Deracemisation of aromatic β-hydroxy esters using immobilised whole cells of Candida parapsilosis ATCC 7330 and determination of absolute configuration by 1H NMR

Article abstract of DOI:10.1016/j.tetasy.2005.07.017

Deracemisation of aryl and substituted aryl β-hydroxy esters using immobilised whole cells of Candida parapsilosis ATCC 7330 yields the corresponding (S)-enantiomer in >99% enantiomeric excess and good yield (up to 68%). The absolute configuration of ethyl 3-(2,4-dichlorophenyl)-3-hydroxy propanoate and ethyl 3-hydroxy-5-phenyl-pent-4-enoate as determined by ¹H NMR using MTPA chloride was found to be 'S'. The chemical shifts of the methoxy groups of the two diastereomeric MTPA esters were used as diagnostic signals to determine the absolute configuration.

Full text of DOI:10.1016/j.tetasy.2005.07.017

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 2790–2798
Deracemisation of aromatic b-hydroxy esters using
immobilised whole cells of Candida parapsilosis ATCC 7330
1
and determination of absolute configuration by H NMR
Santosh Kumar Padhi and Anju Chadha*
Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology, Madras, Chennai 600036, India
Received 23 June 2005; accepted 14 July 2005
Available online 15 August 2005
Abstract—Deracemisation of aryl and substituted aryl b-hydroxy esters using immobilised whole cells of Candida parapsilosis ATCC
7330 yields the corresponding (S)-enantiomer in >99% enantiomeric excess and good yield (up to 68%). The absolute configuration
of ethyl 3-(2,4-dichlorophenyl)-3-hydroxy propanoate and ethyl 3-hydroxy-5-phenyl-pent-4-enoate as determined by 1H NMR using
MTPA chloride was found to be ÔSÕ. The chemical shifts of the methoxy groups of the two diastereomeric MTPA esters were used as
diagnostic signals to determine the absolute configuration.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
enantiomeric excess of the product can also be achieved
by kinetic resolution,10 which is a widely used method. A
maximum of 50% yield of each enantiomer and the
unwanted enantiomer is the limitation of kinetic reso-
lution. An alternative enantiomerically pure product
in high ee and quantitative yield is deracemisation.12
The use of isolated enzymes13 for the deracemisation of
mandelic acid and a two-biocatalytic system14 for the
stereoinversion of L-pantoyl lactone to its D-form are
known. However, a single biocatalyst (whole cells) medi-
ated deracemisation is more advantageous. Backvall and
Huerta15 used a ruthenium-catalyst along with Pseudo-
monas cepacia lipase in a chemo-enzymatic deracemisa-
tion of b-hydroxy esters. Deracemisation of ethyl
3-hydroxy butanoate using aged cultures of Geotrichum
candidum resulted in the corresponding (R)-enantiomer
in 96% ee and 80% yield.16 Nakamura et al.17 reported
the stereoinversion of methyl 3-hydroxy butanoate
and methyl 3-hydroxy pentanoate using G. candidum
IFO 5767 to produce the corresponding (R)-enantiomer
in 97–99% ee and 26–48% isolated yield. The deracemisa-
tion of b-hydroxy esters is restricted to the above
examples, both of which use the same microbe, G. candi-
dum to give the deracemised product, the (R)-antipode.
Enantiopure b-hydroxy esters are important chiral syn-
thons1 and are widely used in the preparation of many
pharmaceutical intermediates such as L-carnitine,2 fluox-
etine3 and inhibitors of HMG-CoA reductase4 among
others. In addition to routine chemical methods5 for
the synthesis of enantiomerically pure b-hydroxy esters,
biocatalytic methods have been developed to a great
advantage.6–8 Biocatalytic approaches for the synthesis
of these target molecules broadly involve (a) an asym-
metric reduction6–9 and (b) a kinetic resolution.10 Both
these methods can be carried out by commercially avail-
able enzymes and microbial whole cells. In the case of
asymmetric reduction, the use of free whole cells is pre-
ferred over isolated enzymes in order to avoid the use
of expensive cofactors and the immobilised whole cells
are preferred over free cells as they are more robust
and can be reused. The use of whole cells often results
in the competitive action of multiple enzymes on the sub-
strate molecules, which reduces the enantiomeric excess
of the desired product. A solution to this problem was
reported by Stewart et al. who engineered whole cells
of Escherichia coli by expressing an oxido-reductase from
bakerÕs yeast for the reduction of b-ketoesters.11 High
The activity of oxidoreductases in Candida parapsilosis
is well illustrated by Kula et al.8 Different strains of this
species were later used for the deracemisation of 1,2-
diols,18 1,3-diols19 and a-hydroxy esters.20 We have
*
Corresponding author. Tel.: +91 44 2257 4106; fax: +91 44 2257
4202; e-mail: anjuc@iitm.ac.in
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.07.017

S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
2791
previously reported the deracemisation of some b-hy-
droxy esters using free cells of C. parapsilosis ATCC
7330.21 Bearing in mind the advantages of using immo-
bilised biocatalysts,22 that is, easy separation, reusability
of the cells, downstream processing, use of higher cell-
concentration and stabilisation of several cell functions,
we herein report the use of immobilised cells of C. par-
apsilosis ATCC 7330 for the deracemisation of a variety
of b-hydroxy esters, thus illustrating the versatility of
this biocatalyst. Experimental unequivocal proof that
the (S)-enantiomer is formed on deracemisation is also
included in this report.
and 42% yield. Apart from the above biocatalytic meth-
ods, asymmetric reduction using chiral catalysts are also
reported. An (o-BINAPO)-Ru catalysed asymmetric
hydrogenation of b-aryl and b-substituted aryl b-keto
esters gave the corresponding (R)-enantiomer in 90–
98% ee in 20 h reaction time, at 50 ꢁC and 80 psi of
H2.5a A catalytic combination of (1S,2R)-ephedrine
and a Ru-complex, for the asymmetric reduction of
ethyl benzoylacetate and its aryl-substituted derivatives
resulted in the corresponding (S)-enantiomers in 99%
yield and 72–94% ee.25 Another Ru (biaryl phosphine
ligands) catalysed asymmetric hydrogenation of b-aryl b-
keto esters gave 95–98% ee and 100% conversion in 20 h
reaction time, at 65 ꢁC and 30 atm of H2.26 The other
synthetic methods reported have also used harsh reac-
tion conditions and different metal catalysts. Substrates
having different para-substituents (Table 1, entries 3, 4
and 5) on deracemisation gave 98–99% ee and 41–51%
isolated yield of their corresponding (S)-enantiomers.
Irrespective of the electronic nature of the substituents
(methyl, chloro and nitro), the ee of the deracemised
product remained very high (98–99%). The standard
substrate with an ortho substituent (Table 1, entry 2)
showed a poor ee (9%) of the product. The deracemisa-
tion of b-hydroxy esters by C. parapsilosis ATCC 7330
possibly follows a stereoinversion mechanism,27 that is,
enantioselective oxidation of one antipode to the keto-
ester intermediate followed by a complementary enan-
tioselective reduction of the intermediate ketoester to
give a single enantiomer in high yield and ee. The poor
ee in the case of ethyl 3-hydroxy-3-(2-methylphenyl)
propanoate could be due to the steric hindrance and
the hydrophobic nature of the ortho-substituent to the
reaction centre. Ethyl 3-(2,4-dichlorophenyl)-3-hydroxy
propanoate (Table 1, entry 6) on deracemisation gave
82% ee and 53% yield of its (S)-enantiomer. The abso-
lute configuration of this molecule as determined by
1H NMR using MTPA chloride is discussed here in
detail. Interestingly, the ee of this o,p-disubstituted sub-
strate (entry 6) was found to be slightly lower than the
para-substituted substrates (entries 3, 4 and 5) and much
higher than the ortho-substituted substrate (entry 2).
The hydrophilic nature of the chloro substituent possi-
bly favours deracemisation as opposed to the steric
effect at the ortho-position in the case of substrate 6.
Deracemisation of ethyl 3-hydroxy-5-phenyl-pent-4-
enoate (Table 1, entry 7) resulted in >99% ee and 28%
yield of its (S)-enantiomer, the absolute configuration
of which was also determined by 1H NMR using MTPA
2. Results and discussion
2.1. Deracemisation of racemic b-aryl and b-substituted
aryl b-hydroxy esters
Deracemisation of aromatic b-hydroxy esters with
immobilised whole cells of C. parapsilosis ATCC 7330
(ICp) was standardised using ethyl 3-hydroxy-3-phenyl
propanoate as the standard substrate. The (S)-enantio-
mer of the product was obtained in 99% ee and 57%
yield. To prove the generality of this deracemisation
reaction with respect to the substrate structure, a variety
of aryl substituted b-hydroxy esters were deracemised
using ICp (Scheme 1), which resulted in the formation
of their (S)-enantiomers in >99% ee and up to 68% yield,
irrespective of the nature of substituents on the aromatic
ring. Kinetic resolution of ethyl 3-hydroxy-5-phenyl-
pent-4-enoate (Table 1, entry 7) by Pseudomonas sp.
gave 30% conversion and 98% ee of the (R)-enantio-
mer.23 Recently, Xu and Yuan24 reported the Candida
rugosa catalysed kinetic resolution of b-hydroxy-b-aryl-
propionates, which also gave the (R)-enantiomer of the
b-hydroxy esters. Using this method, ethyl 3-(2,4-
dichlorophenyl)-3-hydroxy propanoate was obtained in
93.7% ee and 46% yield, ethyl 3-hydroxy-3-(4-nitro-
phenyl) propanoate in 91.6% ee and 43% yield and ethyl
3-hydroxy-3-(4-methylphenyl) propanoate in 94.5% ee
Immobilized
OH
O
OH O
Candida parapsilosis
ATCC 7330
25 oC, 6 h
R
O
R
O
*
(S)
Scheme 1.
Table 1. Immobilised Candida parapsilosis ATCC 7330 mediated deracemisation of aryl and aryl substituted b-hydroxy esters
25
Entry
R
ee (%)
Yield (%)
½aꢀD
Abs Conf.
1
2
3
4
5
6
7
Ph
>99
09
98
99
99
57
68
51
42
41
53
28
ꢁ 50.1 (c 1.5, CHCl3)5a
Sa
Nd
Sa
Sa
Sa
Sb
Sb
o-MePh
p-MePh
p-ClPh
p-NO2Ph
o,p-ClPh
PhCH@CH
Nd
ꢁ44.6 (c 1.2, CHCl3)5a
ꢁ43.7 (c 1.38, CHCl3)5a
ꢁ59.5 (c 1.5, CHCl3)24
ꢁ39.8 (c 1.5, CHCl3)
ꢁ2.57 (c 2.7, CHCl3)
82
>99
ee: Enantiomeric excess measured by HPLC; Nd: not determined.
a The absolute configurations were assigned on the basis of the sign of rotation.5a,24
b The absolute configurations were assigned by 1H NMR using MTPA chloride.

2792
S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
chloride. The hydrolysed by-product (3-hydroxy-5-phen-
yl-pent-4-enoic acid) formed during the deracemisation
of substrate 7 possibly due to the action of a hydrolase,
accounts for the low yield of the product. The low yield
of the deracemised product is also because the oxidised
product which is a b-keto ester intermediate is hydroly-
sed and easily decarboxylated. Another deracemisation
experiment was carried out starting with (R)-ethyl 3-
hydroxy-3-phenyl propanoate. Chiral HPLC analysis
of the product mixture indicates the inversion of config-
uration from (R)-1 to (S)-1.27 It is noteworthy that the
use of different strains of C. parapsilosis reported so
far for deracemisation reactions were limited to sub-
strates with a single functional group, except in the case
of a-hydroxy esters reported from our laboratory.20
method was reported.32 This method involves the deri-
vatisation of the starting substrate with (R)- and (S)-
MTPA chloride followed by the separation of the
1
formed diastereomers and their characterisation by H
NMR. Herein, we report the assignment of the absolute
configuration of deracemised b-hydroxy esters using a
single enantiomer of MosherÕs acid chloride.
The validity of MosherÕs method for determining the
absolute configuration of aryl b-hydroxy esters was first
established by using known enantiomers of standard
substrate-1 viz. ethyl 3-hydroxy-3-phenyl propanoate.
Racemic-1, (R)-1 and (S)-1 were treated with (S)-MTPA
chloride (Scheme 2) to give the corresponding MTPA
esters that is, MTPA esters of (S,R)- and (R,R)-1,
(R,R)-1 and (S,R)-1, respectively. The two diastereomers
of MTPA ester of (S,R)- and (R,R)-1 are represented in
2.2. Assignment of the absolute configuration of the
deracemised products 6 and 7
1
Figure 1a and b and the H NMR of the three MTPA
esters of (RS)-1, (R)-1 and (S)-1 are represented in
The absolute configuration of substrates 1 and 3–5 was
assigned based on the sign of specific rotation of their
deracemised products in comparison with the literature
values.5a,24 Ethyl 3-(2,4-dichlorophenyl)-3-hydroxy pro-
panoate 6 and ethyl 3-hydroxy-5-phenyl-pent-4-enoate 7
were the two substrates, which on deracemisation gave
82% and >99% ee, respectively, whose specific rotations
are not reported in the literature and had to be deter-
mined. Several methods were used for the determination
of absolute configuration of secondary alcohols, for
example, X-ray crystallography (for which a crystalline
sample is a prerequisite28), and NMR-techniques among
others.29 Assignment of the absolute configuration of b-
hydroxy esters have also been reported using X-ray crys-
tallography28b and 1H NMR characterisation of MTPA-
derivatives—directly30 or with lanthanide shift re-
agents.31 The secondary alcohol group of the b-hydroxy
ester was derivatised with a chiral auxiliary of already
known absolute configuration.30 The first and most
accepted derivatising chiral compound for secondary
alcohols was MTPA or a-methoxy-a-(trifluoromethyl)
phenylacetic acid, also known as MosherÕs acid.29b
Recently, the assignment of the absolute configuration
of deracemised a-hydroxy esters using the MosherÕs
Figure 2a–c, respectively.
According to MosherÕs method, the chemical shift differ-
ence between any two similar protons of the b-hydroxy
ester moiety (preferably the methylene protons for the
present case) of the two diastereomers (Fig. 1a and b)
are used to assign the absolute configuration. It can be
1
inferred from (the H NMR spectra) Figure 2a–c that
the chemical shifts of the methylene protons of MTPA
esters of racemate-1 and that of a single enantiomer
[(R) or (S)] are almost identical. However, closer
examination of the 1H NMR of these diastereomers
more shielded
O
less shielded
MeO
MeO
O
O
O
CF3
CF3
H
H
O
O
O
O
MTPA plane
(b) (R,R)-MTPA-1
(a) (S,R)-MTPA-1
Figure 1. MTPA esters of (S,R)- and (R,R)-1.
CF3
MeO
O
Ph
OMe
O
O
Cl
DMAP, CH2Cl2
1.5 h, rt
CF3
Ph
(R or S)
n
R
+
O
O
OH
O
O
n
R
(R,R or S,R or R,S or S,S)-MTPA esters
(R or S or RS)
R=H
n=0,
n=0,
n=0,
(RS)-1
R=H
R=H
R=H
n=0,
n=0,
n=0,
MTPA -(SR and RR)-1
MTPA -(R,R)-1
R=H
R=H
(R)-1
(S)-1
(RS)-6
6
MTPA -(S,R)-1
R=o,p-diCl, n=0,
R=o,p-diCl n=0,
R=H
R=H
R=o,p-diCl, n=0,
R=o,p-diCl n=0,
MTPA -(S,S and R,S)-6
MTPA -6
n=2 (CH2=CH2), (RS)-7
n=2 (CH2=CH2),
R=H
R=H
n=2 (CH2=CH2), MTPA -(S,S and R,S)-7
n=2 (CH2=CH2), MTPA -7
7
Scheme 2.

S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
2793
Figure 2. 1H NMR of –OCH3 and methylene in (a) MTPA-(S,R)- and (R,R)-1, (b) MTPA-(R,R)-1 and (c) MTPA-(S,R)-1.
Table 2. Chemical shift (d)-values in the 1H NMR of MTPA esters of (RS)-1, (R)-1 and (S)-1 from Figure 2a–c
Entry
Product—Mosher ester
Abs Conf.
d-values
Methylenea
OMe
MeO
O
MTPA-(S,R) and (R,R)-1
(R,R) and (S,R)
3.41 d
3.51 d
2.73–2.78 and 3.00–3.07 d
CF3
H
O
O
O
O
MeO
O
O
MTPA-(R,R)-1
MTPA-(S,R)-1
(R,R)
(S,R)
3.42 d
—
2.73–2.78 and 3.00–3.06 d
CF3
H
O
MeO
O
—
3.51 d
2.73–2.78 and 3.00–3.07 d
CF3
H
O
O
O
a No difference in the chemical shift values was observed for all the above three cases.
[MTPA esters of (S,R)- and (R,R)-1, Fig. 1a and b]
shows that the chemical shift difference between the
methoxy signals of the two diastereomers is more signif-
icant than any other set of protons (Table 2).

2794
S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
This chemical shift difference of the methoxy signals is
because in one structure, the methoxy group and the
phenyl ring of the b-hydroxy ester are on the same side
of the MTPA plane29a (Fig. 1b), while in the other dia-
stereomer they are opposite to each other (Fig. 1a). The
methoxy signals for the two diastereomers (Fig. 1a and
b) appeared at 3.41 d and 3.51 d (Fig. 2a). The dia-
magnetic effect of the phenyl ring of the b-hydroxy ester
is more pronounced in the structure where the methoxy
group is on the same side of the phenyl ring (3.41 d,
shielded) as compared to the structure where the phenyl
ring of the b-hydroxy ester and the methoxy group
are on opposite sides of the MTPA plane (3.51 d,
deshielded). Consequently, the 3.41 d methoxy signal
should belong to the diastereomer (R,R)-MTPA-1
(Fig. 1b) and 3.51 d should belong to the diastereomer
(S,R)-MTPA-1 (Fig. 1a). This chemical shift difference
of the methoxy signals is thus used to assign the absolute
configuration of substrate 1 and subsequently for sub-
strates 6 and 7—a fact borne out by experiments carried
out using (S)-MTPA chloride with (R)-1 (authentic
sample, >99% ee), which shows the methoxy signal at
3.42 d (Fig. 2b) and (S)-MTPA chloride with standard
(S)-1 (authentic sample, 87% ee), which shows the meth-
oxy signal at 3.51 d (Fig. 2c). Thus, b-aryl-b-hydroxy
propanoates lend themselves to this special observa-
tion where the chemical shift values of the methoxy
signals can be used to determine the absolute
configuration.
also prepared using the (R)-MTPA chloride with the
deracemised products of 6 and 7 (Scheme 2). The H
1
NMR analysis (Table 3) of Figure 4a shows a difference
in the chemical shift between the two methoxy signals of
MTPA-(R,S)- and (S,S)-6 (Fig. 3a and b), which
appears at 3.46 d and 3.55 d, respectively. The shielded
methoxy signal (3.46 d) belongs to the diastereomer in
which the methoxy group and the aromatic ring of the
b-hydroxy ester lie on the same side that is, MTPA-
(S,S)-6 (Fig. 3a). The 3.55 d methoxy signal belongs to
the MTPA-(R,S)-6 (Fig. 3b). The methoxy signal for
MTPA-6 prepared as shown in Scheme 2 appears at
3.46 d (Fig. 4b) (shielded region) and matches the meth-
oxy group of MTPA-(S,S)-6 (Fig. 4a). This confirms
that MTPA-6 has the same configuration as that of
MTPA-(S,S)-6 and the unknown pure enantiomer 6
prepared from the deracemisation reaction (Table 1) is
the (S)-enantiomer.
In the diastereomers of MTPA-(S,S)- and (R,S)-7
(Fig. 5a and b) the methoxy signals appeared at 3.52 d
and 3.56 d (Fig. 6a and Table 3). This chemical shift dif-
ference between the methoxy signals is less because of
the increased distance of the phenyl ring of the b-hydr-
oxyl ester from the methoxy group in MTPA-(S,S)- and
(R,S)-7 as compared to the corresponding esters of 1
and 6. The shielded methoxy signal (3.52 d) should
belong to the diastereomer in which the methoxy group
and the phenyl ring of the b-hydroxy ester lie on the
same side, that is, MTPA-(S,S)-7 (Fig. 5a). In addition
to these diagnostic signals, a difference in the chemical
shift of the olefinc protons was also observed for both
the diastereomers (Fig. 5a and b), which was used in
characterising the individual diastereomers (Fig. 5a
and b). The Ha signals appeared at 6.03–6.1 and 6.17–
6.23 d while the Hb signals appeared at 6.64–6.69 and
6.77–6.81 d (Fig. 6a and Table 3) indicating that one
pair of Ha and Hb are shielded (6.03–6.1 d and 6.64–
6.69 d) as compared to the other pair, that is, Ha
(6.17–6.23 d) and Hb (6.77–6.81 d) (Fig. 6a). According
to MosherÕs method this shielding is due to the diamag-
netic effect of the phenyl ring of the MosherÕs ester and
hence, the shielded pair should belong to the diastereo-
mer, where Ha and Hb of the b-hydroxy ester and the
phenyl ring of the MosherÕs ester lie on the same side,
that is, MTPA-(R,S)-7 (Fig. 5b) and the deshielded pair,
Ha and Hb 6.17–6.23 d and 6.77–6.81 d belong to the
Having proved the absolute configuration of the known
enantiomers of (R)-1 and (S)-1, this method was used to
assign the absolute configuration of the enantiomerically
pure unknown enantiomers of 6 and 7 obtained after
deracemisation. The MTPA-(S,S)- and (R,S)-6 and
MTPA-(S,S)- and (R,S)-7 were prepared from (R)-
MTPA chloride with (R,S)-6 and (R,S)-7, respectively
(Scheme 2, Figs. 3 and 5). MTPA-6 and MTPA-7 were
more shielded
O
Cl
OMe
O
O
OMe
CF3
O
H
Cl
less shielded
O
CF3
Cl
O
1
H
diastereomer, that is, MTPA-(S,S)-7 (Fig. 5a). The H
O
Cl
NMR analysis of MTPA-7 revealed that the methoxy
signal appeared at 3.52 d, the Ha signal at 6.17–6.23 d
and the Hb signal at 6.77–6.81 d (Fig. 6b), which exactly
matches that of MTPA-(S,S)-7 (Fig. 5a) as discussed
O
(R,S)-MTPA-6 (b)
(S,S)-MTPA-6 (a)
Figure 3. MTPA esters of (S,S)- and (R,S)-6.
Table 3. Chemical shift (d)-values in the 1H NMR of MTPA esters of (R,S)-6 and 6 from Figure 4a and b and MTPA esters of (R,S)-7 and 7 from
Figure 6a and b
Entry
Abs Conf.
d-values
OMe OMe Methylene
MTPA-(S,S)- and (R,S)-6 (S,S) and (R,S) 3.46 d 3.55 d 2.76–2.91 d
MTPA-6 (unknown) (S,S) or (R,S) 3.46 d 2.79–2.91 d
Hb
Ha
—
—
—
—
—
MTPA-(S,S)- and (R,S)-7 (S,S) and (R,S) 3.52 d 3.56 d 2.68–2.75 and 2.81–2.9 d 6.64–6.69 and 6.77–6.81 d 6.03–6.1 and 6.17–6.23 d
MTPA-7 (unknown) (S,S) or (R,S) 3.52 d 2.68–2.73 and 2.81–2.87 d 6.77–6.81 d 6.17–6.23 d
—

S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
2795
Figure 4. 1H NMR of –OCH3 and methylene in (a) MTPA (S,S)- and (R,S)-6 and (b) MTPA-6.
MTPA chloride was found to be S. The chemical shift
difference between the methoxy signals of the two diaste-
reomers of the MTPA esters (substrates 1, 6 and 7) were
used to assign the absolute configuration of the aromatic
b-hydroxy esters.
more shielded
O
Ha
O
OMe
CF3
O
Hb
OMe
CF3
O
Hb
less shielded
H
O
H
O
4. Experimental
Ha
O
O
4.1. General methods
(a) (S,S)-MTPA-7
(b) (R,S)-MTPA-7
Figure 5. MTPA esters (S,S)- and (R,S)-7.
1H and 13C NMR spectra were recorded in CDCl3 solu-
tion on a Bruker AV-400 spectrometer operating at 400
and 100 MHz, respectively. Chemical shifts are
expressed in ppm values using TMS as an internal stan-
dard. HPLC analysis was carried out on a Jasco PU-
1580 liquid chromatograph with a PDA detector using
Chiralcel OD-H and Chiralcel OJ-H chiral columns
(Daicel, 4.6 · 250 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 varies for
different compounds. Ethyl 3-oxo-3-phenyl propanoate
and ethyl 3-oxo-3-(4-nitro phenyl) propanoate were
bought from Fluka, Buchs SG, Switzerland. All other
chemicals used were of analytical grade and glass
distilled.
above. This proves that the unknown optically pure
enantiomer 7 obtained from the deracemisation reaction
(Table 1) is the (S)-enantiomer.
3. Conclusion
Deracemisation of ethyl 3-hydroxy-3-phenyl propano-
ate, the standard substrate, and aryl substituted b-hy-
droxy esters by immobilised whole cells of C.
parapsilosis ATCC 7330 gave their corresponding (S)-
enantiomer in 68% isolated yield and >99% ee. The nat-
ure and the position of the substituents on the aromatic
ring affect the deracemisation. High ee (98–99%) and
good isolated yield (41–51%) of product was observed
with substrates having different para substituents on
the aromatic ring irrespective of their electronic nature.
A hydrophobic substituent present at the ortho-position
of the standard substrate obstructs the process of derac-
emisation while the substrate with an ortho, para-
dichloro substitution is poorly affected (82% ee). The
absolute configuration of the deracemised products as
determined by 1H NMR using an enantiomerically pure
4.2. Synthesis of racemic b-hydroxy esters
Racemic b-hydroxy esters (Table 1, entries 1 and 5) were
synthesised by the sodium borohydride reduction of the
corresponding b-keto esters. Ethyl 3-oxo-3-(4-methyl-
phenyl) propanoate and ethyl 3-oxo-3-(2-methylphenyl)
propanoate were synthesised using a reported method.33
The keto esters were reduced using sodium borohydride,

2796
S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
Figure 6. 1H NMR of –OCH3, methylene, Ha and Hb in (a) MTPA-(S,S)- and (R,S)-7 and (b) MTPA-7.
ethanol and the corresponding racemic b-hydroxy esters
were used for deracemisation. Ethyl 3-hydroxy-3-(4-
chlorophenyl) propanoate, ethyl 3-(2,4-dichlorophen-
yl)-3-hydroxy propanoate and ethyl 3-hydroxy-5-phen-
yl-pent-4-enoate were synthesised by Reformatsky
reaction.34 All the above-synthesised racemic b-hydroxy
esters were characterised by 1H and 13C NMR and com-
pared with the literature reported data (Table 4).
4.4. Immobilisation of biocatalyst
In a 500 mL Erlenmeyer flask containing sterilised water
(204 mL), sodium alginate (4.08 g, 2% w/v) was added
and the suspension was stirred at 50 ꢁC for 1 h. Wet cells
of C. parapsilosis ATCC 7330 (1.7 · 24 = 40.8 g) ob-
tained after centrifugation were suspended in 43.2 mL
(1.8 · 24) of distilled water and added to the above
mixture. A homogeneous suspension was obtained after
stirring for another hour. This uniform slurry was
added dropwise to a pre-chilled CaCl2 aqueous solution
(2% w/v), which resulted in the formation of beads of
average 1.5–2.0 mm diameter (240–250 mL). The beads
were kept in CaCl2 aqueous solution for 12 h and then
thoroughly washed [3 · 300 mL of distilled water] and
stored at 10–15 C before use in biotransformation.
40.8 g wet cells (from 24 flasks) of C. parapsilosis gave
240–250 mL of ICp. 1.7 g of wet cells from one culture
flask contributes ꢂ10 mL of ICp.
4.3. Microorganism and cultivation
C. parapsilosis ATCC 7330 was procured from ATCC
and was grown in a yeast malt broth medium (50 mL)
in 250 mL Erlenmeyer flasks incubated at 25 ꢁC,
200 rpm. The cells were harvested by centrifuging the
44 h culture broth at 3214g for 15 min and washed with
sterile water. The process was repeated thrice and
the wet cells were used for preparing the immobilised
beads.
Table 4. HPLC and NMR characterisation of the deracemised products
Sub. no. (Table 1)
HPLC
NMR—Ref. (1H and 13C)
Column used
Solvent (Hex: IPA)
Ret. time (min)
1
2
3
4
5
6
7
Chiralcel OD-H
Chiralcel OJ-H
Chiralcel OJ-H
Chiralcel OJ-H
Chiralcel OJ-H
Chiralcel OJ-H
Chiralcel OD-H
95:05
90:10
98:02
99.5:0.5
95:05
20.4 (S) and 25.7 (R)
15.5 (minor); 19.1 (major)
125.3 (R) and 134.8 (S)
198.1 (R) and 215.8 (S)
77.7 (R) and 84.0 (S)
41.4 (R) and 50.8 (S)
33.5 (R) and 48.6 (S)
21
5a
5a
5a
24
24
99.5:0.5
95:05

S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
2797
4.5. Deracemisation
4.7. Preparation of MTPA esters
Racemic b-hydroxy esters (504 lmol, 96 lL) dissolved
in the appropriate amount of ethanol (0.04% v/v) was
added to a suspension of 250 mL of ICp in 125 mL
of distilled water, equally distributed in four 250 mL
Erlenmeyer flasks. The biotransformation was carried
out for 6 h at 25 ꢁC and 150 rpm in a water bath orbi-
tal shaker. The beads were filtered and the reaction
mixture extracted using ethyl acetate, dried over anhy-
drous sodium sulfate and concentrated. The crude reac-
tion mixture after column purification was analysed by
chiral HPLC to determine the enantiomeric excess of
the deracemised product. Appropriate control experi-
ments with the reaction mixture containing all the com-
ponents (i) racemic b-hydroxy ester (ii) except the
immobilised whole cells of C. parapsilosis established
the optical purity of the product and the chemical
yield.
To a solution of racemic ethyl 3-hydroxy-3-phenyl
propanoate (10 lL, 0.054 mmol) in CH2Cl2 (0.85 mL),
Dimethylamino pyridine (26 mg, 0.214 mmol) and (S)-
(+)-a-(trifluoromethyl) phenyl acetyl chloride (20 lL,
0.107 mmol) were added at room temperature. Water
was added to the reaction mixture after 1.5 h. The organ-
ic phase was separated and the aqueous phase extracted
with CH2Cl2. The combined organic layer was washed
with brine, dried over anhydrous sodium sulfate and
evaporated under reduced pressure. The residue was
purified by preparative thin layer chromatography (n-
hexane/EtOAc = 95:05) to give the product (16 mg,
1
73% yield). The product was characterised by H NMR
spectroscopy. The MTPA esters of (R)-1, (S)-1, (RS)-6,
6, (RS)-7 and 7 were also synthesised by this method.
1
4.8. H NMR characterisation of the MTPA derivatives
1
4.6. Determination of the reusability of ICp
(i) MTPA-(S,R)- and (R,R)-1: H NMR (400 MHz,
CDCl3): 1.18–1.23 (2t, 6H, 2 · –OCH2CH3),
2.73–2.78 and 3.00–3.07 (dd and d of dd, 4H,
2 · –CH2CO), 3.41 (s, 3H, –OCH3), 3.51 (s, 3H,
–OCH3), 4.04–4.12 (2q, 4H, 2 · –OCH2CH3),
6.33–6.44 (2 dd, 2H, –CHOCO), 7.26–7.71 (m,
20H, 4 · C6H5–).
To a suspension of 250 mL of freshly prepared ICp in
125 mL of distilled water was added a solution of
504 lmol of ethyl 3-hydroxy-3-phenyl propanoate
pre-dissolved in 2400 lL of ethanol. The reaction was
carried out at 25 ꢁC and 150 rpm for 6 h. After filter-
ing the ICp, the reaction mixture was extracted with
ethyl acetate, dried over anhydrous sodium sulfate
and concentrated. The crude reaction mixture after
column purification was analysed for enantiomeric
excess by chiral HPLC. The filtered beads were thor-
oughly washed in distilled water and stored at 10–
15 ꢁC. This procedure was repeated by reusing the
same ICp to determine the recyclability of these beads
by calculating the isolated yield and the ee of the
product.
(ii) MTPA-(R,R)-1: 1H NMR (400 MHz, CDCl3):
1.16–1.19 (t, 3H, –OCH2CH3), 2.73–2.78 and
3.00–3.06 (d of dd, 2H, –CH2CO), 3.42 (s, 3H,
–OCH3), 4.02–4.08 (q, 2H, –OCH2CH3), 6.4–6.44
(dd, 1H, –CHOCO), 7.26–7.71 (m, 10H, 2 ·
C6H5–).
(iii) MTPA-(S,R)-1: 1H NMR (400 MHz, CDCl3):
1.21–1.24 (t, 3H, –OCH2CH3), 2.73–2.78 and
3.00–3.07 (d of dd, 2H, –CH2CO), 3.51 (s, 3H,
–OCH3), 4.04–4.09 (q, 2H, –OCH2CH3), 6.33–
6.37 (dd, 1H, –CHOCO), 7.23–7.69 (m, 10H, 2 ·
C6H5–).
A gradual decrease in activity was observed with
increasing number of cycles (Table 5). The activity of
the reaction was determined by the ee of the deracemised
product that is, ethyl 3-hydroxy-3-phenyl propanoate.
The immobilised cells could be re-employed thrice after
first use with a loss of 5% activity. A loss of ꢂ10% activ-
ity was observed for fifth to seventh cycle beyond which
the loss is ꢂ15%. The gradual decrease in deracemisa-
tion activity could be due to cell leakage through the
immobilisation matrix.
1
(iv) MTPA-(S,S)- and (R,S)-6: H NMR (400 MHz,
CDCl3): 1.18–1.26 (2t, 6H, 2 · –OCH2CH3),
2.76–2.91 (m, 4H, 2 · –CH2CO), 3.46 (s, 3H,
–OCH3), 3.55 (s, 3H, –OCH3), 4.05–4.19 (2q, 4H,
2 · –OCH2CH3), 6.64–6.73 (2 dd, 2H, –CHOCO),
7.08–7.72 (m, 16H, 2 · C6H5–, 2 · –o,p-diClC6H3).
1
(v) MTPA-6: H NMR (400 MHz, CDCl3): 1.18–1.25
(t, 3H, –OCH2CH3), 2.79–2.91 (d of dd, 2H,
–CH2CO), 3.46 (s, 3H, –OCH3), 4.04–4.12 (q,
2H, 2 · –OCH2CH3), 6.69–6.73 (dd, 1H, –CHO-
CO), 7.24–7.72 (m, 8H, C6H5–, –o,p-diClC6H3).
(vi) MTPA-(S,S)- and (R,S)-7: 1HNMR (400 MHz,
CDCl3): 1.17–1.26 (2t, 6H, 2 · –OCH2CH3),
2.68–2.75 and 2.81–2.9 (2d of dd, 4H, 2 ·
–CH2CO), 3.52 (s, 3H, –OCH3), 3.56 (s, 3H,
–OCH3), 4.04–4.15 (2q, 4H, 2 · –OCH2CH3),
6.03–6.1 (m, 2H, –CHOCO), 6.03–6.1 and 6.17–
6.23 (2 dd, 2H, PhCH@CH–CH–), 6.64–6.69 and
6.77–6.81 (2d, 2H, PhCH@CH–CH–), 7.28–7.52
(m, 20H, 4 · C6H5–).
Table 5. Determination of the reusabilitya of immobilised whole cells
of Candida parapsilosis ATCC 7330
Cycle no.
ee (%)
Yield (%)
% Loss of activity
1
2
3
4
5
6
7
8
99
96
94
94
89
87
87
84
57
56
59
55
57
56
49
55
0
3
5.1
5.1
10.1
12.1
12.1
15.1
1
(vii) MTPA-7: HNMR (400 MHz, CDCl3): 1.17–1.25
(t, 3H, –OCH2CH3), 2.68–2.73 and 2.81–2.87 (d
of dd, 2H, –CH2CO), 3.52 (s, 3H, –OCH3), 4.03–
4.11 (q, 2H, –OCH2CH3), 6.05–6.1 (m, 1H,
a The substrate used was ethyl-3-hydroxy-3-phenyl propanoate for the
deracemisation reaction.

2798
S. K. Padhi, A. Chadha / Tetrahedron: Asymmetry 16 (2005) 2790–2798
ski, P., Eds.; Kluwer Academic Publishers: Dordrecht,
–CHOCO), 6.17–6.23 (dd, 1H, PhCH@CH–CH–),
The Netherlands, 2000, pp 1–23.
6.77–6.81 (d, 1H, PhCH@CH–CH–), 7.28–7.52
(m, 10H, 2 · C6H5–).
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Acknowledgement
We thank CSIR, Government of India for financial
support.
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