Deracemisation of aryl substituted α-hydroxy esters using Candida parapsilosis ATCC 7330: Effect of substrate structure and mechanism

Article abstract of DOI:10.1016/j.tet.2005.09.104

Candida parapsilosis ATCC 7330 was found to be an efficient biocatalyst for the deracemisation of aryl α-hydroxy esters (65-85% yield and 90-99% ee). A variety of aryl and aryl substituted α-hydroxy esters were synthesized to reflect steric and electronic effects on biocatalytic deracemisation. The mechanism of this biocatalytic deracemisation was found to be stereoinversion.

Full text of DOI:10.1016/j.tet.2005.09.104

Tetrahedron 61 (2005) 12296–12306
Deracemisation of aryl substituted a-hydroxy esters using
Candida parapsilosis ATCC 7330: effect of substrate structure
and mechanism
*
B. Baskar, N. G. Pandian, K. Priya and Anju Chadha
Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
Received 29 June 2005; revised 2 September 2005; accepted 22 September 2005
Available online 25 October 2005
Abstract—Candida parapsilosis ATCC 7330 was found to be an efficient biocatalyst for the deracemisation of aryl a-hydroxy esters
(65–85% yield and 90–99% ee). A variety of aryl and aryl substituted a-hydroxy esters were synthesized to reflect steric and electronic
effects on biocatalytic deracemisation. The mechanism of this biocatalytic deracemisation was found to be stereoinversion.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Geotrichumcandidum15,16 andSphingomonaspausimobilis.17
Plant cell cultures were used for the deracemisation of various
aromatic alcohols.18 1,2-Diols were deracemised using
Corynosporium cassiicola DSM 624752019 and (S)-1,2-
pentanediol was produced from the racemic 1,2-diol using
Candida parapsilosis.20 We have used C. parapsilosis ATCC
7330 successfully to deracemise 2-hydroxy-4-phenylbutanoic
esters,21 which are versatile chiral synthons given the fact that
they have a –COOR’ functional group in addition to the –OH
group. The only other a-hydroxy acid/ester studied as a
substrate for biocatalytic deracemisation so far is mandelic
acid and its derivatives, and this was achieved using a two-
enzymesystem.13 Tostudythe scopeofC. parapsilosis ATCC
7330 as a general biocatalyst for the deracemisation of
a-hydroxy esters, different types of a-hydroxy esters were
synthesized for this study. Methods reported in the literature
weremodifiedtoprepare2-hydroxy-4-arylbutanoicandbut-3-
enoic esters with a view to increase the number of functional
groups in the substrates and study the selectivity of this
biocatalytic reaction. This paper reports the modifications
made for the synthesis of the racemic substrates, their
separation using chiral HPLC, deracemisation and the
mechanism of deracemisation by C. parapsilosis ATCC
7330 to give optically pure a-hydroxy esters.
Optically pure a-hydroxy esters are important building blocks
for the asymmetric synthesis of a wide variety of bioactive
molecules.1 In particular, enantiomerically pure 2-hydroxy-4-
phenylbutanoic acid/ester is an important pharmaceutical
intermediate, which finds widespread use in the synthesis of
drugs like anti-hypertensives and ACE inhibitors.2 Several
methods, chemical3,4 and enzymatic5 have been reported for
the synthesis of aryl a-hydroxy esters. Biocatalytic prepar-
ation of optically pure a-hydroxy esters by lipase mediated
resolution of racemic a-hydroxy esters6,7 and biocatalytic
asymmetric reduction of a-oxo esters8 is known. Enzymatic
resolution can result in a maximum theoretical yield of only
50% for each enantiomer. In order to increase the yield of one
enantiomer beyond 50%, the starting racemate can be
deracemised. Deracemisation is a method by which a single
enantiomer is obtained from a racemic mixture.9 Preparation
of enantiomerically pure compounds by dynamic kinetic
resolution is one method for deracemisation.10,11 Backvall
et al. reported enzymatic resolution in combination with
metal-catalysed racemisation of a-hydroxy esters.12 Biocata-
lytic deracemisation of a-hydroxy acids and secondary
alcohols can employ either whole cells or isolated
enzymes.13–20 Racemic mandelic acid was deracemised
using a two enzyme system13 and (R)-3-pentyn-2-ol was
obtained from the corresponding racemic alcohol by the use of
Nocardia fusca.14 Arylethanols were deracemised using
2. Results and discussion
2.1. Synthesis of substrates
Keywords: Deracemisation; Chiral HPLC; Candida parapsilosis ATCC
7330; Stereoinversion; Alpha hydroxy esters.
During our attempts to synthesise racemic a-hydroxy
esters, which were used as substrates for biocatalytic
*
Corresponding author. Tel.: C91 44 2257 4106; fax: C91 44 2257 8241;
e-mail: anjuc@iitm.ac.in
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.09.104

B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
12297
Table 1. Esterification of 2-oxo-4-arylbut-3-enoic acids (1a–1n) and 2-
hydroxy-4-arylbut-3-enoic acids (3a–3n) using phosphotungstic acid as
catalyst
deracemisation, methods were developed by modifications
to the available literature to give the great advantage of
reduced reaction times and high yields of the target esters.
Thus, the modifications mainly include the use of micro-
wave irradiation for the synthesis and the use of
phosphotungstic acid—a solid acid for esterification.
R
R1
Yield (%)
Yield (%)
H
H
p-Cl
p-Cl
p-Me
p-Me
o-Cl
o-Cl
m-NO2
m-NO2
2,4-DiCl
2,4-DiCl
2,5-DiOMe
2,5-DiOMe
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
75
77
81
78
85
81
64
63
68
67
69
75
55
50
4a
4b
4c
4d
4e
4f
4g
4h
4g
4j
80
78
75
73
70
73
71
65
75
77
75
79
63
64
2.2. Esterification of 2-oxo-4-arylbut-3-anoic acid and
2-hydroxy-4-arylbut-3-enoic acid using phosphotungstic
acid
Use of diazomethane for esterification is known for the
preparation of a-oxo esters22a but these reactions require
careful handling and dry conditions. a-Hydroxy esters can
be prepared by Grignard reaction, which also requires
stringent conditions and expensive reagents.22b Both these
problems were overcome by employing a suitable catalyst
viz. a heteropoly acid, which has been reported for a number
of esterification reactions.23
4k
4l
4m
4n
reported by Dalla et al.,25 utilizes azalactones, which are
convenient starting materials, but the reaction takes 4–24 h
and results in low yields of the target esters. Azalactones can
be obtained by microwave irradiation of hippuric acid as the
starting material.26 The second step, that is, hydrolysis of the
azalactones, gives a-keto acids27 from which the target
a-hydroxy esters can be obtained by two different methods—
(i) reduction of the a-keto-enol acid using Zn–Hg
metal27 followed by esterification and (ii) esterification of
the a-keto-enol acid followed by reduction of the
corresponding a-keto ester using sodium borohydride.25
Both these methods have disadvantages—the first uses toxic
metals while the second method lacks chemoselectivity,
that is, the reduction of the ester and keto groups, which
results in the diol. In our approach, the synthesis of various
substituted 2-hydroxy-4-phenylpropionic esters using aza-
lactones (1a–1k) resulted in good yields of the product
(73–86%). Briefly, the procedure involves treating a
mixture of aldehydes with N-acetyl glycine in the presence
of sodium acetate and acetic anhydride under microwave
irradiation using a domestic microwave oven. The major
advantage of this method is the short reaction time of
3–5 min. Reduction of the keto-enol of the acid using
sodium borohydride in alkaline medium at 5–10 8C for 1–
2 h results in the formation of the corresponding saturated
a-hydroxy acid (3a–e and 3h) in good yields (68–87%). The
a-hydroxy acids thus obtained were treated with alcohols in
the presence of phosphotungstic acid metal catalyst in
toluene at 90 8C for 6–10 h to give the corresponding esters
(4a–e and 4h) (Scheme 2) in good yields (73–86%)
(Table 2).
The main reason for employing a heteropoly acid in
esterification reactions is its Keggin structure, which helps
in the controlled release of protons.23 Heteropoly acids act
as solid catalysts for both homogeneous and heterogeneous
reactions due to their high acid strength and high thermal
stability. These catalysts can be recovered after the reactions
and reused for multiple cycles without loss in activity.
Phosphotungstic acid—a heteropoly acid was used for
esterifying various 2-oxo-4-arylbut-3-enoic acids (1a–1n)
and 2-hydroxy-4-arylbut-3-enoic acids (3a–3n) with different
alcohols. The reaction was carried out using a Dean–Stark
apparatus in dry toluene at 90 8C for 6–10 h (Scheme 1). The
yields of the final 2-oxo esters (2a–2n) and 2-hydroxy esters
(4a–4n) ranged from 50–85 and 63–80%, respectively
(Table 1).
Scheme 1.
2.3. Synthesis of alkyl 2-hydroxy-3-arylpropionates
3. Separation of racemic a-hydroxy esters by chiral
HPLC
Alkyl 2-hydroxy-3-arylpropionates have been prepared by
various methods.24a–h The most common methods are
reduction of a-keto esters,24a,b hydrolysis of a-halo
esters,24c rearrangement of a-keto acetals,24d oxidation of
ketene silyl acetals,24e ring opening of epoxy esters with
subsequent reduction of the resulting iodohydrins24f,g and
regioselective deoxygenation of cyclic thionocarbonates of
2,3-dihydroxy esters.23h The main drawback in the reported
methods is the difficulty in obtaining starting materials.24d–h
3.1. Direct separation
All the racemic a-hydroxy esters synthesized for biocata-
lytic deracemisation were resolved on a chiral column
using HPLC. The direct separation of enantiomers using a
chiral stationary phase (CSP) by high-performance liquid
chromatography (HPLC) is a powerful, versatile and
convenient method.28 Solutions of racemic a-hydroxy esters
(1 mg mlK1) were prepared in the eluent for the separations.
The preparation of 2-hydroxy-3-arylpropanoic esters as

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B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
Scheme 2.
Table 2. Synthesis of alkyl 2-hydroxy-3-arylpropionates
S. no.
R
Time (min)
Yield (%) 1
R1
Yield (%) 3
Yield (%) 4
a
b
c
d
e
f
g
h
i
H
p-Cl
2,4-DiCl
o-NO2
m-NO2
p-NO2
p-OCOCH3
p-Me
4
4
5
3
3
3
4
4
5
5
5
92
94
65
85
93
95
84
90
60
65
60
Et
85
82
68
75
79
—
—
87
—
—
—
86
83
79
75
73
—
—
85
—
—
—
Me
Me
Me
Me
—
—
Et
—
—
2,5-DiOMe
o-OMe
1-Naphthyla
j
k
—
a Aryl, 1-naphthyl.
The chiral columns used in this study for the direct
separation of racemic a-hydroxy acid esters (3a–3v)
(Table 3) (Fig. 1) were cellulose tris(3,5-dimethylphenyl-
carbamate) (Chiralcel OD-H) column and cellulose tris(4-
methylbenzoate) (Chiralcel OJ-H) column.
As can be inferred from Table 4, the OJ-H column gave
good separation of unsaturated p-substituted; 2,4- and 2,5-
substituted 2-hydroxy-4-arylbut-3-enoates (3c–3f, 3g–h, 3l
and 3m), whereas for meta-substituted 2-hydroxy-4-arylbut-
3-enoates (3m and 3o), OD-H column proved to be better.
For unsubstituted saturated 2-hydroxy-4-arylbutanoates
(3q–3t), and 2-hydroxy-3-arylpropanoates (3u–3v), ortho-
substituted 2-hydroxy-4-arylbut-3-enoates (3a–b and 3j),
enantiomeric separation was achieved on both OJ-H and
OD-H columns (Table 4). In all the cases, base line
separation was achieved with good separation factors (aO1).
Twenty two racemic a-hydroxy esters were resolved by
chiral HPLC. Compounds 3a, 3b, 3i, 3j0, 3m, 3o, 3p, 3s, 3u
and 3v on OD-H, 3c, 3d, 3e, 3f, 3g, 3g0, 3h, 3j, 3k, 3l, 3n, 3q
and 3t on OJ-H (Table 4). The retention factor (k0),
separation factor (a) and resolution (Rs) of every solute
was regulated over a wide range by the addition of
2-propanol. Enantiomeric separation of 3a–3v was achieved
by varying the composition of hexane/isopropanol in the
range 99:1–93:7. Increasing the concentration of 2-propanol
in the mobile phase decreased the retention times (e.g., 3g
and 3g0) and the column behaved as a normal-phase column.
Compounds (3c–3p) are resolved for the first time in detail
with complete optimization.
3.2. Effect of polar modifiers
In order to study the effect of polarity of the eluent on
separation of racemic 2-hydroxy esters, hexane was used
with either 2-propanol or ethanol as polar modifiers. Both,
CSPs of cellulose derivatives and the polar modifier
(alcohols) have the ability to form hydrogen bonds.29

B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
12299
Competition takes place between the analyte and alcohol for
the chiral sites of the CSP. The enantiomeric separation of
3b, 3c and 3e on OD-H and OJ-H columns with hexane/
alcohol (ethanol or isopropanol) (98:2) as the mobile phase
eluent showed the following results. Good enantiomeric
separation for compound 3b was achieved on the OD-H
column while compounds 3c and 3f were resolved on the
OJ-H column. Isopropanol improved the separation factor
(a) for all three compounds 3b, 3c and 3f when separated on
Chiralcel OD-H and/or OJ-H columns. This can be clearly
understood from the considerable decrease in retention time
of the analyte when ethanol was used as polar modifier as
compared to 2-propanol, on OD-H and OJ-H columns.
Enantiomeric resolution (a) was higher for isopropanol
(aO1) than for ethanol (aw1 or !1). This improvement in
resolution as the size of the alcohol is increased at constant
composition of mobile phase has been attributed to the
decrease in the capacity of larger alcohols to compete for
hydrogen bonding sites because of steric hindrance.30
Table 3. Ethyl and methyl esters of racemic 2-hydroxy-4-arylbut-3-enoic
acids (1–16), 2-hydroxy-4-arylbutanoic acids (17–20) and 2-hydroxy-3-
arylpropanoic acids (21–22)
S. no.
C. no.a
R
R0
1
2
3
4
3a
3b
3c
3d
H
H
p-Cl
CH3
CH2CH3
CH2CH3
CH3
p-Cl
5
6
7,7a
8
3e
3f
p-Me
p-Me
2,4-Di Cl
2,4-Di Cl
o-Cl
CH3
CH2CH3
CH3
CH2CH3
CH3
CH2CH3
CH3
CH2CH3
CH2CH3
CH3
3g,3g0
3h
3i,3i0
3j,3j0
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
9,9a
10,10a
11
12
13
14
15
16
17
18
19
20
21
22
o-Cl
1-Naphthyl
2,5-OMe
m-NO2
2,5-OMe
m-NO2
p-Clb
p-Cl
CH3
CH2C6H5
CH2CH3
CH3
p-Cl
p-Me
p-Me
Hb
CH3
CH2CH3
CH2CH3
CH2CH3
4. Deracemization of various aryl a-hydroxy esters by
the whole cells of C. parapsilosis ATCC 7330
3v
p-Me
a Compound number.
b Acylated on hydroxy functional group.
4.1. Substrate specificity
The fact that deracemisation can theoretically deliver 100%
Figure 1. Ethyl and methyl esters of racemic 2-hydroxy esters.
Table 4. Retention factors (k0 and k00), separation factor (a) and resolutions (Rs) of racemic a-hydroxy esters (3a–3v)
S.No
C. no.a
Column used
Hex: IPA (ml/min)
a
Rs
5.02
k0
7.02
k00
9.67
6.91
7.58
13.37
12.82
7.92
1
2
3
4
5
6
7
7a
8
3a
3b
3c
3d
3e
3f
3g
3g0
3h
3i
3i0
3j
3j0
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
OD-H
OD-H
OJ-H
OJ-H
OJ-H
OJ-H
OJ-H
OJ-H
OJ-H
OD-H
OD-H
OJ-H
OD-H
OJ-H
OJ-H
OD-H
OJ-H
OD-H
OD-H
OJ-H
OJ-H
OD-H
OJ-H
OD-H
OD-H
98:2; 1 ml
98:2; 1 ml
98:2; 1 ml
98:2; 1 ml
98:2; 1 ml
98:2; 1 ml
96:4; 1 ml
98:2; 1 ml
96:4; 1 ml
98:2; 0.5 ml
99:1; 0.5 ml
98:2; 1 ml
99:1; 1 ml
98:2; 1 ml
95:5; 1 ml
93:7; 0.5 ml
95:5; 1 ml
95:5; 1 ml
95:5; 1 ml
96:4; 0.5 ml
96:4; 0.5 ml
98:2; 1 ml
98:2; 1 ml
95:5; 1 ml
98:2; 1 ml
1.38
1.37
1.15
1.20
1.11
1.12
1.12
1.13
1.10
1.90
1.92
1.22
1.94
1.40
1.22
1.09
1.16
1.08
1.38
1.32
1.39
1.11
1.10
1.15
1.20
4.72
2.75
4.07
2.36
2.11
1.95
2.13
1.26
7.36
9.84
2.76
11.21
2.08
3.01
1.32
3.64
1.52
3.88
3.50
3.92
1.90
1.68
1.56
2.16
5.04
6.57
11.14
11.53
7.14
3.76
9.154
2.29
5.59
8.07
6.27
6.20
7.92
10.68
9.41
13.17
7.28
4.79
3.42
4.85
7.71
4.27
0.86
1.69
4.19
10.2
2.51
9
10.62
15.52
7.59
12.03
11.08
12.98
10.21
15.22
7.83
6.62
4.51
6.75
8.55
9a
10
10a
11
12
13
14
15
16
17
18
19
20
21
22
4.64
0.99
2.08
a C. no., Compound number.

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B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
yield and O99% ee of one enantiomer from a racemate
makes it a very important strategy to prepare chiral
synthons. We have earlier reported the deracemisation of
racemic ethyl 2-hydroxy-4-phenylbutanoate into the corre-
sponding optically pure (S)-2-hydroxy ester by whole cells
of C. parapsilosis ATCC 7330.21 In the present study, the
substrate tolerance (wrt electronic and steric effects of the
aryl substituents) of this biocatalyst was studied. Derace-
misation of substituted p-chloro and p-methyl esters by the
whole cells of C. parapsilosis ATCC 7330 resulted in the
optically pure (S)-enantiomers in good yields (66–70%) and
high ee’s of 91–95% (Scheme 3, Table 5). The time of the
reaction was 1.5 h. Introduction of a chloro-substituent at
the para position in the aromatic ring of 2-hydroxy-4-
phenylbutanoic methyl and ethyl esters (1–4) gave slightly
lower chemical yields (68–69%) as well as ee (93–95%) as
compared to the corresponding unsubstituted substrate.
Presence of electron donating groups such as para methyl
(3–4, Scheme 3) also gave slightly less chemical yield
(66–70%) than for unsubstituted a-hydroxy ester.21 The ee
was slightly lower (91–93%). Incubation times of these
reactions were also 1.5 h.
Scheme 4.
ortho position and the other one at the para position on the
aromatic ring yielded lower chemical and optical yields of the
final product (14b–17b) as compared to monosubstituted
substrates (5–13) possibly due to steric reasons. The polar
substrates (14–17) do seem to lend themselves to the action of
other enzymes resulting in lower yield of the product. The
benzyl ester of 2-hydroxy-4-(p-chlorophenyl)but-3-enoic acid
(19) and the methyl ester of 2-hydroxy-4-(1-naphthyl)but-3-
enoic acid (18) were not deracemised by C. parapsilosis
ATCC 7330. The reason for this could also be steric due to the
bulky groups present in these two esters, that is, benzyl ester
group in the former case and the 1-naphthyl group in the latter
case. Incubation of these two reactions was extended to 4 h at
25 8C without any resulting deracemisation.
4.2. Deracemisation of racemic 2-hydroxy-3-arylpropa-
noic esters (20a–22a) by the whole cells of C. parapsilosis
ATCC 7330
Deracemisation of racemic 2-hydroxy-3-arylpropanoic
esters (20a–22a) resulted in the formation of (S)-2-
hydroxy-3-propanoic esters (20b–22b) (Table 7) using
C. parapsilosis ATCC 7330. At the end of 1 h, only 15%
ee was obtained. The reaction was prolonged to 4 h, which
resulted in 62–70% chemical yield and 34–60% ee
(Scheme 5). In essence it seems that 2-hydroxy 4-phenyl-
butanoic ethyl ester is the substrate, which gives the highest
yield and ee of the deracemised (S)-product. Mandelates are
also deracemised but propionates are not.
Scheme 3.
For the unsaturated compounds 5b and 6b, chemical and
optical yields were 74–75 and 98–99%, respectively. The
time of the reaction was 1.5 h. C. parapsilosis ATCC
7330 was found to deracemise aryl substituted (E)-2-
hydroxy-4-arylbut-3-enoic esters that have electron with-
drawing or electron donating substituents at the ortho,
para and meta positions in the aromatic ring (7b–13b)
(Scheme 4, Table 6) with slightly lower chemical yields
(65–70%) and optical purity ranging from (52–99% ee)
as compared to unsubstituted a-hydroxy esters. Optical
rotation was measured for 5b and compared with the
value reported in literature31 while for compounds 6b, 7b
and 11b; optical rotations were measured after hydro-
genation and compared with those reported in the
literature.32 For compounds 7b, 9b–10b and 12–17b
optical rotations are reported here for the first time.
When the reaction was carried out with di-substituted
compounds such as 2,4-dichloro and 2,5-dimethoxy
compounds (14–17), the ee and chemical yield of the
reaction dropped to 50–65 and 52–72%, respectively.
5. Mechanistic studies
The deracemisation of various racemic aryl a-hydroxy esters
(1b–13b) to the corresponding S-isomer was achieved by
using the whole cells of C. parapsilosis ATCC 7330 in good
chemical yields (65–85%) and with high ee (92–99%) (Tables
5–7). There were few exceptions viz. 14b–17b and 20b–22b
for which the chemical yields were 55–70% and ee were 50–
65% as discussed. Significantly, all the deracemisation
reactions irrespective of the substitution in the aryl group,
chain length and ester group resulted exclusively in the
formationof‘S’isomer. Themechanismofthederacemisation
of a-hydroxy esters can be explained in two different ways
(i) abstraction of the a-methine proton via enolisation
Substrate molecules possessing two substituents, one at the
Table 5. Deracemisation of a-hydroxy esters (1a–4a) by the whole cells of C. parapsilosis ATCC 7330
Entry
R
R1
ee (%)
Yield (%)
Time (h)
[a]2D7
Lit. value
Abs config.
1b
2b
3b
4b
p-Cl
p-Cl
p-Me
p-Me
Me
Et
Me
Et
95
93
91
93
69
68
66
70
1.5
1.5
1.5
1.5
C22.2 (c 0.57 CHCl3)
C17.4 (c 1 CHCl3)
C28.8 (1.05 CHCl3)
C18 (c 0.6 CHCl3)
Nr
K18.6 (c 1.02 CHCl3)a
Nr
Nr
S
S
—
S
a Ref. 32.

B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
12301
(Scheme 6) or a redox mechanism (stereoinversion) mediated
byoxidoreductases(Scheme 7). Thechoiceofethylmandelate
(23) for the mechanistic study using C. parapsilosis ATCC
7330 was based on the easy availability of (R)- and (S)-ethyl
mandelates in sufficient amounts to carry out experiments to
elucidate the mechanism of deracemisation. There are only
two reports in the literature for deracemisation of racemic
mandelic acid.33,34 It is important to note that neither of these
reports has made use of microbial whole cells to bring about
the reaction in one pot.
secondary alcohols.35,36 In order to verify that the mechanism
of deracemisation of racemic a-hydroxy esters proceeds
through stereoinversion, the following experiments were
performed. Optically pure antipodes of ethyl mandelate [(R)-
23a and (S)-23b] were also employed in these experiments.
5.1. Stereoinversion of (R)-ethyl mandelate (23a) using
whole cells of C. parapsilosis ATCC 7330
Stereoinversion of (R)-ethyl mandelate 23a to the (S)-ethyl
mandelate 23b is to be expected in order to obtain the (S)-
enantiomer in high ee and yield. Incubation of (R)-ethyl
mandelate with whole cells of C. parapsilosis ATCC 7330
Stereoinversion seemed the most plausible mechanism for the
deracemisation of a-hydroxy esters as is seen in other
Table 6. Deracemisation of (E)-2-hydroxy-4-arylbut-3-enoic esters (5a–19a) by C. parapsilosis ATCC 7330
Entry
R
R1
ee (%)
Yield (%)
Time (h)
[a]2D7
Lit. value
Abs config.
5b
6b
7b
8b
H
H
p-Cl
p-Cl
p-Me
p-Me
o-Cl
m-NO2
m-NO2
2,4-DiCl
2,4-DiCl
2,5-DiOMe
2,5-DiOMe
1-Naphthyl
p-Cl
Me
Et
Me
Et
Me
Et
Et
Et
Me
Me
Et
Me
Et
98
99
98
98
92
90
95
95
93
50
54
53
65
10
03
74
75
69
65
70
68
68
70
69
70
72
55
52
75
93
1.5
1.5
1.5
1.5
2
2
2
1.5
1.5
4
C67.1 (c 1 CHCl3)
K67.7 (c 1 CHCl3)a
K21.3 (c 1.1 CHCl3)c
S
S
S
S
—
S
S
—
—
—
—
—
—
—
—
C21.3 (c 1.1 CHCl3)b
C55.4 (c 1.25 CHCl3) Nr
C18.9 (c 1 CHCl3)b
C50.5 (c 1.1 CHCl3)
C40.2 (c 1.2 CHCl3)
C13 (c 0.75 CHCl3)b
C46 (c 0.51 CHCl3)
C55 (c 0.63 CHCl3)
C15 (c 0.75 CHCl3)
C11 (c 1.1 CHCl3)
C13 (c 1.3 CHCl3)
C9 (c 1.1 CHCl3)
—
K18.6 (c 1.02 CHCl3)c
Nr
Nr
K12.4 (c 0.70 CHCl3)c
Nr
Nr
Nr
Nr
Nr
Nr
—
—
9b
10b
11b
12b
13b
14b
15b
16b
17b
18bd
19b
4
4
4
4
Me
CH2 Ph
4
—
Nr, not reported.
a Ref. 31.
b Optical rotation was taken after hydrogenation.
c Ref. 32.
d Aryl, 1-naphthyl.
Table 7. Deracemisation of 2-hydroxy-4-arylpropanoic esters (20a–22a) by the whole cells of C. parapsilosis ATCC 7330
Product
R
R1
Time (h)
ee (%)
Yield (%)
Abs config.
20b
21b
22b
H
Et
Me
Et
4
4
4
60
34
45
70
65
62
S
Nr
S
2,4-DiCl
p-Me
Nr, not reported.
Scheme 7.
Scheme 5.
resulted in complete conversion to the (S)-enantiomer (99%
ee). The reaction was carried out for 1 h at 25 8C (Scheme 8).
Scheme 6.
Scheme 8.

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B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
Figure 2. HPLC chromatogram of stereoinversion of RS-23a using C. parapsilosis ATCC 7330.
Figure 3. HPLC chromatogram of stereoinversion of R-23a into S-23b using C. parapsilosis ATCC 7330.
Having established that the (R)-enantiomer (R-23a) is
converted to the (S)-enantiomer (S-23b), the time course
of the above reaction was determined in order to
trap any intermediate, which may be formed. Aliquots
of reaction mixture were monitored by HPLC on a Sil
C-18 column. The formation of a keto intermediate was
detected at 30 min (Fig. 2). The conversion of the
R-23a to the S-23b was followed by HPLC (Fig. 3)
using a Chiralcel OD-H column.
Incubation of (S)-23b with C.parapsilosis ATCC 7330 gave
unchanged starting material (S)-23b in 95% yield and 99%
ee (Scheme 9) which was confirmed by chiral HPLC (Fig. 4)
using a Chiralcel OD-H column.

B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
12303
5.2. Asymmetric reduction of ethyl benzoylformate (24)
Since the keto intermediate was detected during the
stereoinversion of the (R)-23a isomer to (S)-23b, the
reduction of this keto intermediate (24) viz. ethyl benzoyl
formate prepared synthetically was attempted using
C. parapsilosis ATCC 7330.
Scheme 9.
Figure 4. HPLC chromatogram of biotransformation of S-23b after 1 h using C. parapsilosis ATCC 7330.
Figure 5. HPLC chromatogram of asymmetric reduction of keto ester 24 using C. parapsilosis ATCC 7330.

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B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
The reduction of ethyl benzoylformate (24) to the expected
product ethyl mandelate (23b) was monitored by HPLC on a
Sil-C18 column (Fig. 5). The absolute configuration of ethyl
mandelate thus obtained was established by comparing with
standard using HPLC details of which are given in Section
7.4.2. (S)-ethyl mandelate (23b) was obtained in 65%
chemical yield and 99% ee (Scheme 10).
oxidation of one of the antipodes by an (R)-oxidase to the
keto intermediate followed by its subsequent reduction by a
(S)-specific reductase enzyme.
7. Experimental
7.1. General methods
HPLC analysis was done on a Jasco PU-1580 liquid
chromatograph equipped with a manual injector (20 ml)
and a PDA detector. The columns used were Chiralcel OD-H
and Chiralcel OJ-H (Daicel, 4.6!250 mm). The enantio-
meric excesses (% ee) of 1b–23b were determined by HPLC
analysis. The eluent used was hexane–isopropanol (98/2) at
a flow rate of 1 ml minK1 and the absorbance monitored
using a PDA detector at 254 nm. Optical rotations were
determined on a Jasco Dip 370 digital polarimeter. TLC was
done on Kieselger 60F 254 aluminium sheets (Merck
1.05554).
Scheme 10.
In addition to the above experiments, appropriate controls
were also carried out. The control experiments were (a) only
cells of C. parapsilosis ATCC 7330 and (b) only substrate
[i.e., (RS)-23, (R)-23a and (S)-23b] under identical
conditions as done for the experiments 5.1 and 5.2. These
experiments confirmed that (R,S)-ethyl mandelate is
deracemised by C. parapsilosis ATCC 7330 to (S)-ethyl
mandelate possibly by a combination of two enzymes, (R)-
specific oxidase and (S)-specific reductases (Scheme 11).
Given this mechanism, the observation that propionates are
not deracemised by C. parapsilosis ATCC 7330 may be
attributed to the preponderance (50–80%) of the enol form
in the unsubstituted, p-methyl and 2,4-diCl arylpropionic
a-keto esters, which cannot be substrates for the (S)-specific
reductases.
7.2. Synthesis of substrates
7.2.1. Preparation of alkyl (E)-2-oxo-4-arylbut-3-enoates
(2a–2n). Typical reaction procedure of compound 2d.
Esterification of 2-oxo-4-(p-chlorophenyl)but-3-enoic acid
(5 mmol, 950 mg) was carried out with ethanol (5 ml) in the
presence of phosphotungstic acid (95 mg) as catalyst in dry
toluene (10 ml) for 8 h at 95 8C. After esterification was
complete, toluene and excess ethanol were removed.
Compound 2d was obtained as a yellow colour solid in
78% yield (850 mg, 3.9 mmol) after column purification
using hexane–ethyl acetate (95/5) as eluent.
The same procedure was followed for compounds (2a–c and
2e–1n) (Scheme 1, Table 1).
7.2.2. Preparation of alkyl (E)-2-hydroxy-4-arylbut-3-
enoates (4a–4n). Typical reaction procedure of compound
4d. Esterification of 2-hydroxy-4-(p-chlorophenyl)but-3-
enoic acid (3 RZp-Cl) (5 mmol, 960 mg) was carried out
with ethanol (5 ml) in the presence of phosphotungstic acid
as catalyst (96 mg) in dry toluene (10 ml) for 9 h at 95 8C.
After esterification, toluene and excess ethanol were
removed by evaporation. Upon column purification,
compound 4d was obtained as a white solid in 73% yield
(803 mg, 3.65 mmol).
Scheme 11.
The same procedure was followed for the rest of the
compounds (4a–c and 4e–4n) and the yields ranged from
44–85% (Table 1, Scheme 1).
6. Conclusion
A series of racemic aryl and aryl substituted a-hydroxy
esters were synthesized by modifying known literature
methods to obtain good yields. The racemic esters syn-
thesized were resolved by chiral HPLC on chiral columns
OD-H and OJ-H using hexane and IPA as eluents. Subse-
quently, the racemic aryl a-hydroxy esters sythesised were
deracemised by the whole cells of C. parapsilosis ATCC
7330 to give the corresponding optically pure (S)-hydroxy
esters in good yields (65–85%) and ee (90–99%). To
elucidate the mechanism of deracemisation, a detailed study
was carried out on deracemisation of mandelic ester and was
found to involve two different enzymatic reactions viz.
7.2.3. Preparation of alkyl 2-hydroxy-3-arylpropanoates
(4a–f,h). Typical reaction procedure for compound
p-methyl benzylidine-2-methyl oxazol (4H)-5-ones (1h). A
mixture of N-acetyl glycine (10 mmol, 1.15 g) and
p-methylbenzaldehyde (10 mmol, 1.19 g) in acetic anhy-
dride (40 mmol, 4.0 g) was taken in a 100 ml Erlenmeyer
conical flask. The conical flask containing the reaction
mixture was uniformly mixed using a vortex mixer and then
the reaction mixture was irradiated using a microwave oven
for 4 min. On cooling, the reaction solidified. The residue
was washed with ethanol/water 50:50 mixture and dried

B. Baskar et al. / Tetrahedron 61 (2005) 12296–12306
12305
under vaccum. The product (1h) was obtained in 90% yield
(8.9 mmol, 1.57 g) as a pale yellow solid. Compounds 1a–g
and 1i–k were prepared by following the procedure
described above for compound 1h (Scheme 2) and the
yields obtained are given in Table 2.
methyl (S)-2-hydroxy-4-(p-chlorophenyl)butanoate (1b),
was 69% (207 mg, 0.97 mmol).
7.4. Mechanistic studies
7.4.1. Stereoinversion of 23a using whole cells of
C. parapsilosis ATCC 7330. To a test tube containing
0.3 g of pelleted C. parapsilosis ATCC 7330 cells sus-
pended in sterile distilled water, 1.5 mg of 23a dissolved in
40 ml of ethanol was added. The total volume of the reaction
mixture was made up to 1 ml by adding sterile distilled
water. The reaction was carried out in a water bath shaker at
150 rpm and 25 8C for 1 h. After incubation, the reaction
mixture was centrifuged at 5000 rpm for 10 min. The pro-
duct formed was isolated using ethyl acetate (3!40 ml) and
the organic layer dried over anhydrous sodium sulfate. The
solvent was removed by evaporation and the ee was found to
be 99% as determined using HPLC on a Chiralcel OD-H
column [hexane–isopropanol (99/1)].
Typical reaction procedure for compound (3h). The sodium
salt of (p-methylphenyl)pyruvic acid 2h (10 mmol, 2 g) in
water (2 ml) was cooled to 5–10 8C and sodium borohydride
(15 mmol, 540 mg) was added portionwise. The reaction
mixture was stirred for 2 h at 5–10 8C. A foamy white
precipitate appeared upon neutralization with dil HCl,
which was extracted with ethyl acetate (4!20 ml). The
combined layers of ethyl acetate were dried over anhydrous
sodium sulfate and on evaporation of solvent, resulted in the
hydroxy acid (3h) as a colourless liquid in 87% yield
(8.7 mmol, 1.566 g). Compounds 3a–e were also prepared
by the method described above for compound 3h (Scheme 2)
and the yields obtained are given in Table 2.
Typical reaction procedure for compound 4h. Esterification of
2-hydroxy-3-(p-methylphenyl)propanoic acid (3h) (5 mmol,
900 mg) was carried out with ethanol (5 ml) in the presence of
phosphotungstic acid (90 mg) as catalyst in dry toluene
(10 ml) for 6 h at 95 8C. After completion of reaction, toluene
and excess ethanol were removed to afford compound 4h as a
colourless liquid in 85% yield (884 mg, 4.376 mmol) after
column purification using hexane and ethyl acetate (95:5) as
the mobile phase eluent. Compounds 4a–e were prepared by
following the procedure described above for compound 4h
(Scheme 2) and the yields obtained are given in Table 2.
The same procedure was adapted for compound 23b.
7.4.2. Asymmetric reduction of 24 using whole cells of
C. parapsilosis ATCC 7330. To a 500 ml conical flask
containing 30 g of pelleted C. parapsilosis ATCC 7330 cells
suspended in sterile distilled water, 150 mg (0.842 mmol) of
24 dissolved in 3.6 ml of ethanol was added. The total
volume of the reaction mixture was made up to 100 ml by
adding sterile distilled water. The reaction was carried out in
a water bath shaker at 150 rpm and 25 8C for 1 h. After
incubation, the reaction mixture was centrifuged at
5000 rpm for 10 min. The product formed was isolated
using ethyl acetate (3!40 ml) and the organic layer dried
over anhydrous sodium sulfate. The solvent was removed by
evaporation and 23b was obtained as a low melting solid
after purification by silica gel column chromatography
using hexane–ethyl acetate (98/2) as a mobile phase eluent.
The product was characterized by 1H and 13C NMR
(400 MHz) spectroscopy. The ee was found to be 99% as
determined using HPLC on a Chiralcel OD-H column
[hexane–isopropanol (98/2)]. The yield of the isolated
product, (S)-ethyl mandelate 23b was 65% (98 mg,
0.64 mmol). The absolute configuration of (S)-enantiomer
23b was confirmed by comparing with standards (RS)-23,
(R)-23a and (S)-23b using Chiral HPLC on a Chiralcel OD-H
column [hexane–isopropanol (98/2)]. The retention times of
(R)-23a and (S)-23b were 20.41 and 10.99 min, respectively.
7.3. Deracemisation of a-hydroxy esters
7.3.1. Culture medium of the microorganism. The yeast,
C. parapsilosis (ATCC 7330) was grown in YMB medium
(60 ml) in 250 ml Erlenmeyer flasks. The flasks were
incubated at 25 8C with a shaking speed of 200 rpm for 40 h.
The cells were pelleted down by centrifugation at 3214!g
for 15 min, washed with distilled water and used for the
biotransformation reaction.
7.3.2. Typical procedure for biotransformation of 1
using free cells of C. parapsilosis ATCC 7330. To a 500 ml
conical flask containing 30 g of pelleted C. parapsilosis
ATCC 7330 cells suspended in sterile distilled water,
300 mg (1.41 mmol) of methyl 2-hydroxy-4-(p-chlorophenyl)
butanoate (1) dissolved in 7.5 ml of ethanol was added. The
total volume of the reaction mixture was made up to 100 ml
by adding sterile distilled water. The reaction was carried
out in a water bath shaker at 150 rpm and 25 8C for 1.5 h.
After incubation, the reaction mixture was centrifuged at
5000 rpm for 10 min. The product formed was isolated
using ethyl acetate (3!40 ml) and the organic layer dried
over anhydrous sodium sulfate. The solvent was removed by
evaporation and 1b was obtained as a pale yellow solid after
purification by silica gel column chromatography using
hexane–ethyl acetate (98/2) as a mobile phase eluent. The
product was characterized by 1H and 13C NMR (400 MHz)
spectroscopy. The ee was found to be 95% as determined
using HPLC on a Chiralcel OJ-H column [hexane–
isopropanol (96/4)]. The yield of the isolated product,
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