BIOSINTESIS LUPINin

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Biosynthesis of the lupine alkaloids I Lupinine

W.

MAREK OLEBIEWSKI'

N D

IAND. SPENSER

Department of Chemistry, McMaster University, Hamilton, Ont., Canad a

L8S

4M

Received February 5, 1985

W . MAREK OLEBIEWSKInd IAN . SPENSER.an.

J

Chem. 63, 2707 (1985).

Th e mode of incorporation into lupinine of cadaverine, intramolecularly doubly labelled with I5N and with C at the C-atom

adjacent to N, i.e.,

13C,15~- bond-labelled ,

as determined by I3C nmr spectr oscopy ; lupinine is generated from two

cadaverine-derived C5-units by a route which excludes a dimeric intermediate with C2, symm etry. The mode of incorporation

of 'H from L-(2-'H)lysine, from (R)- and (S)-(1-'H)cadaverine, and from (2-2H)-A1-piperideine nto lupinine was determined

by 'H nmr spectroscopy. T he results corroborate the conclusions from the I3C, N experiment and they establish the stereo-

chemistry of six of the steps in the biosynthetic conversion of L-lysine into lupinine.

W .

MAREK OLEBIEWSKI

t

IAN

D.

SPENSER.

an.

J

Chem. 63, 2707 (1985).

Faisant appel la spectroscopic rmn du I3C et utilisant de la cad avtr ine doublement marquee d 'une faqon intram oltculaire

par du ' 5 ~t du 13C sur le carbone voisin du ' 5 ~liaison I3C, 5N marquee), on a ttud ie le mode d'incorporation de la cadavt rine

la lupinine. Les r6sultats obtenus suggkrent que la lupinine se forme partir de deux unites en C5 dCrivtes de la cad avtr ine

par le biais d'une v oie reactionnelle qui exclut la formation d'un in termtd iaire dimkre posstdan t une symetrie C2,. Faisant

appel la rmn du 'H, on a determ int le mode d'incorporation du 'H de la L-('H-2) lysine, des ('H-2) cad avtr ines -(R ) et -(S )

et de la (ZH-2)-A1-pip~ridinela lupinine. Les rtsultats confirment les conclusions tirees sur la base des experiences conduites

avec le I3C et le I5N; de plus, elles permettent d'etablir la sterkochimie de six des Ctapes conduisant

2

la transformation

biosynthttique de la L-lys&e en lupinhe.

[Traduit par le journal]

Introduction

Speculations concerning the biosynthesis of the lupine alka-

loids go back more than 50 years. In 1931 Schopf suggested

(1, 2) that the ring skeleton of lupinine (20) might be generated

by condensation of two lysine (1) derived fragments, 5-amino-

pentanal

(3) e

'-piperideine (4) (3, 4)) and glutardialdehyde

(S),

followed by reduction (Scheme 1Bd). Schopf later aban-

doned this notion in favour of the idea (3) that the im-

inodialdehyde (7) might be the intermediate between lysine and

lupinine (Schemes 1Ba and 1Bb). This intermediate was

favoured also by Robert Robinson (4). In a further variation of

this theme it was suggested (5, 6) that lupinine arose by

rearrangement of tetrahydroanabasine (14), a dimer of

A'-piperideine (4) (3, 5) (Scheme 1Be).

The first tracer experiments to test these ideas were canied

out twenty-five years ago. Radioactivity from ~~-[2-'~C]lysine

(7) and from its decarboxylation product, [l-'4C]cadaverine

(6, 8), was indeed incorporated into lupinine. With either sub-

strate, approximately one quarter of the total activity of the

alkaloid was present within the hydroxymethyl carbon, C-11,

of lupinine. Also, the two methylene carbon atoms, C-4 and

C-6, together, accounted for one half of the total alkaloid

activity (7, 8). If the assumption is made that this activity is

distributed equally over C-4 and C-6, and that each of these two

C atoms contains one quarter of the label,' it follows that four

sites within lupinine, C- 11, C-4, C-6, and, presumably, C- 10,

each account for one quarter of the label of the intact alkaloid

(20). This conclusion was substantiated by the results of a

recent feeding experiment (9) with ~~-(4,5- '~C~)lysine.n-

'

On leave from the Department of Chemistry, University of War-

saw, 02-093 Warsaw, Poland.

'Another possible interpretation of these results is that one of the

two cen tres, C-4 or C- 6, accounts for 50% of the activity of the intact

lupinine, while the other centre is free of activity.

richment was detected at four pairs of neighbouring carbon

atoms of lupinine, C- 1 -2, C-2,-3, C-7,-8, and C-8,-9. This

labelling pattern serves as evidence that the carbon skeleton of

lupinine is generated from two lysine-derived C5-units, via an

intermediate with CZvymmetry (e.g., either cadaverine (2) or

the iminodialdehyde (7), or both). However, since the distribu-

tion of label observed in all these experiments is that which is

predicted by every one of the five hypothetical routes (Schemes

1Ba-1Be) that have been advanced to account for the deri-

vation of lupinine from lysine, available tracer evidence cannot

serve to discriminate among the biogenetic proposals.

Nor is it certain that cadaverine (2) is an intermediate

between lysine (1) and lupinine (20): Neither the observation

that incorporation of the two substrates, DL-[2-'4C]lysine nd

[l-'4C]cadaverine, leads to the same distribution of label within

lupinine, nor the labelling pattern from DL-(4,5- C,)lysine con-

stitute conclusive evidence for the obligatory intermediacy of

cadaverine. Other interpretations are possible which account

for such a distribution of label (cf. ref. 10).

The tracer experiments with doubly I3C, N- and with

'H-labelled substrates which are here reported serve as critical

tests of the hypotheses that have been advanced to account for

the origin of lupinine. 'The results, which controvert all but one

of the hypothetical sequences, provide support for the tetra-

hydroanabasine route (Scheme 1Be). Furthermore, the results

of the tracer experiments with 2H-labelled substrates clarify

stereochemical aspects of this route. Preliminary accounts of

parts of this work have appeared (1

1 ,

12).

Methods and results

In six tracer experiments, compounds, specifically labelled

with stable (Experiments 1-5) and with radioactive isotopes

(Experiment 6), were administered by the wick method to

intact plants of

upi nu s luteus

(yellow lupine) over a period of

3-6 days. Details of the experiments are summarized in Table

1.



27 8

CAN J CHEM.

OL

63

1985

CHO FHO CHO

C+>

HO

-3 c;3

c f

CHO

CHO

[?>

HO

NH2

CHO FHO

J

c g

- QCHO h

CHO CHO

CHO CHO CHO

cg Jsds )

rom

Bc

CHO

2 7 [ H p

lg

SCHEME

.

Five alternative hypothetical schemes o f the b iogenesis

of lupinine. A. The initial steps.

B -Be.

Variants of the intermediate

steps. CI-Ch. Variants of the final steps.

The sample of cadaverine, intramolecularly doubly labelled

( bond-labelled ) with I3C and N (Experiment 5) was pre-

pared in three steps from sodium ( C, N)cyanide and

1 bromo 4 phthalimidobutane

y the reaction sequence shown

in Scheme 2, in analogy with the preparation of a simila

labelled sample of putrescine (10).

The samples of ~~-(2-~H)lysineExperiment 3), (2-2H)-

piperideine (Experiment 4), S)- +)- l-2H)cadaverine di

drochloride (Experiment 2), and R)- -)- l-2H)cadaverine

hydrochloride (Experiment 1) were prepared by chemical a

enzymic methods, respectively, which we have previou

described (13). The samples of L-[4-3H]lysine monohyd

chloride and ~~- [6 -' ~C ]l ys in eonohydrochloride (Experim

6) were commercial products.

When the administration of tracer was complete, the pla

to which labelled substrates had been administered were mac

ated with cold methanol in a Waring blendor and the alkalo

were isolated by methanol extraction. Lupinine and sparte

were separated by chromatography on silica gel, lupinine w

converted into the hydrochloride, sparteine into the sulfate

The present paper presents a discussion of the results

tained with lupinine. The sparteine results will be dealt with

the second paper of this series.

Lupinine hydrochloride consists of a 1.7: 1 mixture of

trans and cis ring fused stereoisomers, as shown by the natu

abundance

C nmr spectrum (Fig. 1B). The C nmr spectr

(Fig. 1A) of the sample of lupinine hydrochloride from Exp

ment 5, in which (~-'~C,'~N)cadaverinen admixture w

[l-14C]cadaverine had been administered (Experiment

shows that four of the ten C-atoms, C-4, -6, -10, and -1 1, w

enriched in I3C, to an equal extent. The enrichment, abo

natural abundance, calculated for each of these four carb

atoms (Tables 2 and 3) is in agreement with the specific inc

poration of I4C (Table 1).

The signals in the proton noise decoupled

C nmr spectr

due to two of the C-enriched carbon atoms, C- 10 and C-

appear as singlets, the signals due to the two others, C-6 a

C-4, as multiplets (Fig. 1A). The coupling constants of

doublet component of the multiplets are different from o

another (J13~~,15,:rans isomer 4.1 Hz, cis isomer 4.1 H

trans isomer 7.0 Hz, cis isomer 6.4 Hz; isotope s

C-4, cis +0.02, trans $0.02; C-6, cis -0.04, trans, -0.04

Each of the two sets of multiplets consists of a doublet sup

imposed on a singlet. The doublet (87 11%) (I3C-6, N)

the signals due to C-6 (trans, 6 55.7, cis, 52.9 ppm) is m

more intense than the singlet (13 2%) (I3C-6,I4N) Table

and 3), so that the latter is not apparent in the unexpan

spectrum. In the signals due to C-4 (trans, 6 54.8, cis, 6 4

ppm) the doublet (36

+

5% of signal area, Tables 2 and

(I3C-4, N) appears as a shoulder straddling the singlet (64

8%) (I3C-4,I4N).The doublet/singlet ratios at C-6 ((87

11)/(13 + 2)

=

6.7 1.3) and at C-4 ((36 + 5)/(64 + 8

0.6

+

0.1) thus differ from one another by

an

order of m

nitude (Tables 2 and 3).

Lupinine hydrochloride obtained from the feeding exp

ment with [~-4-~H/~~-6-'~C]lysineExperiment 6) was c

verted into the methiodide and crystallized to constant rad

activity and constant tritium/14C ratio. The 'H/I4C ratio of

purified lupinine methiodide was 8.4 + 0.1, approximat

double that of the intermolecularly doubly 'H/I4C labe

lysine that had served as the substrate ('H/I4C of lysine, 4.1

0.1 (Table 1)).

The deuterium nmr spectra of the samples of lupinine (f

base) from the experiments with deuterium labelled substra

(Experiments 1-4, Table 1) are shown in Fig. 2. The d

terium chemical shifts observed in the spectra of these f



GOLEBIEWSKI AND SPENSER

TABLE . Experiments with upinus luteus

a)

Stable isotopes

Plant material Lupinine Sparteine

Fresh Specific Specific

Expt Wt. fed Enrichment weight No. of Weight incorporation Weight incorporation

no. Substrate (mg) (at. ) (g) plants (mg) per C5 unit ( ) (mg) per C5 unit ( )

1 (R)-( l -2H)Cadaverine 150 99d 300 40 9.5 3.0 4 8

dihydrochloridea

(March, 1983)

2 (s)-(1 -2H)Cadaverine 116 93d 21 1 51 13 2.3 4 2

dihydrochloride

(Dec. 1982)

3 ~~-(2-'H)L ysine ono- 500 95d 41' 100 5 2.3 4 3

hydrochloride

(July, 1983)

4 (2-2~)-A'-Piperideine 300 9Sd 247 100 9 5.4 4 8

(Nov. 1983)

5

(~- '~C ,l- '~~ )Ca dav eri ne 40 99.99 28

dihydrochloride

105 50 10 5 47

[I '4C]Cadaverine

dihydrochlorideb

(Aug. 1982)

(b)

Radioactive isotopes

Plant material

Nominal activity

Fresh Sparteine

Expt Total Specific 3H/'4C weight No. of Lupinine 'H/14C sulfate 3 ~ / ' 4 C

no. Substrate (mCi) (mCi/mmol) ratio (g) plants (mg) ratio (mg) ratio

6 ~ ~ - [ 6 - ' ~ C ] ~ y s i n e 0.20 45

monohydrochloride'

4.1 O l h 104 36 4 8.4 0.1' 3 6. 8 0.2

L-[(Rs)-4-3H]Lysine 1.0 2 2 x 1 0 ~

monohydrochloride'

(Dec. 1983)

See Experimental.

'New England Nuclear (nominal total and specific activities, 25 pC i, 105 mCi per mmol, respectively).

'CEA France.

dMeasured by means of computer assisted peak height analysis.

'This experiment was d one during the hot summer of 1983 when the greenhouse was very hot and dry.

'Net specific activity, determined for the mixture of [I ,5-'4C]cadaverine dihydrochloride, after dilution with 1 40 mg (I- C , I-15N) cadaverin e ihydro chloride,

which was fed to the plant: 5.3 X 10' dpm per mmol.

*Specific acitivity of lupinine hydrochloride, after crystallization to constant specific activity: 3.0

X

10' dpm per mmol.

A A-mL sample of the feeding solution (25 m L) containing the doubly labelled lysine, which w as administered to the plants, was set asid e and from it lysine

hydrochloride was re-isolated by carrie r dilution and purified by ion exchange chromatography and crystallization to constant 3H /14 C atio.

'Lupinine was converted into the methiodide which was crystallized to constant 'H/I4C ratio.

N a C N

20 H9

DMSO Ac20

SCHEME . Synthesis of (1-I3C,1-I5N)cadaverine.



2710

CAN.

1 CHEM.

VOL.

63, 1985

TABLE. Incorporation of ( I-I3C, 1-ISN)cadaverine into trans-lupinine hydrochloridea

Natural abundance Enriched in

Chemical

13

C ,

normalized I3C, normalized Percent enrichment

Carbon

shiftb

Type of

peak areac peak areac

0.94 above natural

atom

(ppm)

signal

(ANA)

(AE)

AE/ANA~ abundancee,'

Relative percent exc

13

C above natura

abundance at

individual C atom

The sample in

D2

as contained in a 2-mm tube. Acquisition time 3.41 s, 0.293 Hz/data point; 1.6 ps pulses.

bRelative to acetone (29.8 ppm).

'Peak areas are normalized relative to C-3 100%. Estimated standard deviation *8% for singlet signals, 10% for composite signals.

dThe ratio AE/ANAs normalized so that the average value for carbon atoms 1, 2, 3, 7, 8, and 9 equals 1 O.

'((0.94 AE/ANA)

I X

1.1% for singlets; (0.94 AE/ANA) I. 1% for doublets.

'The average specific incorporation per C5 unit 1/4(15.2(? 1.6)

+

13.9(* 1.9)

+

16.4(22.0)

+

12.5(* l.5))/(99/2)

X

100% 29

rt_

2%, where 9912

I3C is the average enrichment at each terminal position of cadaverine.

TABLE.

Incorporation of (1 I3C, 1 ISN)cadaverine into cis-lupinine hydrochloridea

Natural abundance Enriched in

Chemical

13

C, normalized I3C, normalized Percent enrichment

Carbon shiftb Typ e of peak areac peak areac 1.08 above natural

atom (ppm) signal (ANA) (AE) abundancee.'

Relative percent ex

13

C above natura

abundance at

individual C atom

See footnote

a,

Table

2.

bSee footnote

b,

Table 2.

'Peak areas

are

normalized relative to C-8 100%. Estimated standard deviation 8 % or singlet signals,

k

10% for composite signals.

dThe ratio AE/ANAs normalized so that the average value for carbon atoms 1,

2,

3, 7 , 8, and 9 equals 1 O.

'((1.08 A€/ANA) )

X

1.1% for singlets; (1.08 AE/ANA)

X I . I

for doublets.

'The average specific incorporation per C5 unit 1/4(12.7(2 1.3) 12. l(f 1.5) + 15.1(21.8) 9.9(&1.2))/(99/2) X 100% 25 rt_ 2%.

lupinine samples are summarized in Table 4 and are compared

with the corresponding proton chemical shifts (Table 5). The

2H nmr spectra of two lupinine samples, that derived from

(S)-(l-2H)cadaverine (Experiment 2) and that derived from the

L-component of DL-(2-2H)lysineExperiment 3), were identi-

cal, each showing two signals, at i 1.72 (+0.01) and 1.54

ppm. The chemical shifts of the two signals (6 4.11 and 1.80

ppm) in the spectrum of the sample derived from

(2-2H)-A'-piperideine Experiment 4) corresponded to those of

two of the signals (6 4.14 and 1.77) in the spectrum of the

sample from (R)-(l-2H)cadaverine (Experiment 1). The latter

spectrum showed an additional signal at 6 2.50 ppm. The

assignment of these spectra will be discussed in the appropriate

context of the next section.

iscussion

The results of the tracer experiments with 14C-labelled y

(7) and cadaverine

6,

8) and with ~~-(4,5-'~C~)lys

9)

not discriminate among the various biogenetic hypotheses

have been advanced to account for the biosynthesis of lupin

20) (Scheme lA, B, C). The first task in a reexamination

the problem was to devise experiments capable of narrow

down the possibilities.

All the hypotheses that have been advanced (Scheme

share a common initial phase, decarboxylation of lysine 1

cadaverine 2),followed by oxidative deamination of the la



GOLEBIEWSKI

AND

SPENSER

T R A N S IS

LUPlNlNE HCl

l r om

1- 3C,1?5~-

CADAVERINE

A

R e . 1. I3C nmr spectra (20 .15 MHz) of lupin ine hydrochloride. The spectra were recorded in the Fourier mode on a Bruker WP 80

spectrometer, in 2-mm tubes, with the natural abundance methyl signal of acetone (29.8 ppm) as internal reference. The acquisition time was

3.41 s. For further details see Tables 2 and 3. Top: Fig. ]A . Proton-noise decoupled (P.N.D.) spectrum (20 000 scans) of the 3C , 5~ -e nr ich

sample of lupinine hydrochloride (10 mg in 10

p

H20) derived from (l- 3C,1-15 N)cadaverine Experiment 5). Bottom: Fig. 1B. P.N.D .

Spectrum (18 112 scans) of a natural abundance sample of lupinine hydrochloride (17 mg in 20

p

H20).

TABLE. Incorporation of deuteriated substrates into lupinine. H nmr analysis

H nmr chemical shiftsb (ppm)

Experiment 1 Experiment 2 Experiment 3 Experiment 4

H nmr lupinine from lupinine from lupinine from lupinine from

Hydrogen chemical shiftsa R-(1- H)cadaverine S-(1- H)cadaverine ~L -( 2- ~) ly si ne (2- H)-A -piperideine

atom ( P P ~ ) 2) 3)

413 s (eq) 2.57

4 P r e ( a x ) 1 . 7 5

6 a r e ( e q ) 2.51 2.50

6P s (ax) 1.57

lo p R (ax) 1.78 1.77

11 re 3.75

11

s

4.16 4.14

The

H

nmr spectrum. Recorded in C6D6 at 250

MHz

and

400 MHz

in the Fourier mode on a Bruker WM 250 and WH 400 spectrometer,

respectively. For full assignment of the proton spectrum, see Table 5.

'The

H

nmr spectra. Recorded in

CJ-16

t 38.40

MHz

in the Fourier mode on a Bruker WM 250 spectrometer, in 10 mm tubes with

natural abundance

C6DHs (7.19

ppm) as internal reference. Acquisition time

2.048

s .

to 5-aminopentanal (3), an am inoaldehyde in equilibrium with

its intramolecular Schiff base, A -piperideine (4) (Sche me

1A).

The hypotheses also agree on the final step in the biogenetic

process, reduction of lupinal (19) to lupinine

20)

(Scheme

1C). The hypotheses differ in the proposed routes leading from

5-aminopentanal (3) SA -piperideine (4)) to lupinal (19)

(Schemes lB, 1C).

What was required for a critical examination of the bio-

genetic process was a method w hich could distinguish the dif-

ferent paths between Saminopentanal (3), the last early inter-

mediate common to all the biogenetic proposals, and lupinal

(19), the common intermediate of the final stage. One way of

differentiating among the routes would be by an experiment

cap able of tracing the fate of the intact C-N bond of

5-aminopentanal (3) into the product.

A successful experiment, leading to incorporation of such a

C-N bond into lupinine, ca n have one of three differen

outcome s. Tw o of the routes (Schemes 1Bd and 1Be) lead from

3

into 1 9, via the bicyclic compound (18) and other inter-

mediates, in such a way that the intact C-N bond of 3 is

destined to become the C-6,N bond of lupinine (Scheme 3C).

A third route (Scheme 1Bc) leads from 3 into 19, via the

bicyclic compound (17) and other intermediates, in such a way

that the intac t C-N bond of 3 is destined to become the C-4,N

bond of lupinine (Sch eme 3B ). It can be predicted that each o

these routes leads to a sample of lupinine in which only o ne o

the three C-N bonds of the product is derived from the intac

C-N bond of 3.

Th e other two routes (Schemes 1Ba and 1Bb) differ from the

above in that they give rise to a sample of lupinine which



2712

CAN. J.

CHEM.

VOL. 63

1985

LUPlNlN

rom

FIG

2. 'H nmr spectra (38 .40 MHz) of deuterium-enriched sam-

ples of lupinine derived from deuterium labelled substrates (Experi-

ments 1 -4). Th e spectra were recorded in the Fourier mod e on a

Bruker

WM

250 spectrometer, in 10-mm tubes, with natural abun-

dance C a H 5 (7.19 ppm) as internal reference. Th e acquisition time

was 2.048 s (see Table 4). 2A. Lupinine from (R)-(I-'~)cadaverine

(Experiment 1). 2B. Lup inine from (S)-(1 -2H)cadaverine (Experiment

2). 2C. Lupinine from the L-component of DL-(2-'H)lysine (Experi-

ment 3). 2D. Lupinine from (2-2H)-A'-piperideine (Experiment 4).

contains an equimolar mixture of two species, one of which

contain s the intact C-N bond of

3

at C-4,N while the other

contains it at C-6 ,N. This is the consequence of the postulated

intermediacy of the dialdehydeimine (7). T his comp ound con-

tains two C-N bonds, but only on e of these is derived intact

from

3.

However, the compound possesses CZ v ymm etry. Ring

closure of 7 by an intramolecular Mannich reaction to yield

lup inal (19 ) can take place in two equivalent ways. T he C-N

bond that is derived intact from 3 has an equal chance of

becoming either C-4,N or C -6,N within the product. Th e prod-

uct will then comprise an equimolar mixture of two chem ically

indistinguishable species, which differ solely in the o rigin of

the two C-N bonds, C-4,N and C-6,N (Scheme 3A).

The origin of the two C-N bonds of lupinine, C-4,N and

C-6,N, can be determined by a tracer experiment with C-N

bond-labelled 5-aminopentanal (3) or one of its precursors,

lysine (1) or cadaverine (2).

A sample of the latter, intra-

molecularly doubly I3C,lSN labelled (NH2-CH2-CH2-

C H ~ - C H ~ - ' ~ C H ~ - ' ~ N H ~ ) , was employed to probe the in-

TAB LE . The I3C and

H

mr spectra of trans-lup inine (free ba

'H nmrb

I3C n m f chemical shifts (ppm)'

chemical shifts

C arb on ( P P ~ a xia l p ro to ns e qu ato ria l p ro t

C-1 1 65.8 3.75(re) 4.16(si)

Recorded n C6D6 (128.4 ppm)

at

100.7 M z in the Fourier mode o

Bmker

W 400

spectrometer.

Recorded in C6D6at 400 M z in the Fourier mode on a Bmker W

spectrometer.

'Reference 19.

dCf. references 14- 17.

corporation of an intact C-N bond into lupinine (Experim

5) (1 1). A similar experiment, with essentially identical res

and conclusions, was reported in an independent investiga

(14). Entry of this bond-labelled cadaverine, via 5-am

pentanal, by route Bd or Be (Scheme l) , should yield a sin

species of intramolecularly I3C,lSNdoubly labelled produc

which the I3C,l5N moiety is located at the C-6,N position

lupin ine. Entry by Route Bc (Sche me 1 ) should similarly y

lupinine enriched at C-4,N. Entry via the nondissymme

intermediate 7 (Routes Ba, B b, Sch eme 1) must yield an e

molar mixture of the above two enriched species (Scheme 3

Non-equim olar enrichment at the two sites, C-4,N and C-6

is not consistent with the intermediacy of a dimeric c

pound with Cz, symmetry, such as 7.

The proton noise decoupled I3C nmr spectrum of the sam

of lupinine hydrochloride (a mixture of the trans and the

isomers, in the ratio 1.7 : 1) isolated from plants to wh

(1 I3C,1 15N)cadaverine dihydrochloride had been admi

tered in admixture with [l-'4 C]cad averine dihydrochlo

(Experiment 5, Table 1) is shown in Fig. 1A.

Lupinine hydrochloride rather than the fre e base was cho

as the compound for I3C nmr analysis since, even though

salt consists of a mixture of the trans and cis ring fused

mers, in the I3C nmr spectrum of the cis isomer in D 2 0

signals due to C-4, -6, -10, and -1 1, the carbon atoms cru

for the interpretation of the biosynthetic experiment, are w

resolved (15) (A6 C-4,-6: 8 .3; C-10,-1 1: 2.4 ppm ) (Table

The free base, on the other hand, is

trans

ring fused, and

corresponding signals in the spectrum of the free base s

poor resolution (A6 C-4,-6:

0 (15.1 MHz (15, 16), 5.0 M

(17)), 0.1 (22.5 M Hz (18)) in CDC13; 0. 1 (50.3 MH z (14)

C a 6 ; A6 C-10,-11: 0 (16), 0.4 (15) (15.1 MH z), 0.3 (5.0 M

(17)), 0. 9 (22.5 MHz (18)) in CDC13; 0. 3 (50.3 M Hz (14)

c a 6 ) .

Spectral assignments of the crucial carbon atoms, C-4,

-10, and -11, were made as follows: The natural abunda

proton noise decoupled I3C spectrum of lupinine hy drochlo

(Fig. 1B) shows two sets of 10 signals, corresponding to

stereoisomers of lupinine hydrochloride that differ in

stereochemistry of the ring fusion. Since the two sets of sig

show different signal areas, the two isomers are presen



GOLEBIEWSKl AND SPENSER

oute

1B.

C

vi

outes

106

or 10

SCHEME Incorporation of (l-'3C,1-1SN)cadaverinento lupinine. Labelling patterns predicted from the five biogenetic schemes outlined in

Scheme 1.

A

Via routes B or Bb Scheme 1.B. Via route B Scheme 1. C. Via routes BdorBe Scheme 1. Left columns: I3C,l5N ond-labelled

species. Central columns: Doubly 13C-labelled pecies. Right columns: Singly '3C-labelled pecies. = I3C; = 13C-15N.

unequal amounts (1.7 1). The less intense set of signals shows

two signals at 6 40.0 and 44.6 ppm. Signals in this spectral

region are absent from the spectra of the

trans

fused free base

(14- 18) or the trans fused salt (15) but are present in the is

fused salt (15). The signal set of lower intensity is thus due to

the is fused salt, which is the less abundant (ca. 37%), and the

signals at 6 40.0 and 44.6 pprn must be assigned (19) to C-1

and C-4, respectively, of this isomer. This assignment is con-

firmed by the off-resonance spectrum in which the signal at

6

40.0 appears as a doublet and the signal at 6 44.6 pprn as a

triplet. A second signal, which in the low intensity set appears

as a doublet, is that at 6 58.9 ppm, which must therefore be

assigned to C-10. This leaves the downfield signal at

6

61.3

pprn to be assigned to the hydroxymethyl carbon, C-11, and the

signal at

6

52.9 pprn to C-6. The corresponding resonances in

the more intense signal set, due to the

trans

fused stereoisomer,

which appear as doublets in the off-resonance spectrum, are

those at 6 36.8 pprn (C-1) and at 6 64.7 pprn (C-10). The signal

at 6 59.4 must then be assigned to C-1 1 and those at 54.8 and

55.7 pprn to the aminomethyl carbons, C-4 and C-6. Since in

the spectrum of the enriched sample of lupinine hydrochloride

(Fig. 1A) the high intensity signal at 6 55.7 appears as a doublet

and shows a similar doublet/singlet area (12521 194 = 6.4,

Table 2) as the low intensity doublet due to C-6 at 6 52.9 pprn

(10391156 = 6.7, Table 3), the signal at 6 55.7 must also be

due to C-6. Similarly, the signal at

6

54.8 pprn in the high

intensity set corresponds in doublet/singlet area (5341941 =

0.6, Table 2) to that at 6 44.6 pprn (4341786 = 0.6, Table 3)

in the low intensity set, due to C-4.

The distribution of label within the I3C,l5N nriched alkaloid

is evident from the spectrum (Fig. 1A). As expected, only four

of the ten C-atoms (C-4, C-6, C-10, C-1 1) are enriched in I3C,

to an equal extent, within experimental error (Tables 2 and 3).

The average specific incorporation of label per C5 unit (C,

unit A: C-6, -7, -8, -9, -10, C enrichment in either C-6 or

C-10 but not in both within the same molecule; C5 unit B: C-11,

-1, -2, -3, -4, I3C enrichment in either C-1 1 or in C-4, but not

in both), based on the I3C nmr data (Tables 2 and 3), was ca.

30 at.% I3C. This corresponds to an incorporation of 30199

100 = 30% of cadaverine per C5 unit, a value which is in good

agreement with that obtained from I4C measurements ((3.0

107)/(5.3 lo7)) 100 112 = 28%, Table

1



27 4 CAN. J CHEM.

VOL. 63 1985

The signals due to C-6 and C-4 appear as multiplets. Since

the coupling constants of the doublet component of these mul-

tiplets differ from one another, the multiplets do not arise from

I3C-6,I3C-4coupling, but are due to I3C,l5Ncoupling.

The intense I3C-6,I5Ndoublet indicates intact incorporation

of the I3C-I5N unit of the administered cadaverine into C-6,N

of lupinine. The low intensity doublet due to I3C-4,I5Narises

as a consequence of the remarkably high efficiency of incor-

poration, into the alkaloid, of the administered (l-'3C,l-15N)-

cadaverine. It can be calculated from the I3C nmr data (Tables

2 and 3) that the enriched lupinine which was biosynthesized in

the course of the experiment was diluted by natural abundance

lupinine present in the plants prior to the experiment by approx-

imately 68%. It can also be calculated that the administered

doubly enriched cadaverine was diluted by no more than 25%

of endogenous natural abundance cadaverine, prior to con-

version into lupinine. The intensity of the I3C-4,I5Ndoublet is

fully accounted for by intermolecular I3C,l5N oupling between

two monomer units, derived from this highly enriched cadav-

erine, which are incorporated into lupinine after dimerization.

The observed difference in the doublet/singlet ratios of the

signals due to C-6 and due to C-4 serves as evidence that an

intermediate with C2, symmetry, such as

7

cannot be impli-

cated in lupinine biosynthesis. The biogenetic proposals shown

in Schemes 1Ba and 1Bb are thus eliminated.

Furthermore, since the intact

l3C-I5N unit of the adminis-

tered cadaverine is maintained at C-6,N and not at C-4,N of

lupinine, the sequence shown in Scheme lBc, according to

which the precursor C-N bond enters C-4,N of lupinine, is no

longer in contention.

The biogenetic schemes which need to be examined further

are those shown in Schemes 1Bd and 1Be. These two schemes

differ in the timing of the loss of the nitrogen atom of one of

the two cadaverine-derived aminopentanal units. Scheme 1Bd

postulates that this nitrogen atom is lost at an early stage, so

that the aminopentanal-derived intermediate which serves as

the precursor of the C5 chain, C-4, -3, -2, -1,

-

11, of lupinine

is glutardialdehyde (5), a compound wih C2 symmetry.

Scheme lBe, on the other hand, postulates that this nitrogen

atom is eliminated at a late stage of biosynthesis, following

formation of the CI dimers, 14 and 15.

An experiment with 5-aminopentanal or A'-piperideine, la-

belled at the sp2 carbon atoms with I3C or I4C, would serve as

a critical test of these two suggested routes. In one instance

(Scheme lBd), three of the carbon atoms of lupinine, C-10,

C-11, and C-4, should carry label. In the other instance

(Scheme

lBe), label should be present at two of these carbons,

C-10 and C-11, but not at the third, C-4. Similarly, an experi-

ment with 5-aminopentanal or A'-piperideine labelled with

deuterium at the sp2 carbon atom would distinguish these two

routes, provided that it can be demonstrated that the integrity

of the C-D bond is maintained in the course of the biogenetic

process. The results of an experiment with

(2-2H)-A'-piperi-

deine (Experiment

4

that fulfils this condition will be dis-

cussed later. On the basis of this experiment and of other

experiments with deuteriated substrates (Experiments 1-3)

(see later) the hypothesis based on the intermediacy of glu-

tardialdehyde (Scheme 1Bd) can be discounted.

The biogenetic sequence shown in Scheme 1Be remains. It

is consistent with all available experimental evidence.

The initial step in the biosynthetic process leading to lupinine

from primary metabolites is the decarboxylation of lysine

1)

to

yield cadaverine

2).

Two stereochemical questions concerning

this step must be answered. First, it must be establ

whether L-(i.e., (S)-)lysine or D-(i.e., (R)-)lysine serves a

substrate. Secondly, it must be established whether the d

boxylation process which converts this lysine into cadav

takes place with net retention of configuration or with

inversion of configuration. We have answered both

questions.

The result of the experiment with intermolecularly do

labelled [~- ~H /~ ~- '~ C] ly s i neExperiment 6) demonstrate

L-lysine, rather than D-lysineor DL-lysine, erves as the pr

sor of lupinine in L luteus. The 3H/14C atio of the do

labelled lysine that was administered to the plants was 4

0.1. The 3H/14C atio of the lupinine methiodide that

obtained was 8.4 0.1 (Table 1). From these results it c

calculated that, within experimental error, lupinine is de

entirely (102 3%) from L-lysine( of product derived

L-substrate 50 (3H/14C atio of pr ~d uc t) /( ~H /' ~Cat

substrate)) (20).

Demonstration that the decarboxylation of

L-lysine o ca

erine takes place with net retention of configuration c

from two of the experiments with deuteriated substrates.

These experiments (Experiments 1-4), which solve

only this stereochemical problem, but which also answer

other stereochemical questions concerning the biosynt

steps of the route from lysine into lupinine that involve t

formations at those carbon atoms of lupinine which orig

from the terminal carbon atoms of cadaverine, will no

discussed.

In the first two of these experiments, samples of cadave

chirally deuteriated at C- I, were used as substrates. The

ples of lupinine obtained from these experiments were e

ined by 'H n'mr. The 'H nmr spectra are shown in Figs. 2A

2B. Chemical shifts were assigned by comparison with

corresponding 'H nmr chemical shifts (Table 4).

Unambiguous assignment of the resonances due to th

protons of lupinine was not possible with one-dimensiona

spectroscopy, even at 400 MHz, because of substantial ov

of some of the signals. Homo- and heteroscalar corre

two-dimensional 'H and 'H,I3C nmr spectroscopy (COSY

J-resolved 2D 'H nmr spectroscopy were employed for a

plete assignment of the spectrum. Assignment of the down

signals, due to the 11 re and 1 si protons, had been rep

(21). A full discussion of the 'H nmr spectrum of lupinine

appear elsewhere (19). Assignments of the signals due t

protons of importance in this study are given in Table 4. T

assignments were originally made on the basis of a spec

determined at 250 MHz and subsequently confirmed o

basis of 400-MHz two-dimensional spectra (Table 5). But

at this frequency the signals for two pairs of protons, 4 a

6a, and 4P and lop, which appeared as two unresolved si

at 250 MHz, were insufficiently separated for unequi

assignment of the deuterium signals at chemical shifts c

sponding to these positions:

4a (eq) 2.57 ,6a (eq) 2.51; 4P

1.75, 10P (ax) 1.78 ppm. Assignment of the other three

tons of interest, 6P (ax), 1.57; 11-re, 3.75; 1-si, 4.16

did not pose a problem.

On the basis of the proton resonances, the deuterium si

in the spectrum of lupinine derived from (R)-(I-'H)cadav

(Fig. 2A) can be assigned as follows:

6

4.14, H- 11 si (c

sponding to the signal at

6

4.16 ppm in the 'H spectru

2.50, either H-6a (2.51) or H-4a (2.57); 6 1.77, either

(1.75) or H-1OP (1.78 ppm). Similarly, the signals in the

trum of lupinine from (S)-(1-'H)cadaverine (Fig. 2B) ar



GOLEBIEWSKl

AND

SPENSER

2715

to the following protons: 8 1.73, either H-4P (1.75) or H-1OP

(1.78); 1.54, H-6P (1.57 ppm).

These spectral assignments lead to four possible sets of deu-

teriated positions in the sample of lupinine from (R)-(1-'H)-

cadaverine, and to two possible sets in the sample of lupinine

from (S)-(1-'H)cadaverine, a total of eight possib le com bina-

tions for the assignment of the two spectra:

Spectrum 2A:

Spectrum

2B:

lupinine from

lupinine from

R) -

1-'H)cadaverine S) - 1 'H)cadaverine

6 4.14 6 2.50 6 1.77 6 1.54 6 1.73

A num ber of these combinations of assignments can be elim-

inated on the basis of considerations w hich follow from the fact

that the samples of lupinine from (R)- and from

(S)-(l-2H )cadave rine give different 'H nmr spectra and that

incorporation is thus stereospecific.

Firstly, it can be assumed that, since incorporation is stereo-

specific, the and protons at any one carbon atom cannot

both be deuteriated in a given lupinine sample. Thus, assign-

ment 3 for spectrum 2A, in which H-4a and H-4P both carry

deuterium, can be discounted.

Secondly, it can be assumed that since incorporation is

I

stereospecific, the two samples of lupinine derived from the

two enantiomers of (1-'H)cadaverine cannot both bear deu-

terium at the sam e site. Thus, the pair (1 a) for the assign -

ment of the spectra of the lupinine samples derived from (R)-

and (S)-(l-2H)cadaverine, respectively, can

be

discounted,

since assignment 1 and assignment a both have a resonance

assigned to H-4P. Similarly, the pairs (2 b) and (4 b) are

eliminated, since in each pair both assignments have a reso-

nance due to H-1OP.

A further restriction follows from the mode of incorporation

of I3C- and I4C-labelled substrates into cad averine . F rom these

experim ents it was concluded that the two C5 chains of lu -

pinine, C-6, -7, -8, -9 -10 and C -4, -3 , -2, -1, -1 1, ultimately

originate from one and the same C5 precursor. The o bserved

localization of label in all experiments with I4C-labelled racers

was consistent with equimolar distribution of radioactivity be-

tween the two C 5 chains , and the mode of incorpo ration of I3C

from l-13C,l-15N)cadaverine,ssdem onstrated by I3C nmr,

proved this equimolar distribution vide upra). It is quite un-

likely, therefore, that a sample of lupinine formed bio-

synthetically from a specifically deuteriated C5-substrate would

contain two d euteriated sites in one of the two C5 units, while

the other C 5 unit is free of deuterium . Yet this is the situation

presented by assignment b for spectrum 2B. According to this

assignment, both deuterium sites are located on the C5-unit,

C-6, -7, -8, -9 -10, while the other C,-unit, C-4, -3, -2, -1,

-1 1, is devoid of deu terium. In the light of the results of earlier

biosynthetic experiments such a distribution is improbable.

With these restrictions, the assignment of spectrum 2B is

unambiguous. The signals at 1.7 3 and 1.54 ppm can be

assigned to deuterium at H-4P and H-6P, respectively. Two

possible assignments remain for spectrum 2A. While the sig-

nals at 4.14 and 1.77 ppm can be unequivocally assigned to

deuterium at H-11-si and H-lOP, respectively, the signal at S

2.50 ppm is due either to H-6a or to H-4 a.

Notwithstanding this remaining ambiguity in the assignment

of one of the 'H nI tr spectra of the 'H-labelled samp les of

lupinine, the stereochemistry of every step of the biosynthetic

process from cadaverine into lupinine can now be deduced

from the available evidence.

As sho wn by the I3C nmr spectrum (Fig. 1A) of lupinine

derived from (1-I3C,1-I5N)cadaverine Experiment 5), four car-

bon atom s of lupinine, C-6 and C -10 of one of the C5 units and

C-4 and C-11 of the other, are derived from an a-carbo n atom

of cadaverine. As shown further by this spectrum, only on e of

the three C-N bonds of lupinin e, N, C- 6, represents an intact

C-N bond of cadav erine, whereas the three carbon atoms,

C-4, C -10, and C-1 1 of lupinine represent cadaverine carbon

atoms from which a nitrogen atom had been detached in the

course of the biosynthetic process.

Biochemical separation of a primary amino group from a

carbon atom is invariably accompanied by loss of hydrogen

atom from the a-carbon, either by oxidation (catalyzed by a

dehydrogenase, E .C. 1.4.1 ., or by an oxidase, E.C. 1.4 .3 or

by transamination (catalyzed by an aminotransferase, e.g.,

E.C.2.6.1.). These processes are stereospecific, i.e., the

a-hyd rogen atom must be in the correct steric environment in

order to be removed.

If, in the course of lupinine biosynthesis, removal of a hy-

drogen atom from the cadaverine carbon that is destined to

become C-4, C-10, and C-1 1 of lupinine indeed takes place

stereospecifically, then only one or the other, but not both, of

the samples of lupinine derived from the two enantiomers of

(l-2H)cad averine should carry d euterium at these positions.

The carbon atom destined to become C-6 of lupinine, on the

other hand, is shown by the experiment with '3C,15N-labelle

cadaverine to remain attached to the original nitrogen atom

throughout the biosynthetic process. Deuterium should then be

found at C-6 in both samples of lupinine. It follows that the

signal at

8

2.5 0 ppm in the spectrum of the sample of lupinine

derived from (S)-(l-2H )cadave rine (the S-lupinine ) is due to

a 6 proton rather than to a 4 proton.

The stereospecificity of incorporation of deuterium from

(R)- and from (S)-(l-2H)cadaverine into the two proton sites at

C- 6 of lup inine serves as clear evidence that the integrity of the

C-D bond of cadaverine is maintained on route into lupinin e.

Deu terium from (R)-(1-'H)cadaverine enters the re-site (a ) at

C-6, whereas deuterium from (S)-(1-'H)cadaverine enters the

si-site (P).

With the complete assignment of the signals in the 2H nmr

spectra of the deuteriated sam ples of lupinine (Figs. 2A and B ),

the stereochemical course of the biosynthetic steps from cadav-

erine into lupinine becomes clear.

The carbon atoms destined to become C-10 and C-11 of

lupinine are derived, according to Schemes 1Bd and 1B e (and

also lBc), from the aldehyde group of the intermediate

5-aminopentanal (3), a compound that is formed, early in the

biosynthetic sequence, directly from cadaverine by loss of an

amino group. Deuterium is present at C-10 and C-1 1 in the

lupin ine sam ple derive d from (R)-(1-'H)cadaverine ( R-

lupinine ) (Scheme 4) but not in that derived from

(S)-(l-2H)c adave rine ( S-lupinine ) (Scheme 5). It follow s that

removal of the amino group in the course of conversion of

cadaverine 2)

into 5-aminopentanal (3) is accompanied by

stereospecific loss of the si-proton from the a-carbon atom of

cadaverine (step a, Schemes 4 and 5).



2716

CAN. J CHEM.

VOL.

63

1985

attack from

C a f a c e

C D O

entry of H-lrom

- r c ; Fee

h N y l

Ndsintry of n

H ~ , D ~

rCHsi

H r e from C-re f

SCHEME

The biosynthetic route from cadaverine into lupinine

and its stereochemistry: incorporation of (R)-(1-'H)cadaverine.

The observation that a signal due to deuterium at C-4 is

foun d in the spectrum of S-lupinine (Fig . 2B) but not in that

of R-lupinine (Fig. 2A) proves that glutardialdeh yde (5)

(Schem e 1B d) cannot be implicated in the biosynth etic process:

Loss of the amino group of to yield 5 can, in principle, be

accompanied by loss of the si proton or the re proton from the

carbon to nitrogen. If the si proton were lost, then the sample

of S-lupinine should show only one deuterium signal, due to

deuterium at carbon-6 , while the sample of R-lupinine should

show four signals, due to deuterium at carbons 6, lO ,4, and 11.

If the re-proton were lost then, as a consequence of the C2

symmetry of glutardialdeh yde, the sample of S-lupinine

should show signals due to deuterium at carbons 6, 4, and 1 1,

in the ratio 2: 1 : 1, while the sample of R-lupinine should

show signals due to deuterium at carbons 6, 10, 4, and 11, in

the ratio 2: 2: 1 1. This is not observed. Scheme 1Bd is thereby

disproven . The amin o group is thus shed at a later stage of the

biosynthetic process (step c, Schemes 4 and 5). Since deu-

terium at C-4 is preserved in S-lupinine (Scheme 5) and not

in R-lupinine (Scheme 4), it is the re-proton which is lost,

together with the amino group, in this step.

Since the deuterium at C-4 of the S-lupinine is found in the

re position, reduction of the iminium bond (step d, Scheme 5)

must take place by entry of the reducing hydride ion from the

si-face at C-4. Finally, since deuterium at C-11 of the

R-lupinine is found in the si position, reduction of the carbo-

nyl carbon (step e, Sch eme 4), must take place by entry of th e

hydride ion from the re-face. The two experiments with en-

antiotopically deuteriated (l-2H)cadaverin e thus clarify the hid-

den stereoch emistry of fou r steps in the bio synthetic sequence.

The ov ert stereochemistry at C-1 and C-10 of lupinine is deter-

mined in the dimerization step (step b, Schemes 4 and 5) that

leads from A'-piperideine (4) into tetrahydroanabasine (14).

The proposed reaction sequence (Schemes lBe, 4, and 5)

postulates that A'-piperideine serves as an intermediate be-

tween cadaverine and lupinine. The validity of the proposed

sequence and its stereochemistry can be further tested by means

of an experim ent with deuterium -labelled A'-piperidein e (Ex-

attack from

C a f a c e

enlry of H-froni

Ci/e pf face

from C-re or

-

C-si face

SCHEME. The biosynthetic route from cadaverine into lupi

and its stereochemistry: incorporation of (S)-(I-'H)cadaverine.

periment 4). If the conclusions drawn from the experim

with chirally deuteriated cadaverine (Experim ents 1 and 2)

correct, it can be predicted that deuterium from (2-

A'-piperideine must enter at C-10 and at C-11-si of lupin

The 2H nrnr spectrum of a sam ple of lup inine derived f

(2-2H)-A l-piperideine s shown in Fig. 2D . The predictions

fully substantiated.

The final stereoch emical question that was answered in

investigation concerns the prochirality of the decarboxyla

of lysine in L. luteus. The evidence which shows that L-ly

is the precursor of lupinine in

L.

luteus (Experiment 6)

already been discussed (vide supra). Decarboxylation

L-lysine yields cadaverine. In princip le, this decarboxyla

can take place either with net retention or with net inversio

configuration. Decarboxylation of

L-(i.e., (S)-)(2-2H)ly

with net retention of configuratio n yields (S)-(l-2H )cad aver

whereas decarboxylation with net inversion yields

(I-'H)cadaverine. Incorporation into lupinine of the L-comp

ent of ~ ~ - ( 2 - ~ H ) l y s i n eields a sample of lupinine whose

nmr spectrum (Fig. 2C) is identical with that of the lupin

sample obtained when (S)-(l-2H )cadaverine served as the

strate (Fig. 2B).

It follows that, en rou te to lupinine, L-(2 -2H)lys ine s c

verted into (S)-(l-2H)cadaverine, i.e., in the process lead

from L -lysine to cadaverine the carboxyl gro up of ly sin

replaced by a proton w ith net retention of configuration . T

stereochem ical course is consisten t with th e prochirality of

reactions catalyzed by o ther L-amino acid decarbo xylases

(E.C.4.1.1.).

Th e experimental evidence here presented serves as a crit

test of the biogenetic hypotheses of lu pinine biosynthesis

have been proposed. All but one of the hypotheses are

proven on the basis of the results of the six biosy nthetic exp

ments which are here reported. The remaining hypoth

(Scheme 1Be) which is presented in full detail in Schem

and 5 , is entirely consistent with all the experimental evide

This evidence throws light on the steps from lysine into

pinine and on the stereochem istry of several of these step



GOLEBIEWSKI AND SPENSER 2717

xperimental

Extraction of lupinine and sp arteine from Lupinus luteus

The fresh plant m aterial was imm ediately ground in a blendor with

methanol and extracted in a Soxh let apparatus for 8 h. T he extract was

concentrated in vacuo and sulfuric acid (1 M , 1 mL) was added to the

residue. The mixture was extracted with ether (4

x

10 mL) and the

aqueous phase filtered through Celite and extracted with chloroform

(3 10 mL). The aqueous phase was neutralized with solid potassium

carbonate, basified with potassium hydroxide (50% w/v, 2 mL), and

extracted with methylene chloride (4 x 15 mL). The solvent was

evaporated in vacuo and the residue once again taken through an

acidlb ase cycle (sulfuric acid, potassium hydroxide, as above), yield-

ing a basic fraction (25 mg) which contained lupinine (20) and spar-

teine as the major components.

The alk aloid fraction (25 mg) was applied to a silica gel column (10

185 mm, 4 g, 230-400 mesh, BDH). The column was washed, in

turn, with methylene chloride (10 mL), 3% methanol in methylene

chloride/0.880 ammonia, 50 0: 1 (20 mL) and 5% methanol in meth-

ylene chloride/0.880 ammonia, 333 : 1 (40 mL). The lupinine fraction

was eluted with 8% methanol in methylene chloride/0.880 amm onia,

166: 1 (120 m L), the spa rteine fraction with 12% methanol in methyl-

ene chloride/0.880 ammonia, 100 : 1 (80 mL). Yield of crude alka-

loids: lupinine, 10 mg; sparteine, 4.5 mg. Lupinine was dissolved in

hydrochloric acid (0.1 M, 0.7 mL), water and excess of acid were

removed in vacuo, and the residue dried in vacuo. Recrystallization

from a mixture of methanol-acetone gave lupinine hydrochloride, mp

208-209 °C (lit. (23) mp 207-209 °C). A small sample of lupinine was

converted into lupinine methiodide, lit. (23) mp 295-296°C.

Sparteine was dissolved in acetone (2 drops) and a solution of

sulfuric acid in absolute ethanol (I M, 60 FL freshly prepared) was

added. Crystals of the sulfate derivative formed immediately, were

filtered off, and washed with a mixture of acetone and absolute ethanol

(3 , 3 drops), mp 265-266°C (dec.) (lit. (24) mp 264.5-265.5 C).

Radioactive materials

~ ~ - [ 6 - ' ~ C ] L y s i n end ~ - [ 4- ,H ] l~s in e ere obtained from commer-

cial sources (see Table 1).

Administration of radioactive tracers to L. luteus

Plants were grown from seed and used for tracer experiments 6

weeks after germination. The exp eriment was carried out in the growth

chambers (Experiment 6). Tracer solution was administered to 36

plants by wick in a single dose. The plants were allowed to grow 3

days in contact with tracer.

Materials labelled with stable isotopes

(1 13C,1 N)Cadav erine dihy drochloride (24)

5-Phthalimido pentano(1 -'3C,1 -ISN)nitrile 22)

A solution of

1-bromo-4-phthalimidobutane

21) (0.85 g) in di-

methyl sulfoxide (1.6 m L) was added dropwise over 2 min to a stirred

solution of sodium (I3C,l5N)cyanide 99 at.% I3C, 99 at.% I5N, MSD

Isotopes, M ontreal) (0.1 5 g) in aqueous dimethyl sulfoxide (1.6 mL

DM SO, 0 .2 mL H2 0) . The solution was stirred 4 h at 65°C and left

overnight at room temperature. Water (12 mL) was added and the

mixture extracted with benzene (4 5 mL). The benzene extract was

reextracted with water, dried, and evaporated to dryness in vacuo,

yielding crystalline product (680 mg). A small sample was re-

crystallized from absolute ethanol, mp 69-70°C; 'H nmr (CDC1,) 6:

8.13-7.87 (4H, m), 3.83 (2H, t,

J

6 Hz, H-5) 2.65-2.35 (2H, m,

H-4), 2.1-1.9 (4H, m); I3C nmr (CDCI,) 6: 119 .2 (d, J 1 3 ~ l s 16.7

HZ), 36.7 (C-5), 27.6 (C-4), 22.7 (C -3), 16 .6 (d, J 55.2 (HZ, C-2);

ms m z : 230 (6.5%), 188 (31, M CH 2- 13 C'5 ~)) ,60 (100, M

(C3H5- CHN)), 149 (21), 104 (98), 77 (12), 76 (13).

l-N-Acetyl-5-N-phthaloyl-l,5-diamino(l-13~,11 5 ~ )entane (23)

Crude

5-phthalimid0pentano(l-~~~,l- ~~)nitrile

22) (0.67 g) in

acetic anhyd ride (3 mL) was ad ded to a suspension of freshly prepared

Raney n ickel catalyst (0.5 g) in acetic anhydride (7 mL). (The cataly st

had been freshly prepared imm ediately before use by gradual addition

of sodium hydroxid e pellets to a suspension of nickel aluminum alloy

(1 1 w/ w, BDH ) in water, without cooling. This mixture was left for

30 min and heated on the steam bath for 1 h. The solid was then

washed with water, 95% alcohol, absolute alcohol, and acetic anhy-

dride.) Hyd rogenation was carried out at 80-90°C and 1 atm for 3 h.

The mixture was centrifuged, the supernatant solution was decanted,

and the metallic residue washed with acetic anhydride. The solvent

was evaporated in vacuo to yield the acetyl derivative as a solid (yield

0.8 g, m p 134-136 C, after recrystallization from absolute ethanol);

'H nmr (CDCI,) 6: 7.9-7.7 (4H, m), 6.0 (I H, dtd,

J I ~ N H

9.5 Hz,

J C H ~ - I N H

1 .2 H Z , J I ~ C . N H.8 HZ ), 3.67 (2H , t, J 6.6 HZ, H-5), 3.23

(2H, dm, J I ~ ~ . ~ .39 HZ), 1.94 (3H, d , J 12 HZ), 1.8--1.3 (6H, m);

I3C nmr (CDCl,) 6: 170.2 (1 5 ~ C 0 - ), 68.6 (NCO-), 134.1 (C-3',-

4'), 132.3 (C-1',-6'), 123 .3 (C-2',-5'), 39.5 (d,

J l3 C .1 .1 5 ~

10.2 HZ,

C-I ) , 37.7 (C-5), 29.0 (d,

J

33 HZ, C-2), 28.2 (d ,

J

7.1 HZ, C-4), 24.1

(CH,), 23.3 (d, J 8.5 HZ, C-3).

(1 C,

1

~)Ca dave rine dihydrochloride (24)

Hydroch loric acid (6 M , 14 mL) was added to the crude acetyl

derivative (23) and the mixture was stirred for 18 h at 100°C. Crys-

tallization of phthalic acid started on cooling and was complete after

1 h at 0°C. The phthalic acid was removed by centrifugation and the

supernatant solution was extracted with ethyl acetate (3

x

10 mL).

The aqueous layer was evaporated in vacuo. The residue was dried

(0.45 g) and recrystallized from 95% ethanol. After the first crop of

crystals (0.17 g), mp 255-257 C, was filtered off, the mother liquors

were chromatographed on an ion exchange column (Dowex 50-X4,

H + form, 2.1 mequiv./mL, 1.3

x

cm). The column was washed

with water (20 mL) and the produ ct was eluted with hyd rochloric acid

(1.5 M, 200 mL), yielding a further crop (0.13 g) of

(1-'3C,I-15N)cadaverine ihydrochloride. Total yield 57% (relative to

NaI3Cl5N); H nmr (DzO) 6: 3.76 (I H, t, J 7.2 Hz), 2.97 (2H , t,

J

7.2

Hz) , 2.18 ( lH , t ,

J

7. 2 Hz), 1.45- 1.75 (6H, m); J13C.H.I144 HZ; he

signals at 6 3.76 and 2.18 ppm are doublets of triplets, due to I3C,H-1

coupling of the 1-I3CH2 roup. The signal at 6 2.97 ppm is due to the

5-CH2 group; I3C nmr (D,O) 6: 40.0 (d,

J I ~ ~ . ~ . ~ ~ N

.4 HZ), 27.0 (s, d,

J13c.2 13c.l 35 .4 HZ,), 23.4 (s, C-3).

D L - ( Z - ' H ) L ~ S ~ ~ ~onohyd rochloride was prepared (13) in two steps

from diethyl 2-acetamidomalonate and

1-bromo-4-phthalimidobutan

(21). Condensation yielded diethyl 2-acetamido-2- 4-phthalimido

butyl)malonate, whose hydrolysis in D CI/ D2 0 yielded the desired

product. D~-(2 -'H)L ~sin e onohydrochloride: IH nmr (D2 0) 6: 3.7

(ca. 0.05 H, H-2), 3.05 (2H, t,

J

7.2 Hz, H-6), 1.3-2.0 (m, 6H).

( 2 - ' ~ ) - A ' - ~ i ~ e r i d e i n end (S)-(+)-(I -'H)cadaverine hydrochlo ride

were prepared from the above DL-(2-'H)lysine (vide infra).

(2-'H)-A1-Piperideine (cf. ref. 13)

Freshly crystallized N-bromosuccinimide (0.32 g) was added to a

so lu t ion of D ~- ( 2- ' ~ ) l~s ineonohydrochloride (0.64 g) in water (90

mL) and the solution was heated at 50°C in a nitrogen atmosphere on

a rotary evaporator under mild suction until colorless. Three more

portions of N-bromo succinimide (3 0.3 2 g) were added and reaction

continued in the same way. T he total reaction time was 55 min. The

solution was used for feeding without further purification.

A small portion of the solution was co ncentrated and the 'H and 'H

nmr spectra of the mixture were determined; 'H nmr 6: 7.5 (s), 6. 9 (s),

6.3 (s) , 4.5 (H 20 ), 3.5 (bm), 3.2 (s) , 3.0 (m), 2.6 (s) ,

(CH2-CO),NH, succinimide);

'H

nmr 6: 8.7 (rel. area I ), 4. 9 (rel.

area 2.4), 4.5 (DHO, natural abund ance) ppm . When the solution was

evaporated and redissolved in water, the 'H nmr spectrum changed

considerably: 'H

n r

6: 8.3 (rel. area 5.7), 8.1 (1.3), 6.7 (1.0), 4.9

(1.8), 3 .8 (4 .3). When the solution was basified (K2C0 3) and ex-

tracted six times with chloroform , the chloroform evaporated, and the

residue redissolved in water, a further spectral change was observed:

'H nmr 6: 7.9 (rel. area 0. I), 3. 1 (0.9).

(R)-(-)- and (S)-(+)-(l-ZH)Cadaverineydrochloride

(R)-(-)-(l- ~)Cadaverine

hydrochloride was prepared by decar-

he

value, 18.4 Hz, for

J13c.z.13c.l

that was reported in our pre-

liminary com municatio n (I 1) was in error. We thank D r.

E

Leete for

pointing out this mistake.



2718 CAN.

J

CHEM.

VOL.

63 1985

boxylation of the L-com ponent of DL-lysine in deuterium oxid e, cata-

lyzed by L-lysine decarboxylase (13). 'H nmr ( D2 0) : 2.95 (3.01 H,

t, J 7.2 Hz, H-1,5)

1.45-1.75 (m, 6H); ca. 1 nondeuteriated

product; ms mlz: 89 (M'

I ,

9 ) 88 (M ', 100 +NHsCHD (CH2)4),

87 (M' 1, 14).

s)- +)- l-2~)~adaverine ydrochloride was obtained (13) by

decarboxylation, catalyzed by L-lysine decarboxylase, of the

L -co mp on ent of ~ ~ - ( 2 - ~ H ) l ~ s i n e95 at. 2-'H). (S)-(+)-(1-2H )-

Cadaverine dihydrochloride (93 at. L2H): 'H nmr (D 20 ) similar to

that of

(R)-(-)-(1- H)cadaverine

hydrochloride, above; ms mlz: 89

(M' 1 , 10.6 ), 88 (M ', loo ), 87 (M' 1, 16), 7 non-deuteriated

product, based on computer assisted peak height analysis.

Administration of enriched substrates to L. luteus

The plants used in these experiments were grown from seed and

were used in feeding experiments 3-6 weeks after germination. The

feeding experiments were carried out in the greenhouse (summer

months, Experiments 3 , 5) or in the growth chambers (winter mon ths,

Experiments 1, 2, 4). Tracer solution was administered by wick over

a period of 5-6 days; the plants were then grown for a further period

of 3-4 days before being harvested.

Acknowledgements

We are grateful to Professor Dr. M. W iewiorow ski, Univer-

sity of Pozn an, for a gift of seeds of

Lupinus luteus

o Thelma

Leech, Greenhouse Supervisor, McMaster University, for

providing facilities for our experiments, and to

J.

Ian A.

Thompson and Brian G. Sayer, Department of Chemistry, for

recording nmr spectra. We are greatly indebted to Dr. R. E.

Lenkinsk i, South Western Ontario NMR Facility, Un iversity of

Guelph , for determinin g COSY and 2-D resolved spectra.

This investigation was supported by a grant from the Natural

Sciences and Engineering Research Council of Canada.

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BIOSINTESIS LUPINin