Biocatalytic deracemisation of α-hydroxy esters: High yield preparation of (S)-ethyl 2-hydroxy-4-phenylbutanoate from the racemate

Article abstract of DOI:10.1016/S0957-4166(02)00403-2

Biocatalytic deracemisaton of the racemic ethyl ester of 2-hydroxy-4-phenylbutanoic acid gives the (S)-enantiomer exclusively in >99% e.e. and 85-90% yield. Ethyl and methyl esters of mandelic acid and the methyl ester of 2-hydroxy-4-phenylbutanoic acid also gave the (S)-enantiomer exclusively. Whole cells of Candida parapsilosis (ATCC 7330) were used to effect this biotansformation.

Full text of DOI:10.1016/S0957-4166(02)00403-2

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 1461–1464
Biocatalytic deracemisation of a-hydroxy esters: high yield
preparation of (S)-ethyl 2-hydroxy-4-phenylbutanoate from the
racemate
Anju Chadha* and Baburaj Baskar
Department of Chemistry, IIT-Madras, Chennai 600036, India
Received 15 February 2002; accepted 15 July 2002
Abstract—Biocatalytic deracemisaton of the racemic ethyl ester of 2-hydroxy-4-phenylbutanoic acid gives the (S)-enantiomer
exclusively in >99% e.e. and 85–90% yield. Ethyl and methyl esters of mandelic acid and the methyl ester of 2-hydroxy-4-
phenylbutanoic acid also gave the (S)-enantiomer exclusively. Whole cells of Candida parapsilosis (ATCC 7330) were used to effect
this biotansformation. © 2002 Published by Elsevier Science Ltd.
1. Introduction
Geotrichum candidum16,17 and Sphingomonas pausimo-
bilis.18 Plant cell cultures have also been used for the
deracemisation of various alcohols.19 1,2-Diols have
been deracemised using Corynosporium cassicola DSM
6247520 and (S)-1,2-pentanediol has been produced
from the racemic 1,2-diol using Candida parapsilosis.21
In our attempts to prepare enantiomerically pure 2-
hydroxy-4-phenylbutanoic acid/esters we explored the
possibility of deracemising the racemic acid/ester using
C. parapsilosis. The only a-hydroxy acid/ester studied
as a substrate for biocatalytic deracemisation so far is
mandelic acid and its derivatives and this was done
using a two enzyme system.14 We report herein the first
example of a-hydroxy acid esters deracemised by C.
parapsilosis. The racemic ethyl ester of 2-hydroxy-4-
phenylbutanoic acid afforded the (S)-enantiomer (>
99% e.e., 85–90% isolated yield) upon treatment with C.
parapsilosis (ATCC 7330). The ethyl and methyl esters
of mandelic acid and the methyl ester of 2-hydroxy-4-
phenylbutanoic acid also gave the (S)-enantiomer
exclusively.
Optically active a-hydroxy acids and their esters are
important building blocks for the asymmetric synthesis
of a wide variety of bioactive molecules.1 Several meth-
ods, chemical2–4 and enzymatic,5 have been reported for
the synthesis of these compounds. Enantiomerically
pure 2-hydroxy-4-phenylbutanoic acid/ester is an
important pharmaceutical intermediate used in the syn-
thesis of anti-hypertensive drugs and ACE inhibitors6
and can be prepared by the asymmetric reduction of the
corresponding a-oxo acid by both chemical and biocat-
alytic methods.6,7 We have reported the reduction of
the 2-oxo-4-phenylbutanoic acid ethyl ester to the 2-
hydroxy compound (e.e. >99%) using cells of D.
carota.8 Enzymatic resolution of the racemate is also
known,9,10 but, as in every resolution the maximum
possible yield of each enantiomer is only 50%. In order
to increase the yield of one enantiomer beyond 50%,
there have been attempts to prepare enantiomerically
pure compounds based on the principle of dynamic
kinetic resolution.11,12 Enzymatic resolution in combi-
nation with ruthenium-catalysed racemisation of some
a-hydroxy acid esters has been reported.13 The der-
acemisation of racemic mandelic acid has been effected
using a two enzyme system.14 Deracemisation by a
single biocatalyst has also been reported. (R)-3-Pentyn-
2-ol was obtained enantioselectively from the corre-
sponding racemic alcohol by the use of Nocardia
fusca,15 while arylethanols have been deracemised using
2. Results and discussion
Racemic 2-hydroxy-4-phenylbutanoic acid ethyl ester
was converted into the (S)-enantiomer ([h]2D5=+7.5 (c 1,
EtOH), >99% e.e. as compared to the literature value10)
as the sole product on incubation for 1 h with resting
cells of C. parapsilosis (ATCC 7330). The time of the
reaction was optimised based on the enantiomeric
* Corresponding author. E-mail: anjuc@iitm.ac.in
0957-4166/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0957-4166(02)00403-2

1462
A. Chadha, B. Baskar / Tetrahedron: Asymmetry 13 (2002) 1461–1464
excess (e.e.) of the product (Table 1), which shows the
data for the formation of (S)-2-hydroxy-4-phenylbu-
tanoic acid ethyl ester from the racemate. The other
a-hydroxy acid esters studied were the methyl and ethyl
esters of mandelic acid which also showed complete
conversion after 1 h to the (S)-enantiomer ([h]2D5=+143
(c 1, MeOH); [h]2D5=+134 (c 3, CHCl3)), respectively,
which is >99% e.e. as compared to the reported val-
ues.22,23 The methyl ester of 2-hydroxy-4-phenyl
butanoic acid was also converted into the (S)-enan-
tiomer as evidenced by HPLC (Scheme 1). The prod-
ucts were analysed by HPLC using a chiral (Chiracel
OD) column and solvent composition reported by us.10
In order to rule out the decomposition of the ‘other’
enantiomer, i.e. the (R)-enantiomer, separate experi-
ments were done on a larger scale with 2-hydroxy-4-
phenylbutanoic acid ethyl ester and the isolated yield
was determined (85–90%) as described in Section 4. For
the other three esters the blank runs did not show any
degradation products from the substrate.
isms including the Candida sp.,25 it is likely that the
mechanism involves an enantioconvergent
stereoinversion26,27 as proposed for (RS)-1,2-diols, i.e.
the (R)-hydroxy enantiomer in the racemate is oxidised
to the corresponding oxo-compound by a (R)-specific
NAD+-linked dehydrogenase and then further reduced
to the (S)-a-hydroxy ester by an (S)-specific NADPH-
dependent reductase. Thus, the only enantiomer formed
by this biotransformation is the (S)-enantiomer. When
2-oxo-4-phenylbutanoic acid ethyl ester was incubated
with the resting cells of C. parapsilosis, the product
formed was the (S)-hydroxy ester in >99% e.e. In fact
in a related study we are trying to establish the general-
ity of this asymmetric reduction. In the case of G.
candidum,26 however, stereoinversion (aerobic condi-
tions) in racemic arylethanols results in the formation
of (R)-alcohols, suggesting the involvement of an alco-
hol oxidase rather than an alcohol dehydrogenase. We
are currently in the process of elucidating the mecha-
nism for the C. parapsilosis-mediated deracemisation
reaction of a-hydroxy acid esters.
Table 1. Time course of the deracemisation reaction of
(RS)-2-hydroxy-4-phenylbutanoic acid ethyl ester
3. Conclusion
Entry
Reaction time % e.e. of
In conclusion, we have shown that racemic a-hydroxy
acid esters, i.e. methyl and ethyl esters of 2-hydroxy-4-
phenyl butanoic acid and mandelic acid in the presence
of C. parapsilosis produce valuable chiral synthons—
the (S)-enantiomer in enantiomerically pure form
(>99% e.e.). The isolated yield in the case of ethyl
2-hydroxy-4-phenylbutanoate (85–90% isolated yield)
proved that this one-step reaction is in fact a deracemi-
sation process, whereby the ‘other’ enantiomer is con-
verted to its mirror image and not destroyed. It is
important to note that this reaction was not seen in free
acids, i.e. 2-hydroxy-4-phenylbutanoic acid and man-
delic acid even though the hydroxy group in these
compounds is a secondary alcohol group. This suggests
that other functional groups on the a-carbon influence
the enzymatic deracemisation.
(min)
(S)-2-hydroxy-4-phenylbutanoic acid
ethyl ester
1
2
3
4
10
20
40
60
30
58
86
\99
4. Experimental
4.1. General
Scheme 1. Deracemisation of racemic a-hydroxy acid esters
using whole cells of C. parapsilosis (ATCC 7330).
The racemic esters were dissolved in the minimum
amount of water-miscible organic solvent (10% of the
final reaction volume), which was added to the aqueous
cell suspension. The use of water immiscible solvents
led to prolonged reaction times, e.g. in hexane it takes
5 h to reach 97% e.e. With ethanol, >99% e.e. is
achieved in 1 h. The work up is simple, involving
centrifugation of the cells and straightforward extrac-
tion of the product using an organic solvent. The free
acids, 2-hydroxy-4-phenylbutanoic acid and mandelic
acid, did not show any deracemisation reaction. It is
interesting to note that in an earlier study21,24 C. parap-
silosis (IFO 0708) produced (S)-1,2-pentanediol from
the racemic 1,2-diol. The exact mechanism of this reac-
tion is not known as yet but, given the fact that
oxidoreductases are abundantly present in microorgan-
The strain of C. parapsilosis (ATCC 7330) used was
bought from ATCC, USA. All chemicals used for
media preparation were bought locally. The racemic
and enantiomerically pure samples of mandelic acid
esters were bought from Aldrich Chemical Co., USA
and used as such. Racemic 2-hydroxy-4-phenylbutanoic
acid ethyl and methyl esters were prepared from ben-
zaldehyde and pyruvic acid by standard procedures.
The (R)-enantiomer of this acid ester was bought from
Aldrich Chemical Co., USA. 1H NMR spectra were
measured on a 400 MHz Jeol instrument.
4.2. Growth conditions of C. parapsilosis
Cells of C. parapsilosis were grown in optimised YMB
medium in shake flasks and were grown for 24 h in a

A. Chadha, B. Baskar / Tetrahedron: Asymmetry 13 (2002) 1461–1464
1463
Table 2. Deracemisation of a-hydroxy acid esters with C.
parapsilosis (ATCC 7330)
shaker at 200 rpm, 25°C. Resting cells used for the
biotransformation were harvested after 24 h, cen-
trifuged and used as a suspension in sterile distilled
water.
Entry Compound
Yield (%) Time (h) Configuration
1
2
3
4
n=0; R=Me* 70
n=0; R=Et* 74
n=2; R=Me* 73
n=2; R=Et 85–90
1
1
1
1
S
S
S
S
4.3. A typical biotransformation experiment
Racemic 2-hydroxy-4-phenylbutanoic acid ethyl ester (3
mg; 0.014 mmol)) in organic solvent (0.1 ml) was added
to an aqueous (sterile distilled water) suspension (0.2g/
ml wet weight) of resting cells of C. parapsilosis (ATCC
7330). The final reaction volume was 1.0 ml. The
reaction mixture was incubated at 25°C in an orbital
shaker (200 rpm) for 1 h after which time the cells were
separated and the product extracted into ether, concen-
trated and analysed for enantiomeric purity and chemi-
cal yield. Appropriate control experiments were carried
out using (a) cells in the reaction medium without the
substrate and (b) substrate in the reaction medium
without cells. The HPLC profile of the first control
experiment revealed that nothing from the cells on
extraction co-eluted with the product. The second con-
trol experiment indicated that the substrate did not give
any products in water. The other three esters were also
used as substrates in a similar manner.
* 100 mg of the substrate was used.
esters, was analysed on HPLC using a chiral column,
Chiracel OD from Daicel, Japan. The mobile phase
used was hexane:isopropanol:trifluoro acetic acid
(99:1:0.1) @ 2 ml/min monitored at 254 nm for all the
four acid esters. The retention times for the four
racemic acid esters were: 2-hydroxy-4-phenylbutanoic
acid ethyl ester (9.05 and 13.99 min); 2-hydroxy-4-
phenylbutanoic acid methyl ester (10.05 and 14.65 min);
ethyl mandelate (10.27 and 19.14 min); and methyl
mandelate (10.27 and 19.23 min). For comparison, an
enantiomerically pure standard was used and it was
found that the (S)-enantiomer was the early eluting
enantiomer in all the four acid esters.
4.4. Time course of the reaction
The reaction mixture as given above in a typical bio-
transformation experiment was incubated at 25°C in an
orbital shaker (200 rpm) for different intervals of time.
After incubation the samples were withdrawn, the cells
separated by centrifugation and the supernatant
extracted and analysed for enantiomeric purity. The
time of the reaction was optimised based on the enan-
tiomeric purity of the product (Table 1).
Acknowledgements
This work is supported by the Department of Science
and Technology, India (SP/SI/G-06/99). We thank Dr.
Uday Avalakki for discussions regarding the
microorganism.
4.5. Isolation and characterisation of the products
References
The biotransformation of 2-hydroxy-4-phenylbutanoic
acid ethyl ester was carried out for 1 h (>99% e.e.) on
300 mg (1.44 mmol) and 500 mg (2.4 mmol) scale. The
product was extracted into diethyl ether, dried and
concentrated. This gave isolated yields of 255 mg (1.22
mmol) and 450 mg (2.16 mmol), respectively (85–90%
yield). This concentrated sample was used for HPLC
1. Copolla, G. M.; Schuster, H. F. h-Hydroxy acids in
Enantioselective Syntheses; VCH: Weinheim, 1997.
2. Zhang, W.; Wang, P. G. J. Org. Chem. 2000, 65, 4732–
4735.
3. Aladro, F. J.; Guerra, F. M.; Moreno-Dorado, F. J.;
Bustamante, J. M.; Jorge, Z. D.; Massanet, G. M. Tetra-
hedron Lett. 2000, 41, 3209–3213.
1
analysis to determine its enantiomeric purity. The H
NMR of ethyl ester of 2-hydroxy-4-phenyl butanoic
acids is as follows: (TMS in CDCl3): l 1.25 (t, 3H),
1.85–2.22 (m, 2H), 2.75 (t, 2H), 4.25 (dd, 1H), 4.10–
4.30 (q, 2H) and 7.10–7.30 (m, 5H). The other three
substrates were compared with the standards by HPLC
and their yields are reported in Table 2. NMR of the
standard methyl ester of 2-hydroxy-4-phenylbutanoic
acid is as follows: (TMS in CDCl3): l 1.75–2.15 (m,
2H), 2.7 (t, 2H), 3.8 (s, 3H), 4.12 (dd, 1H) and 7.1–7.4
4. Burk, M. J.; Kalberg, C. S.; Pizzano, A. J. Am. Chem.
Soc. 1998, 120, 4345–4353.
5. (a) Adam, W.; Lazarus, M.; Saha-Moller, C. R.; Schreier,
P. Acc. Chem. Res. 1999, 32, 837–845; (b) Groger, H.
Adv. Synth. Catal. 2001, 343, 547–558.
6. Schmidt, E.; Blaser, H. U.; Fauquex, P. F.; Seidelmeier,
G.; Spindler, F. In Microbial Reagents in Organic Synthe-
sis; Servi, S., Ed.; Kluwer Academic: The Netherlands,
1992; pp. 377–388.
7. Dao, D. H.; Kawai, Y.; Hida, K.; Hornes, S.; Nakamura,
K.; Ohno, A.; Okamura, M.; Akasaka, T. Bull. Chem.
Soc. Jpn. 1998, 71, 425–432.
1
(m, 5H). The H NMR data for the standard methyl
and ethyl esters of mandelic acid were as reported.28,29
4.6. Determination of e.e. of the product formed
8. Chadha, A.; Manohar, M.; Soundararajan, T.;
Lokeswari, T. S. Tetrahedron: Asymmetry 1996, 7, 1571–
1572.
The product from the biotransformation of 2-hydroxy-
4-phenylbutanoic acid ethyl ester and the other three

1464
A. Chadha, B. Baskar / Tetrahedron: Asymmetry 13 (2002) 1461–1464
20. Page, P. C. B.; Carnell, A.; McKenzie, M. J. Synlett 1998,
9. Sugai, T.; Ohta, H. Agric. Biol. Chem. 1991, 55, 293–
294.
774–776.
21. Hasegawa, J.; Ogura, M.; Tsuda, S.; Maemoto, S.; Kut-
suki, H.; Ohashi, T. Agric. Biol. Chem. 1990, 54, 1819–
1827.
22. Meyers, A. I.; Oppenlaender, T. J. Am. Chem. Soc. 1986,
108, 1989–1996.
23. Mukaiyama, T.; Tomimori, K.; Oriyama, T. Chem. Lett.
1994, 16, 485–495.
24. Matsumara, S.; Kawai, Y.; Takahashi, Y.; Toshima, K.
10. Chadha, A.; Manohar, M. Tetrahedron: Asymmetry 1995,
6, 651–652.
11. Noyori, R.; Tokumaga, M.; Kitamura, M. Bull. Chem.
Soc. Jpn. 1995, 68, 36–56.
12. Stecher, H.; Faber, K. Synthesis 1997, 1–16.
13. Huerta, F. F.; Santosh Laxmi, Y. R.; Back-vall, J.-E.
Org. Lett. 2000, 2, 1037–1040.
14. (a) Takahashi, E.; Nakamichi, K.; Furui, M. J. Ferment.
Bioeng. 1995, 80, 247–250; (b) Strauss, U. T.; Faber, K.
Tetrahedron: Asymmetry 1999, 10, 4079–4081.
15. Xie, S. X.; Ogawa, J.; Shimizu, S. Biosci. Biotech.
Biochem. 1999, 63, 1721–1729.
16. Nakamura, K.; Inoue, Y.; Matsuda, T.; Ohno, A. Tetra-
hedron Lett. 1995, 36, 6263–6266.
17. Nakamura, K.; Fujii, M.; Ida, Y. Tetrahedron: Asymme-
try 2001, 12, 3147–3153.
18. Allan, G. R.; Carnell, A. J. J. Org. Chem. 2001, 66,
6495–6497.
19. Takemoto, M.; Matsuoka, Y.; Achiwa, K.; Kutney, J. P.
Tetrahedron Lett. 2000, 41, 499–502.
Biotechnol. Lett. 1994, 16, 485–495.
25. Hata, H.; Shimizu, S.; Hattori, S.; Yamada, H. J. Org.
Chem. 1990, 55, 4377–4380.
26. Strauss, U. T.; Faber, K. In Bio- and Chemo-Catalytic
Deracemisation Techniques; Zwanenburg, B.; Mikota-
jczyk, M.; Kielbasinski, P., Eds. Enzymes in Action,
NATO Science Series, 1999; Vol. 33, pp. 1–23.
27. Azerad, A.; Buisson, D. Curr. Opin. Biotechnol. 2000, 11,
565–571.
28. Aldrich Library of 13C and 1H FT-NMR Spectra, 1(2),
1220 C.
29. Aldrich Library of 13C and 1H FT-NMR Spectra, 1(2),
1221 A.