Stereochemistry of the 1,2-migration of the carboxyl group

that occurs during the biosynthesis of tropic acid from phenylalanine112

EDWARD
LEETE
Natural Products Laboratory, School of Chemistry, University of Minnesota, Minneapolis, MN 55455 U.S.A.
Received June 3, 1986
Thispaper is dedicated to Dr. 0.E. (Ted) Edwards

EDWARD LEETE.Can. J . Chem. 65, 226 (1 987).

(2S,3R)- and (2S,3S)-[1-'~C,3-~H]~hen~lalanine were fed in separate experiments to Daturn innoxia plants. The distribution
of isotopes in the resultant labeled hyoscyamine and scopolamine indicates that the biosynthesis of the acid moiety of these ester
alkaloids, namely (S)-tropic acid, involves the migration of the carboxyl group of phenylalanine to its prochiral C-3 position with
retention of configuration.

EDWARD LEETE.Can. J. Chem. 65, 226 (1987).

Can. J. Chem. Downloaded from cdnsciencepub.com by 223.25.122.19 on 12/06/23

Au cours d'expkriences skparkes, on a fait croitre des plants de Datura innoxia en prksence de [I4C-1,3H-3]-phknylalanines
soit (2S,3R) soit (2S,3S). En se basant sur la distribution des isotopes dans les hyoscyamine et scopolamine marqukes qui en
rksultent, on peut en conclure que la biosynthkse de la portion acide de ces esters d'alcaloi'des, soit l'acide tropique-(S), implique
la migration du groupement carboxyle de la phknylalanine vers sa position prochirale-3 et que cette migration se fait avec
rktention de configuration.
[Traduit par la revue]

Introduction The interpretation of the results is complicated by the fact that

It has been established that the biosynthesis of (S)-tropic acid
(I), the acid moiety of the tropane alkaloids hyoscyamine (2)
and scopolamine (3), involves the intramolecular 1,2-migration
of the carboxyl group of phenylalanine, from C-2 to C-3 (2).
Recently, it has been shown that the biosynthesis of tropic
acid also involves a 1,2-migration of hydrogen from C-3 of
phenylalanine to C-2, which ultimately becomes the hydroxy-
methyl group of tropic acid (3). This preliminary result was
obtained by feeding a mixture of all four possible stereoisomers
of [1-14C,3-3H]phenylalanine(2R,3R; 2R,3S; 2S,3R; and
2S,3S) to Datura innoxia plants. The stereochemistry of the
migration of the carboxyl group has now been probed by
carrying out feeding experiments with (2s)-phenylalanine
stereospecifically labeled with one tritium at its prochiral
C-3 position. These substrates were obtained as previously
described (4). The stereochemical purity at the C-3 position was
determined by conversion to trans-cinnamic acid with the
enzyme phenylalanine ammonia-lyase (PAL). This enzyme
catalyses the trans elimination of ammonia. From the retention
of tritium relative to carbon-14 (located at C-1), it was
calculated that both the (3R)- and (3S)-[1-'4C,3-3H]phenyl-
alanine were 89% enantiomerically pure at their prochiral
C-3 positions. The doubly labeled phenylalanines were fed to
Datura innoxia plants by the wick method. The alkaloids were
isolated and separated as previously described (5) from plants
that were harvested 10 days after the initial feeding.
Results and discussion
Scheme 1 illustrates the fate of hydrogen at C-3 of phenyl-
alanine when tropic acid is formed. If the migration of the
carboxyl group occurs with retention of configuration, (2S,3R)-
[1-14C,3-3H]phenylalanine (5) will afford (S)-tropic acid (4),
retaining tritium at its chiral center. If the migration occurs with
inversion, the tritium will be displaced and will be found on the
hydroxymethyl group of (S)-tropic acid (6).
The results recorded in Table 1 are consistent with the first
mechanism, that is, migration with retention of configuration.
'This article is Part 35 in the series "Chemistry of the Tropane
Alkaloids." For Part 1, see ref. 1.
2~ommunicationNo. 202 from this laboratory.
the tropic acid moiety of hyoscyamine and scopolamine is
readily racemized. Such racemization will result in loss of
tritium from the chiral center of tropic acid. Thus the hyoscya-
mine isolated from the feeding of (2S,3R)-[1-14C,3-3fl]pheny
alanine (Expt. 1) had 50% enantiomeric excess of the (-)-isomer.
However, it retained 65% of tritium relative to carbon-14.
When this hyoscyamine was racemized (by addition of a trace of
sodium hydroxide to a solution of the alkaloid in ethanol), the
reisolated racemic hyoscyamine (atropine) retained only 10% of
its tritium, indicating that most of the tritium was located at the
chiral center of the tropic acid. Hydrolysis of the racemized
hyoscyarnine with aqueous barium hydroxide yielded racemic
tropic acid with the same relative amount of tritium. The
scopolamine, which is optically more stable than hyoscyamine,
retained 83% of tritium relative to carbon-14. However,
hydrolysis of this alkaloid with aqueous barium hydroxide
yielded tropic acid that was almost racemic (18% enantiomeric
excess), and retained only 13% of tritium relative to carbon- 14.
The residual tritium found in the racemic tropic acid presumably
arises from the (2~,3~)-[1-'~C,3-~~]phenylalanine (1 1%) pre-
sent in the administered precursor.
Complementary results were obtained with (2S,3S)- [ 1-
'4~,3-3~]phenylalanine (Expt. 2). The alkaloids isolated from
this feeding experiment retained most of the tritium relative to
carbon-14. Racemization of the alkaloids caused little change in
their 3 ~ 14C / ratios, indicating that the tritium was not located at
the chiral center of tropic acid. Basic hydrolysis of the alkaloids
yielded racemic tropic acid that retained 85% of the tritium,
which was shown to be present on the hydroxymethyl group
using the degradation previously described (3).
Independently, Haslam and co-workers (6) fed (2S,3R)- and
( 2 ~ , 3 S-)[2- 14C,3-3H]phenylalanine to Datura stramonium
plants. They considered that their results were consistent
with a stereospecific loss of the pro-R hydrogen at C-3 of
phenylalanine, the carboxyl group migrating with inversion of
configuration at this position. This conclusion is the direct
opposite of the one reported in the present article. However, the
authors apparently failed to realize that tritium located at the
chiral center of tropic acid is very labile. The 3 ~ 14C / ratio of the
isolated alkaloids was not reported. The mixture of alkaloids
 NMe NMe

HOOC,,,
"C 7
retention
% s COOH - inversion HOOC,,,,
"c
H
,
Can. J. Chem. Downloaded from cdnsciencepub.com by 223.25.122.19 on 12/06/23

Ph/ 'CH~OH P~'R\C / ~h' 'CHOH

Z~ N H ~ I
T
H
4 5 6
SCHEME
1

TABLE1. Feeding of labeled phenylalanines to Datura innoxia

Experiment No.

Exp. No. 1 2

Precursor fed: (2S,3R)-[1-~~~,3-~H]~hen~lalanine

( 2 ~ , 3 ~ ) -'4~,3-3H]phenylalanine
-[l
Time of feeding April June
Fresh weight of plants (g) 220 350
I4c '4c
(dpm/mmol) 3 ~ I4c
/ (dpm/mmol) 3H/ I4C

Activity 1.83 x lo7 10.6

Amount fed 0.5 mmol

Alkaloids isolated
Hyoscyamine.HC1
Weight (mg)
Enantiomeric excessa
Activity
Absolute inc. (I")
Tropic acid
Scopolamine. HCl
Weight (mg)
Enantiomeric excessa
Activity
Absolute inc. (I")
Tropic acid

"The optical purity was obtained by determination of the specific rotations of the alkaloid hydrochlorides. Optically pure
hyoscyamine.HC1 has [a]: -24.2" (c 3.5, EtOH) and scopolamine.HC1 has [a]: - 17.7" ( c 4.5, EtOH).
bAbsolute inc. = total activity (dpm) found in the isolated alkaloid divided by the total activity (dpm) fed to the plants.

was hydrolyzed by refluxing with aqueous 10% sodium hydrox-
ide for 30 min, conditions which would cause racemization of
tropic acid. The tropic acid was assayed for radioactivity after
conversion to methyl-3-0-acetyltropate. It was reported that
this tropic acid derived from (2S,3R)- [2-14C,3-3H]phenyl-
alanine (88% enantiomeric excess at C-3) had lost 88-93% of
its tritium relative to carbon-14. The tropic acid derived from
(2~,3S)-[2-'~~,3-~~]phen~lalanine (83% enantiomeric excess
at C-3) lost 30-35% of the tritium. These experimental results
are thus entirely consistent with ours, the retention of tritium in
the tropic acid derived from the [3S-3H]phenylalanine being
due to its migration to the hydroxymethyl group, and not
because it remained at the chiral center of tropic acid.
The 1,2-migration of the carboxyl group of phenylalanine to
ultimately yield (S)-tropic acid is analogous to the biochemical
conversion of (2R)-methylmalonyl coenzyme A (7) to succinyl
coenzyme A (8) (7), which is illustrated in Scheme 2. In this
reaction, it is the thioester group that undergoes an intramole-
 CAN. J. CHEM. VOL. 65, 1987
intramolecular, with retention of configuration
fl
intermolecular
7
cular migration with retention of configuration. The displaced
hydrogen also migrates in the opposite direction; however, this
migration is intermolecular. It has not yet been determined
whether the 1,2-migration of the 3-pro-S hydrogen of phenyl-
Can. J. Chem. Downloaded from cdnsciencepub.com by 223.25.122.19 on 12/06/23
alanine that occurs during tropic acid biosynthesis is inter- or
intra-molecular. Another point of interest is the fact that the
conversion of 7 to 8 is catalyzed by coenzyme B12.However,
the presence of vitamin B12or its derivatives in higher plants is
tenuous and controversial (8-10). Twenty years ago I sent an
extract of Datura plants to Professor R. H. Abeles (then at
the University of Michigan), but he was unable to detect any
vitamin B12 in this material.
Experimental
Radioactivity assay
Radioactivity measurements were carried out in a Tracor Analytic
Mark I11 liquid scintillation counter. Samples (assayed in duplicate)
were dissolved in water or EtOH (1 mL), which was then diluted with
liquid scintillation solution (10 mL). The liquid scintillation solution
was made by dissolving naphthalene (50 g), 2,5-diphenyloxazole
(3.5 g), and 1,4-(5-phenyloxazol-2-y1)-benzene(0.25 g) in spectro-
scopic grade dioxane (500 mL). The backgrounds in the I4cand 3~
channels were 8 and 14 dpm, respectively. In unquenched samples the
efficiency of counting in the 14cand 3~ channels was 60 and 45%,
respectively, and spillover of 14cinto the 3~ channel was 10%.
(2 S ,3R)- and (2s ,3~)-[1-'~~,3-~~]~hen~lalanine
The (2S,3R)- and (2~,3~)-[1-'~~,3-~~]~henylalanine
were obtained
from [formyl-3~]benzaldehydeand [ l - 1 4 ~ ] g i y c i n eas previously
described (4). The enantiomeric excess at C-3 was determined by
incubation with phenylalanine ammonia-lyase (EC 4.3.1.5) from
Rhodotorula glutinis (Sigma Chem. Co.), as previously described (4).
Administration of the labeled phenylalanine to Datura innoxia plants
and isolation of the alkaloids
The plants were 3 months old, growing in soil in a greenhouse at the
time of feeding, which was done by means of cotton wicks inserted into
the stems of the plants near to ground level. After 10 days the plants
(both the roots and aerial parts) were harvested and the alkaloids,
hyoscyamine and scopolamine, isolated and separated by thin-layer
chromatography (tlc) as previously described (5).
Racemization of hyoscyamine and scopolamine
The following is a typical procedure, the optical rotations being
I
determined in a Perkin-Elmer 242 polarimeter. The hyoscyamine
I hydrochloride (I 13.7 mg) from Expt. 1 was dissolved in absolute EtOH
(10 mL) at 25OC. Aqueous 10% NaOH (0.05 mL) was added. Under
these conditions the rotation was reduced by half in 12.5 min. After 2 h
the solution was acidified with a drop of acetic acid and then evaporated
to dryness. The residue was subjected to tlc o n silica gel PF 254
(Merck), developing with a mixture of CHC13, EtOH, concentrated
NH3 (80: 20: 1). The zone (R 0.3) corresponding to atropine (detected
by uv) was extracted with MeOH. This extract was neutralized with
HC1, evaporated to dryness, and the residue crystallized from arnixture
of EtOH and ethyl acetate to afford atropine hydrochloride. This salt
had essentially the same I4C specific activity as the initial hyoscyamine
hydrochloride, but its 3H/'4C ratio changed from 6.9 to 1.1.
Scopolamine hydrochloride was similarly racemized; however, its
half-life under the same conditions was longer (22 min).
Hydrolysis of the alkaloids
The alkaloid hydrochloride (200 mg) was dissolved in aqueous
saturated Ba(OH)2 solution (10 mL) and the mixture refluxed for
30 min. The cooled solution was acidified with HCl and extracted with
Et20 (3 x 30 mL). The residue obtained on evaporation of the dried
(Na2S04)Et20 extract was crystallized from a mixture of benzene and
hexane, affording tropic acid (50-60 mg), mp 118°C. Optically pure
tropic acid has mp 129°C (1 1).
Acknowledgements
This research was supported by a research grant GM-13246
from the National Institutes of Health, U.S. Public Health
Service. Seeds of Datura innoxia were obtained from the
Zentral Institut fiir Genetik und Kulturpflanzenforschung,
Gatersleben, D.D.R.
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