Biosynthesis of the lupine alkaloids. I.

Lupinine
W. MAREK
GOLEBIEWSKI'
AND IAND. SPENSER

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Department of Chemistry, McMaster University, Hamilton, Ont., Canada L8S 4M1

Received February 5, 1985

GOLEBIEWSKI
and IAND. SPENSER.
Can. J. Chem. 63, 2707 (1985).
W. MAREK
The 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",
was determined by I3C nmr spectroscopy; lupinine is generated from two
cadaverine-derived C5-units by a route which excludes a "dimeric" intermediate with C2, symmetry. The mode of incorporation
of 'H from L-(2-'H)lysine, from (R)- and (S)-(1-'H)cadaverine, and from (2-2H)-A1-piperideineinto lupinine was determined
by 'H nmr spectroscopy. The results corroborate the conclusions from the I3C,"N experiment and they establish the stereochemistry of six of the steps in the biosynthetic conversion of L-lysine into lupinine.
W. MAREK
GOLEBIEWSKI
et IAND. SPENSER.
Can. J . Chem. 63, 2707 (1985).
Faisant appel a la spectroscopic rmn du I3C et utilisant de la cadavtrine doublement marquee d'une faqon intramoltculaire
par du ' 5 et~ du 13Csur le carbone voisin du ' 5 (liaison
~
I3C,I5N marquee), on a ttudie le mode d'incorporation de la cadavtrine
2 la lupinine. Les r6sultats obtenus suggkrent que la lupinine se forme partir de deux unites en C5 dCrivtes de la cadavtrine
par le biais d'une voie reactionnelle qui exclut la formation d'un intermtdiaire "dimkre" posstdant une symetrie C2,. Faisant
appel a la rmn du 'H, on a determint le mode d'incorporation du 'H de la L-('H-2) lysine, des ('H-2) cadavtrines-(R) et -(S)
et de la (ZH-2)-A1-pip~ridine
a la 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 21 la transformation
biosynthttique de la L-lys&e en lupinhe.
[Traduit par le journal]

richment was detected at four pairs of neighbouring carbon

Introduction
atoms of lupinine, C- 1,-2, C-2,-3, C-7,-8, and C-8,-9. This
Speculations concerning the biosynthesis of the lupine alkalabelling pattern serves as evidence that the carbon skeleton of
loids go back more than 50 years. In 1931 Schopf suggested
lupinine is generated from two lysine-derived C5-units, via an
(1, 2) that the ring skeleton of lupinine (20) might be generated
intermediate with CZvsymmetry (e.g., either cadaverine (2) or
by condensation of two lysine (1) derived fragments, 5-aminothe iminodialdehyde (7), or both). However, since the distribupentanal (3) (e
A'-piperideine (4) (3, 4)) and glutardialdehyde
tion of label observed in all these experiments is that which is
(S), followed by reduction (Scheme 1Bd). Schopf later abanpredicted by every one of the five hypothetical routes (Schemes
doned this notion in favour of the idea (3) that the im1Ba-1Be) that have been advanced to account for the deriinodialdehyde (7) might be the intermediate between lysine and
vation of lupinine from lysine, available tracer evidence cannot
lupinine (Schemes 1Ba and 1Bb). This intermediate was
serve to discriminate among the biogenetic proposals.
favoured also by Robert Robinson (4). In a further variation of
Nor is it certain that cadaverine (2) is an intermediate
this theme it was suggested (5, 6) that lupinine arose by
between lysine (1) and lupinine (20): Neither the observation
rearrangement of tetrahydroanabasine (14), a dimer of
that incorporation of the two substrates, DL-[2-'4C]lysineand
A'-piperideine (4) (3, 5) (Scheme 1Be).
[l-'4C]cadaverine, leads to the same distribution of label within
The first tracer experiments to test these ideas were canied
lupinine, nor the labelling pattern from DL-(4,5-"C,)lysine conout twenty-five years ago. Radioactivity from ~~-[2-'~C]lysine stitute conclusive evidence for the obligatory intermediacy of
(7) and from its decarboxylation product, [l-'4C]cadaverine
cadaverine. Other interpretations are possible which account
(6, 8), was indeed incorporated into lupinine. With either subfor such a distribution of label (cf. ref. 10).
strate, approximately one quarter of the total activity of the
The tracer experiments with doubly I3C,"N- and with
alkaloid was present within the hydroxymethyl carbon, C-11,
'H-labelled substrates which are here reported serve as critical
of lupinine. Also, the two methylene carbon atoms, C-4 and
tests of the hypotheses that have been advanced to account for
C-6, together, accounted for one half of the total alkaloid
the origin of lupinine. 'The results, which controvert all but one
activity (7, 8). If the assumption is made that this activity is
of the hypothetical sequences, provide support for the tetradistributed equally over C-4 and C-6, and that each of these two
hydroanabasine route (Scheme 1Be). Furthermore, the results
C atoms contains one quarter of the label,' it follows that four
of the tracer experiments with 2H-labelled substrates clarify
sites within lupinine, C- 11, C-4, C-6, and, presumably, C- 10,
stereochemical aspects of this route. Preliminary accounts of
each account for one quarter of the label of the intact alkaloid
parts of this work have appeared (1 1, 12).
(20). This conclusion was substantiated by the results of a
Methods and results
recent feeding experiment (9) with ~~-(4,5-'~C~)lysine.
EnIn six tracer experiments, compounds, specifically labelled
with stable (Experiments 1-5) and with radioactive isotopes
' On leave from the Department of Chemistry, University of War(Experiment 6), were administered by the wick method to
saw, 02-093 Warsaw, Poland.
intact plants of Lupinus luteus (yellow lupine) over a period of
'Another possible interpretation of these results is that one of the
3-6 days. Details of the experiments are summarized in Table
two centres, C-4 or C-6, accounts for 50% of the activity of the intact
lupinine, while the other centre is free of activity.
1.
-

CAN. J. CHEM. VOL. 63, 1985

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2708

"

CHO

FHO

CHO

'C+> - -3 " c;3

CHO

-cf

CHO

[?>

CHO

CHO

NH2
11

CHO

cg

trom

Bc

- QC)FHO

CHO

CHO

CHO

CHO

CHO

J.
c g

ch

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

labelled sample of putrescine (10).
The samples of ~~-(2-~H)lysine
(Experiment 3), (2-2H)-A'piperideine (Experiment 4), (S)-(+)-(l-2H)cadaverine dihydrochloride (Experiment 2), and (R)-(-)-(l-2H)cadaverine dihydrochloride (Experiment 1) were prepared by chemical and
enzymic methods, respectively, which we have previously
described (13). The samples of L-[4-3H]lysine monohydrochloride and ~~-[6-'~C]lysine
monohydrochloride (Experiment
6) were commercial products.
When the administration of tracer was complete, the plants
to which labelled substrates had been administered were macerated with cold methanol in a Waring blendor and the alkaloids
were isolated by methanol extraction. Lupinine and sparteine
were separated by chromatography on silica gel, lupinine was
converted into the hydrochloride, sparteine into the sulfate.
The present paper presents a discussion of the results obtained with lupinine. The sparteine results will be dealt with in
the second paper of this series.
Lupinine hydrochloride consists of a 1.7: 1 mixture of the
trans and cis ring fused stereoisomers, as shown by the natural
abundance "C nmr spectrum (Fig. 1B). The "C nmr spectrum
(Fig. 1A) of the sample of lupinine hydrochloride from Experiin admixture with
ment 5, in which (~-'~C,'~N)cadaverine
[l-14C]cadaverine had been administered (Experiment 5),
shows that four of the ten C-atoms, C-4, -6, -10, and -1 1, were
enriched in I3C, to an equal extent. The enrichment, above
natural abundance, calculated for each of these four carbon
atoms (Tables 2 and 3) is in agreement with the specific incorporation of I4C (Table 1).
The signals in the proton noise decoupled "C nmr spectrum
due to two of the "C-enriched carbon atoms, C- 10 and C- 11,
appear as singlets, the signals due to the two others, C-6 and
C-4, as multiplets (Fig. 1A). The coupling constants of the
doublet component of the multiplets are different from one
trans isomer 4.1 Hz, cis isomer 4.1 Hz;
another (J13~~,15,:
trans isomer 7.0 Hz, cis isomer 6.4 Hz; isotope shift
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 superimposed on a singlet. The doublet (87 2 11%) (I3C-6,"N) in
the signals due to C-6 (trans, 6 55.7, cis, 6 52.9 ppm) is much
more intense than the singlet (13 2%) (I3C-6,I4N)(Tables 2
and 3), so that the latter is not apparent in the unexpanded
spectrum. In the signals due to C-4 (trans, 6 54.8, cis, 6 44.6
ppm) the doublet (36 + 5% of signal area, Tables 2 and 3)
(I3C-4,"N) appears as a shoulder straddling the singlet (64 2
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 magnitude (Tables 2 and 3).
Lupinine hydrochloride obtained from the feeding experiysine
6) was conment with [ ~ - 4 - ~ H / ~ ~ - 6 - ' ~ C ] l(Experiment
verted into the methiodide and crystallized to constant radioactivity and constant tritium/14C ratio. The 'H/I4C ratio of the
purified lupinine methiodide was 8.4 + 0.1, approximately
double that of the intermolecularly doubly 'H/I4C labelled
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 (free
base) from the experiments with deuterium labelled substrates
(Experiments 1-4, Table 1) are shown in Fig. 2. The deuterium chemical shifts observed in the spectra of these four

CHO

&Jsds&)
CHO
12

17

[Hp

lgL

SCHEME 1. Five alternative hypothetical schemes of the biogenesis

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 prepared in three steps from sodium ("C,"N)cyanide and
1-bromo-4-phthalimidobutane
by the reaction sequence shown

GOLEBIEWSKI AND SPENSER

TABLE1. Experiments with Lupinus luteus

(a) Stable isotopes
Plant material

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Expt .
no.

Substrate

Wt. fed
(mg)

Enrichment
(at. %)

Lupinine

Sparteine

Fresh
weight
(g)

No. of
plants

Weight
(mg)

Specific
incorporation
per C5 unit (%)

Weight
(mg)

Specific
incorporation
per C5 unit (%)

9.5

3.0

(R)-( l -2H)Cadaverine
dihydrochloridea
(March, 1983)

150

99d

300

40

(s)-(1 -2H)Cadaverine
dihydrochloride"
(Dec. 1982)

116

93d

21 1

51

13

2.3

~~-(2-'H)Lysinemonohydrochloride"
(July, 1983)

500

95d

41'

100

2.3

(2-2~)-A'-Piperideine"
(Nov. 1983)

300

9Sd

247

100

5.4

( ~ - ' ~ C , l - ' ~ ~ ) C a d a v e r i n e140

dihydrochloride"

47

99.99

28
105

[I -'4C]Cadaverine
dihydrochlorideb
(Aug. 1982)

50

10

(b) Radioactive isotopes

Plant material
Nominal activity
Expt .
no.
6

Total
(mCi)

Specific
(mCi/mmol)

~~-[6-'~C]~ysine
monohydrochloride'

0.20

45

L-[(Rs)-4-3H]Lysine
monohydrochloride'
(Dec. 1983)

1.0

Substrate

3H/'4C
ratio

4.1

+ O.lh

Fresh
weight
(g)

No. of
plants

Lupinine
(mg)

'H/14C
ratio

Sparteine
sulfate
(mg)

104

36

8.4 2 0.1'

3~/'4C
ratio

6.8

+ 0.2

22x10~

"See Experimental.
'New England Nuclear (nominal total and specific activities, 25 pCi, 105 mCi per mmol, respectively).
'CEA France.
dMeasured by means of computer assisted peak height analysis.
'This experiment was done 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 140 mg (I-"C, I-15N)cadaverinedihydrochloride,
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 mL) containing the doubly labelled lysine, which was administered to the plants, was set aside and from it lysine
hydrochloride was re-isolated by carrier dilution and purified by ion exchange chromatography and crystallization to constant 3H/14Cratio.
'Lupinine was converted into the methiodide which was crystallized to constant 'H/I4C ratio.

NaCN
"20
DMSO

SCHEME2. Synthesis of (1-I3C,1-I5N)cadaverine.

H9
Ac20

CAN. 1. CHEM. VOL. 63, 1985

2710

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

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Chemical
Carbon
shiftb
(ppm)
atom

Type of
signal

Natural abundance
13
C, normalized
peak areac
(ANA)

Enriched in
I3C, normalized
Percent enrichment
0.94
above natural
peak areac
AE/ANA~ abundancee,'
(AE)

Relative percent excess

13
C above natural
abundance at
individual C atoms

"The sample in D20was 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, 2 10% for composite signals.
dThe ratio AE/ANAis 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)X 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 at.%
I3C is the average enrichment at each terminal position of cadaverine.

TABLE
3. Incorporation of (1-I3C, 1-ISN)cadaverine into cis-lupinine hydrochloridea
Chemical
Carbon
shiftb
atom
(ppm)

Type of
signal

Natural abundance
13
C , normalized
peak areac
(ANA)

Enriched in
I3C, normalized
peak areac
(AE)

1.08

Percent enrichment
above natural
abundancee.'

Relative percent excess

13
C above natural
abundance at
individual C atoms

"See footnote a, Table 2.

bSee footnote b, Table 2.
'Peak areas are normalized relative to C-8 = 100%. Estimated standard deviation 8 % for singlet signals, k 10% for composite signals.
dThe ratio AE/ANA
is normalized so that the average value for carbon atoms 1, 2, 3, 7, 8, and 9 equals 1.O.
'((1.08 A/ANA)- 1) 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)lysine(Experiment 3), were identical, each showing two signals, at i3 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.

Discussion
The results of the tracer experiments with 14C-labelledlysine
(7) and cadaverine (6, 8) and with ~ ~ - ( 4 , 5 - ' ~ C ~ ) l y(9)
s i ndo
e
not discriminate among the various biogenetic hypotheses that
have been advanced to account for the biosynthesis of lupinine
(20) (Scheme lA, B, C). The first task in a reexamination of
the problem was to devise experiments capable of narrowing
down the possibilities.
All the hypotheses that have been advanced (Scheme 1)
share a common initial phase, decarboxylation of lysine (1)to
cadaverine (2), followed by oxidative deamination of the latter

GOLEBIEWSKI AND SPENSER

TRANS

CIS

LUPlNlNE HCl
lrom

1-'3C,1?5~CADAVERINE

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Re. 1. I3C nmr spectra (20.15 MHz) of lupinine 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~-enriched
sample of lupinine hydrochloride (10 mg in 10 pL 'H20) derived from (l-'3C,1-15N)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 pL 'H20).
TABLE4. Incorporation of deuteriated substrates into lupinine. 'H nmr analysis
-

'H nmr chemical shiftsb (ppm)

Hydrogen
atom
413 si (eq)
4Pre(ax)
6are(eq)
6P si (ax)
lop R (ax)
11 re
11 si

'H nmr
chemical shiftsa
(PP~)
2.57
1.75
2.51
1.57
1.78
3.75
4.16

Experiment 1
lupinine from
R-(1-'H)cadaverine

Experiment 2
lupinine from
S-(1-'H)cadaverine

(2)

(3)

Experiment 3
Experiment 4
lupinine from
lupinine from
~L-(2-'~)lysine (2-'H)-A'-piperideine

2.50
1.77
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-16at 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 aminoaldehyde in equilibrium with

its intramolecular Schiff base, A'-piperideine (4) (Scheme 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) (S A'-piperideine (4)) to lupinal (19)
(Schemes lB, 1C).
What was required for a critical examination of the biogenetic process was a method which could distinguish the different paths between Saminopentanal (3), the last early intermediate 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
capable 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, can have one of three different
outcomes. Two of the routes (Schemes 1Bd and 1Be) lead from
3 into 19, via the bicyclic compound (18) and other intermediates, 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 intact C-N bond of 3 is destined to become the C-4,N
bond of lupinine (Scheme 3B). It can be predicted that each of
these routes leads to a sample of lupinine in which only one of
the three C-N bonds of the product is derived from the intact
C-N bond of 3.
The other two routes (Schemes 1Ba and 1Bb) differ from the
above in that they give rise to a sample of lupinine which

2712

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CHEM. VOL. 63,

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LUPlNlNE
from

1985

TABLE5. The I3C and 'Hnmr spectra of trans-lupinine (free base)

'H nmrb
chemical shifts (ppm)'

Carbon

I3C nmf
chemical shifts
( P P ~

axial protons

equatorial protons

C-1 1

65.8

3.75(re)

4.16(si)

"Recorded in C6D6 (128.4 ppm) at 100.7 MHz in the Fourier mode on a

Bmker WH 400 spectrometer.
Recorded in C6D6at 400 MHz in the Fourier mode on a Bmker WH 400
spectrometer.
'Reference 19.
dCf. references 14- 17.

FIG. 2. 'H nmr spectra (38.40 MHz) of deuterium-enriched samples of lupinine derived from deuterium labelled substrates (Experiments 1-4). The spectra were recorded in the Fourier mode on a
Bruker WM 250 spectrometer, in 10-mm tubes, with natural abundance C a H 5 (7.19 ppm) as internal reference. The acquisition time
was 2.048 s (see Table 4). 2A. Lupinine from (R)-(I-'~)cadaverine
(Experiment 1). 2B. Lupinine from (S)-(1 -2H)cadaverine (Experiment
2). 2C. Lupinine from the L-component of DL-(2-'H)lysine (Experiment 3). 2D. Lupinine from (2-2H)-A'-piperideine (Experiment 4).

contains an equimolar mixture of two species, one of which

contains 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). This compound contains two C-N bonds, but only one of these is derived intact
from 3. However, the compound possesses CZvsymmetry. Ring
closure of 7 by an intramolecular Mannich reaction to yield
lupinal(19) can take place in two equivalent ways. The 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. The product will then comprise an equimolar mixture of two chemically
indistinguishable species, which differ solely in the origin 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, intramolecularly doubly I3C,lSN labelled (NH2-CH2-CH2CH~-CH~-'~CH~-'~NH~), was employed to probe the in-

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

5) (1 1). A similar experiment, with essentially identical results
and conclusions, was reported in an independent investigation
(14). Entry of this bond-labelled cadaverine, via 5-aminopentanal, by route Bd or Be (Scheme l), should yield a single
species of intramolecularly I3C,lSNdoubly labelled product in
which the I3C,l5N moiety is located at the C-6,N position of
lupinine. Entry by Route Bc (Scheme 1) should similarly yield
lupinine enriched at C-4,N. Entry via the nondissymmetric
intermediate 7 (Routes Ba, Bb, Scheme 1) must yield an equimolar mixture of the above two enriched species (Scheme 3A).
Non-equimolar enrichment at the two sites, C-4,N and C-6,N,
is not consistent with the intermediacy of a "dimeric" compound with Cz, symmetry, such as 7.
The proton noise decoupled I3C nmr spectrum of the sample
of lupinine hydrochloride (a mixture of the trans and the cis
isomers, in the ratio 1.7: 1) isolated from plants to which
(1-I3C,1-15N)cadaverine dihydrochloride had been administered in admixture with [l-'4C]cadaverine dihydrochloride
(Experiment 5, Table 1) is shown in Fig. 1A.
Lupinine hydrochloride rather than the free base was chosen
as the compound for I3C nmr analysis since, even though the
salt consists of a mixture of the trans and cis ring fused isomers, in the I3C nmr spectrum of the cis isomer in D 2 0 the
signals due to C-4, -6, -10, and -1 1, the carbon atoms crucial
for the interpretation of the biosynthetic experiment, are well
resolved (15) (A6 C-4,-6: 8.3; C-10,-1 1: 2.4 ppm) (Table 3).
The free base, on the other hand, is trans ring fused, and the
corresponding signals in the spectrum of the free base show
poor resolution (A6 C-4,-6: 0 (15.1 MHz (15, 16), 5.0 MHz
(17)), 0.1 (22.5 MHz (18)) in CDC13; 0.1 (50.3 MHz (14)) in
C a 6 ; A6 C-10,-11: 0 (16), 0.4 (15) (15.1 MHz), 0.3 (5.0 MHz
(17)), 0.9 (22.5 MHz (18)) in CDC13; 0.3 (50.3 MHz (14)) in
ca6).
Spectral assignments of the crucial carbon atoms, C-4, -6,
-10, and -11, were made as follows: The natural abundance
proton noise decoupled I3C spectrum of lupinine hydrochloride
(Fig. 1B) shows two sets of 10 signals, corresponding to two
stereoisomers of lupinine hydrochloride that differ in the
stereochemistry of the ring fusion. Since the two sets of signals
show different signal areas, the two isomers are present in

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GOLEBIEWSKl AND SPENSER

Route
1B.

3C
via
Routes
106
or 10,

SCHEME
3. Incorporation of (l-'3C,1-1SN)cadaverine
into 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 Bd or Be, Scheme 1. Left columns: I3C,l5Nbond-labelled
species. Central columns: Doubly 13C-labelledspecies. Right columns: Singly '3C-labelledspecies. = I3C; W = 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 cis
fused salt (15). The signal set of lower intensity is thus due to
the cis 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 confirmed 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 6 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,l5Nenriched 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; C5unit 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 X
100 = 30% of cadaverine per C5unit, a value which is in good
agreement with that obtained from I4C measurements ((3.0 X
107)/(5.3 X lo7)) X 100 X 112 = 28%, Table 1.

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2714

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 multiplets 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 incorporation, 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 approximately 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 conversion into lupinine. The intensity of the I3C-4,I5Ndoublet is
fully accounted for by intermolecular I3C,l5Ncoupling between
two monomer units, derived from this highly enriched cadaverine, 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 implicated in lupinine biosynthesis. The biogenetic proposals shown
in Schemes 1Ba and 1Bb are thus eliminated.
Furthermore, since the intact l3C-I5N unit of the administered 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, labelled 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 experiment 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'-piperideine (Experiment 4) that fulfils this condition will be discussed 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 glutardialdehyde (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 established

whether L-(i.e., (S)-)lysine or D-(i.e., (R)-)lysine serves as the
substrate. Secondly, it must be established whether the decarboxylation process which converts this lysine into cadaverine
takes place with net retention of configuration or with net
inversion of configuration. We have answered both these
questions.
The result of the experiment with intermolecularly doubly
labelled [ ~ - ~ H / ~ ~ - ' ~ C ] l(Experiment
ysine
6) demonstrates that
L-lysine, rather than D-lysineor DL-lysine,serves as the precursor of lupinine in L. luteus. The 3H/14Cratio of the doubly
labelled lysine that was administered to the plants was 4.1 2
0.1. The 3H/14Cratio of the lupinine methiodide that was
obtained was 8.4 2 0.1 (Table 1). From these results it can be
calculated that, within experimental error, lupinine is derived
entirely (102 2 3%) from L-lysine (% of product derived from
~ C of
L-substrate = 50 (3H/14Cratio of p r ~ d u c t ) / ( ~ H / ' ratio
substrate)) (20).
Demonstration that the decarboxylation of L-lysineto cadaverine takes place with net retention of configuration comes
from two of the experiments with deuteriated substrates.
These experiments (Experiments 1-4), which solved not
only this stereochemical problem, but which also answered all
other stereochemical questions concerning the biosynthetic
steps of the route from lysine into lupinine that involve transformations at those carbon atoms of lupinine which originate
from the terminal carbon atoms of cadaverine, will now be
discussed.
In the first two of these experiments, samples of cadaverine,
chirally deuteriated at C- I, were used as substrates. The samples of lupinine obtained from these experiments were examined by 'H n'mr. The 'H nmr spectra are shown in Figs. 2A and
2B. Chemical shifts were assigned by comparison with the
corresponding 'H nmr chemical shifts (Table 4).
Unambiguous assignment of the resonances due to the 19
protons of lupinine was not possible with one-dimensional nmr
spectroscopy, even at 400 MHz, because of substantial overlap
of some of the signals. Homo- and heteroscalar correlated
two-dimensional 'H and 'H,I3C nmr spectroscopy (COSY) and
J-resolved 2D 'H nmr spectroscopy were employed for a complete assignment of the spectrum. Assignment of the downfield
signals, due to the 11-re and 11-si protons, had been reported
(21). A full discussion of the 'H nmr spectrum of lupinine will
appear elsewhere (19). Assignments of the signals due to the
protons of importance in this study are given in Table 4. These
assignments were originally made on the basis of a spectrum
determined at 250 MHz and subsequently confirmed on the
basis of 400-MHz two-dimensional spectra (Table 5). But even
at this frequency the signals for two pairs of protons, 4 a and
6a, and 4P and lop, which appeared as two unresolved signals
at 250 MHz, were insufficiently separated for unequivocal
assignment of the deuterium signals at chemical shifts corresponding to these positions: 4 a (eq) 2.57,6a (eq) 2.51; 4P (ax)
1.75, 10P (ax) 1.78 ppm. Assignment of the other three protons of interest, 6P (ax), 1.57; 11-re, 3.75; I 1-si, 4.16 ppm,
did not pose a problem.
On the basis of the proton resonances, the deuterium signals
in the spectrum of lupinine derived from (R)-(I-'H)cadaverine
(Fig. 2A) can be assigned as follows: 6 4.14, H- 11-si (corresponding to the signal at 6 4.16 ppm in the 'H spectrum); 6
2.50, either H-6a (2.51) or H-4a (2.57); 6 1.77, either H-4P
(1.75) or H-1OP (1.78 ppm). Similarly, the signals in the spectrum of lupinine from (S)-(1-'H)cadaverine (Fig. 2B) are due

GOLEBIEWSKl AND SPENSER

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 deuteriated 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 possible combinations for the assignment of the two spectra:

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Spectrum 2A:
lupinine from
(R)-(1-'H)cadaverine
6 4.14

'
/

6 2.50

6 1.77

Spectrum 2B:
lupinine from
(S)-(1-'H)cadaverine
6 1.54

6 1.73

A number of these combinations of assignments can be eliminated on the basis of considerations which follow from the fact
that the samples of lupinine from (R)- and from
(S)-(l-2H)cadaverine give different 'H nmr spectra and that
incorporation is thus stereospecific.
Firstly, it can be assumed that, since incorporation is stereospecific, the a and P protons at any one carbon atom cannot
both be deuteriated in a given lupinine sample. Thus, assignment 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
stereospecific, the two samples of lupinine derived from the
two enantiomers of (1-'H)cadaverine cannot both bear deuterium at the same site. Thus, the pair (1 + a) for the assignment 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 resonance due to H-1OP.
A further restriction follows from the mode of incorporation
of I3C- and I4C-labelled substrates into cadaverine. From these
experiments it was concluded that the two C5 chains of lupinine, C-6, -7, -8, -9, -10 and C-4, -3, -2, -1, -1 1, ultimately
originate from one and the same C5 precursor. The observed
localization of label in all experiments with I4C-labelledtracers
was consistent with equimolar distribution of radioactivity between the two C5 chains, and the mode of incorporation of I3C
from (l-13C,l-15N)cadaverine,
assdemonstrated by I3C nmr,
proved this equimolar distribution (vide supra). It is quite unlikely, therefore, that a sample of lupinine formed biosynthetically from a specifically deuteriated C5-substrate would
contain two deuteriated sites in one of the two C5 units, while
the other C5 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 deuterium. 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 6 1.73 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 signals at S 4.14 and 1.77 ppm can be unequivocally assigned to

2715

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-4a.
Notwithstanding this remaining ambiguity in the assignment
of one of the 'H nItr spectra of the 'H-labelled samples of
lupinine, the stereochemistry of every step of the biosynthetic
process from cadaverine into lupinine can now be deduced
from the available evidence.
As shown by the I3C nmr spectrum (Fig. 1A) of lupinine
derived from (1-I3C,1-I5N)cadaverine(Experiment 5), four carbon atoms 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-carbon atom
of cadaverine. As shown further by this spectrum, only one of
the three C-N bonds of lupinine, N,C-6, represents an intact
C-N bond of cadaverine, 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-hydrogen atom must be in the "correct" steric environment in
order to be removed.
If, in the course of lupinine biosynthesis, removal of a hydrogen 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)cadaverine should carry deuterium 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-labelled
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 2.50 ppm in the spectrum of the sample of lupinine
derived from (S)-(l-2H)cadaverine (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 lupinine serves as clear evidence that the integrity of the
C-D bond of cadaverine is maintained on route into lupinine.
Deuterium 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 samples of lupinine (Figs. 2A and B),
the stereochemical course of the biosynthetic steps from cadaverine into lupinine becomes clear.
The carbon atoms destined to become C-10 and C-11 of
lupinine are derived, according to Schemes 1Bd and 1Be (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
lupinine sample derived from (R)-(1-'H)cadaverine ("Rlupinine") (Scheme 4) but not in that derived from
(S)-(l-2H)cadaverine ("S-lupinine") (Scheme 5). It follows 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

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CDO

attack from
C a f a c e

entry of H - l r o m
c-rc

;:Fee

.,

e n l r y of H-froni
Ci/e pf%face

hN.yl
LNdsi
entry of

Hre

n H ~ , HrCHsi
D ~
Pf

from C-re

f r o m C-re or
&-

C-si face

SCHEME
4. The biosynthetic route from cadaverine into lupinine
and its stereochemistry: incorporation of (R)-(1-'H)cadaverine.

SCHEME
5. The biosynthetic route from cadaverine into lupinine
and its stereochemistry: incorporation of (S)-(I-'H)cadaverine.

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

found in the spectrum of "S-lupinine" (Fig. 2B) but not in that
of "R-lupinine" (Fig. 2A) proves that glutardialdehyde (5)
(Scheme 1Bd) cannot be implicated in the biosynthetic process:
Loss of the amino group of 3 to yield 5 can, in principle, be
accompanied by loss of the si proton or the re proton from the
carbon a 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 glutardialdehyde, the sample of "S-lupinine"
should show signals due to deuterium at carbons 6, 4, and 11,
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 amino group is thus shed at a later stage of the
biosynthetic process (step c, Schemes 4 and 5). Since deuterium 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 carbonyl carbon (step e, Scheme 4), must take place by entry of the
hydride ion from the re-face. The two experiments with enantiotopically deuteriated (l-2H)cadaverine thus clarify the hidden stereochemistry of four steps in the biosynthetic sequence.
The overt stereochemistry at C-1 and C-10 of lupinine is determined 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 between cadaverine and lupinine. The validity of the proposed
sequence and its stereochemistrycan be further tested by means
of an experiment with deuterium-labelled A'-piperideine (Ex-

periment 4). If the conclusions drawn from the experiments

with chirally deuteriated cadaverine (Experiments 1 and 2) are
correct, it can be predicted that deuterium from (2-2H)A'-piperideine must enter at C-10 and at C-11-si of lupinine.
The 2H nrnr spectrum of a sample of lupinine derived from
(2-2H)-Al-piperideineis shown in Fig. 2D. The predictions are
fully substantiated.
The final stereochemical question that was answered in this
investigation concerns the prochirality of the decarboxylation
of lysine in L. luteus. The evidence which shows that L-lysine
is the precursor of lupinine in L. luteus (Experiment 6) has
already been discussed (vide supra). Decarboxylation of
L-lysine yields cadaverine. In principle, this decarboxylation
can take place either with net retention or with net inversion of
configuration. Decarboxylation of L-(i.e., (S)-)(2-2H)lysine
with net retention of configuration yields (S)-(l-2H)cadaverine,
whereas decarboxylation with net inversion yields (R)(I-'H)cadaverine. Incorporation into lupinine of the L-component of ~ ~ - ( 2 - ~ H ) l y syields
i n e a sample of lupinine whose 'H
nmr spectrum (Fig. 2C) is identical with that of the lupinine
sample obtained when (S)-(l-2H)cadaverine served as the substrate (Fig. 2B).
It follows that, en route to lupinine, L-(2-2H)lysineis converted into (S)-(l-2H)cadaverine, i.e., in the process leading
from L-lysine to cadaverine the carboxyl group of lysine is
replaced by a proton with net retention of configuration. This
stereochemical course is consistent with the prochirality of the
reactions catalyzed by other L-amino acid decarboxylases (22)
(E.C.4.1.1.).
The experimental evidence here presented serves as a critical
test of the biogenetic hypotheses of lupinine biosynthesis that
have been proposed. All but one of the hypotheses are disproven on the basis of the results of the six biosynthetic experiments which are here reported. The remaining hypothesis
(Scheme 1Be) which is presented in full detail in Schemes 4
and 5, is entirely consistent with all the experimental evidence.
This evidence throws light on the steps from lysine into lupinine and on the stereochemistry of several of these steps.

GOLEBIEWSKI AND SPENSER

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Experimental
Extraction of lupinine and sparteine from Lupinus luteus
The fresh plant material was immediately ground in a blendor with
methanol and extracted in a Soxhlet apparatus for 8 h. The 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 X 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
acidlbase cycle (sulfuric acid, potassium hydroxide, as above), yielding a basic fraction (25 mg) which contained lupinine (20) and sparteine as the major components.
The alkaloid fraction (25 mg) was applied to a silica gel column (10
X 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, 500: 1 (20 mL) and 5% methanol in methylene chloride/0.880 ammonia, 333: 1 (40 mL). The lupinine fraction
was eluted with 8% methanol in methylene chloride/0.880 ammonia,
166: 1 (120 mL), the sparteine fraction with 12% methanol in methylene chloride/0.880 ammonia, 100: 1 (80 mL). Yield of crude alkaloids: 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
208-209C (lit. (23) mp 207-209C). A small sample of lupinine was
converted into lupinine methiodide, lit. (23) mp 295-296C.
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 :1, 3 drops), mp 265-266C (dec.) (lit. (24) mp 264.5-265.5"C).
Radioactive materials
~ ~ - [ 6 - ' ~ C ] L y sand
i n e~-[4-,H]l~sine
were obtained from commercial 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 experiment 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)Cadaverine dihydrochloride (24)
5-Phthalimidopentano(1 -'3C,1 -ISN)nitrile(22)
A solution of 1-bromo-4-phthalimidobutane(21) (0.85 g) in dimethyl sulfoxide (1.6 mL) was added dropwise over 2 min to a stirred
solution of sodium (I3C,l5N)cyanide(99 at.% I3C, 99 at.% I5N, MSD
Isotopes, Montreal) (0.15 g) in aqueous dimethyl sulfoxide (1.6 mL
DMSO, 0.2 mL H20). 'The solution was stirred 4 h at 65C and left
overnight at room temperature. Water (12 mL) was added and the
mixture extracted with benzene (4 X 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 recrystallized from absolute ethanol, mp 69-70C; '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);
160 (100, M ms m / z : 230 (6.5%), 188 (31, M - (CH2-13C'5~)),
(C3H5-"CHN)), 149 (21), 104 (98), 77 (12), 76 (13).
l-N-Acetyl-5-N-phthaloyl-l,5-diamino(l-13~,1
- 1 5 ~pentane
)
(23)
(22) (0.67 g) in
Crude 5-phthalimid0pentano(l-~~~,l-'~~)nitrile
acetic anhydride (3 mL) was added to a suspension of freshly prepared
Raney nickel catalyst (0.5 g) in acetic anhydride (7 mL). (The catalyst
had been freshly prepared immediately before use by gradual addition
of sodium hydroxide pellets to a suspension of nickel aluminum alloy

2717

(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 anhydride.) Hydrogenation was carried out at 80-90C 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, mp 134-136"C, after recrystallization from absolute ethanol);
'H nmr (CDCI,) 6: 7.9-7.7 (4H, m), 6.0 (IH, dtd, J I ~ N89.5
, H Hz,
JCH~-I,NH
11.2 HZ, J I ~ C . N
2.8
H HZ), 3.67 (2H, t, J 6.6 HZ, H-5), 3.23
(2H, dm, J I ~ ~139
. ~HZ),
. , 1.94 (3H, d, J 12 HZ), 1.8--1.3 (6H, m);
I3C nmr (CDCl,) 6: 170.2 ( 1 5 ~ C 0 - ) ,168.6 (NCO-), 134.1 (C-3',4'), 132.3 (C-1',-6'), 123.3 (C-2',-5'), 39.5 (d, J l 3 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-"~)Cadaverine dihydrochloride (24)
Hydrochloric acid (6 M, 14 mL) was added to the crude acetyl
derivative (23) and the mixture was stirred for 18 h at 100C. Crystallization of phthalic acid started on cooling and was complete after
1 h at 0C. 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 I I cm). The column was washed
with water (20 mL) and the product was eluted with hydrochloric acid
(1.5 M, 200 mL), yielding a further crop (0.13 g) of
(1-'3C,I-15N)cadaverinedihydrochloride. Total yield 57% (relative to
NaI3Cl5N); 'H nmr (DzO) 6: 3.76 (IH, 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; the
signals at 6 3.76 and 2.18 ppm are doublets of triplets, due to I3C,H-1
coupling of the 1-I3CH2group. The signal at 6 2.97 ppm is due to the
5-CH2 group; I3C nmr (D,O) 6: 40.0 (d, J I ~ ~4.4
. ~HZ),
. ~27.0
~ N
(s, d,
J13c.2,13c.l 35.4 HZ,), 23.4 (s, C-3).
D L - ( Z - ' H ) L ~ monohydrochloride
S~~~
was prepared (13) in two steps
from diethyl 2-acetamidomalonate and 1-bromo-4-phthalimidobutane
(21). Condensation yielded diethyl 2-acetamido-2-(4-phthalimidobutyl)malonate, whose hydrolysis in DCI/D20 yielded the desired
product. D~-(2-'H)L~sinemonohydrochloride: IH nmr (D20) 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~erideine
and (S)-(+)-(I -'H)cadaverine hydrochloride
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
solution of D ~ - ( 2 - ' ~ ) l ~ s imonohydrochloride
ne
(0.64 g) in water (90
mL) and the solution was heated at 50C in a nitrogen atmosphere on
a rotary evaporator under mild suction until colorless. Three more
portions of N-bromosuccinimide (3 X 0.32 g) were added and reaction
continued in the same way. The total reaction time was 55 min. The
solution was used for feeding without further purification.
A small portion of the solution was concentrated 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 (H20), 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 abundance) ppm. When the solution was
evaporated and redissolved in water, the 'H nmr spectrum changed
considerably: 'H nmr 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 (K2C03) and extracted 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)Cadaverine
hydrochloride
(R)-(-)-(l-'~)Cadaverine hydrochloride was prepared by decar-

he value, 18.4 Hz, for J13c.z.13c.l that was reported in our preliminary communication (I 1) was in error. We thank Dr. E. Leete for
pointing out this mistake.

2718

CAN. J. CHEM. VOL. 63, 1985

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boxylation of the L-component of DL-lysine in deuterium oxide, catalyzed by L-lysine decarboxylase (13). 'H nmr (D20) 6: 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~)~adaverinehydrochloride was obtained (13) by
decarboxylation, catalyzed by L-lysine decarboxylase, of the
s i n eat.% 2-'H). (S)-(+)-(1-2H)L-component of ~ ~ - ( 2 - ~ H ) l ~ (95
Cadaverine dihydrochloride (93 at.% L2H): 'H nmr (D20) similar to
hydrochloride, above; ms mlz: 89
that of (R)-(-)-(1-'H)cadaverine
(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 months,
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. Wiewiorowski, University of Poznan, for a gift of seeds of Lupinus luteus,to 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.
Lenkinski, South Western Ontario NMR Facility, University of
Guelph, for determining COSY and 2-D J resolved spectra.
This investigation was supported by a grant from the Natural
Sciences and Engineering Research Council of Canada.
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