Deracemization of racemic 4-pyridyl-1-ethanol by Catharanthus roseus cell cultures

Article abstract of DOI:10.1016/S0031-9422(98)00241-6

Immobilized cells of Catharanthus roseus synthesized (R)-4-pyridyl-1- ethanol from the corresponding racemate in 100% yield by stereoinversion of the (S)-alcohol in the racemate to the (R)-alcohol. Furthermore, in the reduction of 4-acetylpyridine, we could synthesize both (R) and (S)-4- pyridyl-1-ethanol during a short or a long incubation.

Full text of DOI:10.1016/S0031-9422(98)00241-6

Phytochemistry Vol. 49, No. 6, pp. 1627±1629, 1998
#
1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
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031-9422/98/$ - see front matter
PII: S0031-9422(98)00241-6
DERACEMIZATION OF RACEMIC 4-PYRIDYL-1-ETHANOL
BY CATHARANTHUS ROSEUS CELL CULTURES
MASUMI TAKEMOTO* and KAZUO ACHIWA
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan
(
Received in revised form 9 March 1998)
Key Word IndexÐCatharanthus roseus; Apocynaceae; cell cultures; stereoinversion; re-
duction; chiral 4-pyridyl-1-ethanol.
AbstractÐImmobilized cells of Catharanthus roseus synthesized (R)-4-pyridyl-1-ethanol from the corre-
sponding racemate in 100% yield by stereoinversion of the (S)-alcohol in the racemate to the (R)-alcohol.
Furthermore, in the reduction of 4-acetylpyridine, we could synthesize both (R) and (S)-4-pyridyl-1-ethanol
during a short or a long incubation. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
When the racemic alcohols (R,S)-a-phenyl-3-
pyridylmethanol (1a) or (R,S)-4-pyridyl-1-ethanol
In recent years, much attention has been paid to
the ability of cultured plant cells to transform enan-
tioselectively not only secondary metabolites, but
also organic foreign substrates [1±6]. Synthesis of
chiral compounds in high enantiomeric excess (ee)
is also very important in synthetic organic chem-
istry. Chemical or biological methods are known
for the preparation of optically active alcohols from
racemates: optical resolution, selective oxidation of
one of the enantiomers, and deracemization of race-
mates. Among these methods, deracemization is the
most promising method, because it permits 100%
conversion of the starting racemates to the corre-
sponding chiral compound, whereas the theoretical
maximum yield is 50% for enzymatic or chemical
resolution. However, relatively little work has been
done using this approach [7±9]. In our paper, we
report on the novel deracemization of 4-pyridyl-1-
ethanol using Catharanthus roseus cell cultures
(
B5 medium [10], 93% of (R,S)-1a and 100% of
1b) were exposed to immobilized CRC (ICRC) in
(
(
R,S)-1b were converted to (-)-1a [optical yield
OY), 100% ee] and (R)-(-)-1b (OY, 87% ee), re-
spectively (Method 1). When the racemic alcohol
R,S)-a-phenyl-2-pyridylmethanol (1c) was subjected
to ICRC, after a long incubation, the oxidation of
c occurred to a€ord the corresponding ketone 2c,
leaving unreacted (R)-1c [2c: CY, 59%, (R)-1c: CY,
1%, OY, 92% ee] (Method 2). From these obser-
(
1
4
vations, we expected the following mechanism in
the case of 1b (deracemization of racemic alcohol),
as shown in Fig. 2.
At ®rst, (S)-1b in the racemate is converted to the
corresponding ketone, 2b, which is rapidly reduced
to (R)-1b; in contrast, (R)-1b is resistant to oxi-
dation. Then, we did experiments using (S)-1b or
(
R)-1b and ketone 2b as the substrates with ICRC
as shown in Table 1. In the case of (S)-1b, the ee
decreased with incubation times and (S)-1b was
inverted to (R)-1b, reaching maximum values of
(
CRC) in detail.
8
0% ee [(R)-1b]. On the other hand, the recovered
RESULTS AND DISCUSSION
yields were consistent from beginning to end [(R)-1b
or (S)-1b: CY, 100%]. We succeeded in the stereo-
inversion of (S)-1b to (R)-1b (OY, 80% ee) in 100%
yield after incubation for 8 days; in this reaction,
ketone 2b was not detected as an intermediate.
Next, the biotransformation of (R)-1b was per-
formed. The ee and the recovered yield of (R)-1b
were consistent from beginning to end, respectively
Recently, we developed novel methods [7] for
producing optically active pyridyl alcohols from the
corresponding racemates by CRC (Fig. 1): (1) dera-
cemization of racemic alcohol in near 100% yield
(Method 1), and (2) highly enantioselective resol-
ution of racemic alcohol via oxidation (Method 2).
[(R)-1b: OY, 97±100% ee, CY, 99±100%]. In these
experiments, the ketone as an intermediate was not
*
Author to whom correspondence should be addressed.
1627

1
628
M. TAKEMOTO and K. ACHIWA
Fig. 1. Synthesis of optically active pyridyl alcohols 1a±c from the corresponding racemates by
Catharanthus roseus cell cultures. ee = enantiomeric excess.
detected in the reaction mixtures. Then, reduction ify whether the ketone exists as an intermediate in
of 2b with ICRC was performed. Unexpectedly, the stereoinversion. Further studies are in progress.
ketone 2b was reduced not to (R)-1b but to
(
S)-1b (OY, 87% ee, CY, 97%) after 2 days of
incubation. However, the ee of (S)-1b decreased
with incubation time and (S)-1b was inverted to
EXPERIMENTAL
(
R)-1b, reaching maximum values of 80% ee in General
9
1% yield after 15 days of incubation. Nevertheless,
Structures of all products were determined by
1
interpretation of their MS and HNMR, and by
the chemical yields of 1b were consistent from
beginning to end (91±100%). Although the mechan-
ism of the reduction of 2b are not clear at present,
it is reasonable to expect, from our results, that this
reduction is performed by a two-step process. The
comparison with reported data [7]. Chemical yields
refer to compounds puri®ed by CC on silica gel.
Optical purities (% ee) of (S)-1b and (R)-1b were
determined by HPLC using columns packed with
Chiralcel OB-H (Daicel Chemical Industries Ltd),
®
The second is stereoinversion of (S)-1b to (R)-1b.
rst is the stereo-selective reduction of 2b to (S)-1b.
using Et NH±2-PrOH±hexane, 0.01:1:10 as eluant.
2
From these observations, the mechanism in derace- All compounds were located by spraying TLC
mization of racemic alcohol [Method (1)] may be plates with a 10% soln of phosphomolybdic acid in
explained by the stereoinversion of (S)-1b in the EtOH and heating.
racemate to (R)-1b. In this stereoinversion, we pre-
sumed ketone 2b to be the intermediate. But, in the
Cultivation of CRC
reduction of 2b with ICRC, the ketone 2b was not
reduced to (R)-1b but to (S)-1b (OY, 87% ee, CY, every 7 days by transferring 1-week-old cultures
7%). In these experiments, we were unable to clar- (8 ml) into B5 medium (80 ml) containing 2,4-D
Suspension cells of C. roseus were subcultured
9
Fig. 2. Proposed mechanism of deracemization.

Stereoinversion of (S)-4-pyridyl-1-ethanol by Catharanthus roseus
1629
Table 1. Bioreaction of (S)-1b, (R) = 1b and 2b with Preparation of (R)-1b and (S)-1b
ICRC
(R)-1b and (S)-1b were prepared according to
the method of ref. [11]. (R)-1b was synthesized by
hydrolysis of racemic acetate (10 mmol) in the pre-
sence of 200 mg lipase SAM II and 20 ml P bu€er
i
(
pH 7). (S)-1b was synthesized by esteri®cation of
1b (10 mmol) in the presence of 20 ml tert-butyl-
methylether, 30 mmol vinyl acetate and 200 mg
2
0
lipase SAM II. (R)-(+)-1b : [a]
c 1.25). O.Y. 100% ee {Ref. [11] (R)-(+)-1b:
D
+45.1 (MeOH;
2
0
D
[a] +42.5 (MeOH; c 1.04). O.Y. >95% ee}. (S)-(-
-1b : [a] +44.8 (MeOH; c 1.23). O.Y. 98% ee
D
2
0
)
2
0
{Ref. [11] (S)-(-)-1b : [a]
O.Y. >95% ee}.
D
�43.0 (MeOH; c 1.24).
Biotransformation of substrates [(S)-1b, (R)-1b or
2b] with ICRC
The substrate [(S)-1b, (R)-1b or 2b] (35 mg) was
administered to precultured B5 medium (80 ml)
containing ICRC and the mixt. incubated at 258 on
a rotary shaker (110 rpm) in the dark. At the ter-
mination of the reaction, the mixt. was ®ltered and
the ICRC beads washed with CH Cl . The ®ltrate
2
2
(
cultured medium from ICRC beads) was extracted
with CH Cl and the combined organic layer was
washed with brine, dried (MgSO ) and concd in
vacuo. The residue was subjected to CC on silica gel
with CH Cl to give the corresponding compound.
2
2
4
2
2
Reaction time, chemical yields and optical yields
are listed in Table 1.
REFERENCES
1
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N. M. and Takemoto, M., Heterocycles, 1996,
4
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2
3
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D. and Kutney, J. P., Phytochemistry, 1996, 42,
4
23.
(
1 ppm) and 2% sucrose (pH 5.5) on a rotary
4
5
. Suga, T., Hirata, T. and Izumi, S.,
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Kawabe, S., Plant Cell Rep., 1988, 7, 493.
shaker (110 rpm) at 258 kept in the dark.
Preparation of ICRC
A 5% Na alginate soln (80 ml) was added to
freely suspended C. roseus cells in the stationary
phase (80 ml of B5 medium, 10 days). The mixt.
was stirred until it became homogeneous. The Na
6. Naoshima, Y. and Akakabe, Y., J. Org. Chem.,
1989, 54, 4237.
7. Takemoto, M. and Achiwa, K., Tetrahedron
Asymmetry, 1995, 6, 2925.
alginate mixt. was added dropwise to a 0.6% CaCl
soln (11). The resulting ICRC beads, ca 3±4 mm
2
8. Hasegawa, J., Ogura, M., Tsuda, S., Maemoto,
S. and Kutsuki, H., Agric. Biol. Chem., 1990,
54, 1819.
9. Nakamura, K., Inoue, Y., Matsuda, T. and
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diam, were allowed to stand for 1 hr and washed
with H O. ICRC prepared from 20 ml of cells and
broth, as described, was added to freshly prepared
2
B5 medium (80 ml per ¯ask) containing 2.4-D 10. Gamborg, O. L., Miller, R. A. and Ojima, K.,
1 ppm) and 2% sucrose, and the medium was Experimental Cell Research, 1968, 50, 151.
shaken on a rotary shaker (110 rpm) in the dark at 11. Seemayer, R. and Schneider, M. P.,
58 for 2 days. Tetrahedron Asymmetry, 1992, 3, 827.
(
2