 12 Mp 990 ˚C []25D = +75 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 140 (s 3H Me) 165 (s
3H ester-Me) 191 (dd 1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 197 203 204
207 (s 3H x 4 Ac) 231 (dd 1H J3a3e=126 Hz J3a4=124 Hz H-3aI) 247 (dd 1H
J3a3e=126 Hz J3e4=40 Hz H-3eI) 286 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII)
338 (dd 1H J45=96 Hz J56=20 Hz H-5III) 343 (s 3H OMeI) 381 (s 3H OMeII)
386 (dddd 1H J=16 16 34 134 Hz OCH2-) 397 (dddd 1H J=16 16 32
134 Hz OCH2-) 405 (dd 1H J56=nd Hz J67=86 Hz H-6I) 409 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aIII) 411 (brs 1H J45=24 Hz J56=nd Hz H-5I) 415 (dd
1H J67b=48 Hz J7a7b=116 Hz H-7bIII) 421 (dd 1H J56=18 Hz J67=70 Hz
H-6II) 431 (dd 1H J45=76 Hz J56=18 Hz H-5II) 433 (ddd 1H J3a4=124 Hz
J3e4=40 Hz J45=24 Hz H-4I) 447 (ddd 1H J3a4=22 Hz J3e4=36 Hz J45=76 Hz
H-4II) 450 (dd 1H J12=22 Hz J23=38 Hz H-2III) 460 (dd 1H J78a=46 Hz
J8a8b=124 Hz H-8aII) 464 (dd 1H J7 8a=34 Hz J8a 8b=126 Hz H-8aI) 465 (d 1H
J12=22 Hz H-1III) 494 (dddd 1H J=14 14 30 106 Hz =CH2) 500 (dd 1H J7
8b=24 Hz J8a 8b= 126 Hz H-8bI) 502 (dd 1H J23=38 Hz J34=100 Hz H-3III)
505 (dddd 1H J=16 16 34 172 Hz =CH2) 511 (dd 1H J34=100 Hz J45=96
47
Hz H-4III) 514 (ddd 1H J56=20 Hz J67a=78 Hz J67b=48 Hz H-6III) 524 (dd 1H
J78b=24 Hz J8a8b =124 Hz H-8bII) 538 (ddd 1H J67=86 Hz J78a=34 Hz
J78b=24 Hz H-7I) 559ndash564 (m 1H -CH=) 565 (ddd 1H J67=70 Hz J78a=46 Hz
J78b=24 Hz H-7II) 739-747 (m 8H ArI ArII) 754-755 (m 4H ArI ArII)
795ndash801 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 206 206 207
(Ac-CH3) 247 and 253 (Isop-Me) 263 (ester-Me) 326 (C-3II) 343 (C-3I) 522
(OMeI) 525 (OMeII) 622 (C-8II) 624 (C-8I) 625 (C-7III) 642 (OCH2-) 646
(C-4III) 668 (C-6III) 675 (C-5I) 692 (C-4I) 698 (C-4II) 702 (C-6II) 703 (C-3III)
704 (C-7I) 7096 (C-7II) 7096 (C-6I) 711 (C-5III) 721 (C-5II) 762 (C-2II) 972
(C-1III) 985 (C-2I) 988 (C-2II) 1097 (Cisop) 1156 (=CH2) 1248 (eater-C) 12840
12843 1285 1295 1297 1298 1299 1300 1304 1330 13307 13314 (ArI
and ArII) 1337 (-CH=) 1651 1653 1659 1662 (Bz C=O) 1675 (C-1I) 1694
(C-1II) 1701 17017 17024 and 1706 (Ac C=O) IR 1746 1724 1279 1248
1219 cm-1 ESI-HRMS for C69H76O30 14074319 [M+Na]+ Found 14074302
65 (2346-Tetra-O-acetyl--D-mannopyranosyl)-(1-5)-[methyl O-[methyl (78-
di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyranosyl)onate]]
-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyranosid)onate (13)
A reaction mixture of imidate 9 (752 mg 1526 mol) 6 (500 mg 510 mol)
and MS-AW 300 (578 mg) was suspended in dichloromethane (14 mL) The reaction
was stirred for 1 h under argon and then cooled to 0 degC 001 M TMSOTf (2000 L
200 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of triethylamine and
saturated sodium hydrogen carbonate The reaction solution was diluted with
dichloromethane and then filtered through Celite The filtrate was extracted twice
with dichloromethane The combined organic layer was dried over Na2SO4 filtrated
and concentrated The residue was purified by BioRad S-X3 (tolueneethyl acetate
11) to give 13 (609 mg 91) as colorless powder Mp 855 ˚C []25D = +375 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 121 (s 3H Me) 138 (s 3H Me) 185 (dd
1H J3a3e=154 Hz J3a4=22 Hz H-3aII) 200 202 205 216 (s 3H x 4 Ac) 223
(dd 1H J3a3e=126 Hz J3a4=122 Hz H-3aI) 228 (dd 1H J3a3e=126 Hz J3e4=48
48
Hz H-3eI) 295 (dd 1H J3a3e=154 Hz J3e4=36 Hz H-3eII) 342 (s 3H OMeI)
356 (s 3H OMeII) 378 (brs 1H J45=22 Hz J56=16 Hz H-5I) 392 (dddd 1H
J=16 16 50 130 Hz OCH2-) 397 (dddd 1H J=14 14 54 130 Hz OCH2-)
409 (dd 1H J56a=20 Hz J6a6b=122 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=94
Hz H-6I) 418 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (ddd 1H J45=100 Hz
J56a=20 Hz J56b=32 Hz H-5III) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II) 439
(dd 1H J56b=32 Hz J6a6b=122 Hz H-6bIII) 452 (ddd 1H J3a4=22 Hz J3e4=36
Hz J45=78 Hz H-4II) 463 (dd 1H J78a=52 Hz J8a8b =126 Hz H-8aII) 463 (dd
1H J7 8a=38 Hz J8a 8b=126 Hz H-8aI) 473 (ddd 1H J3a4=118 Hz J3e4=48 Hz
J45=22 Hz H-4I) 482 (d 1H J12=22 Hz H-1III) 482 (dd 1H J7 8b=24 Hz J8a
8b=126 Hz H-8bI) 492 (dddd 1H J=14 16 28 106 Hz =CH2) 501 (dddd 1H
J=14 16 32 172 Hz =CH2) 530 (dd 1H J78b=24 Hz J8a8b =126 Hz H-8bII)
534 (dd 1H J34=34 Hz J45=100 Hz H-4III) 540 (dd 1H J23=110 Hz J34=34
Hz H-3III) 542 (dd 1H J12=34 Hz J23=110 Hz H-2III) 546 (ddd 1H J67=94 Hz
J78a=38 Hz J78b=24 Hz H-7I) 567 (ddd 1H J67=72 Hz J78a=52 Hz J78b=24 Hz
H-7II) 563ndash570 (m 1H -CH=) 737ndash744 (m 8H ArI ArII) 752ndash757 (m 4H ArI
ArII) 794ndash799 (m 8H ArI ArII) 13C NMR (150 MHz CDCl3) 207 2076 2078
and 209 (Ac-CH3) 246 and 251 (Me) 246 (C-3II) 251 (C-3I) 522 (OMeI) 524
(OMeII) 618 (C-6III) 622 (C-8II) 623 (C-8I) 648 (OCH2-) 660 (C-4III) 675
(C-4I) 687 (C-5III) 689 (C-7I) 692 (C-2III) 696 (C-4II) 698 (C-3III) 705 (C-6I)
706 (C-6II) 708 (C-7II) 722 (C-5II) 725 (C-5I) 967 (C-2II) 985 (C-1III) 987
(C-2I) 1098 (Cisop) 1163 (=CH2) 12836 12841 12846 12850 1290 12962
12964 1297 1298 13017 and 13315 (ArI and ArII) 13347 (-CH=) 1648 1653
1658 1662 (Bz C=O) 1673 (C-1I) 1687 (C-1II) 16956 16961 1698 and 1708
(Ac C=O) IR (neat) 1746 1724 1278 1249 1217 cm-1 ESI-HRMS calcd for
C66H72O28 13354108 [M+Na]+ Found 13354097
66 (346-Tri-O-acetyl-2-azido-2-deoxy--D-galactopyranosyl)-(1-5)-[methyl O-
[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octulopyr
-anosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulopyrano
-sid)onate (14)
49
A reaction mixture of 6 (500 mg 510 mol) imidate 10 (726 mg 1526
mol) and MS-AW 300 (578 mg) in dichloromethane (14 mL) was stirred for 1 h
under argon and cooled to 0 degC Then 001 M TMSOTf (200 L 200 mol) in
dichloromethane was added dropwise to the reaction mixture and stirred for 4 h The
solution was neutralized by the addition of triethylamine and saturated NaHCO3 and
diluted with dichloromethane The reaction mixture was filtered through Celite and
the filtrate was extracted with dichloromethane The organic phase was dried over
Na2SO4 filtered and concentrated Purification of the residue by BioRad S-X3
(tolueneethyl acetate 11) obtained compound 14 (380 mg 58) as colorless
powder Mp 960 ˚C []25D = +510 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3)
121 (s 3H Me) 139 (s 3H Me) 192 (dd 1H J3a3e=154 Hz J3a4=22 Hz
H-3aII) 196 206 211 (s 3H x 3 Ac) 221 (dd 1H J3a3e=126 Hz J3a4=124 Hz
H-3aI) 227 (dd 1H J3a3e=126 Hz J3e4=46 Hz H-3eI) 301 (dd 1H J3a3e=156 Hz
J3e4=36 Hz H-3eII) 332 (s 3H OMeI) 359 (s 3H OMeII) 374 (dd 1H J12=34
Hz J23=110 Hz H-2III) 383 (brs 1H J45=20 Hz J56=nd Hz H-5I) 395 (dddd 1H
J=54 and 130 Hz OCH2-) 397 (dddd 1H J=50 and 130 Hz OCH2-) 408 (dd 1H
J56a=55 Hz J6a6b=108 Hz H-6aIII) 414 (dd 1H J56=nd Hz J67=96 Hz H-6I)
415 (dd 1H J56b=90 Hz J6a6b=108 Hz H-6bIII) 421 (dd 1H J56=16 Hz J67=76
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 452 (ddd 1H J3a4=22 Hz
J3e4=36 Hz J45=78 Hz H-4II) 454 (ddd 1H J45=08 Hz J56a=55 Hz J56b=90 Hz
H-5III) 462 (dd 1H J78a=42 Hz J8a8b=126 Hz H-8aII) 464 (dd 1H J7 8a=30 Hz
J8a 8b=120 Hz H-8aI) 479 (ddd 1H J3a4=124 Hz J3e4=46 Hz J45=20 Hz H-4I)
491 (dddd 1H J=100 and 14 Hz =CH2) 499 (dddd 1H J=100 and nd Hz =CH2)
500 (dd 1H J7 8b=26 Hz J8a 8b=120 Hz H-8bI) 505 (d 1H J12=34 Hz H-1III)
532 (dd 1H J23=110 Hz J34=32 Hz H-3III) 533 (dd 1H J78b=25 Hz J8a8b=126
Hz H-8bII) 548 (dd 1H J34=32 Hz J45=08 Hz H-4III) 563-567 (m 1H -CH=)
567 (ddd 1H J67=76 Hz J78a=42 Hz J78b=25 Hz H-7II) 570(ddd 1H J67=96
Hz J78a=30 Hz J78b=26 Hz H-7I) 736ndash739 (m 2H ArI ArII) 742ndash745 (m 3H
ArI ArII) 751ndash758 (m 4H ArI ArII) 794ndash803 (m 8H ArI ArII) 13C NMR (150
MHz CDCl3) 206 and 207 (Ac-CH3) 246 and 251 (Me) 319 (C-3II) 344 (C-3I)
521 (OMeII) 522 (OMeI) 582 (C-2III) 604 (C-6III) 620 (C-8II) 622 (C-8I) 648
(OCH2-) 666 (C-5III) 671 (C-4III) 677 (C-4I) 688 (C-3III) 694 (C-7I) 699 (C-4II)
704 (C-6I C-6II) 707 (C-7II) 720 (C-5I) 722 (C-5II) 966 (C-2II) 979 (C-1III) 982
(C-2I) 1098 (Cisop) 1164 (=CH2) 1284 12847 12852 1295 12965 12972
50
1298 1302 1329 1331 and 1335 (ArI and ArII) 13315 (-CH=) 1652 1658
1662 (Bz C=O) 1671 (C-1I) 1690 (C-1II) 1697 1701 and 1702 (Ac C=O) IR
(neat) 2112 1748 1723 1278 1251 1218 cm-1 ESI-HRMS for C64H69N3O26
13184067 [M+Na] Found 13184072
67 (346-Tri-O-acetyl-2-acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[methy
-l O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-2-octu
-lopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-octulop-
yranosid)onate (16)
Triphenylphosphine (62 mg 237 mol) was added to a solution of 14 (237
mg 183 mol) dissolved in a mixed solvent (THFH2O 191 036 mL) After
stirring for 16 h the reaction mixture was diluted with toluene and concentrated by
evaporation to give a residue 15 Without purification the residue was directly
acetylated with pyridineAc2O (1004 vv 1900 L) in the presence of a catalytic
amount of DMAP over 18 h After removing the solvent the residue was purified by
TLC (CH2Cl2EtOAchexane 331) to give compound 16 (154 mg 64) as
colorless powder Mp 990 ˚C []25D = +621 (c 12 CHCl3)
1H-NMR (600 MHz
CDCl3) 120 (s 3H Me) 137 (s 3H Me) 188 (dd 1H J3a3e=156 Hz J3a4=20
Hz H-3aII) 197 200 214 (s 3H x 3 Ac) 202 (s 3H NHAc) 218 (dd 1H
J3a4=118 Hz J3a3e=130 Hz H-3aI) 223 (dd 1H J3e4=56 Hz J3a3e=130 Hz H-3eI)
302 (dd 1H J3a3e=156 Hz J3e4=34 Hz H-3eII) 335 (s 3H OMeI) 343 (s 3H
OMeII) 374 (brs 1H J45=20 Hz J56=16 Hz H-5I) 383 (dddd 1H J=14 16 48
and 110 Hz OCH2-) 390 (dddd 1H J=16 16 60 and 124 Hz OCH2-) 406 (dd
1H J56a=50 Hz J6a6b=108 Hz H-6aIII) 411 (dd 1H J56=16 Hz J67=98 Hz H-6I)
414 (dd 1H J56b=94 Hz J6a6b=108 Hz H-6bIII) 419 (dd 1H J56=16 Hz J67=80
Hz H-6II) 436 (dd 1H J45=78 Hz J56=16 Hz H-5II) 453 (ddd 1H J3a4=20 Hz
J3e4=34 Hz J45=78 Hz H-4II) 453 (ddd 1H J45=22 Hz J56a=50 Hz J56b=94 Hz
H-5III) 453 (dd 1H J78a=40 Hz J8a8b =124 Hz H-8aI) 460 (dd 1H J7 8a=40 Hz
J8a 8b=126 Hz H-8aII) 471 (dd 1H J12=34 Hz J23=114 Hz JNH 2=100 Hz H-2III)
475 (ddd 1H J3a4=114 Hz J3e4=56 Hz J45=20 Hz H-4I) 485 (dd 1H J78b=26
Hz J8a8b=124 Hz H-8bI) 494 (dddd 1H J=12 176 Hz =CH2) 496 (dddd 1H
J=18 106 Hz =CH2) 503 (d 1H J12=34 Hz H-1III) 534 (dd 1H J23=114 Hz
J34=32 Hz H-3III) 534 (dd 1H J7 8b=24 Hz J8a 8b=126 Hz H-8bII) 540 (dd 1H
J34=32 Hz J45=22 Hz H-4III) 553 (ddd 1H J67=98 Hz J78a=40 Hz J78b=26 Hz
51
H-7I) 566 (ddd 1H J67=80 Hz J78a=40 Hz J78b=24 Hz H-7II) 570 (m 1H
-CH=) 643 (d 1H JNH2=100Hz NHAc) 737ndash738 (m 2H ArI ArII) 740ndash746 (m
6H ArI ArII) 751ndash759 (m 4H ArI ArII) 793ndash807 (m 8H ArI ArII) 13C NMR
(150 MHz CDCl3) 206 2075 2084 (Ac-CH3) 230 (NHAc-CH3) 250 and 251
(Me) 317 (C-3II) 349 (C-3I) 476 (C-2III) 521 (OMeII) 524 (OMeI) 604 (C-6III)
619 (C-8II) 624 (C-8I) 6543 (OCH2-) 6645 (C-5III) 668 (C-4III) 674 (C-4I) 685
(C-3III) 686 (C-7I) 699 (C-4II) 702 (C-6II) 705 (C-7II) 706 (C-5I) 707 (C-6I)
721 (C-5II) 962 (C-2II) 979 (C-1III) 992 (C-2I) 1099 (Cisop) 1170 (=CH2) 12839
12842 1285 1286 1288 1293 1296 12970 12973 1298 1299 and 1301 (ArI
and ArII) 1330 (-CH=) 1649 1651 1657 1662 (Bz C=O) 1683 (C-1I) 1686
(C-1II) 1701 1073 1705 (Ac C=O) 1709 (NHAc-C=O) IR(neat) 1746 1723
1278 1249 1217 cm-1 ESI-HRMS for C66H73NO27 13344268 [M+Na] Found
13344253
68 (L-Glycero--D-manno-heptopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-mann
-o-2-octulopyranosylonate)]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyr
-anosid)onate (17)
Aqueous 80 trifluoroacetic acid (TFA 220 L) was added to a solution of 11
(210 mg 152 mol) in dichloromethane (22 mL) at room temperature After stirring
for 1 h the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (30 mL) and then 01 M sodium hydroxide (24 mL 024
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) to give compound 17 as colorless powder in 87 yield
[]25D = +302 (c 05 H2O) 1H-NMR (600 MHz D2O)162 (dd 1H J3a3e=130 Hz
J3a4=126 Hz H-3aII) 182 (dd 1H J3a3e=124 Hz J3a4=128 Hz H-3aI) 191 (dd
1H J3a3e=128 Hz J3e4=42 Hz H-3eI) 206 (dd 1H J3a3e=130 Hz J3e4=48 Hz
H-3eII) 344 (dd 1H J56=24 Hz J67=88 Hz H-6I) 346 (dd 1H J78a=62 Hz
J8a8b=116 Hz H-8aII) 354 (dd 1H J56=06 Hz J67=80 Hz H-6II) 359-383 (m
12H H-8aI H-8bI H-7I H-8bII H-7II H-7aIII H-7bIII H-5III H-4III H-3III OCH2)
387-390 (m 3H H-6III H-5II H-2III) 396 (ddd 1H J=30 50 122 Hz H-4II) 407
52
(brs 1H H-5I) 411 (ddd 1H J=20 42 124 Hz H-4I) 506 (ddd 1H J=104 Hz
=CH2) 517 (d 1H J=18 Hz H-1III) 519 (dddd 1H J=14 32 172 Hz =CH2)
578-585 (m 1H -CH=) 13C NMR (150 MHz D2O) 343 (C-3I) 345 (C-3II) 629
(C-8I) 632 (C-8II) 639 (C-7III) 640 (OCH2) 661 (C-4II) 662 (C-5II) 663 (C-4III)
690 (C-7I) 692 (C-6III) 695 (C-4I) 700 (C-2III) 702 (C-7II) 704 (C-3III) 719
(C-6I) 720 (C-6II) 726 (C-5III) 728 (C-5I) 1000 (C-2I C-2II) 1011 (C-1III) 1171
(=CH2) 1340 (-CH=) 17487 (C-1I) 17493 (C-1II) IR (neat) 3346 3333 1678
1623 cm-1 ESI-HRMS for C26H41O21 6892149 [M-2Na+H] Found 6892140
69 (-D-Mannopyranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyran
-osylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-octulopyranosid)onate (1
8)
The procedure was similar to that described for compound 17 The reaction was
performed with 513 mg of 13 80 trifluoro acetic acid (17 mL) and 01 M sodium
hydroxide (50 mL) to afford 261 mg (93) of compound 18 []25D = +804 (c 08
H2O) 1H-NMR (600 MHz D2O) 170 (dd 1H J3a3e=128 Hz J3a4=120 Hz
H-3aII) 190 (dd 1H J3a3e=122 Hz J3a4=122 Hz H-3aI) 201 (dd 1H J3a3e=122
Hz J3e4=44 Hz H-3eI) 205 (dd 1H J3a3e=128 Hz J3e4=46 Hz H-3eII) 349 (dd
1H H-6I) 351 (dd 1H H-8aII) 362ndash372 (m 5H H-8aI H-6II H-7I OCH2 H-6aIII)
377ndash395 (m 10H H-4III H-5II OCH2 H-8bII H-8bI H-3III H-7II H-6bIII H-4II
H-5III) 401 (ddd 1H H-4I) 402 (d 1H H-2III) 415 (brs 1H H-5I) 510 (dddd 1H
=CH2) 513 (d 1H J12=16 Hz H-1III) 522 (dddd 1H -CH=) 582ndash588 (m 1H
-CH=) 13C NMR (150 MHz D2O) 344 (C-3I) 346 (C-3II) 605 (C-4III) 627
(C-8I) 633 (C-8II) 640 (OCH2) 659 (C-6III) 661 (C-4II) 667 (C-5II) 692 (C-7I)
701 (C-2III) 703 (C-3III) 705 (C-7II) 707 (C-4I) 717 (C-6II) 722 (C-6I) 727
(C-5III) 732 (C-5I) 10000 (C-2I) 1006 (C-1III) 1015 (C-2II) 1170 (=CH2) 1340
(-CH=) 1750 (C-1I) 1751 (C-1II) IR (neat) 3381 3274 1604 1568 cm-1
ESI-HRMS for C25H39O20 6592035 [M-2Na+H] Found 6592061
610 (2-Acetamido-2-deoxy--D-galactopyranosyl)-(1-5)-[O-(sodium 3-deoxy--
D-manno-2-octulopyranosylonate]]-(2-4)-sodium (allyl 3-deoxy--D-manno-2-oct
-ulopyranosid)onate (19)
53
The procedure was similar to that described for compound 17 The reaction was
performed with 274 mg of 16 80 trifluoro acetic acid (09 mL) and 01 M sodium
hydroxide (25 mL) to afford compound 19 (156 mg) in quantitative yield []25D =
+1165 (c 03 H2O) 1H-NMR (600 MHz D2O) 173 (dd 1H J3a4=126 Hz
J3a3b=130 Hz H-3aII) 194ndash204 (m 2H H-3aI H-3bI) 202 (s 3H Ac) 210 (d 1H
J3b4=44 Hz J3a3b=130 Hz H-3bII) 352 (H-6I) 354 (dd 1H H-8aII) 365ndash377 (m
6H H-6II H-8aI H-7I OCH2 H-6aIII H-6bIII) 381ndash386 (m 3H H-8bII H-8bI
OCH2) 388ndash391 (m 1H H-7II) 394ndash399 (m 4H H-5II H-3III H-4II H-4III) 426
(ddd 1H J=20 54 112 Hz H-4I) 416 (dd 1H J12=34 Hz H-2III) 417 (brs 1H
H-5I) 426 (d 1H J=64 Hz H-5III) 514 (dddd 1H =CH2) 519 (d 1H J12=34 Hz
H-1III) 526 (dddd 1H =CH2) 586-592 (m 1H -CH=) 13C NMR (150 MHz D2O)
220 (CH3) 345 (C-3I) 346 (C-3II) 503 (C-2III) 604 (C-6III) 628 (C-8I) 633
(C-8II) 641 (OCH2) 660 (C-4II) 666 (C-5II) 676 (C-3III) 684 (C-4III) 692 (C-7II)
703 (C-7I) 705 (C-5III) 709 (C-4I) 719 (C-6II) 7260 (C-6I) 7263 (C-5I) 977
(C-1III) 1004 (C-2II) 1012 (C-2I) 1170 (=CH2) 1341 (-CH=) 1747 1750 1752
(3C C-1I C-1II C=O) IR (neat) 3295 1680 1614 cm-1 ESI-HRMS for C27H42NO20
7002300 [M-2Na+H] Found 7002294
611 Methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyran
-oside (23)
A catalytic amount of NN-dimethyl-4-aminopyridine (DMAP) was added to a
solution of methyl 67-di-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranoside
(20 19 g 48 mmol) in acetic anhydride (45 mL)pyridine (97 mL) at 0 degC After
stirring for 2 h at room temperature the mixture was concentrated and purified by
silica gel chromatography (ethyl acetatehexane 45) to give 23 (22 g 94)
[]25D= -192 (c 31 CHCl3)
1H-NMR (600 MHz CDCl3) 196 201 205 213 (s
3H x 4 Ac) 335 (s 3H OCH3) 382 (dd 1H J12=14 Hz J23=32 Hz H-2) 399
(dd 1H J45=102 Hz J56=20 Hz H-5) 425 (dd 1H J67a=76 Hz J7a7b=112 Hz
H-7a) 334 (dd 1H J67b=58 Hz J7a7b=112 Hz H-7b) 463 (d 1H J=124 Hz
OCH2Ph) 469 (d 1H J=124 Hz OCH2Ph) 478 (d 1H J12=14 Hz H-1) 519 (dd
1H J23=32 Hz J34=102 Hz H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=58
54
Hz H-6) 544 (dd 1H J34=102 Hz J45=102 Hz H-4) 729ndash737 (m 5H Ph) 13C
NMR (150 MHz CDCl3) 206 207 208 2084 (Ac-CH3) 553 (OCH3) 620
(C-7) 652 (C-4) 671 (C-6) 685 (C-5) 715 (C-3) 732 (OCH2Ph) 749 (C-2) 994
(C-1) 1280 1284 1299 1376 (Ph) 1696 1702 1705 17051 (Ac C=O)
ESI-HRMS for C23H30O11 5051686 [M+Na]+ Found 5051644
612 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (24)
A mixture of H2SO4AcOHAc2O (90 mL 25025) was added to a solution of
methyl 3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside (23
22 g 45 mmol) in acetic acid and acetic anhydride (12 100 mL) After stirring for
2 h at room temperature the reaction mixture was neutralized by the addition of
sodium acetate (35 g) poured into saturated sodium hydrogen carbonate and
extracted with dichloromethane The combined organic layer was washed with brine
dried over anhydrous sodium sulfate filtered and concentrated The residue was
purified by silica gel chromatography (ethyl acetatehexane 45) to give a syrup (22 g
95) The syrup was treated with hydrazine acetate (05 g 56 mmol) in DMF (300
mL) for 2 h at room temperature The reaction mixture was diluted with ethyl acetate
washed with water and brine dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by silica gel chromatography (ethyl
acetatehexane 11) to give 24 (16 g 80) 1H-NMR (600 MHz CDCl3)198
202 206 214 (s 3H x 4 Ac) 386 (dd 1H J12=18 Hz J23=32 Hz H-2) 414 (dd
1H J67a=70 Hz J7a7b=114 Hz H-7a) 418 (dd 1H J45=100 Hz J56=20 Hz H-5)
440 (dd 1H J67b=54 Hz J7a7b=114 Hz H-7b) 464 (d 1H J=124 Hz OCH2Ph)
467 (d 1H J=124 Hz OCH2Ph) 523 (ddd 1H J56=20 Hz J67a=70 Hz J67b=54
Hz H-6) 527 (dd 1H J23=32 Hz J34=102 Hz H-3) 529 (d 1H J12=18 Hz H-1)
544 (dd 1H J34=102 Hz J45=100 Hz H-4) 730ndash737 (m 5H Ph) 13C NMR (150
MHz CDCl3) 207 208 2085 (Ac-CH3) 628 (C-7) 655 (C-4) 672 (C-6) 687
(C-5) 714 (C-3) 732 (OCH2Ph) 756 (C-2) 930 (C-1) 1279 1280 1284 1377
(Ph) 1698 1703 1706 1713 (Ac C=O) ESI-HRMS for C22H28O11 4911529
[M+Na]+ Found 4911514
613 3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl tri
55
-chloroacetimidate (25)
Compound 24 (15 g 32 mmol) was dissolved in dry dichloromethane (30 mL)
Potassium carbonate (22 g 160 mmol) was added followed by trichloroacetonitrile
(32 mL 320 mmol) and the mixture was stirring for 22 h at room temperature The
reaction mixture was filtered through Celite The solution was concentrated and
purified by silica gel column chromatography (ethyl acetatehexane 23+1 Et3N) to
give 25 (19 g 96) []25D= -134 (c 37 CHCl3)
1H-NMR (600 MHz CDCl3)
196 201 202 213 (s 3H x 4 Ac) 406 (dd 1 H J12=18 Hz J23=34 Hz H-2)
418 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7a) 420 (dd 1H J45=92 Hz J56=20
Hz H-5) 428 (dd 1H J67b=56 Hz J7a7b=114 Hz H-7b) 465 (d 1H J=122 Hz
OCH2Ph) 478 (d 1H J=122 Hz OCH2Ph) 523 (dd 1H J23=34 Hz J34=102 Hz
H-3) 527 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6) 554 (dd 1H
J34=102 Hz J45=92 Hz H-4) 635 (d 1H J12=18 Hz H-1) 727ndash740 (m 5H Ph)
868 (s 1H OC(NH)CCl3) 13C NMR (150 MHz CDCl3) 207 208 2086
(Ac-CH3) 620 (C-7) 646 (C-4) 668 (C-6) 710 (C-5) 713 (C-3) 731 (OCH2Ph)
733 (C-2) 906 (OCNHCCl3) 953 (C-1) 1281 1284 1372 (Ph) 1600
(OCNHCCl3) 1695 1704 1705 (Ac C=O) ESI-HRMS for C24H28Cl3NO11
6340626 [M+Na]+ Found 6340639
614 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-3-O-tert-butyldimethyl-
silyl-L-glycero--D-manno-heptopyranoside (27)
A mixture of 21 (15 g 29 mmol)
(2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-236-tri-O-acetyl--D-glucopyran
osyl trichloroacetimidate (26 56 g 72 mmol) and MS-AW 300 molecular sieves
(70 g) in dry dichloromethane (500 mL) was stirred for 1 h under argon then cooled
to 0 degC TMSOTf (1060 L 06 mmol) in dry dichloromethane (05 mL) was added
dropwise to the reaction mixture and the mixture was stirred for 3 h The solution was
neutralized by the addition of triethylamine and saturated sodium hydrogen carbonate
and diluted with dichloromethane The mixture was filtered through Celite and the
filtrate was extracted with dichloromethane The organic phase was dried over
56
anhydrous sodium sulfate filtered and concentrated Purification of the residue by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and flash column
chromatography (tolueneacetone 51) afforded 27 (26 g 79) []25D= +10 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 008 009 [s 3H x 2 Si(CH3)2C(CH3)3] 090
[s 9H Si(CH3)2C(CH3)3] 196 198 204 205 205 206 209 213 215 (s 3H x
8 Ac) 333 (s 3H OCH3) 355 (ddd 1H J45=100 Hz J56a=52 Hz J56b=20 Hz
H-5II) 356 (brs 1H J12=34 Hz J23=nd Hz H-2I) 364 (dd 1H J45=88 Hz J56=nd
Hz H-5I) 376 (dd 1H J34=92 Hz J45=100 Hz H-4II) 380 (m 1H J34=nd Hz
J45=88 Hz H-4I) 386 (ddd 1H J45=08 Hz J56a=74 Hz J56b=62 Hz H-5III) 406
(m 1H J23=nd Hz J34=nd Hz H-3I) 407 (dd 1H J56a=74 Hz J6a6b=112 Hz
H-6aIII) 409 (dd 1H J56a=52 Hz J6a6b=102 Hz H-6aII) 415 (dd 1H J56b=62 Hz
J6a6b=112 Hz H-6bIII) 421 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 433 (dd
1H J67b=56 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=20 Hz J6a6b=102 Hz
H-6bII) 448 (d 1H J12=80 Hz H-1III) 455 (d 1H J12=80 Hz H-1II) 463 (d 1H
J=118 Hz OCH2Ph) 472 (d 1H J12=34 Hz H-1I) 477 (d 1H J=118 Hz
OCH2Ph) 487 (dd 1H J12=80 Hz J23=94 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 518 (dd 1H
J23=94 Hz J34=92 Hz H-3II) 534 (dd 1H J34=34 Hz J45=08 Hz H-4III) 535 (m
1H J56=nd Hz J67a=74 Hz J67b=56 Hz H-6I) 724ndash736 (m 5H Ph) 13C NMR
(150 MHz CDCl3) -48 -46 (Si(CH3)2C(CH3)3) 181 (Si(CH3)2C(CH3)3) 205
206 2067 207 2085 209 259 297 (Ac-CH3) 258 (Si(CH3)2C(CH3)3) 552
(OCH3) 606 (C-6III) 623 (C-6II) 625 (C-7I) 665 (C-4III) 687 (C-6I) 691 (C-2III)
702 (C-5I) 706 (C-5III) 709 (C-3III) 710 (C-3I) 719 (C-2II) 724 (C-5II) 729
(C-3II CH2Ph) 765 (C-4II) 774 (C-4I) 783 (C-2I) 1003 (C-1I C-1II) 1009 (C-1III)
1273 12737 1281 1385 (Ph) 1690 1695 1697 1700 1701 1702 17023
1703 17036 (Ac C=O) Anal Calcd for C51H74O26Si C 5415 H 659 Found C
5394 H 648
615 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-2-O-benzyl-L-glycero--D-manno-h-
eptopyranoside (28)
57
Compound 27 (4860 mg 4297 mol) was dissolved in a mixture of
trifluoroacetic acid and water (91 vv 100 mL) at room temperature After stirring
for 5 min the mixture was diluted with toluene and concentrated The residue was
purified by flash column chromatography (dichloromethaneacetone 91) to give 28
(4240 mg 97) []25D= +80 (c 10 CHCl3)
1H NMR (600 MHz CDCl3) 197
204 204 205 206 209 212 213 216 (s 3H x 9 Ac) 330 (s 3H OCH3) 364
(m 1H J45=95 Hz H-5I) 366 (dd 1H J34=95 Hz J45=95 Hz H-4I) 373 (ddd
1H J56a=65 Hz J56b=20 Hz H-5II) 376 (dd 1H J34=95 Hz H-4II) 377 (dd 1H
J12=20 Hz J23=33 Hz H-2I) 388 (ddd 1H J45=15 Hz J56a=75 Hz J56b=65 Hz
H-5III) 392 (dd 1H J23=33 Hz J34=95 Hz H-3I) 398 (d 1H J3-OHH-3=25 Hz
3-OH) 404 (dd 1H J56a=65 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=110 Hz H-6aIII) 413 (dd 1H J56b=65 Hz J6a6b=110 Hz H-6bIII) 427 (dd
1H J67a=65 Hz J7a7b=110 Hz H-7aI) 430 (dd 1H J67b=70 Hz J7a7b=110 Hz
H-7bI) 449 (d 1H J12=80 Hz H-1III) 454 (d 1H J12=80 Hz H-1II) 459 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 470 (d 1H J=120 Hz OCH2Ph) 475 (d 1H
J12=20 Hz H-1I) 484 (d 1H J=120 Hz OCH2Ph) 494 (dd 1H J12=80 Hz
J23=95 Hz H-2II) 497 (dd 1H J23=108 Hz J34=35 Hz H-3III) 519 (dd 1H
J12=80 Hz J23=108 Hz H-2III) 522 (dd 1H J23=95 Hz J34=95 Hz H-3II) 525
(ddd 1H J67a=65 Hz J67b=70 Hz H-6I) 535 (dd 1H J34=35 Hz J45=15 Hz
H-4III) 738ndash725 (m 5H Ph) 13C NMR (150 MHz CDCl3) 203 2034 204 205
206 207 (Ac-CH3) 550 (OCH3) 607 (C-6III) 618 (C-6II) 620 (C-7I) 665 (C-4III)
680 (C-6I) 684 (C-5I) 690 (C-2III) 698 (C-3I) 706 (C-5III) 708 (C-3III) 713
(C-2II) 7255 (C-5II) 726 (C-3II) 730 (OCH2Ph) 760 (C-2I) 762 (C-4II) 796
(C-4I) 990 (C-1I) 1004 (C-1II) 1009 (C-1III) 1273 12734 1281 1384 (Ph)
1689 1693 16987 1699 1700 1701 1702 17023 (Ac C=O) ESI-HRMS for
C45H60O26 10393271 [M+Na]+ Found 10393303
616 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno
-heptopyranoside (29)
Compound 28 (2803 mg 2758 mol) was acetylated with pyridineAc2O (11
58
vv 16 mL) in the presence of a catalytic amount of NN-dimethyl-4-aminopyridine
(DMAP) over 2 h After removing the solvent the residue was purified by flash
column chromatography (dichloromethaneethyl acetate 52) to give 29 (1809 mg
73) []25D= +61 (c 08 CHCl3)
1H-NMR (600 MHz CDCl3) 196 197 204
205 206 208 209 215 216 (s 3H x 10 Ac) 334 (s 3H OCH3) 361 (ddd 1H
J45=100 Hz J56a=48 Hz J56b=18 Hz H-5II) 376 (dd 1H J12=24 Hz J23=34 Hz
H-2I) 380 (dd 1H J45=96 Hz J56=10 Hz H-5I) 384 (dd 1H J34=86 Hz
J45=100 Hz H-4II) 386 (ddd 1H J45=08 Hz J56a=76 Hz J56b=66 Hz H-5III)
388 (dd 1H J34=84 Hz J45=96 Hz H-4I) 407 (dd 1H J56a=76 Hz J6a6b=114
Hz H-6aIII) 410 (dd 1H J56a=54 Hz J6a6b=120 Hz H-6aII) 412 (dd 1H J56b=66
Hz J6a6b=114 Hz H-6bIII) 425 (dd 1H J67a=74 Hz J7a7b=112 Hz H-7aI) 432
(dd 1H J67b=60 Hz J7a7b=112 Hz H-7bI) 438 (dd 1H J56b=18 Hz J6a6b=120
Hz H-6bII) 447 (d 1H J12=80 Hz H-1III) 455 (d 1H J=122 Hz OCH2Ph) 457
(d 1H J12=72 Hz H-1II) 464 (d 1H J=122 Hz OCH2Ph) 477 (d 1H J12=24 Hz
H-1I) 482 (dd 1H J12=72 Hz J23=82 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=82 Hz J34=86 Hz H-3II) 527 (dd 1H J23=34 Hz J34=84 Hz H-3I) 534 (dd
1H J34=34 Hz J45=08 Hz H-4III) 539 (ddd 1H J56=10 Hz J67a=72 Hz J67b=60
Hz H-6I) 726ndash733 (m 5H Ph) 13C NMR (150 MHz CDCl3) 205 206 2068
207 2074 208 209 (Ac-CH3) 553 (OCH3) 607 (C-6III) 622 (C-6II) 624 (C-7I)
666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I) 706 (C-5III) 707 (C-5I) 710
(C-3III) 719 (C-2II) 721 (C-5II) 727 (OCH2Ph) 733 (C-3II) 739 (C-4I) 752 (C-2I)
762 (C-4II) 991 (C-1I) 999 (C-1II) 1011 (C-1III) 1278 1279 1284 1377 (Ph)
1691 1697 1699 1702 1703 1704 (Ac C=O) ESI-HRMS for C47H62O27
10673376 [M+Na]+ Found 10813368
617 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyr-
anose (30)
A solution of 29 (1950 mg 1841 mol) in a mixture of H2SO4AcOHAc2O
(01614 40 mL) was stirred for 3 h at room temperature The reaction mixture was
59
neutralized by the addition of sodium acetate poured into saturated sodium hydrogen
carbonate and extracted with dichloromethane The combined organic layer was
washed with brine dried over anhydrous sodium sulfate filtered and concentrated
The residue was purified by flash column chromatography (ethyl acetatehexane 21)
to give a syrup (1245 mg 64) The syrup was treated with hydrazine acetate (130
mg 1204 mol) in DMF (10 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (ethyl acetatehexane 21) to give 30 (891 mg 77) 1H-NMR (600
MHz CDCl3) 196 198 204 206 206 207 215 215 (s 3H x 10 Ac) 337
(brs 1H OH) 361 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 379 (dd
1H J12=28 Hz J23=32 Hz H-2I) 385 (dd 1H J34=90 Hz J45=100 Hz H-4II)
386 (ddd 1H J45=12 Hz J56a=76 Hz J56b=62 Hz H-5III) 389 (dd 1H J34=86
Hz J45=98 Hz H-4I) 398 (dd 1H J45=98 Hz J56=nd Hz H-5I) 407 (dd 1H
J56a=76 Hz J6a6b=110 Hz H-6aIII) 409 (dd 1H J56a=46 Hz J6a6b=120 Hz
H-6aII) 415 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII) 415 (dd 1H J67a=70 Hz
J7a7b=110 Hz H-7aI) 438 (dd 1H J67b=62 Hz J7a7b=110 Hz H-7bI) 439 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 448 (d 1H J12=80 Hz H-1III) 456 (d 1H
J=122 Hz OCH2Ph) 457 (d 1H J12=72 Hz H-1II) 463 (d 1H J=122 Hz
OCH2Ph) 482 (dd 1H J12=72 Hz J23=84 Hz H-2II) 493 (dd 1H J23=104 Hz
J34=34 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 515 (dd 1H
J23=84 Hz J34=90 Hz H-3II) 527 (d 1H J12=28 Hz H-1I) 533 (dd 1H J34=34
Hz J45=12 Hz H-4III) 533 (dd 1H J23=32 Hz J34=86 Hz H-3I) 537 (ddd 1H
J56=nd Hz J67a=70 Hz J67b=62 Hz H-6I) 727ndash733 (m 5H Ph) 13C NMR (150
MHz CDCl3) 205 206 2065 2066 207 2079 208 209 (Ac-CH3) 607
(C-6III) 624 (C-6II) 626 (C-7I) 666 (C-4III) 681 (C-6I) 690 (C-2III) 693 (C-3I)
705 (C-5III) 706 (C-5I) 710 (C-3III) 719 (C-2II) 720 (C-5II) 727 (OCH2Ph) 732
(C-3II) 740 (C-4I) 757 (C-2I) 762 (C-4II) 924 (C-1I) 998 (C-1II) 1011 (C-1III)
1277 1278 1284 1377 (Ph) 1692 16966 1697 1700 17015 1702 17024
1704 1709 (Ac C=O) ESI-HRMS for C46H60O27 10673220 [M+Na]+ Found
60
10673226
618 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop-
yranosyl trichloroacetimidate (31)
Trichloroacetonitrile (850 L 8421 mol) was added to a solution of 30
(880 mg 842 mol) in dry dichloromethane (08 mL) under argon Potassium
carbonate (580 mg 4210 mol) was added to the reaction mixture After stirring for
13 h at room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 31 (960 mg 100) []25D= -57 (c 13 CHCl3)
1H-NMR (600 MHz
CDCl3) 196 199 200 206 204 204 204 205 206 207 215 215 (s 3H x
10 Ac) 365 (ddd 1H J45=100 Hz J56a=46 Hz J56b=20 Hz H-5II) 384 (dd 1H
J34=92 Hz J45=100 Hz H-4II) 387 (ddd 1H J45=nd Hz J56a=76 Hz J56b=62 Hz
H-5III) 388 (dd 1H J34=56 Hz J45=94 Hz H-4I) 394 (dd 1H J45=94 Hz
J56=08 Hz H-5I) 404 (dd 1H J12=36 Hz J23=28 Hz H-2I) 407 (dd 1H
J56a=76 Hz J6a6b=112 Hz H-6aIII) 409 (dd 1H J56a=48 Hz J6a6b=120 Hz
H-6aII) 414 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd 1H J67a=72 Hz
J7a7b=114 Hz H-7aI) 422 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bII) 449 (d 1H J12=80 Hz H-1III) 461 (d 1H
J12=76 Hz H-1II) 463 (d 1H J=120 Hz OCH2Ph) 468 (d 1H J=120 Hz
OCH2Ph) 485 (dd 1H J12=76 Hz J23=86 Hz H-2II) 494 (dd 1H J23=106 Hz
J34=36 Hz H-3III) 510 (dd 1H J12=80 Hz J23=104 Hz H-2III) 516 (dd 1H
J23=86 Hz J34=92 Hz H-3II) 529 (ddd 1H J56=08 Hz J67a=72 Hz J67b=62 Hz
H-6I) 533 (dd 1H J34=34 Hz J45=nd Hz H-4III) 548 (dd 1H J23=28 Hz J34=56
Hz H-3I) 631 (d 1H J12=36 Hz H-1I) 726ndash734 (m 5H Ph) 868 (NH) 13C
NMR (150 MHz CDCl3) 205 2054 207 2074 2076 2079 208 209 210
(Ac-CH3) 607 (C-6III) 621 (C-7I and C-6II) 666 (C-4III) 679 (C-6I) 693 (C-2III)
699 (C-3I) 706 (C-5III) 710 (C-3III) 715 (C-2II) 716 (C-5II) 724 (-OCH2Ph and
C-5I) 730 (C-3II) 740 (C-4I) 749 (C-2I) 760 (C-4II) 908 (OCNHCCl3) 960
(C-1I) 992 (C-1II) 1011 (C-1III) 1279 1280 1284 1372 (Ph) 1605
61
(OCNHCCl3) 1690 1696 1698 1699 1701 17013 17016 1702 17024 1704
(Ac C=O) ESI-HRMS for C48H60Cl3NO27 12102316 [M+Na]+ Found 12102294
619 Methyl (2346-tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acet
-yl--D-glucopyranosyl)-(1-4)-67-di-O-acetyl-L-glycero--D-manno-heptopyranos
-ide (32)
Compound 28 (2270 mg 02 mmol) was hydrogenated in the presence of 10
PdC (1000 mg) in ethyl acetate (150 mL) under atmospheric pressure of hydrogen
After stirring for 35 h at room temperature the reaction mixture was filtered through
Celite and concentrated The residue was purified by flash column chromatography
(dichloromethaneacetone 31) to give 32 (1980 mg 97) []25D= +260 (c 10
CHCl3) 1H-NMR (600 MHz CDCl3) 197 204 204 205 207 212 214 216
217 (s 3H x 9 Ac) 255 (d 1H J2-OHH-2=15 Hz 2-OH) 335 (s 3H OCH3) 355
(dd 1H J34=80 Hz J45=98 Hz H-4I) 371 (dd 1H J45=98 Hz J56=10 Hz H-5I)
372 (ddd 1H J45=100 Hz J56a=55 Hz J56b=20 Hz H-5II) 378 (dd 1H J34=90
Hz J45=100 Hz H-4II) 381 (dd 1H J23=35 Hz J34=80 Hz H-3I) 396 (dd 1H
J12=10 Hz J23=35 Hz H-2I) 388 (ddd 1H J45=10 Hz J56a=75 Hz J56b=65 Hz
H-5III) 403 (dd 1H J56a=55 Hz J6a6b=120 Hz H-6aII) 408 (dd 1H J56a=75 Hz
J6a6b=113 Hz H-6aIII) 414 (dd 1H J56b=65 Hz J6a6b=113 Hz H-6bIII) 422 (m
1H J56=10 Hz H-6I) 429 (m 2H H-7aI H-7bI) 430 (d 1H J3-OHH-3=10 Hz
3-OH) 446 (d 1H J12=80 Hz H-1II) 451 (d 1H J12=85 Hz H-1III) 465 (dd 1H
J56b=20 Hz J6a6b=120 Hz H-6bII) 480 (d 1H J12=10 Hz H-1I) 493 (dd 1H
J12=80 Hz J23=95 Hz H-2II) 497 (dd 1H J23=105 Hz J34=35 Hz H-3III) 511
(dd 1H J12=85 Hz J23=105 Hz H-2III) 522 (dd 1H J23=95 Hz J34=90 Hz
H-3II) 535 (dd 1H J34=35 Hz J45=10 Hz H-4III) 13C NMR (150 MHz CDCl3)
204 2042 205 2054 206 2064 209 (Ac-CH3) 552 (OCH3) 607 (C-6III) 616
(C-6II) 620 (C-7I) 665 (C-4III) 677 (C-5I) 679 (C-6I) 690 (C-2III) 695 (C-3I)
695 (C-2I) 708 (C-3III) 710 (C-5III) 712 (C-2II) 725 (C-3II) 728 (C-5II) 760
(C-4II) 790 (C-4I) 1001 (C-1I) 1006 (C-1II) 1009 (C-1III) 1690 1693 1700
17004 17016 1702 17026 1703 (Ac C=O) ESI-HRMS for C38H54O26 9492801
[M+Na]+ Found 9492766
62
620 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-12367-penta-O-acetyl-L-glycero--D-manno-heptopyrano
-se (33)
Compound 32 (3420 mg 04 mmol) was treated with acetic anhydride (10 mL)
and pyridine (20 mL) at room temperature The reaction mixture was stirred
overnight and concentrated The residue was purified by silica gel column
chromatography (dichloromethaneacetone 41) to give a syrup The syrup was
dissolved in a mixture of H2SO4AcOHAc2O (80 mL 01614) at room temperature
After stirring for 15 h the reaction mixture was neutralized by the addition of sodium
acetate (02 g) poured into saturated sodium hydrogen carbonate and extracted with
chloroform Combined organic phase was washed with brine dried over anhydrous
sodium sulfate filtered and concentrated The residue was purified by silica gel
column chromatography (dichloromethaneacetone 41) to give 33 (2570 mg 67)
[]25D= +290 (c 10 CHCl3)
1H-NMR (600 MHz CDCl3) 197 199 203 205
206 207 208 213 214 215 216 218 (s 3H x 12 Ac) 361 (ddd 1H J45=95
Hz J56a=70 Hz J56b=55 Hz H-5II) 382 (dd 1H J34=85 Hz J45=95 Hz H-4II)
385ndash388 (m 2H H-5III H-4I) 391 (dd 1H J45=95 Hz J56=12 Hz H-5I)
405ndash409 (m 2H H-6aIII H-7aI) 413 (dd 1H J56b=62 Hz J6a6b=110 Hz H-6bIII)
419 (dd 1H J56a=70 Hz J6a6b=115 Hz H-6aII) 424 (dd 1H J56b=55 Hz
J6a6b=115 Hz H-6bII) 440 (dd 1H J67b=20 Hz J7a7b=120 Hz H-7bI) 449 (d 1H
J12=80 Hz H-1III) 461 (d 1H J12=75 Hz H-1II) 482 (dd 1H J12=75 Hz J23=80
Hz H-2II) 495 (dd 1H J23=105 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz
J23=105 Hz H-2III) 516 (dd 1H J23=80 Hz J34=85 Hz H-3II) 522 (dd 1H
J12=25 Hz J23=90 Hz H-2I) 532ndash536 (m 3H H-6I H-4III H-3I) 606 (d 1H
J12=25 Hz H-1I) 13C NMR (150 MHz CDCl3) 205 2051 206 2065 207
208 2081 209 211 (Ac-CH3) 607 (C-6III) 622 (C-6II) 623 (C-7I) 666 (C-4III)
679 (C-3I) 684 (C-6I) 689 (C-2III) 691 (C-2I) 706 (C-5III) 710 (C-3III) 714
(C-5I) 719 (C-2II) 722 (C-5II) 731 (C-3II) 732 (C-4I) 760 (C-4II) 904 (C-1I)
998 (C-1II) 1012 (C-1III) 1683 1691 1695 16953 1696 1698 1701 17014
1702 1703 1704 (Ac C=O) ESI-HRMS for C43H58O29 10612961 [M+Na]+
63
Found 10612981
621 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero-D-manno-heptopyranose (3
4)
Compound 33 (2566 mg 2470 mol) was treated with hydrazine acetate (455
mg 4940 mol) in DMF (30 mL) for 8 h at 0 degC The reaction mixture was diluted
with ethyl acetate washed with water and brine dried over anhydrous sodium sulfate
filtered and concentrated The residue was purified by silica gel column
chromatography (acetonedichloromethane 14) to give 34 (2234 mg 90)
1H-NMR (600 MHz CDCl3) 196 198 204 206 207 208 213 216 219 (s
3H x 11 Ac) 327 (brs 1H OH) 362 (ddd 1H J45=100 Hz J56a=46 Hz J56b=16
Hz H-5II) 381 (dd 1H J34=94 Hz J45=100 Hz H-4II) 387 (ddd 1H J56a=78 Hz
J56b=62 Hz H-5III) 388 (dd 1H J45=98 Hz H-4I) 406 (dd 1H J56a=46 Hz
J6a6b=120 Hz H-6aII) 407 (dd 1H J56a=78 Hz J6a6b=112 Hz H-6aIII) 409 (dd
1H J45=98 Hz H-5I) 412 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bIII) 416 (dd
1H J67a=68 Hz J7a7b=114 Hz H-7aI) 438 (dd 1H J56b=16 Hz J6a6b=120 Hz
H-6bII) 439 (dd 1H J67b=58 Hz J7a7b=114 Hz H-7bI) 449 (d 1H J12=80 Hz
H-1III) 460 (d 1H J12=68 Hz H-1II) 480 (dd 1H J12=68 Hz J23=82 Hz H-2II)
494 (dd 1H J23=104 Hz J34=34 Hz H-3III) 511 (dd 1H J12=80 Hz J23=104
Hz H-2III) 515 (dd 1H J23=82 Hz J34=94 Hz H-3II) 522ndash523 (m 2H H-6I
H-4III) 534 (d 1H J12=32 Hz H-1I) 540 (dd 1H H-3I) 541 (dd 1H J12=32 Hz
H-2I) 13C NMR (150 MHz CDCl3) 205 206 208 209 2092 (Ac-CH3) 607
(C-6III) 625 (C-6II) 628 (C-7I) 666 (C-4III) 683 (C-6I) 6896 (C-3I) 690 (C-2III)
691 (C-2I) 704 (C-5I) 706 (C-5III) 710 (C-3III) 719 (C-2II) 7192 (C-5II) 731
(C-3II) 733 (C-4I) 760 (C-4II) 920 (C-1I) 991 (C-1II) 1011 (C-1III) 1692 1697
1698 1700 1701 1702 1704 1705 (Ac C=O) ESI-HRMS for C41H56O28
10192856 [M+Na]+ Found 10192848
622 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-2367-tetra-O-acetyl-L-glycero--D-manno-heptopyranosyl
trichloroacetimidate (35)
64
Trichloroacetonitrile (570 L 5656 mol) was added to a solution of 34 (571
mg 573 mol) in dry dichloromethane (10 mL) under argon Potassium carbonate
(393 mg 2844 mol) was added to the reaction mixture After stirring for 24 h at
room temperature the mixture was filtered through Celite The solution was
concentrated and purified by silica gel column chromatography (ethyl acetatehexane
21) to give 35 (602 mg 92) []25D= +24 (c 10 CHCl3)
1H-NMR (600 MHz
CDCl3) 197 200 200 204 207 212 215 219 (s 3H x 11 Ac) 363 (ddd 1H
J45=100 Hz J56a=40 Hz J56b=20 Hz H-5II) 384ndash389 (m 3H H-4II H-5III H-4I)
401 (dd 1H J45=98 Hz J56=14 Hz H-5I) 406 (dd 1H J56a=72 Hz J6a6b=112
Hz H-6aIII) 409 (dd 1H J56a=40 Hz J6a6b=122 Hz H-6aII) 414 (dd 1H J56b=62
Hz J6a6b=112 Hz H-6bIII) 417 (dd 1H J67a=72 Hz J7a7b=114 Hz H-7aI) 421
(dd 1H J67b=60 Hz J7a7b=114 Hz H-7bI) 447 (dd 1H J56b=20 Hz J6a6b=122
Hz H-6bII) 453 (d 1H J12=80 Hz H-1III) 461 (d 1H J12=70 Hz H-1II) 483 (dd
1H J12=70 Hz J23=78 Hz H-2II) 495 (dd 1H J23=106 Hz J34=34 Hz H-3III)
510 (dd 1H J12=80 Hz J23=106 Hz H-2III) 516 (dd 1H J23=78 Hz J34=104
Hz H-3II) 534 (dd 1H J45=08 Hz J34=34 Hz H-4III) 537 (ddd 1H J56=14 Hz
J67a=72 Hz J67b=60 Hz H-6I) 543 (dd 1H J12=22 Hz J23=34 Hz H-2I) 544
(dd 1H J23=34 Hz H-3I) 623 (d 1H J12=22 Hz H-1I) 874 (NH) 13C NMR (150
MHz CDCl3) 205 2057 206 207 2074 208 (Ac-CH3) 607 (C-6III) 620
(C-6II) 621 (C-7I) 666 (C-4III) 678 (C-2I) 679 (C-6I) 690 (C-3I and C-2III) 706
(C-5III) 710 (C-3III) 716 (C-2II) 717 (C-5II) 721 (C-5I) 731 (C-3II) 733 (C-4I)
758 (C-4II) 905 (OCNHCCl3) 946 (C-1I) 999 (C-1II) 1011 (C-1III) 1600
(OCNHCCl3) 1691 1694 16942 16967 1697 1700 1701 1702 1703 1704
(Ac C=O) ESI-HRMS for C43H56Cl3NO28 11621952 [M+Na]+ Found 11621940
623 Methyl (3467-tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyra-
nosyl)-(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranoside
(36)
A mixture of 25 (1880 mg 3080 mol) and 22 (1350 mg 3080 mol) and 4
Aring molecular sieves (1350 mg) was suspended in dry dichloromethane (09 mL) The
reaction mixture was stirred for 1 h under argon and then cooled to -78 degC
65
TMSOTf (22 μL 123 mol) in dichloromethane was added dropwise to the reaction
mixture The reaction was warmed to room temperature and stirred for 2 h The
reaction was neutralized by the addition of a few drops of triethylamine and aqueous
saturated sodium hydrogen carbonate The reaction mixture was diluted with
dichloromethane and filtered through Celite The filtrate was extracted twice with
dichloromethane The combined organic phase was dried over anhydrous sodium
sulfate filtered and concentrated The residue was purified by BioRad S-X3 size
exclusion beads (tolueneethyl acetate 11) to give 36 (1366 mg 50) []25D=
+412 (c 10 CHCl3) 1H-NMR (600 MHz CDCl3) 177 194 196 204 205
209 213 (s 3H x 7 Ac) 331 (s 3H OCH3) 366 (dd 1H J12=16 Hz J23=30 Hz
H-2I) 376 (dd 1H J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz
J23=30 Hz H-2II) 387 (dd 1H J45=100 Hz J56=20 Hz H-5I) 402 (dd 1H
J67a=64 Hz J7a7b=112 Hz H-7aII) 407 (dd 1H J23=30 Hz J34=98 Hz H-3I)
411 (dd 1H J67b=68 Hz J7a7b=112 Hz H-7bII) 423 (dd 1H J67a=76 Hz
J7a7b=112 Hz H-7aI) 432 (dd 1H J67b=56 Hz J7a7b=112 Hz H-7bI) 459 464 (d
2H J=122 Hz OCH2Ph) 476 (d 1H J12=16 Hz H-1I) 482 484 (d 2H J=128
Hz OCH2Ph) 503 (d 1H J12=16 Hz H-1II) 505 (ddd 1H J56=18 Hz J67a=64
Hz J67b=68 Hz H-6II) 521 (ddd 1H J56=20 Hz J67a=76 Hz J67b=56 Hz H-6I)
531 (dd 1H J23=30 Hz J34=100 Hz H-3II) 540 (dd 1H J34=98 Hz J45=100 Hz
H-4II) 547 (dd 1H J34=98 Hz J45=100 Hz H-4I) 728ndash745 (m 10H Ph) 13C
NMR (150 MHz CDCl3) 204 2066 2069 207 208 2083 (Ac-CH3) 551
(OCH3) 615 (C-7II) 620 (C-7I) 655 (C-4II) 669 (C-6I) 671 (C-6II) 678 (C-4I)
688 (C-5I) 692 (C-5II) 708 (C-3II) 726 (OCH2Ph) 734 (OCH2Ph) 744 (C-3I)
751 (C-2I) 755 (C-2II) 992 (C-1II) 994 (C-1I) 1280 1281 1282 1284 1287
1290 1374 1376 (Ph) 1693 1694 1697 1701 1703 1705 1706 (Ac O=C)
ESI-HRMS for C43H54O20 9133106 [M+Na]+ Found 9133093
624 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranose (37)
Compound 36 (1585 mg 1780 mol) was dissolved in a mixture of
H2SO4AcOHAc2O (40 mL 01614) and stirred for 2 h at room temperature The
66
reaction mixture was neutralized by the addition of sodium acetate (12 g) poured into
saturated sodium hydrogen carbonate and extracted with dichloromethane The
combined organic layer was washed with brine dried over anhydrous sodium sulfate
filtered and concentrated to a syrup (1565 mg) The crude syrup was treated with
hydrazine acetate (204 mg 2210 mol) in DMF (12 mL) for 7 h at 0 degC The
reaction mixture was diluted with ethyl acetate washed with water and brine dried
over anhydrous sodium sulfate filtered and concentrated The residue was purified
by silica gel column chromatography (ethyl acetatehexane 11) to give 37 (1224 mg
78) 1H-NMR (600 MHz CDCl3) 182 195 196 205 206 210 215 (s 3H x
7 Ac) 303 (s 1H OH) 370 (dd 1H J12=20 Hz J23=26 Hz H-2I) 372 (dd 1H
J45=100 Hz J56=18 Hz H-5II) 381 (dd 1H J12=16 Hz J23=30 Hz H-2II) 407
(dd 1H J45=102 Hz J56=20 Hz H-5I) 410 (dd 1H J67a=52 Hz J7a7b=114 Hz
H-7aII) 414 (dd 1H J67b=nd Hz J7a7b=114 Hz H-7bII) 414 (dd 1H J67a=nd Hz
J7a7b=114 Hz H-7aI) 416 (dd 1H J23=26 Hz J34=70 Hz H-3I) 438 (dd 1H
J67b=54 Hz J7a7b=114 Hz H-7bI) 459 464 (d 2H J=122 Hz OCH2Ph) 482
485 (d 2H J=128 Hz OCH2Ph) 504 (ddd 1H J56=18 Hz J67a=52 Hz J67b=nd
Hz H-6II) 506 (d 1H J12=16 Hz H-1II) 518 (ddd 1H J56=20 Hz J67a=nd Hz
J67b=54 Hz H-6I) 530 (d 1H J12=20 Hz H-1I) 531 (dd 1H J23=30 Hz
J34=102 Hz H-3II) 540 (dd 1H J34=102 Hz J45=100 Hz H-4II) 547 (dd 1H
J34=70 Hz J45=102 Hz H-4I) 730ndash746 (m 10H Ph) 13C NMR (150 MHz
CDCl3) 206 207 2075 209 215 (Ac-CH3) 617 (C-7II) 626 (C-7I) 655
(C-4II) 671 (C-6I) 6716 (C-6II) 680 (C-4I) 689 (C-5I) 693 (C-5II) 707 (C-3II)
726 (OCH2Ph) 734 (OCH2Ph) 742 (C-3I) 754 (C-2I) 755 (C-2II) 929 (C-1I)
994 (C-1II) 1280 1282 12825 1285 1287 1291 1374 1376 (Ph) 1694 1695
1698 1704 1707 1711 (Ac O=C) ESI-HRMS for C42H52O20 8992950 [M+Na]+
Found 8992930
625 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-467-tri-O-acetyl-2-O-benzyl-L-glycero-D-manno-heptopyranosyl trichloro-
acetimidate (38)
Trichloroacetonitrile (1920 L 19 mmol) was added to a solution of 37 (1399
67
mg 1600 mol) in dry dichloromethane (16 mL) under argon Potassium carbonate
(1130 mg 08 mmol) was added to the reaction After stirring for 21 h at room
temperature the mixture was filtered through Celite The solution was concentrated
and purified by flash column chromatography (ethyl acetatehexane 11) to give 38 as
a mixture of anomers (1295 mg 80 =61) -isomer []25D= +186 (c 15
CHCl3) 1H-NMR (600 MHz CDCl3) 190 194 197 201 205 206 213 (s 3H
x 7 Ac) 340 (dd 1H J45=100 Hz J56=20 Hz H-5II) 375 (dd 1H J12=14 Hz
J23=30 Hz H-2II) 389 (dd 1H J67a=40 Hz J7a7b=116 Hz H-7aII) 394 (dd 1H
J12=16 Hz J23=32 Hz H-2I) 405 (dd 1H J23=32 Hz J34=66 Hz H-3I) 408 (dd
1H J45=100 Hz J56=20 Hz H-5I) 411 (dd 1H J67b=72 Hz J7a7b=116 Hz
H-7bII) 416 (dd 1H J67a=76 Hz J7a7b=114 Hz H-7aI) 427 (dd 1H J67b=54 Hz
J7a7b=114 Hz H-7bI) 460 462 (d 2H J=122 Hz OCH2Ph) 473 491 (d 2H
J=124 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=40 Hz J67b=86 Hz H-6II)
502 (d 1H J12=14 Hz H-1II) 520 (dd 1H J23=30 Hz J34=102 Hz H-3II) 521
(ddd 1H J56=20 Hz J67a=76 Hz J67b=54 Hz H-6I) 531 (dd J34=102 Hz
J45=100 Hz H-4II) 552 (dd J34=66 Hz J45=100 Hz H-4I) 640 (d 1H J12=16
Hz H-1I) 727ndash750 (m 10H Ph) 880 (s 1H NH) 13C NMR (150 MHz CDCl3)
206 207 2076 208 (Ac-CH3) 621 (C-7I) 626 (C-7II) 653 (C-4II) 667 (C-6I)
669 (C-4I) 672 (C-6II) 698 (C-5II) 708 (C-3II) 715 (C-5I) 722 (OCH2Ph) 735
(OCH2Ph) 737 (C-2I) 753 (C-3I) 758 (C-2II) 906 (OCNHCCl3) 950 (C-1I) 1002
(C-1II) 1278 1280 1282 1285 1286 1289 1369 1374 (Ph) 1595
(OCNHCCl3) 1694 1697 1702 1705 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422051 -isomer []25D= -126 (c 03 CHCl3)
1H-NMR (600 MHz CDCl3) 175 188 190 197 198 202 205 (s 3H x 7 Ac)
352 (dd 1H J45=98 Hz J56=20 Hz H-5II) 364 (dd 1H J45=100 Hz J56=26 Hz
H-5I) 374 (dd 1H J12=20 Hz J23=30 Hz H-2II) 378 (dd 1H J23=28 Hz
J34=96 Hz H-3I) 389 (dd 1H J12=nd Hz J23=28 Hz H-2I) 400 (dd 1H J67a=78
Hz J7a7b=116 Hz H-7aI) 403 (dd 1H J67a=66 Hz J7a7b=112 Hz H-7aII) 411 (dd
1H J67b=64 Hz J7a7b=112 Hz H-7bII) 438 (dd 1H J67b=50 Hz J7a7b=116 Hz
H-7bI) 453 455 (d 2H J=128 Hz OCH2Ph) 498 (ddd 1H J56=20 Hz J67a=66
68
Hz J67b=64 Hz H-6II) 493 498 (d 2H J=126 Hz OCH2Ph) 497 (d 1H J12=20
Hz H-1II) 520 (ddd 1H J56=20 Hz J67a=78 Hz J67b=50 Hz H-6I) 525 (dd 1H
J23=30 Hz J34=102 Hz H-3II) 532 (dd 1H J34=102 Hz J45=98 Hz H-4II) 546
(dd 1H J34=96 Hz J45=100 Hz H-4I) 572 (d 1H J12=nd Hz H-1I) 719ndash748 (m
10H Ph) 869 (s 1H NH) 13C NMR (150 MHz CDCl3) 195 197 1971 198
1983 199 (Ac-CH3) 600 (C-7I) 613 (C-7II) 644 (C-4II) 656 (C-6I) 660 (C-4I)
665 (C-6II) 681 (C-5II) 695 (C-3II) 724 (C-5I) 725 (OCH2Ph) 7253 (OCH2Ph)
731 (C-2I) 745 (C-3I) 759 (C-2II) 894 (OCNHCCl3) 960 (C-1I) 984 (C-1II)
1268 1270 1270 1274 1276 1363 1364 (Ph) 1597 (OCNHCCl3) 1682
1684 1687 1691 1692 16961698 (Ac O=C) ESI-HRMS for C44H52Cl3NO20
10422046 [M+Na]+ Found 10422012
626 (2346-Tetra-O-benzyl--D-galactopyranosyl)-(1-4)-(236-tri-O-benzyl--D
-glucopyranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylide-
ne-3-deoxy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3
-deoxy--D-manno-2-octulopyranosid)onate (41)
A mixture of 6 (100 mg 102 mol) and 40 (341 mg 305 mol) and
MS-AW 300 molecular sieves (100 mg) was suspended in diethyl ether and
dichloromethane (31 04 mL) The reaction mixture was stirred for 1 h under argon
and cooled to 0 degC Then 001 M TMSOTf (600 L 06 mol) in dichloromethane
was added dropwise to the reaction mixture After stirring for 1 h the reaction was
warmed to room temperature and stirred for 1 h The reaction solution was neutralized
by the addition of a few drops of triethylamine and aqueous saturated sodium
hydrogen carbonate The reaction mixture was diluted with dichloromethane and was
filtered through Celite The filtrate was extracted twice with dichloromethane The
combined organic phase was dried over anhydrous sodium sulfate filtered and
concentrated The residue was purified by BioRad S-X1 size exclusion beads
(tolueneethyl acetate 11) and TLC chromatography(ethyl acetatehexane 13) to
give 41 (40 mg 20) []25D= +410 (c 05 CHCl3)
1H-NMR (600 MHz CDCl3)
098 (dd 1H J3a3b=154 Hz J3a4=24 Hz H-3aII) 118 (s 3H Me) 134 (s 3H Me)
223 (dd 1H J3a3b=124 Hz J3a4=50 Hz H-3aI) 226 (dd 1H J3a3b=124 Hz
69
J3b4=118 Hz H-3bI) 259 (dd 1H J3a3b=154 Hz J3b4=34 Hz H-3bII) 331 (ddd
1H J45=nd Hz J56a=50 Hz J56b=nd Hz H-5IV) 332 (dd 1H J23=98 Hz J34=28
Hz H-3IV) 338 (dd 1H J56a=50 Hz J6a6b=88 Hz H-6aIV) 338 (s 3H OMeI) 348
(s 3H OMeII) 351 (dd 1H J12=34 Hz J23=98 Hz H-2III) 356 (dd 1H J56b=nd
Hz J6a6b=88 Hz H-6bIV) 364 (dd 1H J56a=16 Hz J6a6b=92 Hz H-6aIII) 369 (dd
1H J45=30 Hz J56=12 Hz H-5I) 374 (dd 1H J12=78 Hz J23=98 Hz H-2IV)
386 (dd 1H J56=16 Hz J67=76 Hz H-6II) 386 (dddd 1H J=14 32 48 130 Hz
OCH2-) 390 (dd 1H J34=28 Hz J45=nd Hz H-4IV) 390 (dd 1H J23=98 Hz
J34=nd Hz H-3III) 394 (dd 1H J56b=nd Hz J6a6b=92 Hz H-6bIII) 394 (ddd 1H
J3a4=24 Hz J3b4=34 Hz J45=nd Hz H-4II) 395 (dddd 1H J=nd Hz OCH2-) 403
(dd 1H J56=12 Hz J67=94 Hz H-6I) 404 (dd 1H J45=nd Hz J56=16 Hz H-5II)
405 (dd 1H J34=nd Hz J45=100 Hz H-4III) 411 (ddd 1H J45=100 Hz J56a=16
Hz J56b=nd Hz H-5III) 425 437 (d 2H J=118 Hz CH2Ph) 428 (dd 1H J78a=34
Hz J8a8b=126 Hz H-8aI) 435 (d 1H J12=78 Hz H-1IV) 436 460 (d 2H J=120
Hz CH2Ph) 446 (dd 1H J78b=26 Hz J8a8b=126 Hz H-8bI) 455 497 (d 2H
J=112 Hz CH2Ph) 458 (dd 1H J78a=46 Hz J8a8b=128 Hz H-8aII) 462 466 (d
2H J=120 Hz CH2Ph) 470 (ddd 1H J3a4=50 Hz J3b4=118 Hz J45=30 Hz H-4I)
470 501 (d 2H J=104 Hz CH2Ph) 477 500 (d 2H J=124 Hz CH2Ph) 486
487 (d 2H J=nd Hz CH2Ph) 486 (dddd 1H J=nd Hz =CH2) 488 (d 1H J12=34
Hz H-1III) 493 (dddd 1H J=16 16 32 172 Hz =CH2) 530 (dd 1H J78b=26
Hz J8a8b=128 Hz H-8bII) 558-564 (m1H -CH=) 559 (ddd 1H J67=76 Hz
J78a=46 Hz J78b=26 Hz H-7II) 575 (ddd 1H J67=94 Hz J78a=34 Hz J78b=26
Hz H-7I) 703ndash756 (m 47H Ar) 784ndash800 (m 8H Ar) 13C NMR (150 MHz
CDCl3) 246 and 251 (Isop-Me) 314 (C-3II) 344 (C-3I) 520 (OMeI) 521
(OMeII) 621 (C-8I) 621 (C-8II) 647 (OCH2-) 677 (C-4I) 679 (C-6III) 681
(C-6IV) 693 (C-7I) 698 (C-4II) 699 (C-6II) 7066 (C-7II) 707 (C-6I) 710 (C-5III)
7203 (C-5I) 7206 (C-5II) 723 726 730 734 744 747 753 (OCH2Ph) 729
(C-5IV) 737 (C-4IV) 762 (C-4III) 789 (C-2III) 802 (C-2IV) 803 (C-3III) 823
(C-3IV) 962 (C-2II) 983 (C-1III) 988 (C-2I) 1027 (C-1IV) 1096 (Cisop) 1162
(=CH2) 1263 1267 1269 1273 1274 1275 1277 1278 1279 1280 1281
70
12814 1282 1283 12831 1284 12840 12845 1286 1296 12965 1297
12975 1298 1300 1303 1329 1330 1331 1334 (Ar) 1336 (-CH=) 13814
1382 1385 1390 1391 1393 1399 (Ar) 1648 1651 1658 1661 (Bz C=O)
1677 (C-1I) 1687 (C-1II) MALDI-TOF MS for C113H116O29 1959750 [M+Na]+
Found 1959300
627 (2346-Tetra-O-acetyl--D-galactopyranosyl)-(1-4)-(236-tri-O-acetyl--D-g
-lucopyranosyl)-(1-4)-(367-tri-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptop
-yranosyl)-(1-5)-[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-de
o-xy--D-manno-2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy
--D-manno-2-octulopyranosid)onate (43)
A mixture of 6 (420 mg 427 mol) 31 (960 mg 841 mol) and MS-AW
300 molecular sieves (430 mg) was suspended in dry dichloromethane (16 mL) The
reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf (2600 L
25 mol) in dichloromethane was added dropwise to the reaction mixture After
stirring for 2 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 21) to give 43 (228 mg 26) []25D= +29
(c 19 CHCl3) 1H-NMR (600 MHz CDCl3) 122 (s 3H Me) 141 (s 3H Me)
190 191 195 199 201 202 203 203 205 214 (s 3H x 10 Ac) 202 (dd 1H
J3a3b=156 Hz J3a4=22 Hz H-3aII) 218 (dd 1H J3a3b=126 Hz J3a4=124 Hz
H-3aI) 238 (dd 1H J3a3b=126 Hz J3b4=44 Hz H-3bI) 291 (dd 1H J3a3b=156 Hz
J3b4=34 Hz H-3bII) 349 (s 3H OMeI) 355 (s 3H OMeII) 354 (ddd 1H J45=96
Hz J56a=50 Hz J56b=16 Hz H-5IV) 379 (dd 1H J45=nd Hz J56=nd Hz H-5I)
379 (dd 1H J34=90 Hz J45=96 Hz H-4IV) 382 (ddd 1H J45=06 Hz J56a=72 Hz
J56b=62 Hz H-5V) 382 (dd 1H J12=16 Hz J23=28 Hz H-2III) 385 (dd 1H
J34=94 Hz J45=102 Hz H-4III) 391 (dd 1H J=nd Hz OCH2-) 392 (dd 1H
71
J67a=78 Hz J7a7b=114 Hz H-7aI) 395 (dd 1H J45=102 Hz J56=nd Hz H-5III)
397 (dd 1H J=nd Hz OCH2-) 407 (dd 1H J56a=72 Hz J6a6b=112 Hz H-6aV)
408 (dd 1H J56=nd Hz J67=94 Hz H-6I) 408 (dd 1H J56a=50 Hz J6a6b=120 Hz
H-6aIV) 411 (dd 1H J56b=62 Hz J6a6b=112 Hz H-6bV) 422 (dd 1H J67b=52 Hz
J7a7b=114 Hz H-7bI) 430 (dd 1H J56=16 Hz J67=60 Hz H-6II) 438 (dd 1H
J45=78 Hz J56=16 Hz H-5II) 447 (d 1H J12=78 Hz H-1IV) 435 (dd 1H
J56b=16 Hz J6a6b=120 Hz H-6bIV) 443 (d 1H J12=80 Hz H-1V) 445 454 (d
2H J=120 Hz CH2Ph) 446 (ddd 1H J3a4=124 Hz J3b4=44 Hz J45=nd Hz H-4I)
455 (ddd 1H J3a4=22 Hz J3b4=34 Hz J45=78 Hz H-4II) 462 (dd 1H J78a=62
Hz J8a8b=124 Hz H-8aII) 463 (dd 1H J78a=38 Hz J8a8b=124 Hz H-8aI) 479 (dd
1H J12=78 Hz J23=88 Hz H-2IV) 488 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bI)
491 (dd 1H J23=104 Hz J34=34 Hz H-3V) 493 (dd 1H J=14 nd Hz =CH2)
501 (d 1H J12=16 Hz H-1III) 505 (dd 1H J=16 16 32 172 Hz =CH2) 508
(dd 1H J12=80 Hz J23=104 Hz H-2V) 512 (dd 1H J23=88 Hz J34=90 Hz
H-3IV) 518 (dd 1H J78b=22 Hz J8a8b=124 Hz H-8bII) 530 (ddd 1H J56=nd Hz
J67a=78 Hz J67b=52 Hz H-6III) 533 (dd 1H J34=34 Hz J45=06 Hz H-4III) 534
(dd 1H J23=28 Hz J34=94 Hz H-3III) 558 (ddd 1H J67=94 Hz J78a=38 Hz
J78b=24 Hz H-7I) 563-569 (m1H -CH=) 575 (ddd 1H J67=60 Hz J78a=62 Hz
J78b=22 Hz H-7II) 727ndash757 (m 17H Ar) 794ndash800 (m 8H Ar) 13C NMR (150
MHz CDCl3) 205 2054 207 208 2084 209 230 238 (Ac-CH3) 246 and
251 (Isop-Me) 320 (C-3II) 347 (C-3I) 522 (OMeII) 523 (OMeI) 645 (OCH2-)
607 (C-6V) 623 (C-6IV) 624 (C-8I) 632 (C-8II) 635 (C-7III) 666 (C-4V) 682
(C-6III) 6822 (C-4I) 690 (C-2V) 6967 (C-7I) 697 (C-3III) 700 (C-4II) 705 (C-6I)
706 (C-5V) 7091 (C-7II) 709 (C-5III) 710 (C-3V) 710 (C-6II) 714 (C-5I) 718
(C-2IV) 721 (C-5IV) 723 (OCH2Ph) 725 (C-5II) 733 (C-3IV) 739 (C-4III) 769
(C-2III) 763 (C-4IV) 9788 (C-2II) 979 (C-1III) 987 (C-2I) 1002 (C-1IV) 1011
(C-1V) 1095 (Cisop) 1160 (=CH2) 1274 1276 1282 1283 1284 1285 1286
1288 1294 1297 1298 1301 1302 1309 1328 1330 1332 1335 (Ar)
13353 (-CH=) 1384 (Ar) 1652 1653 1660 1662 (Bz C=O) 1676 (C-1I) 1691
(C-1II) 1696 1696 1697 1701 1702 1703 1704 1704 (Ac C=O)
72
MALDI-TOF MS for C98H112O45 2031638 [M+Na]+ Found 2030629
628 (3467-Tetra-O-acetyl-2-O-benzyl-L-glycero--D-manno-heptopyranosyl)-
(1-3)-(2-O-benzyl-467-tri-O-acetyl-L-glycero--D-manno-heptopyranosyl)-(1-5)-
[methyl O-[methyl (78-di-O-benzoyl-45-O-isopropylidene-3-deoxy--D-manno-
2-octulopyranosyl)onate]]-(2-4)-(allyl 78-di-O-benzoyl-3-deoxy--D-manno-2-oct
-ulopyranosid)onate (44)
A mixture of 6 (623 mg 634 mol) 38 (1295 mg 1268 mol =61) and
MS-AW 300 molecular sieves (62 mg) was suspended in dichloromethane (27 mL)
The reaction mixture was stirred for 1 h under argon and then 001 M TMSOTf
(3800 μL 38 mol) in dichloromethane was added dropwise to the reaction mixture
After stirring for 1 h the reaction was neutralized by the addition of a few drops of
triethylamine and aqueous saturated sodium hydrogen carbonate The reaction
mixture was diluted with dichloromethane and filtered through Celite The filtrate was
extracted twice with dichloromethane The combined organic phase was dried over
anhydrous sodium sulfate filtered and concentrated The residue was purified by
BioRad S-X1 size exclusion beads (tolueneethyl acetate 11) and TLC
chromatography (ethyl acetatehexane 32) to give 44 (669 mg 57) []25D= -70 (c
10 CHCl3) 1H-NMR (600 MHz CDCl3) 119 (s 3H Me) 136 (s 3H Me) 188
193 194 197 199 201 211 (s 3H x 7 Ac) 202 (dd 1H J3a3b=156 Hz
J3a4=26 Hz H-3aII) 208 (dd 1H J3a3b=124 Hz J3a4=122 Hz H-3aI) 225 (dd 1H
J3a3b=124 Hz J3b4=40 Hz H-3bI) 296 (dd 1H J3a3b=156 Hz J3b4=36 Hz H-3bII)
338 (s 3H OMeI) 343 (s 3H OMeII) 369 (dd 1H J45=22 Hz J56=nd Hz H-5I)
380 (dd 1H J12=18 Hz J23=30 Hz H-2IV) 389 (dd 1H J=16 30 48 130 Hz
OCH2-) 394 (dd 1H J=14 28 56 nd Hz OCH2-) 395 (dd 1H J45=98 Hz
J56=82 Hz H-5IV) 395 (dd 1H J12=16 Hz J23=24 Hz H-2III) 406 (dd 1H
J23=24 Hz J34=100 Hz H-3III) 409 (dd 1H J56=nd Hz J67=96 Hz H-6I) 410
(dd 1H J45=98 Hz J56=86 Hz H-5III) 421 (dd 1H J67a=60 Hz J7a7b=nd Hz
H-7aIV) 421 (dd 1H J67b=20 Hz J7a7b=nd Hz H-7bIV) 424 (dd 1H J67a=58 Hz
J7a7b=118 Hz H-7aIII) 426 (dd 1H J56=18 Hz J67=72 Hz H-6II) 432 (dd 1H
J67b=34 Hz J7a7b=118 Hz H-7bIII) 435 (dd 1H J45=78 Hz J56=18 Hz H-5II)
73
449 (ddd 1H J3a4=26 Hz J3b4=36 Hz J45=78 Hz H-4II) 459 (dd 1H J78a=38
Hz J8a8b=124 Hz H-8aI) 461 (dd 1H J78a=42 Hz J8a8b=124 Hz H-8aII) 461
468 (d 2H J=nd Hz CH2Ph) 462 470 (d 2H J=nd Hz CH2Ph) 467 (ddd 1H
J3a4=122 Hz J3b4=40 Hz J45=22 Hz H-4I) 480 (dd 1H J78b=26 Hz J8a8b=124
Hz H-8bI) 493 (dd 1H J=12 16 28 106 Hz =CH2) 498 (dd 1H J=16 16
32 172 Hz =CH2) 507 (d 1H J12=16 Hz H-1III) 515 (dd 1H J23=30 Hz
J34=100 Hz H-3IV) 520 (ddd 1H J56=82 Hz J67a=60 Hz J67b=20 Hz H-6IV)
521 (d 1H J12=18 Hz H-1IV) 527 (dd 1H J78b=24 Hz J8a8b=124 Hz H-8bII)
531 (ddd 1H J56=86 Hz J67a=58 Hz J67b=34 Hz H-6III) 541 (dd J34=100 Hz
J45=98 Hz H-4IV) 552 (dd J34=100 Hz J45=98 Hz H-4III) 557 (ddd 1H
J67=96 Hz J78a=38 Hz J78b=26 Hz H-7I) 563ndash569 (m1H -CH=) 568 (ddd 1H
J67=72 Hz J78a=42 Hz J78b=24 Hz H-7II) 721ndash760 (m 22H Ar) 794ndash804 (m
8H Ar) 13C NMR (150 MHz CDCl3) 206 208 2083 and 2087 209 (Ac-CH3)
246 and 251 (Isop-Me) 320 (C-3II) 345 (C-3I) 521 (OMeII) 522 (OMeI) 624
(C-8II) 626 (C-8I) 635 (C-7IV) 641 (C-7III) 648 (OCH2-) 653 (C-4IV) 669
(C-4III) 676 (C-4I) 677 (C-6III) 680 (C-6IV) 690 (C-7I) 699 (C-4II) 700 (C-5IV)
701 (C-3III) 703 (C-6I) 703 (C-5III) 703 (C-6II) 706 (C-7II) 712 (C-3IV) 721
729 (OCH2Ph) 722 (C-5II) 741 (C-5I) 755 (C-2IV) 772 (C-2III) 970 (C-2II) 988
(C-2I) 990 (C-1IV) 992 (C-1III) 1096 (Cisop) 1164 (=CH2) 1273 1274 1277
1278 1283 12832 1284 1286 1288 1289 1295 1296 1297 12975 1299
1301 1329 1330 1333 (Ar) 1334 (-CH=) 1338 1378 1383 (Ar) 1652 1654
1658 1662 (Bz C=O) 1674 (C-1I) 1694 (C-1II) 1696 1697 1698 1704 1705
1707 1708 (Ac C=O) MALDI-TOF MS for C94H104O38 1863611 [M+Na]+ Found
1862589
629 (L-Glycero--D-manno-heptopyranosyl)-(1-3)-(L-glycero--D-manno-heptop-
yranosyl)-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]-(2-4)-s
-odium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (45)
Compound 44 (100 mg 54 mol) in dry methanol (03 mL) was added to a
suspension of Pd(OH)2-C (20 03 mg) in dry methanol (02 mL) under a H2
atmosphere with stirring at room temperature After stirring for 3 days insoluble
74
materials were removed by filtration through Celite and the filtrate was evaporated
The residue was dissolved in dichloromethane (07 mL) and aqueous 80
trifluoroacetic acid (800 L) was added at room temperature After stirring for 1 h
the solvent was removed by evaporation under an argon stream to give a crude
compound that was not subjected to further purification The crude compound was
dissolved in methanol (10 mL) and then 01 M sodium hydroxide (13 mL 013
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was purified by gel filtration
chromatography (Biogel P-2) and a Sep-Pak C18 column (H2O) to give 45 (45 mg
90) as a colorless powder []25D=+1276 (c 05 H2O) 1H-NMR (600 MHz D2O)
092 (t 3H J=74 Hz CH3) 154ndash161 (m 2H CH2) 176 (dd 1H J3a3b=132 Hz
J3a4=126 Hz H-3aII) 193 (dd 1H J3a3b=126 Hz J3a4=124 Hz H-3aI) 206 (dd
1H J3a3b=126 Hz J3b4=42 Hz H-3bI) 220 (dd 1H J3a3b=132 Hz J3b4=48 Hz
H-3bII) 321ndash324 (m 1H OCH2) 328ndash332 (m 1H OCH2) 357 (dd 1H J67=94
Hz H-6I) 361 (dd 1H J78a=62 Hz J8a8b=118 Hz H-8aII) 366 (dd 1H J67=82 Hz
H-6II) 368 (dd 1H J67a=57 Hz J7a7b=108 Hz H-7aIII) 373ndash377 (m 4H H-7bIII
H-7aIV H-7bIV H-5III) 381 (dd 1H J78a=64 Hz J8a8b=116 Hz H-8aI) 384ndash398
(m 8H H-7I H-3III H-4III H-7II H-8bII H-4IV H-5IV H-8bI) 401ndash406 (m 4H
H-5II H-3IV H-6III H-6IV) 407 (dd 1H J12=12 Hz J23=32 Hz H-2III) 409 (ddd
1H J3a4=126 Hz J3b4=48 Hz J45=30 Hz H-4II) 413 (dd 1H J12=16 Hz J23=30
Hz H-2IV) 422 (brs 1H J45=22 Hz H-5I) 426 (ddd 1H J3a4=124 Hz J3b4=42
Hz J45=22 Hz H-4I) 517 (d 1H J12=12 Hz H-1III) 529 (d 1H J12=16 Hz
H-1IV) 13C NMR (150 MHz D2O) 109 (CH3) 239 (CH2) 352 (C-3I C-3II) 635
(C-7IV C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 664 (C-4III) 668 (C-4II) 6681
(C-5II) 670 (C-4IV) 694 (C-6III) 697 (C-7I) 700 (C-6IV) 701 (C-4I) 706 (C-2III)
708 (C-2IV) 7083 (C-7II) 711 (C-3IV) 724 (C-5III) 726 (C-6I) 7262 (C-6II) 733
(C-5IV and C-5I) 792 (C-3III) 1005 1007 (C-2I C-2II) 1013 (C-1III) 1030 (C-1IV)
1756 1759 (C-1I C-1II) ESI-HRMS for C33H55O27 8832931 [M-2Na+H]- Found
8832929
75
630-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-4)-L-glycero--D-manno
-heptopyranosyl-(1-5)-[O-(sodium 3-deoxy--D-manno-2-octulopyranosylonate)]
-(2-4)-sodium (propyl 3-deoxy--D-manno-2-octulopyranosid)onate (46)
Compound 43 (90 mg 45 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 02 mg) in dry methanol (05 mL) under atmospheric pressure of
hydrogen for 2 days at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was dissolved in dichloromethane (06 mL) and
aqueous 80 trifluoroacetic acid (700 L) was added at room temperature After
stirring for 1 h the solvent was removed by evaporation under an argon stream to give
a crude compound that was not subjected to further purification The crude compound
was dissolved in methanol (10 mL) and then 01 M sodium hydroxide (14 mL 014
mmol) was added at room temperature After stirring for 24 h the mixture was
concentrated by evaporation The residue was passed through a Bio-Gel P-2 column
(25 x 100 cm H2O) and a Sep-Pak C18 column (H2O) to give 46 (25 mg 53) as a
colorless powder []25D = +174 (c 03 H2O) 1H-NMR (600 MHz D2O) 091 (t
3H J=74 Hz CH3) 155ndash162 (m 2H CH2) 177 (dd 1H J3a3b=128 Hz J3a4=126
Hz H-3aII) 192 (dd 1H J3a3b=128 Hz J3a4=120 Hz H-3aI) 207 (dd 1H
J3a3b=128 Hz J3b4=44 Hz H-3bI) 218 (dd 1H J3a3b=128 Hz J3b4=46 Hz H-3bII)
319ndash324 (m 1H OCH2) 326ndash330 (m 1H OCH2) 339 (dd 1H J12=80 Hz
J23=88 Hz H-2IV) 354 (dd 1H J12=78 Hz J23=100 Hz H-2V) 358 (dd 1H
J67=84 Hz H-6I) 359 (dd 1H J78a=62 Hz J8a8b=116 Hz H-8aII) 364ndash384 (m
14H H-3V H-6II H-3IV H-6aIV H-6bIV H-5V H-4IV H-6aV H-6bV H-7aIII H-5III
H-7bIII H-8aI H-7I) 389 (dd 1H J78b=26 Hz J8a8b=116 Hz H-8bII) 391ndash399 (m
4H H-4V H-7II H-5IV H-8bI) 400ndash407 (m 3H H-4III H-3III H-5II) 409 (brs 1H
J12=16 Hz H-2III) 410 (ddd 1H J3a4=126 Hz J3b4=46 Hz J45=30 Hz H-4II)
414 (ddd 1H J67a=78 Hz J67b=48 Hz H-6III) 421 (brs 1H J45=22 Hz H-5I)
424 (ddd 1H J3a4=120 Hz J3b4=44 Hz J45=22 Hz H-4I) 444 (d 1H J12=78 Hz
H-1V) 458 (d 1H J12=80 Hz H-1IV) 532 (d 1H J12=16 Hz H-1III) 13C NMR
(150 MHz D2O) 108 (CH3) 238 (CH2) 350 353 (C-3I C-3II) 606 (C-6IV) 616
(C-6V) 635 (C-8I) 640 (C-8II) 646 (C-7III) 654 (OCH2) 669 (C-4II) 670 (C-5II)
76
692 (C-6III) 697 (C-7I) 699 (C-4V) 702 (C-4I) 703 (C3III) 705 (C-2III) 707
(C-7II) 716 (C-5III) 720 (C-2V) 723 (C-5IV) 726 (C-6I) 727 (C-6II) 731 (C-4III)
734 (C-5I) 746 (C-3IV) 755 (C-2IV) 760 (C-3V) 765 (C-5V) 788 (C-4IV) 1005
1008 (C-2I C-2II) 1010 (C-1III) 1030 (C-1IV) 1036 (C-1V) 1755 1759 (C-1I
C-1II) ESI-HRMS for C38H63O31 10153353 [M-2Na+H]- Found 10153370
631 (-D-Galactopyranosyl-(1-4)--D-glucopyranosyl)-(1-5)-[O-(sodium 3-deox-
y--D-manno-2-octulopyranosylonate)]-(2-4)-sodium (propyl 3-deoxy--D-mann
-o-2-octulopyranosid)onate (47)
Compound 41 (53 mg 27 mol) was hydrogenated in the presence of
Pd(OH)2-C (20 15 mg) in dry methanol (04 mL) under atmospheric pressure of
hydrogen for 1 day at room temperature The reaction mixture was filtered through
Celite and concentrated The residue was treated with aqueous 80 trifluoroacetic
acid (400 L) at room temperature After stirring for 5 min the solvent was removed
by evaporation under an argon stream to give a crude compound that was not
subjected to further purification The crude compound was dissolved in methanol (08
mL) and then 01 M sodium hydroxide (05 mL 005 mmol) was added at room
temperature After stirring for 24 h the mixture was concentrated by evaporation
The residue was purified by a Bio-Gel P-2 column (25 x 100 cm H2O) anda Sep-Pak
C18 column (H2O) to give 47 (14 mg 60) as a colorless powder []25D = +201 (c
01 H2O) 1H-NMR (600 MHz D2O) 090 (t 3H J=74 Hz CH3) 153ndash160 (m
2H CH2) 180 (dd 1H J3a3b=128 Hz J3a4=126 Hz H-3aII) 202 (dd 1H
J3a3b=126 Hz H-3aI) 206ndash208 (m 2H H-3bI H-3bII) 321ndash330 (m 2H OCH2)
355 (dd 1H J12=78 Hz J23=102 Hz H-2IV) 356ndash360 (m 3H H-2III H-6I H-8aII)
366 (dd 1H J23=102 Hz J34=34 Hz H-3IV) 372ndash382 (m 6H H-6II H-4III
H-6aIV H-6bIV H-5IV H-8aI) 389ndash401 (m 9H H-8bI H-7II H-4IV H-6aIII H-6bIII
H-3III H-8bII H-5II H-4I) 406 (ddd 1H J67=90 Hz J78a=30 Hz J7 8b=24 Hz
H-7I) 409ndash412 (m 1H J3a4=126 Hz J3b4=48 Hz J45=22 Hz H-4II) 423 (brs 1H
H-5I) 423ndash426 (m 1H H-5III) 447 (d 1H J12=78 Hz H-1IV) 528 (dd 1H
J12=36 Hz H-1III) 13C NMR (150 MHz D2O) 108 (CH3) 228 (CH2) 352
(C-3I C-3II) 602 (C-6III) 617 (C-6IV) 633 (C-8I) 640 (C-8II) 653 (OCH2) 667
77
(C-4II) 675 (C-5II) 692 (C-7I) 693 (C-4IV) 701 713 716 718 720 723 724
730 732 (C-4I C-7II C-2IV C-5III C-6II C-6I C-2III C-3III C-5I) 740 (C-3IV) 759
(C-5IV) 785 (C-4III) 996 1005 (C-2I C-2II) 1023 (C-1III) 1035 (C-1IV) 1759 and
1761 (C-1I C-1II) ESI-HRMS for C31H51O25 8232719 [M-2Na+H]- Found
8232732
78
References
1 Scherp H Annual review of Microbiology 1955 9 319ndash334
2 Cohn A MacNeil J Clark T Ortega-Sanchez I Briere E Meissner H Baker
C Messonnier N Morbidity and mortality weekly report Recommendations and
reports 2013 62 1ndash28
3 World Health Organization Weekly epidemiological record 2001 76 281ndash288
4 Mola S Nield L Weisse M Infections in Medicine 2008 February 27
5 Acton A Meningitis-Advances in Research and Treatment Scholarly Editions
Atlanta Georgia 2012
6 Aronson J Meylers Side Effects of Antimicrobial Drugs Elsevier Science New
York 2009
7 Chandran A Herbert H Misurski D Santosham M Pediatr Infect Dis J 2011
30 3ndash6
8 Yang Q Jennings H Purification of Capsular Polysaccharide eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 41ndash48
9 Rosenstein N Perkins B Stephens D Popovic T Hughes J N Engl J Med
2001 344 1378ndash1388
10 Feavers I Meningococcal Vaccines and Vaccine Development eds Pollard A
Maiden M Hummer press Totowa New Jersey 2001 pp 1ndash22
11 Nizet V Esko J Bacterial and Viral Infections eds Varki A Cummings R
Esko J Freeze H Hart G Cold Spring Harbor Laboratory Press La Jolla
CA 2009 537ndash551
12 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Holst O Muumlller-Loennies S
Comprehensive Glycoscience 2007 1 123ndash179 (c) Raetz C Whitfield C Annu
Rev Biochem 2002 71 635-700
13 (a) Holst O Ulmer A Brade H Flad H Rietschel E FEMS Immunol Med
Microbiol 1996 16 83ndash104 (b) Caroff M Karibian D Carbohydr Res 2003
338 2431ndash2447
14 Moran A Prendergast M Appelmelk B FEMS Immunol Med Microbiol 1996
79
16 105ndash115
15 (a) Silipo A Molinaro A Subcell Biochem 2010 53 69ndash99 (b) Zaumlhringer U
Lindner B Rietschel E Adv Carbohydr Chem Biochem 1994 50 211ndash276
16 Knirel Y O-Specific polysaccharides of Gram-negative bacteria eds Moran A
Brennan P Holst O Itzstein M Elsevier Amsterdam 2009 pp 57ndash73
17 Yamasaki R Yabe U Kataoka C Takeda U Asuka S Infect Immun 2010 78
3247ndash3257
18 Holst O Brade H Chemical Structure of the Core Region of Lipopolysaccharides
eds Morrison D C amp Ryan J L CRC Press Boca Ranton FL 1929 pp
135ndash170
19 Muumlller-Loennies S Lindner B Brade H Eur J Biochem 2002 269
5982ndash5991
20 Cox A Howard M Inzana T Carbohydr Res 2003 338 1223ndash1228
21 Chalabaev S Kim T H Ross R Derian A Kasper D L J Biol Chem 2010
285 34330ndash34336
22 De C Molinaro A Nunziata R Lanzetta R Parrilli M Holst O Eur J Org
Chem 2004 2427ndash2435
23 Zdorovenko G Yakovleva L Gvodziak R Zakharova I Koshechkina L
Mikrobiol Zh 1982 44 65ndash70
24 Leive L Morrison D Methods Enzymol 1972 28 254ndash262
25 Ribi E Haskins W Landy M Milner K Exp Med 1961 114 647ndash663
26 Westphal O Jann K Methods Carbohydr Chem 1965 5 83ndash91
27 Hitchcock P Brown T Bacteriol 1983 154 269ndash277
28 (a) Luumlderitz O Westphal O Staub AM Nikaido H Microbial toxins IV
Bacteral endotoxins 1971 145ndash233 (b) Wang Q Shi X Leymarie N Madico
G Sharon J Costello C Zaia J Biochemistry 2011 50 10941ndash10950
29 Boltje T Buskas T Boons G Nature Chem 2009 1 611ndash622
30 (a) Kuboki A Tajimi T Tokuda Y Kato D Sugai T Ohira S Tetrahedron
Lett 2004 45 4545ndash4548 (b) Kikelj V Plantier-Royon R Portella C Synthesis
2006 1200ndash1204
80
31 (a) Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J
Carbohydr Chem 2001 20 171ndash180 (b) Ohara T Adibekian A Esposito D
Stallforth P Seeberger P H Chem Commun 2010 46 4106ndash4108
32 (a) Olsson J Oscarson S Tetrahedron Asymm 2009 20 879ndash886 (b) Cox AD
St Michael F Neelamegan D Lacelle S Cairns C Richards J Glycoconj J
2010 27 401ndash417
33 Ichiyanagi T Fukunaga M Tagashira R Hayashi S Nanjo M Yamasaki R
Tetrahedron 2011 67 5964ndash5971
34 (a) Bernlind C Oscarson S J Org Chem 1998 63 7780ndash7788 (b) Yang Y
Oishi S Martin C Seeberger P J Am Chem Soc 2013 135 6262ndash6271
35 Paulsen H Wulff A Brenken M Liebigs Ann Chem 1991 11 1127ndash1145
36 (a) Kosma P Carbohydr Res 1988 180 19ndash28 (b) Kosma P Schulz G Unger
F M Brade H Carbohydr Res 1989 190 191ndash201 (c) Wimmer N Brade H
Kosma P Carbohydr Res 2000 329 549ndash560
37 Imoto M Kusunose N Matsuura Y Kusumoto S Shiba T Tetrahedron Lett
1987 28 6277ndash6280
38 Yu B Tao H J Org Chem 2002 67 9099ndash9102
39 Kondo H Ichikawa Y Wong C H J Am Chem Soc 1992 114 8748ndash8750
40 Nicolaou KC Caulfield TJ Kataoka H Stylianides NA J Am Chem Soc 1990
112 3693ndash3695
41 Shoda S Glycoside Synthesis From Anomeric Halides eds Demchenko A
WILEY-VCH Weinheim 2008 pp 29ndash59
42 Yoshizaki H Fukuda N Sato K Oikawa M Fukase K Suda Y Kusumoto S
Angew Chem Int Ed 2001 40 1475ndash1480
43 Yamasaki R Takajyo A Kubo H Matsui T Ishii K Yoshida M J Carbohydr
Chem 2001 20 171ndash180
44 Kong F Carbohydr Res 2007 342 345ndash373
45 Tvaroska I Taravel FR Adv Carbohydr Chem Biochem 1995 51 15ndash61
46 Kereacutekgyaacutertoacute J Kamerling J P Bouwstra J B Vliegenthart J F G Carbohydr
Res 1989 186 51ndash62
81
47 Grundler G Schmidt R R Liebigs Ann Chem 1984 1826ndash1847
48 Kalikanda J Li Z J Org Chem 2011 76 5207ndash5218
49 Rosen T Lico M I Chu W T D J Org Chem 1988 53 1582ndash1584
50 Mungall W Greene G Heavner G Letsinger R J Org Chem 1975 40
1659ndash1662
51 Holst O Structure of the Lipopolysaccharide Core Region Knirel Y Valvano M
Eds Springer Wien New York 2011 pp 21ndash40
52 (a) Tsai C-M Jankowska-Stephens E Mizanur R Cipollo J J Biol Chem 2009
284 4616ndash4625 (b) Knirel Y Lindner B Vinogradov E Kocharova N
Senchenkova S Shaikhutdinova R Dentovskaya S Fursova N Bakhteeva I
Titareva G Balakhonov S Biochemistry 2005 44 1731ndash1743 (c) Bystrova O
Knirel Y Lindner B Kocharova N Kondakova A Zaumlhringer U Pier G FEMS
Immunol Med Microbiol 2006 46 85ndash99
53 Ishii K Esumi Y Iwasaki Y Yamasaki R Eur J Org Chem 2004 6
1214ndash1227
54 Dean B Oguchi H Cai S Otsuji E Tashiro K Hakomori S Toyokuni T
Carbohydr Res 1993 245 175ndash192
55 Ishii K Synthesis of oligosaccharides expressed in lipooligosaccharides produced
by pathogenic Gram-negative bacteria construction of the branched core
oligosaccharides by using 3-O-silyl-heptose Tottori University 2005
56 Ishii K Kubo H Yamasaki R Carbohydr Res 2002 337 11ndash20
57 Greenberg W Priestley E Sears P Alper P Rosenbohm C Hendrix M Hung
S Wong C J Am Chem Soc 1999 121 6527ndash6541
58 Tvaroska I Bleha T Adv Carbohydr Chem Biochem 1989 47 45ndash123
59 (a) Loumlnn H J Carbohydr Chem 1987 6 301ndash306 (b) Lahmann M Oscarson S
Org Lett 2000 2 3881ndash3882
60 Tamura K Mizukami H Maeda K Watanabe H Uneyama K J Org
Chem 1993 58 32-35
82
Summary
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are the major
glycolipds expressed in the outer membrane of gram-negative bacteria and play an
important role in the pathogenesis of bacterial infections LPS is composed of
O-specific polysaccharide a core oligosaccharide (core OS) and lipid A whereas
LOS lacks an O-antigen The core OS which is a significant target for vaccine
development and diagnostics of pathogenic bacteria can be further subdivided into
the inner core and outer core The inner core OS is composed of a 45-branched
3-deoxy-D-manno-2-octulosonic acid (Kdo) structures However there are few reports
about the synthesis of this branch Kdo structure To synthesize this branch Kdo
structure and extend our previous research a new synthetic approach was developed
In this study chapter 2 described the synthesis of linked Kdo disaccharide
chapter 3 showed a new synthetic approach to synthesize 45-branched Kdo
trisaccharides using Kdo disaccharide as an acceptor in chapter 4 more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor chapter 5 showed the conclusions
In chapter 2 2-4 linked Kdo disaccharide was obtained by glycosidation of Kdo
donor with 45-diol acceptor To optimize the condition of this reaction several types
of Kdo donors with different leaving groups were prepared from the common Kdo
intermediate and were glycosylated with 45-diol acceptor The results showed that all
donors produced the -glycoside as the main product and the stereoselectivity was not
influenced by the type of leaving group Moreover the -fluoride donor with
BF3middotOEt2 as the activator provided the best yield and -selectivity of product
In chapter 3 Kdo(2-4)Kdo as an acceptor was glycosylated with
L-glycero-D-manno-heptosyl (Hep) mannosyl (Man) and 2-azido-2-deoxy-galactosyl
imidates (GalN3) respectively and three corresponding 45-branched trisaccharides
Hep(1-5)[Kdo(2-4)]Kdo Man(1-5)[Kdo(2-4)]Kdo and
GalN3(1-5)[Kdo(2-4)]Kdo were successfully synthesized in good yield and high
-selectivity These results confirmed that glycosylation at the 4-OH position of the
Kdo acceptor followed by a second glycosylation at 5-OH position could produce the
83
target 45-branched Kdo structures and this new synthetic strategy is different from
Paulsenrsquos method
In chapter 4 to extend the utility of the new synthetic strategy more complex
45-branched Kdo structures were synthesized by using the same Kdo disaccharide as
the acceptor To do it firstly the L-glycero-D-manno-heptopyranose (Hep) units
Gal(1-4)Glc(1-4)Hep and Hep(1-3)Hep for the branched core oligosaccharide
were prepared from the corresponding Hep building blocks Then the Hep units were
glycosylated with the common acceptor Kdo(2-4)Kdo to afford 45-branched core
oligosaccharide structures Three complex 45-branched Kdo structures
Gal(1-4)Glc(1-5)[Kdo(2-4)]Kdo and Hep(1-3)Hep(1-5)[Kdo(2-4)]Kdo
Gal(1-4)Glc(1-4)Hep(1-5)[Kdo(2-4)]Kdo were successfully obtained
In all the new synthetic approach using Kdo(2-4)Kdo as an intermediate is
useful for the synthesis of 45-branched core OS structures including Kdo
trisaccharides Kdo tetrasaccharides and Kdo pentasaccharide
84
グラム陰性菌が産生するリポ多糖およびリポオリゴ糖の内部コア糖鎖の合成
研究45 で分岐した 3-デオキシ-D-マンノオクト-2-ウロン酸の構築
要旨
リポ多糖(LPS)やリポオリゴ糖(LOS)はグラム陰性細菌が細胞外膜に産生
する複合糖脂質であり細菌感染において重要な役割を持っているLPS は O
抗原多糖コアオリゴ糖(コア OS)そして Lipid A からなりこれに対して
LOS は O 抗原を欠損した構造をしているコア OS は内部コアと外部コアに分
けられるコア OSは細菌の属によって特に糖鎖構造が保存された領域であり
病原性細菌の診断ツールや感染予防のためのワクチン開発の抗原となる標的
分子として注目されている多くの LPSLOSの内部コア OSには 45-分岐した
3-デオキシ-D-マンノオクト-2-ウロン酸(Kdo)構造が存在しているこのよう
な分岐構造を有する Kdo 構造を含んだ分岐糖鎖の化学合成法についてはわず
かな報告しか存在しない本論文ではこの分岐構造を合成するために新し
い化学合成法を達成した
本論文では第 2 章に 2-4 結合を有する Kdo2 糖の合成について述べてい
る第 3章ではこの 2糖を糖受容体として用いた 45分岐構造を有する3糖の
新しい合成経路での合成について述べている第 4 章では 45-分岐 Kdo 構造
を含むより大きな糖鎖の合成法について述べられ第 5章でこれらについての
総括が述べられている
第 2章では45-ジオール Kdo受容体と様々な Kdo供与体とのグリコシル
化反応について述べているこの反応の反応条件を最適化するために数種類
の脱離基を持った Kdo 誘導体を共通の中間体から調製したそして 45-ジオ
ール受容体とのグリコシルを検討したその結果すべての供与体においてα-
グリコシドを主生成物として得られることそして BF3OEt2を活性化剤に用い
たα体のフッ化糖供与体を用いた反応では最も良い収率とα選択性を与える
ことが明らかになった
第 3 章では第 2 章で合成を達成した Kdoα(2-4)Kdo を糖受容体として用
いL-グリセロ-D-マンノヘプトース(Hep)マンノース (Man)および 2-アジ
ド-2-デオキシガラクトースのイミデート誘導体による 5 位水酸基へのグリコ
シル化反応の検討を行ったそしてα選択的に良好な収率で合成することに成
85
功したこの結果は Kdo の 45-ジオール誘導体に対してまず 4 位水酸基にへ
のグリコシル化続く 5 位水酸基へのグリコシル化反応は目的とする 45-分岐
構造を持つ Kdoの合成が可能であることを示したこの方法はこれまでに唯一
報告されている Paulsenらの方法とは異なる方法でありこのような糖鎖の合
成における新しい合成の方法論を提供するものである
第 4 章では 3 章で達成した新しい 45-分岐 Kdo 糖鎖合成法の有用性を拡
大するためにより複雑な糖鎖の合成を試みた結果について述べたはじめに
Hepを含む供与体ユニット Galβ(1-4)Glcβ(1-4)Hepと Hepα(1-3)Hepの合成
を対応する Hepビルディングブロックから調製したその後共通の受容体であ
る Kdoα(2-4)Kdoを用いたグリコシル化による 45分岐 Kdo糖鎖の合成を行っ
たその結果 3 種類の 45 分岐 Kdo 糖鎖である Galβ(1-4)Glcα(1-5)[Kdoα
(2-4)]Kdo Hepα(1-3)Hepα(1-5)[Kdoα(2-4)]Kdo そして Galβ(1-4)Glcβ
(1-4)Hepα(1-5)[Kdoα(2-4)]Kdoの合成を達成した
Kdoα(2-4)Kdoを中間体として用いる 45分岐 Kdoの新しい合成法はこの
構造を有する LPSLOSの内部コア糖鎖の合成に有用であることを示した
86
Acknowledge
This work was supported by JSPS KAKENHI Grant Number 23580474 and
15K01822 We thank Professors Jun-ichi Tamura and Toshiki Nokami for helpful
discussions and are also grateful to Ms Maki Taniguchi for technical assistance
87
List of publications
1 Ruiqin Yi Atsushi Ogaki Mayumi Fukunaga Hiromitsu Nakajima Tsuyoshi
Ichiyanagi Synthesis of 45-disubstituted-3-deoxy-D-manno-octulosonic acid (Kdo)
derivatives Tetrahedron 70 3675-3682 201406 (The corresponding content is in
chapter 2 and chapter 3)
2 Ruiqin Yi Hirofumi Narimoto Miku Nozoe Tsuyoshi Ichiyanagi Convergent
synthesis of 45-branched inner-core oligosaccharides of lipopoly- and
lipooligosaccharide Bioscience Biotechnology and Biochemistry Accepted at June
13th 2015 (The corresponding content is in chapter 4)