Deracemisation of β-hydroxy esters using immobilised whole cells of Candida parapsilosis ATCC 7330: substrate specificity and mechanistic investigation

Article abstract of DOI:10.1016/j.tet.2006.03.045

Deracemisation of aryl substituted β-hydroxy esters by immobilised whole cells of Candida parapsilosis ATCC 7330 gave >99% ee and up to 75% yield of their corresponding (S)-enantiomers. Mechanistic investigation of the deracemisation reaction carried out using a deuterated substrate, ethyl 3-deutero-3-hydroxy-3-phenyl propanoate revealed that while the (S)-enantiomer remains unreacted the (R)-enantiomer undergoes enantioselective oxidation to its corresponding ketoester, which on complementary enantiospecific reduction gives the (S)-enantiomer in high yield and % ee.

Full text of DOI:10.1016/j.tet.2006.03.045

Tetrahedron 62 (2006) 5133–5140
Deracemisation of b-hydroxy esters using immobilised whole cells
of Candida parapsilosis ATCC 7330: substrate specificity
and mechanistic investigation
*
Santosh Kumar Padhi, D. Titu, N. Ganesh Pandian and Anju Chadha
Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
Received 2 August 2005; revised 21 February 2006; accepted 9 March 2006
Available online 17 April 2006
Abstract—Deracemisation of aryl substituted b-hydroxy esters by immobilised whole cells of Candida parapsilosis ATCC 7330 gave >99%
ee and up to 75% yield of their corresponding (S)-enantiomers. Mechanistic investigation of the deracemisation reaction carried out using
a deuterated substrate, ethyl 3-deutero-3-hydroxy-3-phenyl propanoate revealed that while the (S)-enantiomer remains unreacted the (R)-
enantiomer undergoes enantioselective oxidation to its corresponding ketoester, which on complementary enantiospecific reduction gives
the (S)-enantiomer in high yield and % ee.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
cells of baker’s yeast are reported to carry out a highly
stereoselective synthesis of a-unsubstituted and a-alkyl-b-
hydroxy esters.13 Kinetic resolution is widely used for the
synthesis of optically pure b-hydroxy esters from racemic
b-hydroxy esters.14 The limited yield of each enantiomer
(maximum 50%) and the formation of the ‘unwanted’ iso-
mer are the major drawbacks in the resolution of a racemate.
Deracemisation15 is an attractive alternative approach to
synthesise chiral b-hydroxy esters from their racemates in
high ee and quantitative yield. Use of two-enzyme systems16
and whole cells is known to deracemise secondary alco-
hols.17 Azerad et al. used aged cultures of a local strain of
Geotrichum candidum to deracemise ethyl 3-hydroxy buta-
noate into the (R)-enantiomer in 96% ee and 80%
yield.18 Nakamura et al. reported the stereoinversion of
aliphatic b-hydroxy esters (methyl-3-hydroxy butanoate
and methyl-3-hydroxy pentanoate) using G. candidum IFO
5767 to produce the (R)-enantiomers in 97–99% ee and
26–48% isolated yield.15e Deracemisation of b-hydroxy
esters is restricted to the above examples, both of which use
G. candidum for aliphatic b-hydroxy esters and the deracem-
ised product is the (R)-antipode. Whole cells of Candida
parapsilosis are a rich source of oxidoreductases19 and dif-
ferent strains of this species are known to deracemise 1,2-
diols20 and 1,3-diols.21 More recently, deracemisation of
aryl and aryl substituted a-hydroxy esters by C. parapsilosis
ATCC 7330 to the (S)-enantiomer 17 and asymmetric reduc-
tion of ethyl 4-chloro-3-oxo butanoate using genetically
modified C. parapsilosis (IFO 1396) to the (R)-hydroxy
ester in 99% ee and 95% yield11c were reported. We have
previously reported the deracemisation of some racemic
Optically pure b-hydroxy esters are important chiral
synthons for the synthesis of numerous pharmaceuticals
such as fluoxetine,1 b-lactam antibiotics,2 Tuckolide—an
HMGCoA reductase-inhibitor3 and dihydrokawain (a nar-
cotic).4 The amino derivative of a (S)-b-hydroxy ester is
present in the side chain of TaxolÒ.5 Ethyl 4-chloro-3-hy-
droxy butanoate is used in the preparation of L-carnitine,
a nutraceutical.6 Chiral b-hydroxy esters are also used as
starting materials in the preparation of enantiomerically
pure b-blockers, i.e., propranolol, alprenolol and 1-(isopro-
pylamino)-3-p-methoxy-phenoxy-2-propanol.7 The applica-
tion of (R)-(ꢀ)-sodium b-hydroxy butanoate as a cerebral
function improving agent on cerebral hypoxia, anoxia and
ischeamia in mice and rats has been reported.8 Both the en-
antiomers of ethyl 3-hydroxy butanoate and ethyl 3-hydroxy
pentanoate are extremely useful in the synthesis of phero-
mones.9 Optically pure b-hydroxy esters also play an impor-
tant role in many biological reactions inside the human
body.10 With the varied applications of these molecules,
there is much interest in their asymmetric synthesis. Asym-
metric reduction and kinetic resolution are the two main bio-
catalytic approaches for the synthesis of these compounds.
Asymmetric reduction of b-ketoesters by different microbial
whole cells11 and plant cells is known.12 Engineered whole
Keywords: Deracemisation; b-Hydroxy esters; Candida parapsilosis;
Mechanism.
* Corresponding author. Tel.: +91 44 2257 4106; fax: +91 44 2257 4102;
e-mail: anjuc@iitm.ac.in
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.03.045

5134
S. K. Padhi et al. / Tetrahedron 62 (2006) 5133–5140
b-hydroxy esters using free and immobilised whole cells of
C. parapsilosis ATCC 7330.22 Here we report the deracem-
isation by immobilised C. parapsilosis ATCC 7330 (ICp) of
19 aryl and substituted aryl substrates with different elec-
tronic and steric effects. The substrate specificity of the bio-
catalyst and the investigated mechanism of deracemisation
are discussed.
propanoate, along with acetophenone and 1-phenyl ethanol,
which led us to propose and investigate the stereoinversion
mechanism for deracemisation of b-hydroxy esters. In order
to prove the proposed mechanism of this deracemisation re-
action, the following substrates were incubated with immo-
bilised cells of C. parapsilosis ATCC 7330 and are discussed
in detail.
2.1.1. Substrate (R)-ethyl 3-hydroxy-3-phenyl propa-
noate [(R)-1]. Optically pure (R)-ethyl 3-hydroxy-3-phenyl
propanoate [(R)-1] was incubated with ICp for 6 h and the
product was analysed at the end of every hour for the forma-
tion of (i) the (S)-enantiomer [(S)-1] using chiral HPLC and
(ii) ethyl 3-oxo-3-phenyl propanoate (2) using reverse-phase
HPLC. Chiral HPLC confirmed the presence of (S)-hydroxy
ester (68%) at the end of 6 h. The ketoester (2) intermediate
could be detected from 4 h onwards and was confirmed by
comparison with a standard (Scheme 1).
2. Results and discussion
2.1. Mechanistic investigation of deracemisation
The different mechanisms by which deracemisation can be
carried out are (i) re-racemisation and repeated resolution,
(ii) dynamic kinetic resolution and (iii) stereoinversion.15a
Kato et al. reported the deracemisation of a-alkyl carboxylic
esters, which involves enantioselective esterification, epimer-
ization and then hydrolysis.15b All these deracemisation
mechanisms share a common stereochemical phenomenon,
i.e., one enantiomer of the substrate retains its configuration
throughout the deracemisation process and the other enan-
tiomer crosses the plane of symmetry (or inversion of configu-
ration) to become product.15a Literature reports on the use of
different strains of C. parapsilosis in the deracemisation of
secondary alcohols reveal that oxidoreductases actively
participate in the deracemisation by stereoinversion.15c–e,20
Deracemisation of a-hydroxy esters by whole cells of
C. parapsilosis ATCC 7330 also occurs via stereoinver-
sion.17 Mechanistic investigation of the deracemisation of
b-hydroxy esters was done using ethyl 3-hydroxy-3-phenyl
propanoate (1, Table 1). The deracemised product of racemic
ethyl 3-hydroxy-3-phenyl propanoate (1) on HPLC analysis
(reverse phase) showed the presence of ethyl 3-oxo-3-phenyl
Oxidation of (R)-ethyl 3-hydroxy-3-phenyl propanoate [(R)-
1] to the ketoester intermediate (2) followed by its reduction
to [(S)-1] involves the loss of a methine proton. To prove the
role of the methine proton in the stereoinversion, two exper-
iments were carried out using ethyl 3-deutero-3-hydroxy-
3-phenyl propanoate (3) and ethyl 3-hydroxy-3-phenyl
butanoate (4) as the substrates.
2.1.2. Substrate ethyl 3-deutero-3-hydroxy-3-phenyl
propanoate (3). Ethyl 3-deutero-3-hydroxy-3-phenyl prop-
anoate (3) was incubated with ICp for 6 h and the resulting
1
reaction mixture was analysed by H NMR spectroscopy
after work up. The H NMR spectrum of the reaction mix-
1
ture confirmed the presence of the starting reactant (deuter-
ated, 3) as well as its undeuterated counterpart (1) (dd at
Table 1. Deracemisation of b-hydroxy esters using immobilised whole cells of Candida parapsilosis ATCC 7330
Sub. no.
R
R0
n
ee%
Yield %
Abs. config.
[a]2D5 (This study)
[a]2D5 (Lit. reported)
1
7
8
9
Ph
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
0
0
0
0
0
0
0
0
0
0
0
0
1
2
0
0
0
0
0
99
09
98
>99
>99
99
99
72
99
26
00
00
13
87
>99
93
57
68
51
42
75
48
42
62
41
66
63
62
59
10
48
47
71
38
23
S
Nd
S
S
S
S
S
S
S
Nd
—
—
Nd
S
S
S
S
—
Nd
ꢀ50.1 (c 1.5, CHCl3)
Nd
+43.7 (c 1.4, CHCl3)28
o-MeC6H4
p-MeC6H4
p-EtC6H4
o-OMeC6H4
p-OMeC6H4
p-ClC6H4
m-BrC6H4
p-NO2C6H4
m-NO2C6H4
1-Naphthyl
9-Anthranyl
Ph
Ph
Ph
Ph
Ph
Me
Me
ꢀ44.6 (c 1.2, CHCl3)
ꢀ43.4 (c 1.2, CHCl3)
ꢀ57.7 (c 1.8, CHCl3)
ꢀ43.7 (c 0.52, CHCl3)
ꢀ43.7 (c 1.38, CHCl3)
ꢀ21.4 (c 1.46, CHCl3)
ꢀ59.5 (c 1.5, CHCl3)
Nd
+44.7 (c 1.2, CHCl3)33a
Nr
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ꢀ42.0 (c 2.30, CHCl3)33b
+39.4 (c 0.52, CHCl3)33a
+44.2 (c 1.38, CHCl3)33a
Nr
+23.1 (c, 1.0, CHCl3)28
—
—
Nd
ꢀ2.35 (c 2.71, CH2Cl2)
ꢀ49.9 (c 1.0, CHCl3)
ꢀ51.6 (c 2.61, CHCl3)
ꢀ31.2 (c 3.78, CHCl3)
—
+1.3 (c 1.0, CHCl3)33e
ꢀ52.0 (c 1.0, CHCl3)33c
Nr
n-Propyl
n-Butyl
CH2Ph
CH2CH]CHPh
67
00
79
+35.2 (c 3.78, CHCl3)33d
Nd
Nd: not determined; Nr: not reported.
OH
O
OH
O
OH O
O
O
O
O
O
O
+
(S)-1
(R)-1
(R)-1
2
68:32
Scheme 1.

S. K. Padhi et al. / Tetrahedron 62 (2006) 5133–5140
5135
OH
H
O
OH
D
O
O
+
O
8
6
4
2
0
Figure 1. 1H NMR of the deracemised product of ethyl 3-deutero-3-hydroxy-3-phenyl propanoate.
Immobilized
OH
O
OH
O
OH
O
Candida parapsilosis
ATCC 7330
O
O
*
O
+
D
D
H
1
90% ee
(RS)-3
3
Confirmed by 1H-NMR spectroscopy
Scheme 2.
5.13 ppm, Fig. 1) as the products in the ratio of 94:6, respec-
tively (Scheme 2).
nyl propanoate [(S)-1] in >99% ee and 25% yield (Scheme
3) indicating the presence of a (S)-specific reductase in C.
parapsilosis ATCC 7330.
The formation of the undeuterated product (1) is possible
only by the reduction of the corresponding ketoester—ethyl
3-oxo-3-phenyl propanoate (2) detected earlier. The pres-
ence of the (S)-enantiomer in the product mixture (3 and 1
in 94:6) in 90% ee indicates that the deuterated (S)-3 is
due to the retention of configuration of (S)-3 from the race-
mic substrate while (R)-3 is converted to (S)-1 via the keto-
ester-2, which was detected earlier.
O
O
OH
O
Immobilized
Candida parapsilosis
ATCC 7330
O
O
(S)-1
2
>99% ee and 25% yield
Scheme 3.
2.1.3. Substrate ethyl 3-hydroxy-3-phenyl butanoate (4).
Ethyl 3-hydroxy-3-phenyl butanoate (4) when incubated
with ICp for 6 h resulted in complete recovery of the starting
racemate thus proving that the presence of a methine proton
is mandatory for this deracemisation.
In order to account for the low yield from ICp mediated
reduction of 2, the product mixture was analysed using
reverse-phase HPLC, which confirmed the presence of ethyl
3-hydroxy-3-phenyl propanoate (1), acetophenone (5) and
1-phenyl ethanol (6). Formation of acetophenone could be
due to decarboxylation of the hydrolysed23 product of the
ketoester intermediate (2) while 1-phenyl ethanol could be
the reduced product of acetophenone accounting for w15–
20%, of the yield.22b Loss of another 10–15% of product
can be attributed to the work up of the aqueous product
mixture.22c
The experimental results described in Sections 2.1.2 and
2.1.3 unequivocally prove that ICp mediated deracemisation
of b-hydroxy esters proceeds by a stereoinversion mecha-
nism.
2.1.4. Reduction of ethyl 3-oxo-3-phenyl propanoate (2)
using immobilised whole cells of C. parapsilosis ATCC
7330. Incubation of ethyl 3-oxo-3-phenyl propanoate with
ICp resulted in the production of (S)-ethyl 3-hydroxy-3-phe-
The scheme of reactions during deracemisation as proved
from the above experiments can thus be represented as given
in Scheme 4.

5136
S. K. Padhi et al. / Tetrahedron 62 (2006) 5133–5140
OH O
OH O
Table 1) on deracemisation gave 26% ee and 66% isolated
OH
Remains
yield while ethyl 3-(3-bromophenyl)-3-hydroxy propanoate
(13, Table 1) on deracemisation with ICp gave 72% ee and
62% isolated yield. The nitro group in the meta position
lowers electron density in the aromatic ring, which could af-
fect the removal of a hydride ion from the reaction centre,
which we believe, is the initial step in this deracemisation
reaction. The better optical yield in the case of meta bromo
substituent than the meta nitro (15, Table 1) is because the
bromo group does not have strong negative inductive effect
as compared to the nitro group. Also the mesomeric effect
is not active in the meta position for 15 (Table 1). Ethyl
3-hydroxy-3-naphthalen-1-yl-propanoate and ethyl 3-anthra-
cen-9-yl-3-hydroxy-propanoate (16, 17, Table 1) on ICp
mediated deracemisation, were recovered in racemic form.
The reaction centre in these molecules is sterically hindered
due to the large size of the aromatic ring. Substrate 17 (Ta-
ble 1), ethyl 3-anthracen-9-yl-3-hydroxy-propanoate in its
racemic form is a new compound reported here for the first
time and was used for deracemisation. b-Hydroxy esters with
a spacer between the chiral centre and the phenyl ring, i.e.,
ethyl 3-hydroxy-4-phenyl butanoate and ethyl 3-hydroxy-
5-phenylpentanoate (18, 19, Table 1) on deracemisation
gave 13% ee and 59% yield and 87% ee and 10% yield of
the products, respectively. A similar study on the baker’s
yeast reduction25 of the corresponding ketoesters of 1, 18
and 19 (Table 1), indicates that with increasing number of
carbon atoms between the keto group and the phenyl ring,
the reduction proceeds at a faster rate. In this study substrate
1 without spacer and substrate 19 (Table 1) with two carbons
as the spacer, undergoes deracemisation with high ee. The
low yield of the product (19, Table 1) is due to hydrolysis
of the ester group as a side reaction. The reasons for the
poor ee of 18 (Table 1) are not clear at present. ICp mediated
deracemisation of racemic b-hydroxy esters having ester
moieties with increasing chain length (1, 20–22, Table 1) re-
sulted in a gradual decrease in the ee of the (S)-enantiomer
product. High ee (>99%) of the product was obtained for
methyl-3-hydroxy-3-phenyl propanoate (20, Table 1) with
48% yield. Phenylmethyl-3-hydroxy butanoate did not un-
dergo deracemisation with ICp (23, Table 1). This molecule,
with its aromatic ring on the opposite side of the reaction cen-
tre, is quite different from all the above substrates used for de-
racemisation. 3-Hydroxy-butyricacid 3-phenyl-allyl ester 24
(Table 1) on deracemisation resulted in 79% ee but a low iso-
lated yield of 23%.
O
O
unreacted
O
*
(S)-1
6
(S)-1
(S)-specific
Reductase
+
O
O
2
O
OH O
(R)-specific
O
Oxidase
(R)-1
5
Scheme 4.
It is clear from Scheme 4 that, while the oxidation reaction is
enantioselective and chemospecific, the reduction reaction is
enantiospecific. The enantiospecificity of the reduction reac-
tion is important as it is responsible for the high optical yield
of the deracemised product. Notably, all earlier reported de-
racemisation reactions using different strains of C. parapsi-
losis were limited to substrates without multiple functional
groups except in the case of a-hydroxy esters.17 Thus, the
appearance of the side products during deracemisation of
b-hydroxy esters is not a surprise.
2.2. Deracemisation using immobilised whole cells of
C. parapsilosis ATCC 7330
The number of biocatalytic methods for the preparation of
chiral b-hydroxy esters is limited as compared to synthetic
methods. Biocatalytic reduction of ketoesters of substrates
1,24 18,25 1926 and 2027 (Table 1) to their (S)-enantiomers
has been reported in 87–97% ee and 42–87% yield. Lipase
catalysed kinetic resolution of substrates 8,28 11,28 14,28
18,25 2025 and 2225 (Table 1) has also been reported in 86–
94.5% ee and 42–48% yield. No suitable biocatalytic
methods are reported for the preparation of substrates 7, 9,
10, 12, 13, 15, 16 and 21 (Table 1). Substrate 17 (Table 1)
is a new compound and is used here for the first time for
deracemisation.
Deracemisation of racemic ethyl 3-hydroxy-3-phenyl prop-
anoate (1, Table 1) with ICp under optimised conditions22b
gave its (S)-enantiomer in 99% ee and 57% isolated yield.
In addition, a group of optically pure aryl and substituted
aryl b-hydroxy esters was prepared using the ICp mediated
deracemisation (Scheme 5) in order to study the substrate
specificity of the biocatalyst. Deracemisation of substrates
with electron donating (8, 9, 11 and 12, Table 1) and electron
withdrawing (14, Table 1) substituents at the para position of
the standard substrate resulted in the formation of their (S)-
enantiomers in 98–99% ee and 41–51% yield. This indicates
that the electronic nature of the substituents at the para po-
sition of the standard substrate does not affect the derace-
misation. At the ortho position however, these substituents
can obstruct the process of deracemisation due to their prox-
imity to the reaction centre. Among the meta substituted
substrates ethyl 3-hydroxy-3-(3-nitrophenyl) propanoate (15,
As evidenced from Table 1, the high ee (>99%) and high
yield (up to 75%) of the products during the ICp mediated
deracemisation of varied racemic b-hydroxy esters prove
the broad substrate specificity of the biocatalyst.
Ethanol was used as the auxiliary solvent in the ICp medi-
ated deracemisation to avoid the formation of side products
due to transesterification except for substrates 20, 21 and 22
(Table 1), where methanol, n-propanol and n-butanol were
used, respectively.
Immobilized
OH
*
O
OH
O
Candida parapsilosis
ATCC 7330
R
R'
R
R'
3. Conclusion
O
O
n
n
25 °C
upto 99% ee and 75% yield
The mechanism of ICp catalysed deracemisation of racemic
b-hydroxy esters was proved to be via stereoinversion.
Scheme 5.

S. K. Padhi et al. / Tetrahedron 62 (2006) 5133–5140
5137
Experiments proving the mechanism showed that the (S)-en-
4.3. Culture medium
antiomer of ethyl 3-hydroxy-3-phenyl propanoate retained
its absolute configuration and the (R)-enantiomer underwent
an inversion of configuration via a ketoester intermediate,
when incubated with ICp. Deracemisation of aryl and aryl
substituted b-hydroxy esters by ICp gave >99% ee and up
to 75% yield. High ee (98–99%) of the deracemised products
was observed irrespective of the electronic nature of the sub-
stituents at the para position of the aromatic ring. A nitro
group at the meta position of the aromatic ring decreases
the ee of the deracemised product (26%). A methyl group
at the ortho position of the aromatic ring decreases the ee
of the deracemised product while a methoxy group does
not. Substrates with polyaromatic rings did not undergo
deracemisation. Increasing chain length of the ester moiety
decreases the ee of the product.
The composition of the media used for culturing C. parapsi-
losis ATCC 7330 was: YMB (yeast malt broth) [yeast extract
(3 g Lꢀ1), malt extract (3 g Lꢀ1), peptone (5 g Lꢀ1) and dex-
trose (10 g Lꢀ1) at pH 6.5]. The yeast malt broth was steri-
lised in an autoclave at 121 ꢂC and 15 lb kgꢀ1 pressure for
20 min.
4.4. Cultivation of microorganism
The strain C. parapsilosis ATCC 7330 was routinely main-
tained in agar plates as well as slants (2.1% agar with the
above mentioned culture media). Sub-culturing was per-
formed every 12 weeks. The plates and slants were preserved
at 4 ꢂC.
4.5. Culture conditions
4. Experimental
The pure culture of C. parapsilosis ATCC 7330 was inocu-
lated with a loop, into the YMB media. A working volume
of 50 mL (YMB media, after inoculation) in the 250 mL
Erlenmeyer flasks was cultivated in an orbital shaker at
25 ꢂC, 200 rpm. The cells were harvested by centrifuging
the 44 h culture broth at 3750g followed by washing with
sterile water. The process was repeated thrice and finally
the wet biomass was used for biotransformation.
4.1. General methods
1H and 13C NMR spectra were recorded in CDCl3 solution
on a Bruker AV-400 spectrometer operating at 400 and
100 MHz, respectively. Chemical shifts are expressed in
parts per million values using TMS as an internal standard.
HPLC analysis was carried out on a Jasco PU-1580 liquid
chromatograph with a PDA detector using Chiralcel-ODH
and Chiralcel OJ-H chiral columns (Daicel, 4.6ꢁ250 mm).
Optical rotations were recorded on a Jasco Dip 370 digital
polarimeter. TLC was done on Kieselger 60F254 aluminium
sheets (Merck 1.05554). The mobile phase was hexane/
isopropanol, the proportion of solvents and the flow rate
vary for different compounds.
4.6. Immobilization
Immobilization was done following an earlier reported
procedure.22b
4.7. Mechanistic investigation of deracemisation
4.2. Materials and methods
In order to prove that the deracemisation follows a stereoin-
version mechanism, different substrates were used with ICp.
The details of these experiments are discussed below.
Racemic b-hydroxyl esters 1, 7–11, 14, 15, 18, 23 and 24
were synthesised by sodium borohydride reduction of their
corresponding ketoesters. The ketoesters of 1, 11, 14 and
15 were purchased from Fluka, Buchs SG, Switzerland;
however, for substrates 7, 8, 9, 10 and 18 the ketoesters
were prepared by a literature known method.29 The ketoest-
ers of 23 and 24 were synthesised by a reported microwave
irradiation method.30 Substrates 12, 13, 16, 17 and 19 were
synthesised by Reformatsky reaction.31 Substrates 20, 21
and 22 were synthesised by our reported procedure,32 i.e.,
sodium borohydride mediated reduction cum transesterifica-
tion of the ethyl 3-oxo-3-phenyl propanoate. (S)-Ethyl 3-hy-
droxy-3-phenyl propanoate was prepared by baker’s yeast
reduction of the corresponding ketoester.24 The (R)-ethyl
3-hydroxy-3-phenyl propanoate and the ketoester were pur-
chased from Fluka, Buchs SG, Switzerland and used as such.
Sodium borodeuteride was purchased from Sigma–Aldrich
Chemical Co., Milwaukee, U.S.A. The deuterated hydroxy
ester was prepared by the reduction of the corresponding
ketoester with sodium borodeuteride. Acetophenone used
as a standard was purchased from a local chemical company.
1-Phenyl ethanol was prepared by reducing acetophenone
using sodium borohydride. Ethanol used in the biotransfor-
mation was of spectroscopic grade. C. parapsilosis ATCC
7330 was procured from American Type Culture Collection
(ATCC 7330).
4.7.1. Substrate (R)-ethyl 3-hydroxy-3-phenyl propa-
noate [(R)-1]. To a suspension of 100 mL of immobilised
C. parapsilosis ATCC 7330 in 50 mL of sterile distilled
water taken in a 500 mL Erlenmeyer flask, 40 mL (210 mmol)
of (R)-ethyl 3-hydroxy-3-phenyl propanoate (>99% ee) pre-
dissolved in 1 mL of absolute ethanol was added. The reac-
tion mixture was incubated in a water-bath shaker for 6 h
at 25 ꢂC and 150 rpm. Aliquots were taken at different in-
tervals of time (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0
and 6.0 h), extracted and analysed using both chiral HPLC
(Chiralcel-ODH, hexane/isopropanolT95:05) and reverse-
phase HPLC (Kromasil C-18 column, HPLC grade aceto-
nitrile/water 40:60).
4.7.2. Substrate ethyl 3-deutero-3-hydroxy-3-phenyl
propanoate (3). A suspension of 60 mL of immobilised
beads of C. parapsilosis ATCC 7330 in 30 mL of sterile dis-
tilled water was prepared in a 500 mL Erlenmeyer flask.
Ethyl 3-deutero-3-hydroxy-3-phenyl propanoate (15 mL,
w78 mmol) pre-dissolved in 375 mL of absolute ethanol
was added to the above suspension. The reaction mixture
was incubated in a water-bath shaker at 25 ꢂC for 6 h at
150 rpm. Three similar experiments were carried out simul-
taneously to estimate the yield of the product. The reaction

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S. K. Padhi et al. / Tetrahedron 62 (2006) 5133–5140
mixture after the necessary work up was characterised using
1H NMR. The reaction mixture was also analysed using
chiral HPLC to determine the optical purity of the product.
4.9.2. Ethyl 3-hydroxy-3-(2-methylphenyl) propanoate
(7). Colourless oil; spectroscopic data identical to that re-
ported in literature.22a
4.9.3. (S)-Ethyl 3-hydroxy-3-(4-methylphenyl) propa-
noate (8). Colourless oil; spectroscopic data identical to
that reported in literature,22a [a]D25 ꢀ44.6 (c 1.2, CHCl3).
HRMS (ESI): found 231.1004, C12H16O3Na [M+Na]+ re-
quires 231.0997.
4.7.3. Substrate ethyl 3-hydroxy-3-phenyl butanoate (4).
To a suspension of 10 mL of immobilised C. parapsilosis
ATCC 7330 in 5 mL of sterile distilled water taken in
a 100 mL Erlenmeyer flask, 4 mL (21 mmol) of racemic ethyl
3-hydroxy-3-phenyl butanoate pre-dissolved in 100 mL of
absolute ethanol was added. The reaction mixture was incu-
bated in a water-bath shaker for 6 h at 25 ꢂC and 150 rpm.
After the incubation time, the reaction mixture was filtered
to recover the immobilised beads and the filtrate was ex-
tracted with ethyl acetate (3ꢁ30 mL). The organic layer
was dried over anhydrous sodium sulfate and concentrated.
This concentrated product was dissolved in 2 mL of hexane/
isopropanol, 99:01 (HPLC grade), filtered through a 0.2-mm
filter and analysed using chiral HPLC (Chiralcel-ODH,
hexane/isopropanolT99:01). The chiral HPLC analysis
(Chiralcel-ODH, hexane/isopropanolT99:01) indicated the
presence of only the racemic substrate.
4.9.4. (S)-Ethyl 3-(4-ethylphenyl)-3-hydroxy propanoate
(9). Colourless oil; [a]2D5 ꢀ43.4 (c 1.2, CHCl3). IR nmax
(neat): 3455, 3059, 2966, 1733, 1514, 1372, 1269, 1116,
1
1037, 965, 890, 871, 833, 691 cmꢀ1; H NMR (400 MHz,
CDCl3) d: 1.21 (t, J¼7.6 Hz, 3H), 1.24 (t, J¼7.1 Hz, 3H),
2.63 (q, J¼7.5 Hz, 2H), 2.7 (ddd, J¼16.2, 9.1, 3.8 Hz,
2H), 3.3 (s, 1H), 4.16 (q, J¼7.1 Hz, 2H), 5.09 (dd, J¼9.1,
3.8 Hz, 1H), 7.17 (d, J¼8.0 Hz, 2H), 7.27 (d, J¼8.0 Hz,
2H); 13C NMR (100 MHz, CDCl3) d: 14.1, 15.5, 28.5,
43.4, 60.7, 70.2, 125.7, 127.9, 139.9, 143.81, 172.3.
HRMS (ESI): found 245.1161, C13H18O3Na [M+Na]+ re-
quires 245.1154.
4.7.4. Reduction of ethyl 3-oxo-3-phenyl propanoate (2)
using immobilised whole cells of C. parapsilosis ATCC
7330. The reaction was carried out as given in Section 4.7.3
using ethyl 3-oxo-3-phenyl propanoate as the substrate. The
reaction mixture analysed by a reverse-phase HPLC (Kroma-
sil C-18 column, HPLC grade acetonitrile/water 40:60) illus-
trated the presence of side products as shown in Scheme 4.
4.9.5. (S)-Ethyl 3-hydroxy-3-(2-methoxyphenyl) propa-
noate (10). Colourless oil; spectroscopic data identical to
that reported in literature,34a [a]D25 ꢀ57.7 (c 1.8, CHCl3).
4.9.6. (S)-Ethyl 3-hydroxy-3-(4-methoxyphenyl) propa-
noate (11). Colourless oil; spectroscopic data identical to
that reported in literature,22a [a]D25 ꢀ43.7 (c 0.52, CHCl3).
4.8. Typical procedure for the deracemisation using
immobilised whole cells of C. parapsilosis ATCC 7330
(ICp)
4.9.7. (S)-Ethyl 3-(4-chlorophenyl)-3-hydroxy propa-
noate (12). Colourless oil; spectroscopic data identical to
that reported in literature,34a [a]D25 ꢀ43.7 (c 1.38, CHCl3).
HRMS (ESI): found 251.0457, C11H13O3ClNa [M+Na]+ re-
quires 251.0451.
Racemic b-hydroxy esters (504 mmol, 96 mL) dissolved in
an appropriate amount of solvent (0.06% v/v, ethanol/meth-
anol/n-propanol/n-butanol) were added to a suspension of
250 mL of ICp in 125 mL of distilled water, equally dis-
tributed in four 250 mL Erlenmeyer flasks. The biotransfor-
mation was carried out for 6 h at 25 ꢂC and 150 rpm in a
water-bath orbital shaker. The beads were filtered and the
filtrate was extracted using ethyl acetate, dried over anhy-
drous sodium sulfate and concentrated. The crude product
after column purification (30:70Tethyl acetate/hexane, for
substrate 1) was analysed by chiral HPLC to determine the
optical purity of the deracemised product. The standard sub-
strate 1 under identical conditions gave 99% ee and 57%
yield. Appropriate control experiments with the reaction
mixture containing all the components except (i) racemic
b-hydroxy ester and (ii) the immobilised whole cells of
C. parapsilosis ATCC 7330, established the optical purity
of the product and the chemical yield. Deracemisation of
other aromatic b-hydroxy esters (7–24, Table 1) was carried
out following the same procedure.
4.9.8. (S)-Ethyl 3-(3-bromophenyl)-3-hydroxy propa-
noate (13). Pale yellow liquid; spectroscopic data identical
to that reported in literature,34b [a]D25 ꢀ21.4 (c 1.46,
CHCl3). HRMS (ESI): found 294.9946, C11H13O3BrNa
[M+Na]+ requires 294.9942.
4.9.9. (S)-Ethyl 3-hydroxy-3-(4-nitrophenyl) propanoate
(14). Pale yellow liquid; spectroscopic data identical to
that reported in literature,22a [a]D25 ꢀ59.5 (c 1.5, CHCl3).
4.9.10. Ethyl 3-hydroxy-3-(3-nitrophenyl) propanoate
(15). Pale yellow liquid; IR nmax (neat): 3451, 3073, 2986,
2972, 2938, 1990, 1713, 1538, 1463, 1380, 1346, 1304,
1277, 1226, 1203, 1182, 1073, 1043, 1007, 939, 914, 859,
816, 734, 692, 622, 566 cmꢀ1 1H NMR (400 MHz,
;
CDCl3) d: 1.26 (t, J¼7.1 Hz, 3H), 2.75 (m, 2H), 3.77 (s,
1H), 4.19 (q, J¼7.1 Hz, 2H), 5.25 (m, J¼5.2 Hz, 1H), 7.88
(m, 4H); 13C NMR (100 MHz, CDCl3) d: 14.0, 43.0, 61.1,
69.2, 120.7, 122.6, 129.4, 131.7, 144.7, 148.4, 171.8.
HRMS (ESI): found 262.0692, C11H13NO5Na [M+Na]+ re-
quires 262.0691.
4.9. Spectroscopic characterisation of the deracemised
products
4.9.1. (S)-Ethyl 3-hydroxy-3-phenyl propanoate (1). Col-
ourless oil; spectroscopic data identical to that reported in
literature,22a [a]D25 ꢀ50.1 (c 1.5, CHCl3). HRMS (ESI):
found 217.0837, C11H14O3Na [M+Na]+ requires 217.0837.
4.9.11. Ethyl 3-hydroxy-3-napthalen-1-yl propanoate
(16). Colourless oil; spectroscopic data identical to that
reported in literature,34a HRMS (ESI): found 267.0999,
C15H16O3Na [M+Na]+ requires 267.0997.

S. K. Padhi et al. / Tetrahedron 62 (2006) 5133–5140
5139
4.9.12. Ethyl 3-anthracen-9-yl-3-hydroxy propanoate
(17). Pale yellow liquid; IR nmax (neat): 3486, 3049, 2983,
1707, 1621, 1447, 1332, 1282, 1174, 1077, 1105, 1024,
References and notes
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1
963, 892, 736, 422 cmꢀ1; H NMR (400 MHz, CDCl3) d:
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2H), 3.38 (s, 1H), 4.22 (q, J¼7.1 Hz, 2H), 6.74 (dd,
J¼10.4, 2.4 Hz, 1H), 7.47 (m, 4H), 7.98 (d, J¼8.2 Hz,
2H), 8.4 (s, 1H), 8.66 (m, 2H); 13C NMR (100 MHz,
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128.5, 129.1, 129.3, 131.6, 132.34, 172.6; HRMS
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1394, 1356, 1269, 1195, 1058, 915, 761, 700, 609 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d: 0.89 (t, J¼7.4 Hz, 3H),
1.62 (sextet, J¼7 Hz, 2H), 2.69 (ddd, J¼16.1, 8.9, 3.8 Hz,
2H), 3.56 (s, 1H), 4.03 (t, J¼6.6 Hz, 2H), 5.14 (dd,
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less oil; spectroscopic data identical to that reported in
literature.34c
4.9.19. 30-Phenyl-prop-20-enyl 3-hydroxy butanoate (24).
Pale yellow liquid; IR nmax (neat): 3445, 3069, 3023, 2979,
2937, 1731, 1578, 1494, 1449, 1372, 1275, 1224, 1147,
1
1039, 965, 886, 750, 693, 634, 607, 540 cmꢀ1; H NMR
(400 MHz, CDCl3) d: 1.23 (d, J¼6.3 Hz, 3H), 2.49 (ddd,
J¼16.5, 8.2, 4.1 Hz, 2H), 3.17 (s, br, 1H), 4.21 (m, 1H),
4.75 (d, J¼6.4 Hz, 2H), 6.26 (dt, J¼15.9, 6.4 Hz, 1H),
6.64 (d, J¼15.9 Hz, 1H), 7.3 (m, 5H); 13C NMR
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128.3, 128.5, 134.5, 136.0, 172.6. HRMS (ESI): found
243.0997, C13H16O3Na [M+Na]+ requires 243.0998.
Acknowledgements
We thank CSIR, New Delhi for the financial support.

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