Stereoselective Syntheses of Semiochemicals

- Applications in Ecological Chemistry

ELLEN M. SANTANGELO

Doctoral Thesis

Stockholm 2004

Akademisk avhandling som med tillstånd av Kungl Tekniska Högskolan iStockholm framlägges till offentlig granskning för avläggande av filosofiedoktorsexamen i kemi med inriktning mot organisk kemi torsdagen den9:e december kl 10.00 i sal E3, KTH, Osquars Backe 14, 2 tr, Stockholm.Avhandlingen försvaras på engelska. Opponent är Professor Olov Sterner, Lunds universitet.

ISBN-91-7283-912-0

ISRN KTH/IOK/FR--04/93--SE

ISSN 1100-7974

TRITA-IOK

Forskningsrapport 2004:93

© Ellen M. Santangelo 2004

Santangelo, Ellen M., 2004 “Stereoselective syntheses of

semiochemicals – applications in ecological chemistry”. KTH

Chemistry, Organic Chemistry, SE-100 44, Stockholm, Sweden.

ABSTRACT

This thesis describes the syntheses of semiochemicals and their

applications in the development of control methods for pest insects.

The compounds synthesized are divided into three groups: 1)

Lepidoptera pheromones; 2) methyl substituted chiral pheromones

and 3) aphid pheromones.

Different purification techniques have been explored in order to

provide > 99% pure semiochemicals for field tests. Examples of the

techniques are uses of urea inclusion complexes, argentum

chromatography, low temperature crystallization and what we call the

Baeckström isolation technique.

Iridoids have been produced in a synthetic strategy including an

intramolecular enal-enamine [4+2] cycloaddition, a dynamic

acetylation and an enantioselective transesterification mediated by a

lipase from Pseudomonas cepacia. The use of chiral auxiliaries to

perform the intramolecular [4+2] cycloaddition has also been

investigated. A useful asymmetric route to iridoids has been

developed.

Keywords: synthesis, semiochemical, pheromone, Lepidoptera, diene, Diatraea, Phyllonorycter, Migdolus, Tribolium, aphid, iridoid, nepetalactone, intramolecular [4+2] cycloaddition.

This thesis is dedicated to the memory

of my grandmother Anna,

who was the greatest inspiration of my life.

LIST OF PUBLICATIONS

This thesis is based on the following papers, referred to in the text by their

Roman numerals I-IX.

I. Identification, syntheses, and characterization of the geometric

isomers of 9,11-hexadecadienal from female pheromone glands of

the sugar cane borer Diatraea saccharalis.

Ellen M. Santangelo, Myrian Coracini, Peter Witzgall, Arlene G. Correa and C.

Rikard Unelius. Journal of Natural Products 2002, 65, 909-915.

II. Eletrophysiological studies and identification of possible sex

pheromone components of Brazilian populations of the sugar cane

borer, Diatraea saccharalis.

Luciane G. Batista-Pereira, Ellen M. Santangelo, Kathrin Stein, C. Rikard

Unelius, Alvaro E. Eiras and Arlene G. Correa. Zeitschrift für Naturforschung

2002, 57c, 753-758.

III. Relative attractiveness of (10E)-dodecen-1-yl acetate and

(4E,10E)-dodecadien-1-yl-acetate to male spotted tentiform

leafminers Phyllonorycter blancardella (F).

Ashraf M. El-Sayed, Lena I. Wainman, Ellen M. Santangelo, C. Rikard Unelius

and R. Mitch Trimble. Journal of Chemical Ecology 2004, 30, 1827-1838.

IV. Sex pheromone of a North American population of the spotted

tentiform leafminer, Phyllonorycter blancardella.

Ashraf M. El-Sayed, Lena I. Wainman, Ellen M. Santangelo, C. Rikard Unelius

and R. Mitch Trimble. Entomologia Experimentalis et Applicata (submitted).

V. Synthesis of the four possible stereoisomers of N-2’-methylbutyl-

2-methylbutylamide, the sex pheromone of the longhorn beetle

Migdolus fryanus Westwood.

Ellen M. Santangelo, Paulo H. G. Zarbin, Quézia B. Cass, José T. B. Ferreira

and Arlene G. Correa. Synthetic Communications 2001, 31, 3685-3698.

VI. Diastereoselective synthesis of the common aggregation

pheromone of flour beetles: 4,8-dimethyldecanal.

Ellen M. Santangelo, Arlene G. Correa and Paulo H. G. Zarbin (submitted).

VII. Resolution of an iridoid synthon, gastrolactol, by means of

dynamic acetylation and lipase-catalyzed alcoholysis.

Ellen M. Santangelo, Didier Rotticci, Ilme Liblikas, Torbjörn Norin and C.

Rikard Unelius. Journal of Organic Chemistry 2001, 66, 5384-5387.

VIII. Simplified isolation procedure and interconversion of the

diastereomers of nepetalactone and nepetalactol.

Ilme Liblikas, Ellen M. Santangelo, Johan Sandell, Mats Svensson, Peter

Baeckström, Ulla Jacobsson and C. Rikard Unelius (submitted).

IX. Asymmetric syntheses of iridoids and resolution of (2)-

phenylindoline.

Ellen M. Santangelo, Ilme Liblikas, Anoma Mudalige, Karl W. Törnroos, Per-

Ola Norrby and C. Rikard Unelius. Manuscript.

Published papers were reprinted with permission from:

I and VII: American Chemical Society. II: Verlag der Zeitschrift für Naturforschung. III: Kluwer Academic Publishers. V: Marcel Dekker.

TABLE OF CONTENTS

Abstract List of Publications 1. INTRODUCTION.................................................................... 1

1.1. Pheromones......................................................................1

1.2. Applications of pheromones.................................................2

1.3. Commercialization – problems and benefits of pheromones......3

1.4. Human pheromones? .........................................................4

1.5. Aim of the thesis ...............................................................4

2. LEPIDOPTERA PHEROMONES................................................ 6

2.1. Diatraea saccharalis ...........................................................6

2.2. Phyllonorycter blancardella................................................ 11

3. METHYL SUBSTITUTED CHIRAL PHEROMONES…………………15

3.1. Migdolus fryanus ............................................................. 15

3.2. Tribolium ........................................................................ 19

4. APHIDS……………………………………………………………………….21

4.1. Isolation of nepetalactones from Nepeta species .................. 23

4.2. Synthesis of Iridoids......................................................... 27

4.2.1. Use of chiral auxiliaries to perform the [4+2]-cycloaddition…32

5. CONCLUDING REMARKS ..................................................... 39

ACKNOWLEDGEMENTS............................................................ 41

REFERENCES........................................................................... 43

1

1. INTRODUCTION

1.1. Pheromones

Pheromones are substances which occur in Nature and are used

for chemical communication between animals. The term “pheromone”

coined by Karlson and Lüscher1 is a substance which is secreted by an

individual and received by a second individual of the same species, in

which it releases a specific reaction, for example a definite behaviour

or a developmental process. The name is derived from the Greek

pherein, to carry or transfer, and hormon, to excite or stimulate.

The first chemical identification of a pheromone took place in the

late 1950s, after almost two decades of work by a team led by Adolf

Butenandt.2 The team chose to study the domesticated silk moth

(Bombyx mori) which could be reared in the enormous numbers

needed. They extracted the female pheromone gland in a solvent and

then fractionated the complex mixture. The biological activity in the

extracts was monitored with a simple bioassay to see which fractions

excited the males into fanning their wings. The silk moth pheromone,

which they named ‘bombykol’, was eventually identified as a long

chain alcohol with 16 carbon atoms, (10E,12Z)-hexadecadien-1-ol (1)

(Figure 1). More than half a million moths were required to obtain

some 6 mg of material.3 Today, powerful new tools have

revolutionized the study of pheromones so that much of the task can

now be attempted with a single female insect.4

The first sex pheromone identified was achiral, but in the late

1960s a number of chiral pheromones were discovered, e.g. exo-

brevicomin (2) (Figure 1), the pheromone of the western pine beetle.

2

OH

bombykol (1)

O

O

exo-brevicomin (2) Figure 1. Structures of bombykol (1) and exo-brevicomin (2)

An understanding of the relationship between structure and

biological effect requires that the absolute configurations of naturally

occurring chiral pheromones are determined. It is now well established

that pheromones are not always enantiomerically pure. Therefore,

their enantiomeric composition must be determined.5 Difficulties are

often encountered in stereochemical studies of pheromones, because

they are usually obtained in small quantities.

Pheromone synthesis is important, as it provides material for

rigorous determination of the (absolute) configuration of the

semiochemical, and for carrying out extensive biological tests.6

1.2. Applications of pheromones

The most successful applications of pheromones are for insect

pest managements. Such applications provide significant costs and

environmental benefits to the farmer, the consumer and the society.

Pheromones are the safest of all currently available insect control

products.7 There are many successful pest management schemes

using pheromones for the control of insect pests. Such schemes are as

effective as the conventional pesticides they have replaced.8

The main ways of exploiting pheromones to control pests are

monitoring, mating disruption and mass trapping.

3

1.3. Commercialization – problems and benefits of pheromones

Given the power of pheromones for insect pest control the

question of why they have not been used more still remains. The most

serious challenges to get more frequent use of pheromones in pest

control are economic and political.9

Large agricultural companies are unlikely to develop commercial

pheromone technologies for more than a very few major crops. The

specificity of pheromones, which is their strength for minimal impact

on the environment, is a commercial disadvantage as each species has

its own pheromone blend, and this situation is further complicated by

geographical races using different pheromone mixtures.

Most of the small companies involved in commercialization of

pheromones do not have the resources for basic research; therefore,

much of the research and extension work will need to be supported by

the government.3

Political changes at the level of consumers and governments will

affect the development of pheromones in pest control. Changing

consumer attitudes to pesticide use are already improving the

attitudes to alternatives for conventional pest control and the use of

pheromones is likely to benefit.10 Government influences through

legislation include promising solutions such as the Swedish taxes on

pesticides.11 Legislation restricting pesticide use is increasingly

important. Tougher legislation has, for example, dramatically changed

the patterns of pesticide use in California.12 Government subsidies for

pheromone treatments, such as those to wine-grape growers in

Germany and Switzerland, can also help.13

4

1.4. Human pheromones?

Richard Axel and Linda B. Buck, this year’s Nobel Laureates in

Physiology or Medicine, have shown the basic principles for recognizing

and remembering about 10.000 different odours.14

Odours and the potential existence of human pheromones are

undoubtedly important, but hard to investigate. One of the major

challenges to human pheromone research is that of designing rigorous

experiments that eliminate other cues and variables. Scientific studies

on human pheromones are hampered because of the complexity of

odours and the fact that humans are emotionally complex.3

Demonstrated effects in research on human pheromones include

parent-offspring recognition15 and menstrual synchrony.16 However,

firm conclusions on human pheromones in sexual attraction remain

elusive despite unceasing interest from the popular press.3

1.5. Aim of the thesis

The scientific challenges presented in the chemical

communication between animals have meant that some research

teams have combined the expertise of chemists and biologists.

During a research project on pest insect semiochemicals,

different phases pose different demands on the chemist. In the initial

phase structure elucidation by analysis and synthesis is needed. After

this, samples are needed for electrophysiological and/or laboratory

bioassays. Later, compounds for field tests on a small and,

subsequently, on a large scale are required. In each phase the

5

amounts and purities required of the desired compounds may vary.

Thus flexible synthetic approaches are important.

The aim of this thesis has been to provide chemical support to

an interdisciplinary research program on semiochemicals for

applications in insect pest control.

6

2. LEPIDOPTERA PHEROMONES

Most of the sex pheromones identified from moth species are

unsaturated straight-chain aliphatic alcohols, acetates, and

aldehydes.17 Many insect species use more than one attracting

chemical as an aid to reproductive isolation. The sex pheromone often

consists of a particular blend of a number of components, some

species using various mixtures of Z and E isomers, some utilizing

acetate-alcohol or acetate-aldehyde mixtures, and some using

mixtures of positional isomers.18

For many lepidopterous pheromones a precise mixture of the Z-

and E-isomers of the alkenes is necessary if the synthetic material is to

be an effective attractant in the field.

The purity of the synthetic pheromones is generally of critical

importance. They must not be contaminated with inhibitor chemicals

which can cause a decrease in the response of males to the attractant.

1.2. Diatraea saccharalis

Diatraea saccharalis (Fabricius) (Lepidoptera: Pyralidae) is the

major pest of sugar cane in Brazil.19 The attack of the insect to the

plants results in production loss in the sugar and alcohol industries.

Although D. saccharalis can be efficiently controlled by biological

control using its natural enemy Cotesia flavipes,20 there is a residual

damage of about 10% which justifies the search for an additional

strategy.

The sex pheromone was assigned as (9Z,11E)-hexadecadienal

(3a) (Figure 2) by Hammond in a short conference abstract21 and by

7

Carney in a patent application.22 However, the compound described

had not been found to be attractive for males in field experiments.23

This observation might indicate that the pheromone produced by

females was most probably a multi-component blend.

O

3a

Figure 2. Structure of (9Z,11E)-hexadecadienal (3a)

In a pilot experiment, Peter Witzgall and Miryan Coracini, from

the Department of Plant Protection Sciences, Swedish University of

Agricultural Sciences, Alnarp, found the four isomers of 9,11-

hexadecadienal as well as (Z)-hexadec-11-enal and hexanal in the

gland of D. saccharalis. In order to investigate their effect on male

attraction, we decided to synthesize the four geometric isomers of 3.

A few reports on the synthesis of single isomers of 9,11-

hexadecadienal (3) were found in the literature.18,24 Svirskaya et al.

have reported the synthesis of all four isomers of 9,11-

hexadecadienal using four separate routes.25 One week after the

acceptance of our first publication on this topic (Paper I), Svatos and

co-workers26 reported the synthesis of 3a and a preliminary

examination of hexane extracts of the abdomens of calling sugar cane

females, confirming the identification of (9Z,11E)-hexadecadienal

(3a) as the major component of the sex pheromone of D. saccharalis

and suggesting that additional components might be involved in the

sexual communication of this species.

Our divergent synthetic route is shown in Schemes 1, 2 and 3

and described in Paper I.

8

HO

THPO

OH

THPO

Br

Br

a, b

c, d

4 5

6

(a) Amberlyst-15, Et2O, DHP (b) Br2, CH2Cl2, -78 oC (c) NaNH2, THF, -78 oC (d) n-BuLi, THF, paraformaldehyde

Scheme 1. Synthesis of the key intermediate 6

A large amount (more than 30 grams) of intermediate 6 was

prepared and it was also used in the synthesis of the sex pheromone

of Lampronia capitella, consisting of (9Z,11Z)-tetradecadienal in

combination with the corresponding alcohol and acetate.27

9

THPO H

O

THPO

OTHP

H

O

H

O

6

a, b

7

8

8a

3b

3c

cd

e, f

e, f

(a) LiAlH4, THF (b) MnO2, CH2Cl2 (c) CH3(CH2)4P+Ph3Br-, n-BuLi, THF/Et2O, HCl.Et2O (d) CH3(CH2)4P+Ph3Br-, KHMDS, THF (e) Amerlyst-15, MeOH (f) PDC, CH2Cl2

Scheme 2. Syntheses of the isomers (9E,11E)-3b and (9E,11Z)-3c hexadecadienal

Under normal conditions the Wittig reaction provided a mixture

of E,Z-8a/E,E-8 = 20:1, while the Wittig reaction, performed under

Schlosser conditions, provided E,Z-8a/E,E-8 = 1:10. After removal of

the protective group, the purification of both geometric isomers was

performed using urea inclusion complexes (clathrates).

10

THPO

H

O

THPO

OTHP

H

O

H

O

6

a

b, c

d, c

e, f

e, f

9

8b

8c

3a

3d

(a) MnO2, CH2Cl2 (b) CH3(CH2)4P+Ph3Br-, n-BuLi, TFH/Et2O, HCl.Et2O (c) (cyclohexyl)2BH, H+/EtOH(d) CH3(CH2)4P+Ph3Br-, KHMDS, THF (e) Amberlyst-15, MeOH (f) PDC, CH2Cl2

Scheme 3. Syntheses of the isomers (9Z,11E)-3a and (9Z,11Z)-3d hexadecadienal

The dienic compound Z,E-8b was purified, after deprotection,

by the same methodology as was used for E,E-8 and E,Z-8a. The

deprotected Z,Z-8c was purified by recrystallization at low

temperature (-30 oC). The method works well with dienic alcohols

having 12 or more carbon atoms in their carbon chains.

When all possible geometric isomers of 3 were at hand, a more

extensive investigation of the glands of D. saccharalis females from

three locations in Brazil was done (Paper II) and all four geometric

isomers of 3 were found as well as two, more saturated analogues:

hexadecanal and (Z)-hexadec-11-enal. No significant variations in the

composition of the gland extracts of the three Brazilian populations

11

were observed and only two compounds, (Z)-hexadec-11-enal and

(9Z,11E)-hexadecadienal (3a), elicited antennal responses (GC-EAD).

Unfortunately but interestingly, field tests did not catch any

insect. The hypothesis that the stability of the aldehydes was too low

for Brazilian field conditions could be ruled out as virgin females (used

as control) did not attract any males either. Presumably trap design

or lack of insects in the right part of their life cycle was the correct

explanation of the unsuccessful field trials. Further studies are

needed.

1.3. Phyllonorycter blancardella

The leaf miner moths of the genus Phyllonorycter are very small

(4-8 mm long). A caterpillar of this genus spends most of its life

inside a leaf, eating the inner part without damaging the cuticle, and

in this way a conspicuous mark, i.e. a mine, is formed.28

Some species of the genus Phyllonorycter, such as Ph.

blancardella, Ph. mespilella, Ph. pyrifoliella and Ph. populifoliella, are

known to be serious pests of orchards and recreation parks, and these

species are attracting more and more attention in pest monitoring and

control programmes in Europe and North America.29

Phyllonorycter blancardella is one of the most common pests in

apple orchards within the genus Phyllonorycter. Reissig et al.30 have

reported that a high population density of this moth has resulted in

reduced fruit size, premature fruit ripening and reduced fruit set the

following year.

Roelofs et al.31 found that (10E)-dodecen-1-yl acetate (E10-

12:Ac) was a potent sex attractant for Ph. blancardella in apple

12

orchards in New York, U.S.A. Mozuraitis et al.32 identified E10-12:Ac,

the corresponding alcohol (E10-12:OH) and dodecan-1-yl acetate

(12:Ac) in the extracts of the pheromone gland of female Ph.

blancardella, although they found no behavioural responses to the

latter two compounds in field experiments conducted in Lithuania. In

field experiments conducted in Nova Scotia, Canada, and

Massachusetts, U.S.A., Gries et al.33 found that (4E,10E)-dodecadien-

1-yl acetate (E4,E10-12:AC) (12) was two to four times more

attractive to P. blancardella than the E10-12:Ac earlier reported.

In order to clarify these somewhat contradicting results we

decided to synthesize the attractive diene (12) reported by Gries to

compare its attractiveness with the relative attractiveness of E10-

12:Ac.

The dienic compound 11 was obtained by an unsymmetrical

double Wittig olefination of acetaldehyde and the cyclic hemiacetal 2-

hydroxytetrahydrofuran with the ylide prepared from 1,6-hexane-

bistriphenylphosphonium dibromide (10) using sodium

bis(trimethylsilyl)amide (NaHMDS) as base (Scheme 4). The bis-

phosphonium salt 10 was readily synthesized in a large scale from

1,6-dibromohexane and could be stored for at least one year.

BrPh3PPPh3Br+ -

- + H

O

O O OH

O

O

OHNaHMDS, THF,

1. urea inclusion2. SiO2/AgNO3

3. Ac2O, 0 oC, Pyridine

10 11

12

Scheme 4. Synthesis of (4E,10E)-dodecadien-1-yl acetate (12)

13

In order to achieve trans-selective olefination, we first

performed the Wittig reaction under Schlosser’s conditions,34 but this

route was unsuccessful.

Under normal conditions, the Wittig reaction provided the four

possible geometrical isomers of 11 (Figure 3).

olefinic carbon atoms methyl carbon atoms

Figure 3. Expansion of two regions of the 13C NMR spectrum of a mixture of geometrical isomers of 11

E,E-isomers are known to form urea inclusion complexes

(chlatrates). From a hot solution of urea in methanol, the E,E-isomer

of 11 was co-crystallized with urea while the other geometrical

isomers remained in solution. After filtration and hydrolysis of the

urea complex the recovered dienol (4E,10E)-dodecadien-1-ol (11)

was finally purified by MPLC, using AgNO3-impregnated silica gel

(Figure 4).

Silver nitrate had a long history of use as a chromatographic

support (π acceptor), after Lucas35 and Nichols36 reported that silver

ions could complex with alkenes (π donor).37 Surprisingly this method

was patented only recently.38

A number of silver-loaded chromatography phases are

commercially available, but they are also simple to prepare. However,

14

it is important to note the following facts. First, silver salts are light

sensitive, so that all silver-loaded media must be protected from light

during and after their preparation. Second, AgNO3 is toxic, and it

rapidly stains skin black, so that adequate precautions should be

taken during handling. Third, AgNO3 is corrosive, so that all metal

equipment should be thoroughly flushed with water after use.39

olefinic carbon atoms

Figure 4. Expansion of the 13C NMR spectrum of (4E,10E)-12:OH (11) after purification

Field tests with E4,E10-12:OAc (12) and E10-12:OAc (Paper

III) in Ontario, Canada, showed relatively weak behavioral responses

of the dienic compound and lack of synergism with the monoene,

which demonstrated that the E10-12:OAc can be used in lures for

monitoring the occurrence and activity of this species in Ontario.

There are several possible reasons for the disparity between our

results and those of Gries and co-workers.33 First, a related species,

Phyllonorycter mespilella (Hübner) may have been mistaken for P.

blancardella. E4,E10-12:OAc has been identified as a pheromone

component of P. mespilella. A second possible reason may be a

geographic variation in the pheromone chemistry of P. blancardella.

Such variation is known in the Lepidoptera, with different populations

sometimes utilizing different compounds or ratios in their pheromone

blend.40

15

3. METHYL SUBSTITUTED CHIRAL PHEROMONES

3.1. Migdolus fryanus (Paper V)

Migdolus fryanus (Westwood) (Coleoptera: Cerambycidae) is a

sugarcane pest, restricted to South America. Field tests have shown

that mate-finding is mediated by a female sex pheromone,41 which

has been isolated and identified by Leal and co-workers42 as N-(2’S)-

methylbutyl-2-methylbutylamide (16).

In order to establish the absolute configuration of the

pheromone, we synthesized all four possible stereoisomers of 16

using 2-methylbutan-1-ol (13) as a common starting material

(Scheme 5), commercially available only in its (S)-(-)-form.

OH

R R'

NH2

R R'

Cl

R R'

NH

R R''R' R'''

O

O13

14

15

16

(S)-13, 14 and 15 : R = Me, R' = H

(R)-13, 14 and 15 : R = H, R' = Me

(2'S,2R)-16 : R = R'' = H and R' = R''' = Me(2'S,2S)-16 : R' = R'' = H and R = R''' = Me(2'R,2S)-16 : R' = R''' = H and R = R'' = Me(2'R,2R)-16 : R = R''' = H and R' = R'' = Me

Scheme 5. Synthetic pathway to N-2’-methylbutyl-2-methylbutylamide (16)

Compound (R)-(+)-13 was synthesized from commercially

available methyl (S)-(+)-3-hydroxy-2-propionate (17) in five steps

(Scheme 6). Protection of the hydroxyl group of 17 with 3,4-dihydro-

2H-pyran furnished the tetrahydropyran derivative, in 93% yield,

which was reduced by LiAlH4 in 85% yield. Compound 18 was

converted into its corresponding tosylate, in 95% yield, which was

16

then coupled with methyl magnesium iodide (MeMgI) using lithium

tetrachlorocuprate (Li2CuCl4) as catalyst, affording 19 in 82% yield.

HO OMe

H

O

THPO

H

THPO OH

H

HO

H

1. DHP, THF, p-TsOH

2. LiAlH4, Et2O

1. pyridine, TsCl

2. MeMgI, THFLi2CuCl4, -80 oC

Amberlyst-15

MeOH

17 18

19 (R)-13

Scheme 6. Synthesis of (R)-2-methylbutan-1-ol (13)

The alcohol (R)-13 was always obtained with traces of

methanol. The enantiomeric excess of the synthetic alcohol (R)-13

was determined by 1H NMR spectroscopy analysis of its corresponding

Mosher ester and comparison with Mosher ester derivatives of racemic

and (S)-13 and was shown to be >99%.

The first idea to convert the alcohol (R)-13 into the amine (R)-

14 (Scheme 5) was to mesylate the alcohol (R)-13, then make the

corresponding azide, which finally would be hydrogenated to the

amine (R)-14. The mesylation of (R)-13 never worked, despite

numerous attempts, most probably because of the presence of

methanol.

Compound 19 was oxidized and converted into the acyl chloride

(R)-15. Treatment of (R)-15 with ammonium hydroxide (28% NH3 in

water) afforded the corresponding amide in 74% yield. The amide was

then reduced using LiAlH4 to afford the amine (R)-14 (Scheme 7).

17

OTHP

H

Cl

H

O

NH2

H

1. Jones, acetone

2. SOCl2

1. NH3

2. LiAlH4, THF

19 (R)-15 (R)-14

Scheme 7. Synthesis of (R)-2-methylbutylamine (14)

The enantiomeric purity of the amine (R)-14 was investigated

by 1H NMR analysis of its corresponding Mosher amide (R,R)-21

(Scheme 8). Rac-14 and (S)-14 were commercially available.

Although there was not a baseline separation of the signal

corresponding to the methylene -CH2-NH- in the 1H NMR spectrum,

the enantiomeric excess of (R)-14 could be estimated to be at least

65%. According to this result, a partial racemization of (R)-14 had

probably occurred during the transformation of the acyl chloride (R)-

15 to its corresponding amide.

Cl

O

MeO CF3 HN

O

MeO CF3 H HN

O

MeO HCF3

rac-14

(S)-14(R)-14

(S)-20

(R,S)-21 (R,R)-21+

Scheme 8. Mosher amides 21 prepared from the rac-, (S)- and (R)-2-

methylbutylamines (14)

The stereoisomers (2’R,2R)-16 and (2’S,2R)-16 were obtained

from the acyl chloride (R)-15 and the amines (R) and (S)-14

respectively, and the stereoisomers (2’R,2S)-16 and (2’S,2S)-16

from (S)-(+)-2-methylbutyric anhydride (22) (98% ee, commercially

available) and (R)- and (S)-14 (Scheme 9).

18

O

NH

NH

O O

O

OH

H

H

H

H H

(R)-14

CH2Cl2

(S)-14

CH2Cl2

(S)-22

(2'R,2S)-16

(2'S,2S)-16

Scheme 9. Syntheses of the stereoisomers (2’S,2R)-16 and (2’S,2S)-16

In order to determine the enantiomeric purity of the synthetic

pheromone, the four stereoisomers of compound 16 were analyzed by

GC using several chiral columns, however, no resolution was

obtained.

Based on the optical rotation of (2’S,2S)-16 (prepared from

commercially available reagents), the enantiomeric excess of

(2’R,2R)-16 was determined as > 80%.

Attempts to determine the absolute stereochemistry of the

natural pheromone of Migdolus fryanus are in progress.

19

3.2. Tribolium (Paper VI)

Tribolium castaneum (red flour beetle) and T. confusum

(confused flour beetle) (Coleoptera: Tenebrionidae) are notorious

pests found in stored grain products, including grain products stored in

your kitchen. The adult beetles are about 4 mm in length, and they

can serve as the intermediate hosts for several species of parasites.

Red and confused flour beetles attack stored grain products such

as flour, cereals, meal, crackers, beans, spices, pasta, cake mix, dried

pet food, dried flowers, chocolate, nuts and seeds. These beetles have

chewing mouthparts, but do not bite or sting. They are two of the

most important pests of stored products in the home and grocery

stores. The confused flour beetle apparently carries this name due to

confusion about its identity as it is so similar to the red flour beetle at

first glance.

The aggregation pheromone of this species was isolated and

identified by Suzuki43 as 4,8-dimethyldecanal (27) (Scheme 10). The

absolute configuration was established by Mori and co-workers44 as

(4R,8R), but later bioassays showed that a mixture of (4R,8R) and

(4R,8S), in a 8:2 ratio, was about 10 times more active than (4R,8R)

itself.45

The syntheses of two isomers of 4,8-dimethyldecanal: (4R,8R)-

27 and (4S,8R)-27a was performed by connecting two chiral building

blocks (Scheme 10). The key step was the coupling reaction of

tosylates 25 and 25a with the chiral Grignard reagent, prepared from

(R)-1-bromo-2-methylbutane (23). Compound 23 was obtained

enantiomerically pure from 19, as previously described for Migdolus

fryanus.

20

OH OTs

H

H

H

O

* *

*

*

THPOH

BrHPPh3, Br2

CH2Cl2(R)-19 (R)-23

(R)-24(S)-24a

(R)-25(S)-25a

TsCl, Py, 0 oC

CHCl3, 86 %

(R)-23, Mg, THF

Li2CuCl4, 82 %

(R)-26(S)-26a

Ozone, CH2Cl2,

dimethyl sulfide

(R,R)-27(S,R)-27a

Scheme 10. Syntheses of (4R, 8R)- and (4S, 8R)-4,8-dimethyldecanal (27) and

(27a)

21

4. APHIDS

Aphids (Homoptera: Aphididae) are the main agricultural insect

pests of northern Europe and are also pests in horticulture, causing

damage to crops either directly by feeding or by transmitting plant

virus diseases.46 In the early 1970s, Pettersson47 demonstrated that

aphids in the Schizaphis genus released a sex pheromone from the

hind tibiae of females to attract males. Later studies by Marsh48 also

showed a sex pheromone for the vetch aphid, Megoura viciae.

The sex pheromones of a number of aphids have now been

identified49 as iridoids and principally comprise of 28 and 29, either as

single compounds or as species-specific blends.

O O

H

H

H

H

OHO

28 29

Figure 5. Structures of aphid sex pheromone components: (4aS,7S,7aR)-

nepetalactone (28); (1R,4aS,7aR)-nepetalactol (29)

Iridoids are a large class of naturally occurring compounds with

over 300 members in the family.50 They possess an oxygenated

monoterpenoid skeleton characterized by a functionalized cyclopentane

ring which is cis-fused to a dihydropyran, δ-lactol or δ-lactone ring. The

name “iridoid” originated from the fact that the first iridoids were

isolated from the secretion of ants belonging to the genus

Iridomyrmex.

22

The cis-linked partially hydrogenated cyclopenta[c]pyran

skeletons, which are characteristic of the iridoids, have been numbered

by earlier groups in different ways.51 Figure 6 shows some of the

numbering systems employed in the literature. Nangia and co-

workers47 published that C is consistent with the biosynthesis of

iridoids, which seems dubious (Scheme 11).

The preferred representation used in this text is C which is in

accordance with the IUPAC nomenclature.52

O

H

H

OH

CO2H

1

6

2

45

39

78

O

H

H

OH

CO2H

1

5

1

34

28

67

O

H

H

OH

CO2H

7a

4a

1

34

27

56

A B C

Figure 6. Numbering systems of iridoids from geraniol

Biosynthetic studies carried out to date suggest that the most

probable biosynthetic sequence from geraniol to loganin is shown in

Scheme 11: geranyl pyrophosphate (30) (GPP) is oxidized to 10-

hydroxygeraniol (31) and then isomerised to 10-hydroxynerol 32. The

trialdehyde 33 derived from nerol undergoes a double Michael addition

to yield the iridodial precursor 34 which undergoes cyclisation to lactol

35 (Scheme 11).

23

H O

H O

HH

O

H-

OHC CHO

CHONAD+

OH

OH

CHOH

O

O

H

H

H

H

OH

OH

O

H

H

OH

CHO

OPP

1

4

7

9 10

via

D8,10

via allylic

cation

NADH induced

cyclisation

30GPP

3110-hydroxygeraniol

3210-hydroxynerol

33

3435

loganin

4

5

Scheme 11. Biosynthesis of loganin

Certain iridoids are central intermediates in the biosyntheses of

many important families of plant-derived alkaloids. In addition, many

iridoids themselves have interesting biological activities ranging from

sedative to antimicrobial and antileukemic effects.53

4.1. Isolation of nepetalactones from Nepeta species

The essential oil compositions of some Nepeta (Lamiaceae) plant

species are characterized by the presence of nepetalactone isomers.

The genus Nepeta contains more than 280 species that are distributed

over a large part of Central and Southern Europe, as well as Western,

Central and Southern Asia.54 Many of them are widely used in folk

medicine because of their antispasmodic, diuretic and antiseptic

properties. The feline attractant properties of several Nepeta species

have been known for a long time. Nepetalactone and its isomers are

24

considered to be responsible for the attractant activity of Nepeta

species on cats.55

Birkett and Pickett56 described the isolation of nepetalactone 28

on a commercial scale from the catmint, N. cataria, by steam

distillation. This was the first example of a plant resource being

developed as a feedstock for the production of a commercially

available insect pheromone. 35 ton of biomass yielded over 30 kg of

oil (0.098% yield).

By grinding plant material with sufficient amounts of silica gel to

give a free flowing powder, Baeckström and Jirón57 employed

combined extraction and chromatography for a very efficient

extraction of azadirachtin in a column. We wanted to test whether the

same methodology could be employed for extraction of nepetalactones

from fresh Nepeta plants, and compare this simple methodology with

conventional extraction.

Nepeta faassénii is a very common garden plant in Sweden. It is

a hybrid of N. racemosa and N. nepetella. We collected 800 g of N.

faassénii at the beginning of June 2004. One portion was extracted

according to the the Baeckström procedure in the following way: the

fresh aerial part of the plant was minced with silica (1.5 times the

weight of the plant material) by means of a household meat grinder.

The material was passed through the grinder three times to produce a

free flowing powder. The sample containing powder was added to a 50

mm inner diameter glass column, which was sealed with pistons that

could be adjusted to the resulting bed length, followed by fresh silica

gel (Figure 7A). Gradient elution provided the extraction and partial

separation of nepetalactone isomers.

25

(A) (B)

(C)

Figure 7. (A) Baeckström SEPARO AB column (B) fractions eluted from column A

(C) TLC plate for the fractions showed in B

The second portion of N. faassénii was treated in the following

way: the fresh aerial part of the plant was minced with water by

means of an electric (food) mixer. The plant material was removed by

filtration and extracted with dichloromethane. The crude mixture

extracted from the water phase was subjected to MPLC.

The relative amounts of nepetalactones in both extraction

procedures were 28 (85%) and 36 (15%). When Baeckström’s

methodology was used, the yield per 100 g of dry plant was 1.05 g,

and in the normal extraction it was 0.134 g.

26

O

H

H

O

O

H

H

36 (cis-cis)28 (cis-trans)

O

Figure 8. Nepetalactones present in Nepeta faassénii

Except for being efficient and simple, the Baeckström method of

combined extraction and chromatography had the advantage, that

fewer compounds were collected in less solvent, than in conventional

extraction. In this particular case, fractions 35 to 40 contained

compound 28 (cis-trans) together with an additional, unidentified

compound, and fractions 41 to 45 contained compound 36 (cis-cis)

mixed with a trace amounts of the cis-trans isomer 28 (Figure 7C).

27

4.2. Synthesis of Iridoids Since the discovery of naturally occurring iridoid monoterpene

lactones in the 1950s, numerous synthetic approaches were published

in the literature.58 The first synthesis of rac-nepetalactone-28, in ten

steps and unpublished yield, was reported in 1960 by Sakan and

coworkers59. After that, numerous syntheses of iridoids were

described. Among them, enantioselective syntheses of (-)- and (+)-

nepetalactone using a hetero Diels-Alder approach, independently

published in successive papers by Schreiber60 and Denmark,61 are

interesting ones. The drawback with the Schreiber’s synthesis is that

the product contains a saturated methyl cyclopentanoid ring, which is

almost inert to further reaction that can provide more elaborate

iridoids. Furthermore, the methyl group will always be trans to the cis

fused ring junction.

Our synthetic strategy in the preparation of our iridoid synthon

resembles the one used by Schreiber et al., but it is combined with an

enzymatic resolution.

The key step in our synthetic strategy is an intramolecular enal-

enamine [4+2] cycloaddition within the N-methylindoline derivative of

(8)-oxocitral (Scheme 12). When citral (37) is used as starting

material instead of citronellal used by Schreiber, a double bond is

formed in the five-membered ring (compound 39), permitting further

reactions such as allylic oxidations and halogenations (Scheme 12).

28

O

H

H

NPh

***

CHO

CHO

CHO

HN

SeO2

CH2Cl2, 40% MeOH/Hexane, 46%

37 38 39

Scheme 12. Oxidation of 37 and intramolecular enal-enamine[4+2] cycloaddition

When the dial 38 is treated with N-methylaniline, one can

assume that the intermediate species 40 (Scheme 13) is formed.

Since a stable, uncharged imine cannot be formed from a secondary

amine, compound 40 dehydrates to the corresponding dienamines 41

and 42. The (Z)-isomer of the enamine-enal 41 undergoes an

intramolecular Diels-Alder reaction, forming the cis-fused ring product

39. The assignment of the relative stereochemistry of 39 depicted in

Scheme 11 is based on NMR experiments. Molecular modelling

calculations confirm this assignment.

In the analogous reaction of the N-methylamine with the α-

methylaldehyde group, enamine 43 would be formed, but its

cycloaddition product 46 could not be detected. The methyl group

most probably prevents the formation of the transition state of the

cycloaddition.

Initially, the cycloaddition reaction was performed under

anhydrous conditions (by means of molecular sieves), but a higher

yield was obtained with the two-phase system described in the

experimental part. During anhydrous conditions, reaction of N-

methylaniline with both aldehyde groups should lead to the bis-

enamine 47. Without water being present, the reaction should have

come to an end at this point.

29

O

H

H

NPh

O

Me

Me

NPh

NPh

O

**

*

**

*

OH

NPh

CHO

CHO

CHO

NPh

OH

O

NPh

CHO

NO Ph

O

NPh

38

40

43

42

4139

44

45 46

NPh N

Ph

47

Scheme13: Proposed intermediates in the enal-enamine cycloaddition step

30

Careful hydrolysis of compound 39 with p-toluenesulfonic acid

provided the lactol 48 as a 5:1 mixture of anomeric isomers, the

1R*,4aS*,7aS* form dominating (Scheme 14).

A dynamic chemical acetylation of the four stereoisomers of 48

in pyridine and acetic anhydride yielded two of four possible acetates

(49 and 49a). We assumed that the hemiacetal had equilibrated

during the reaction, resulting in the 49 and 49a configurations shown

in Scheme 14.

O

H

H

OH

O

H

H

OH

O

H

H

OH

O

H

H

OH

O

H

H

OAc

O

H

H

OAc

p-TsOH

aq. TFH, 87%

Ac2O, 0 oC

Pyridine, 86%+

49 49a

39

1R*,4aS*,7aS*-48 1S*,4aS*,7aS*-48

42% 8%

Scheme 14. Hydrolysis of 39 and dynamic chemical acetylation of 48

The enzymatic kinetic resolution of the acetates 49 and 49a was

investigated. The lipase preparation “Amano I, PS-C” (lipase from

Pseudomonas cepacia) catalyzed the alcoholysis of 49a. The reaction

was carried out in tert-butyl methyl ether in the presence of an excess

of butan-1-ol. Under these reaction conditions, this lipase displayed an

excellent enantioselectivity (E>200). The reaction reached 50%

conversion in less than 3 h. The enantiomeric purity of the product was

measured by means of chiral gas chromatography. The substrate 49

and the product 48 were isolated in high enantiopurities (ee >99%

31

and 98%, respectively) and chemical yields (45% and 40%,

respectively) (Scheme 15).

O

H

H

OAc

O

H

H

OAc

+

49 49a

O

H

H

OAc

O

H

H

OH

+

4948

E > 200Amano I, PS-C

butan-1-olt-butyl methylether

Scheme 15. Enzymatic resolution of 49 and 49a

To determine the absolute configuration of the chiral lactol 48

obtained and the acetate 49 remaining after the resolution procedure

(Scheme 15), lactol 48 was oxidized using Fetizon’s reagent62 to

provide gastrolactone (50, Scheme 16). The double bond of the five-

membered ring of 50 was stereoselectively hydrogenated using

rhodium/C as a catalyst, providing cis,cis-nepetalactone (36) together

with some over-reduced material.

The product 36 (Scheme 16) obtained in the hydrogenation, was

analyzed by chiral GC and the chromatogram was compared with the

chromatogrammes of the two enantiomers of racemic nepetalactone

synthesized previously63 and with that of the natural product

(4aR,7S,7aS)-cis,cis-nepetalactone isolated from Nepeta racemosa.

The absolute stereochemistry of 48 and 49 was as depicted in Scheme

15.

32

O

H

H

OH

48

O

H

H

O

50 36

O

OH

H

AgCO3

benzene, 67%

H2, Rh/C

hexane, 42%

Scheme 16. Synthesis of cis-cis-nepetalactone 36

In summary, in a synthetic sequence, including a dynamic

acetylation (Scheme 14) and an enantioselective transesterification

mediated by the lipase Amano I, PS-C (Scheme 15), we have resolved

a lactol with three chiral centers.

4.2.1. Use of chiral auxiliaries to perform the [4+2]-

cycloaddition

In their first publication on the intramolecular enamine/enal

cycloaddition, Schreiber and co-workers52, noted that the reaction

responded to several chiral amines with substantial asymmetric

induction. Two years later, Schreiber and Meyers64 published the

synthesis of brefeldin using a chiral amine in the cycloaddition

reaction. A 1:1 mixture of 51 and 52 (as 3:2 ratio isomers) proceeded

to completion over a 12-h period at room temperature to provide a

17:1 ratio of two cycloadducts 53 (Scheme 17). The relative

stereochemistry of the bicyclic dihydropyran was in accord with

Schreiber’s earlier studies.

33

NH

OPh

Me O

N

OPh

MeH

H

3:2+ 51 + 52 (1:1)

ether, rt, 75 %51 52

53

CHO

CHO

Scheme 17. Schreiber and Meyer’s asymmetric enamine-enal cycloaddition

In order to perform the intramolecular enal-enamine

cycloaddition in an enantioselective manner we first used 2-tert-butyl-

4-methyl-5-phenyl-oxazolidine (52) as a chiral auxiliary (Scheme 18).

CHO

CHO

O

NH

Me

Ph

O

H

H

N

OPh

38THF, 4Å mol. sieves

54

3:2

52

Scheme 18. Intramolecular asymmetric enamine-enal cycloaddition

Only one diastereomer of 54 could be seen in the 1H NMR and

the 13C NMR-spectra, which shows, not only that the product has been

obtained in excellent diastereomeric excess, but also that one

diastereomer of the oxazolidine acetal is selectively reacting or the

oxazolidine acetal is equilibrating during the reaction. Both scenarios

would give rise to the all cis configuration of the substituents on the

oxazolidine ring in the product. The assignment of the relative

stereochemistry of compound 54 depicted in Scheme 18 was based on

34

NMR-experiments. NOEs between the phenyl group and tert-butyl

group plus the CH-CH3 methyl and between the Ph-CH-O proton, the

CH-C(CH3)3 proton and the Me-CH-N proton revealed that the phenyl

group, tert-butyl group and the CH-CH3 methyl were all cis to each

other.

The stereochemistry at the ring junction was concluded to be cis

due to NOEs between the 4a and 7a protons. The anomeric proton has

to be trans to 7a since no NOE was observed and the coupling

constant was over 10 Hz.

NOEs between the 7a and the Me-CH-N proton and between CH3-

CH-N and 9CH3 give the correct orientation of the two rings system in

space.

The yield in this asymmetric cycloaddition was low and it was

difficult to get the cycloaddition reaction to proceed to completion. We

believe that our approach could be useful for iridoid and alkaloid

syntheses and we therefore investigated the key cycloaddition reaction

step in detail in order to optimize the reaction conditions.

One should note that the solvent used in the reaction was THF

and molecular sieves. The two-phase system successfully employed for

the synthesis of racemic gastrolactone56 was not used since we

anticipated that the oxazolidine moiety might be partly solvolysed by

methanol.

Despite numerous attempts to hydrolyze 54, we never

succeeded. We therefore looked for other chiral auxiliaries that could

be used in a two-phase system. Several secondary amines were

screened for their ability to promote the cycloaddition with high levels

of stereoinduction. Optimal results were obtained with racemic 2-

phenylindoline (55), which provided a pure diastereoisomer (syn or

anti in Scheme 19). From these results it was evident that 2-(S)-

35

phenylindoline (55) would give either of the cyclized products 56b or

56c in Scheme 19 when reacted with 8-oxocitral (38).

O

NH

H

O

NH

H

O

NH

H

O

NH

H

Anti Syn

CHO

CHONH

38

55

MeOH/Hexane

or

56a 56c

56d56b

Scheme 19. The stereoisomers that can be formed hypotetically by reaction of 8-oxocitral (38) with rac-2-phenylindoline (55)

With this aim, racemic 2-phenylindoline (55) was subjected to a

resolution procedure (Scheme 20). Racemic 2-phenylindoline (55) was

reacted with (R)-(+)-α-methyl-benzyl-isocyanate (57). The two

diastereoisomers formed, (R,R)-58 and (R,S)-58, were separated by

an unusual variation of column chromatography. The crystals were

dissolved in a large amount of hexane and pumped into a column

saturated with hexane. The compounds absorbed were then eluted by

increasing amounts of ethyl acetate in hexane via another column

coupled in series.

36

(S)

(R)

N

NH Ph

O

H

N

NH Ph

O

H

NH

NCO

PhH

B2H6 / THF

B2H6 / THF

(R)-55

(S)-55

(R)-57

rac-55

(R,R)-58

1.

2. Chromatographic separation

reflux

reflux

(R,S)-58

Scheme 20. Resolution of racemic (2)-phenylindoline (55)

Both isomers of the urea derivative, (R,R)-58 and (R,S)-58,

were crystalline compounds and were sent for X-ray examination in

order to determine the absolute configuration. Unfortunately, one

isomer did not give crystals good enough for X-ray analysis. The other

isomer apparently had several crystalline lattices and the structure

could not be unambiguously assigned.

The geometries of the (R,R)-58 and (R,S)-58 structures were

optimized using the hybrid density functional method B3LYP and the 6-

31G(d) basis set (Figure 9). The NMR chemical shifts were calculated

using the GIAO method as implemented in Gaussian 94 using a larger

basis set: 6-311G (d,p). The chemical shift calculated for the methyl

protons of the (R,S)-58 was 0.3 ppm lower than that for (S,S)-58.

Experimentally we found that the signals for the protons of the methyl

group of one isomer of urea derivative 58 was 0.26 ppm to lower field

than these of the other isomer.

37

Figure 9. The structure of the (R,S)-58 isomer optimized at the B3LYP/6-31G(d)

level of theory

Both diastereoisomers of the urea derivative, (R,R)-58 and

(R,S)-58, were separately transformed into the enantiomeric 2–

phenylindolines by reduction with diborane (Scheme 20).

The enantiomeric purities of the two resolved 2-phenylindoline

(55) enantiomers were > 99.4% and 90.1%, respectively, as analyzed

by chiral gas chromatography. Attempts to crysttalise one enantiomer

of 55 for X-ray analysis is in progress.

Both enantiomers of (2)-phenylindoline (55) are now ready to

be used as chiral auxiliaries in the intramolecular cycloaddion reaction

shown in Scheme 19.

Although the resolution of 2-phenylindoline is an important

achievement, our initial aim was to develope a synthetic procedure for

iridoids. With this objective, a pure enantiomer of (2)-phenylindoline

(55) could be used as chiral auxiliary in the intramolecular

cycloaddition reaction shown in Scheme 19. As we have shown that

this reaction is stereoselective and is giving rise to only one

diastereomer, we have presented a formal enantioselective synthesis

38

of the iridoid gastrolactol, which could be obtained by hydrolysis of the

cycloaddition adduct 56. Gastrolactol is evidently a versatile synthetic

precursor on the route to more elaborate iridoids.

39

5. CONCLUDING REMARKS

A wide variety of compounds are used as pheromones across the

animal kingdom.

In this work many pheromone components were synthesized and

several important questions in chemical ecology were answered. Some

projects gave more of basic understanding and some addressed more

applied issues, like the Phyllonorycter blancardella project.

In order to provide substances in > 99% isomeric purity for field

trials, many purification techniques were explored, e.g. argentum

chromatography, urea inclusion complexes, the Baeckström isolation

technique as well as low temperature crystallization.

In this process quite a number of chemical problems were faced.

The stereoselective syntheses of iridoids were examples of the

elaborated synthetic efforts undertaken during this doctoral work.

40

41

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to all people who made this work possible. My supervisors Assoc. Prof. Rikard Unelius, Prof. Anna-Karin Borg-Karlson and Prof. emeritus Torbjörn Norin for accepting me as a graduate student, for their support and never-ending enthusiasm. All former and present members of Ecological Chemistry group for an inspiring and stimulating working atmosphere. Ilme for helping me in the lab when I started, Raimondas for answering endless biological questions, Marie Pettersson for being so kind, Peter Baeckström for sharing his knowledge in chromatography and for all the nice talks, Monika, Jenny, Pagula, Sture Strömberg, Ulla Jacobsson, Katinka, Martha, Matilda, Astrid, Ashitani, and all the others that have come and gone. I would especially like to thank Johan for being a good friend and for all the fun we had together in Sweden and around the world. Prof. Arlene Correa and the members of her group for the opportunity to work in Brazil during the times I was there. Thanks to all co-workers for a fruitful collaboration. For comments and constructive criticism on the thesis during the writing process I want to thank Prof. Christina Moberg, Dr. Gunhild Aulin-Erdtman, Peter Baeckström, and Dr. Fredrik Rahm. IFS, MISTRA, Sven and Dagmar Saléns Foundation, Aulin-Erdtman foundation, and Royal Institute of Technology are greatly acknowledged for financial support. Lena Skowron, Ingvor Larsson, Ann Ekqvist, Henry Challis and Jan Sidén for all the help with whatever was needed. Thanks to all former and present colleagues at the organic chemistry department at KTH, especially Fredrik R., Åsa, Timo, Oscar, Daniel, Emma, Antonio, Erika, Stina, Katja, Raivis, Sari, and Emelie for the friendship and Erik for helping me get through in a difficult moment. I will never forget that! I cannot fully express my gratitude to my dearest friends Henrik and Marisa, for the wonderful time we shared together and for their generosity. Martinho and Emilie for the “caipirinhas” and “feijoadas” we have enjoyed together. Anders Jakobsson, Fredrik Petersson, Joakim Jarleman, Tony Sjögren and Ulf Lindberg for being the best friends one could ask for. I couldn’t find words to describe how much I appreciate every single moment we have spent together. Most importantly, I would like to thank my family for their endless support and encouragement throughout my years at KTH and earlier. I could not have done any of this without the knowledge that I could always turn to them for advice, at any time. Special thanks to my sister Erika, for encouraging correspondence throughout the years. Fabio for teaching me how to live by myself and for his support. A person’s accomplishments are rarely the product of only him or herself. I owe my happiness to all above mentioned people, and many more.

42

43

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