Biocatalytic reduction of α-keto amides to (R)-α-hydroxy amides using Candida parapsilosis ATCC 7330

Article abstract of DOI:10.1016/j.cattod.2012.03.081

Biocatalytic reduction of primary and secondary α-keto amides was accomplished using whole cells of Candida parapsilosis ATCC 7330. The primary (R)-α-hydroxy amides were obtained in good enantiomeric excess (up to 94%) and conversion (88-99%) as compared to the secondary (R)-α-hydroxy amides.

Full text of DOI:10.1016/j.cattod.2012.03.081

Catalysis Today 198 (2012) 345–352
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Catalysis Today
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Biocatalytic reduction of ␣-keto amides to (R)-␣-hydroxy amides using Candida
parapsilosis ATCC 7330
Selvaraj Stellaa, Anju Chadhaa,b,∗
a Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
b National Centre for Catalysis Research, Indian Institute of Technology Madras, Chennai 600036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Biocatalytic reduction of primary and secondary ␣-keto amides was accomplished using whole cells of
Candida parapsilosis ATCC 7330. The primary (R)-␣-hydroxy amides were obtained in good enantiomeric
excess (up to 94%) and conversion (88–99%) as compared to the secondary (R)-␣-hydroxy amides.
© 2012 Elsevier B.V. All rights reserved.
Received 13 February 2012
Received in revised form 27 March 2012
Accepted 31 March 2012
Available online 27 May 2012
Keywords:
Primary ␣-keto amides
Secondary ␣-keto amides
Biocatalytic reduction
(R)-␣-hydroxy amides
Candida parapsilosis ATCC 7330
1. Introduction
of an optically active hydroxy ester which needs to be synthe-
sized by another biocatalytic route in order to avoid the formation
Optically active ␣-hydroxy amides are important chiral inter-
mediates for biologically active compounds like bradykinin B1
selective antagonists or inverse agonists and chiral ligands for
enantioselective addition reaction [1–4]. Asymmetric oxidation of
racemic ␣-hydroxy amides [5–7], ring opening of chiral ␣,␤-epoxy
amides [8,9], enantioselective Passerini-type reaction [10] and
asymmetric reduction of ␣-keto amides [11,12] are the available
chemical methods to prepare chiral ␣-hydroxy amides. All these
methods involve the use of expensive or toxic metal/chiral cata-
lysts. Further, ␣-keto amides bearing a chiral auxiliary are reduced
using reducing agents like samarium iodide, borohydride, K-
selectride to give the hydroxy amide in high diastereomeric excess
[13–15]. Optically pure ␣-hydroxy amides can also be synthesized
from chiral ␣-hydroxy esters [4] which in turn need to be syn-
thesized by another asymmetric method. Biocatalysts are greener
alternatives to chemical catalysts [16,17]. Biocatalytic prepara-
tions of ␣-hydroxy amides involve reduction of ␣-keto amides
[18–23], aminolysis of ␣-hydroxy esters by lipases [24,25] and
nitrile hydratase mediated hydration of optically active ␣-hydroxy
cyanohydrin [26]. The reported Candida antarctica lipase catalyzed
aminolysis to prepare chiral ␣-hydroxy amides involves the use
of racemic hydroxy amides [25]. For the hydration of cyanohy-
drin, only a small number of enantioselective nitrile hydratases are
known and the ee of the ␣-hydroxy amides depends on the selec-
tivity of oxy nitrilase enzyme which is used for the preparation of
chiral cyanohydrin [27]. Hence, biocatalytic reduction of the prochi-
ral precursor i.e. ␣-keto amide is an efficient and straightforward
method for the preparation of chiral ␣-hydroxy amides. However,
reported methods for the reduction of ␣-keto amides are limited in
contrast to the ubiquitous biocatalytic reduction of ␣-keto esters,
though amides and esters are similar groups. There are only few
specific examples reported for the biocatalytic reduction of ␣-keto
amides. A purified carbonyl reductase from Candida parapsilosis
IF0 0708 is known to reduce two cyclic keto amides, indoline-2,3-
dione and its N-methyl derivative to (R)-hydroxy amide in 28% yield
and >99% ee [18]. An ␣-keto amide reducing enzyme from Saccha-
romyces cerevisiae reduced chloro substituted benzoylformamides
[19] and different actinomycete strains were used for the reduc-
tion of 2-chlorobenzoylformamide to obtain both enantiomers
of 2-chloromandelamide in >99% conversion and ee [20,21].
Catalytic antibodies reportedly reduced an ␣-keto amide, (S)-3-
(4-nitrophenyl)-2-oxo-N-(1-phenylethyl)propanamide, using the
reductant NaBH3CN to give the product in >99% diastereomeric
excess [22]. A complex keto amide was reduced using the cell free
extract of Aerobasidium pullulans SC 13984 and glucose dehydro-
genase in the presence of glucose and NADP with a maximum of
60% yield [23]. Given the fact that there is no general biocatalytic
∗
Corresponding author. Tel.: +91 44 2257 4106; fax: +91 44 2257 4102.
E-mail address: anjuc@iitm.ac.in (A. Chadha).
0920-5861/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cattod.2012.03.081

346
S. Stella, A. Chadha / Catalysis Today 198 (2012) 345–352
Scheme 1. Preparation of primary ␣-keto amides by amidation of ␣-keto acids.
method for the preparation of chiral ␣-hydroxy amides, herein we
report the reduction of ␣-keto amides using the whole cells of C.
parapsilosis ATCC 7330 – a versatile biocatalyst as we have reported
earlier [28–36]. In order to examine the effect of amides in place of
esters in C. parapsilosis ATCC 7330 mediated reduction to produce
optically pure ␣-hydroxy amides, ␣-keto amides were synthesized
and used for the biocatalytic reduction.
synthesized by NaBH4 reduction of the corresponding ␣-keto
amides (1i and 1j) in 90% yield.
2.3. Preparation of secondary ˛-keto amides 1a–1j
2.3.1. Oxidation of the secondary ˛-hydroxy amides using TEMPO
– Ca(OCl)2
Secondary ␣-keto amides (1a–1h) were prepared by the oxida-
tion of racemic ␣-hydroxy amides (rac-2a-2h) using the catalytic
system TEMPO-Ca(OCl)2 as reported by us [43]. Compound 1d and
1e are new compounds and their spectral characterization were
given in our previous paper [43] and the analytical data of remain-
ing ␣-keto amides (1a [5], 1b [5], 1c [44], 1f [45], 1g [5] and 1h [5])
were in accordance with the lit. values.
2. Experimental
2.1. General
C. parapsilosis ATCC 7330 was purchased from ATCC Manas-
sas, VA 201018, USA and maintained at 4 ◦C in yeast malt agar
medium (Himedia product). DL-mandelic acid was purchased from
Lancaster. Ring substituted mandelic acids were purchased from
Sigma. Aliphatic and aromatic amines were bought locally. All
solvents were of analytical grade and used as received. Acetyl chlo-
ride was distilled prior to use. HPLC analysis of the ␣-hydroxy
amides was carried out on a Jasco PU-1580 liquid chromatograph
with a PDA detector using Daicel OJ-H, OD-H chiral columns (for
resolving secondary ␣-hydroxy amides) and Phenomenox Lux-2
chiral column (for resolving primary ␣-hydroxy amides) to deter-
mine the ee. Hexane: isopropanol mixture was used as the mobile
phase with flow rate 1 ml/min. The proportion of solvents varied
for different ␣-hydroxy amides. The conversion of the ␣-hydroxy
amides was determined by reverse phase C18 column using ace-
tonitrile:water solvent mixture with flow rate 0.8 ml/min. Optical
rotations were recorded on a Rudolph, Autopol IV digital polarime-
ter. For microwave irradiation a domestic microwave oven IFB
model-17G1S was used. 1H and 13C NMR spectra were recorded in
CDCl3 or CD3OD solution on a Bruker AV-500 spectrometer oper-
ating at 500 and 125 MHz, respectively.
2.3.2. N-benzyl-2-oxo-4-phenylbutanamide 1i [46]
In a 50 ml RB flask, ethyl 2-oxo-4-phenylbutanoate (2.03 mmol)
and benzylamine (10 equiv.) were refluxed in toluene (6 ml) for 6 h.
After the reaction, the reaction mass was washed with dil. HCl and
the organic solvent was evaporated in rotavap. The crude prod-
uct was purified by column chromatography with hexane: ethyl
acetate mixture to give the product 1i (30% yield).
2.3.3. N-benzyl-3-methyl-2-oxobutanamide 1j [47]
In a 100 ml double neck RB equipped with a magnetic stir-
rer, argon inlet adapter, 3-methyl-2-oxo-butyric acid (8.56 mmol)
was treated with triethylamine (1.5 equiv.) in dry DCM (20 ml).
To this solution, ethylchloroformate (1.2 equiv.) was added at 0 ◦C
and stirred for 1 h. Then benzyl amine (1 equiv.) was added and
stirred for 2 h. After the reaction, the reaction mass was evaporated
and dissolved in ethyl acetate. The organic layer was washed with
water and dil. HCl. The solvent was removed under high vacuum
and the crude product was purified by column chromatography
with hexane: ethyl acetate mixture to obtain the product 1j in 30%
yield.
2.2. Preparation of racemic secondary ˛-hydroxy amides
(rac-2a-2j)
2.4. Preparation of primary ˛-keto amides (3a–3e)
Primary ␣-keto amides were prepared by the amidation of cor-
responding ␣-keto acids. Though amidation method was reported
for the preparation of primary ␣-hydroxy amides [48], it is used
here for the first time to prepare ␣-keto amides 3a–3e (Scheme 1).
N-substituted mandelamides (rac-2a, rac-2b, rac-2e-2h) which
are derived from benzyl amine, aniline, propyl amine and pyrroli-
dine were synthesized by microwave method [37].
In a 50 ml RB flask, DL-mandelic acid (304 mg, 2 mmol) was
taken and mixed with the amine (2 equiv.). Excess amount of amine
(10 equiv.) was used in case of low boiling point amines such as
pyrrolidine and propyl amine. The unsealed RB flask which con-
tains the reaction mixture was kept in a beaker and irradiated
with the microwave radiation (300 W Power) for a specified period
(4–20 min) to get the corresponding ␣-hydroxy amides (50–97%
yield). All the products were purified by column chromatography
and characterized by spectral techniques. The analytical data of rac-
2a [5], rac-2b [5], rac-2e [38], rac-2f [39], rac-2g [5] and rac-2h [5]
were in accordance with the lit. values.
2.4.1. Preparation of ˛-keto acids
(a) The ethyl/methyl esters of mandelic acid and its ring substi-
tuted derivatives (p-OMe, p-Cl and p-Br) were oxidized using
TEMPO-Ca(OCl)2 catalyst system as reported by us [43] to give
the corresponding ␣-keto esters which were hydrolysed by
KOH in THF to give the ␣-keto acids.
(b) ethyl 2-oxo-2-p-tolylacetate was prepared according to lit. pro-
cedure [49] and hydrolysed by KOH in THF to give the ␣-keto
acid (2-oxo-2-p-tolylacetic acid).
N-methyl mandelamide (rac-2c) and N-ethyl mandelamide
(rac-2d) were prepared according to lit. procedure [40,41].
N-benzyl-2-hydroxy-4-phenylbutanamide [42] (rac-2i) and
N-benzyl-2-hydroxy-3-methylbutanamide [39] (rac-2j) were
2.4.2. Amidation of ˛-keto acids
In a 50 ml double neck RB equipped with a magnetic stirrer,
argon inlet adapter, benzoylformic acid (2 mmol) was treated with

S. Stella, A. Chadha / Catalysis Today 198 (2012) 345–352
347
IR(KBr, cm−1) 3423, 3307, 3226, 1682, 1654, 1582, 1480, 1390.
EI/MS 227.2085.
freshly distilled acetyl chloride (2 equiv.) in dry methanol (6 ml) at
0 ◦C and stirred for 6 h. The reaction mass was evaporated and redis-
solved in methanol. To this 30% NH3 solution (15 ml) was added
at 0 ◦C and stirred for 2 h. During the course of the reaction the
product benzoylformamide 3a was formed as white solid. The reac-
tion mass was kept in refrigerator to settle down the product. The
solid was filtered, dried and recrystallized from toluene to obtain
pure ␣-keto amide 3a in 41% yield. The remaining ␣-keto amides
3b–3e were also prepared by the same procedure. ␣-keto amides
are known to exist as both cis and trans isomers. The NMR data
of benzoylformamide 3a taken in CDCl3 was similar to the liter-
ature value [50] and showed a single isomer. The intramolecular
hydrogen bonding between the amide NH-proton and the keto
group in the trans isomer is not disturbed by the non-hydrogen
bonding solvent [51] (CDCl3) and it is the predominant isomer in
CDCl3. When the spectrum was taken in hydrogen bonding sol-
vent CH3OH-D4, the presence of two isomers was observed due
to the slow rotation around the OC–CO single bond [52] which
is restricted in non-hydrogen bonding solvent CDCl3 due to the
intramolecular hydrogen bonding of the trans isomer. The hydro-
gen bonding solvent (CH3OH-D4) stabilizes both the cis and trans
isomers. The trans–cis isomers of 3a were observed in the ratio
1:0.28 as deduced from the integration of aromatic protons. The
ring substituted derivatives of primary ␣-keto amides 3b–3e are
not soluble in chloroform; hence their NMR spectra were recorded
in CH3OH-D4. Halogen substituted ␣-keto amides 3b (p-Cl) and 3c
(p-Br) show 2:1 and 2:1.3 trans-cis ratio respectively while the elec-
tron donating p-CH3 substituted ␣-keto amide 3e exhibited less
amount of the minor cis isomer in the trans–cis ratio 3:0.26 and only
a trace amount of cis isomer was seen in p-OMe substituted ␣-keto
amide 3d. For ␣-keto amides which exhibit cis–trans isomerism
only the 1H NMR values are given below. The major and minor
peaks of the same proton are separately listed, if clear separation
is obtained.
2.5. Preparation of racemic primary ˛-hydroxy amides
(rac-4a–4e)
(i) Racemic mandelamide rac-4a and its ring substituted
derivatives rac-4c–4e were prepared by following the literature
procedure used for the synthesis of (S)-mandelamide [48]. (ii)
2-Oxo-2-p-tolylacetamide 3b (prepared by the amidation of 2-oxo-
2-p-tolylacetic acid) was reduced by NaBH4 in methanol to give
the corresponding racemic ␣-hydroxy amide rac-4b. The spectral
data of rac-4a–4e were similar to its corresponding optically pure
compounds R-4a–4e.
2.6. Growth conditions for C. parapsilosis ATCC 7330 [29]
C. parapsilosis ATCC 7330 was grown in yeast malt broth medium
(50 ml) in 250 ml Erlenmeyer flasks incubated in a shaker at
200 rpm and 25 ◦C. The cells were harvested by centrifuging the
40th hour culture broth at 10,000 rpm for 10 min and subsequent
washing with distilled water. In total, 1.6 g wet cells were obtained
per 50 ml medium in 250 ml Erlenmeyer flask. The wet cells were
suspended in potassium phosphate buffer (pH 7.2, 10 mM) and used
for biotransformation.
2.7. Biocatalytic reduction of secondary ˛-keto amides 1a–1j
using whole cells of C. parapsilosis ATCC 7330
6.4 g wet cells of C. parapsilosis ATCC 7330 was suspended in
16 ml of 10 mM potassium phosphate buffer (pH 7.2). 5 mmol glu-
cose and 10 mg of 1a (0.042 mmol) dissolved in 0.5 ml acetonitrile
were added into the cell suspension; incubated in a shaker at
200 rpm and 25 ◦C for 24 h. The reaction was done in triplicates. The
product R-2a was extracted by ethylacetate (thrice with 10 ml) and
concentrated under high vacuum. The crude product was purified
by column chromatography by hexane: ethylacetate mixture (4:1)
to give 71% yield of R-2a. The ee of R-2a (68%) was determined by
chiral HPLC OJ-H column using 9:1 hexane:isopropanol mixture.
The specific rotation of R-2a {[˛]D23 = −30.5 (c 1, CHCl3) for 68%
ee} was compared with the literature value {Lit. [˛]D25 = −82.4 (c
1.09, CHCl3) for >99% ee [5]} to assign the absolute configuration.
It was found to be ‘R’. The same procedure was followed for the
reduction of remaining secondary ␣-keto amides. The product R-2j
is slightly water soluble and that accounts for the reduced isolated
yield as compared to its conversion (Table 2, entry 5). The products
R-2b [5], R-2c [41], R-2d [54], R-2e and R-2f were not isolated due
to their low conversion/ee.
2-Oxo-2-phenylacetamide 3a [50]
The NMR data of 3a in CDCl3 is similar to lit. [50] value.
1H NMR (500 MHz, CH3OH-D4) ␦ 7.32–7.37 (m, 3H), 7.52–7.55 (m,
2H), 7.65–7.69 (m, 3H), 8.08–8.11 (m, 2H) (major:minor = 1:0.28).
2-Oxo-2-p-tolylacetamide 3b
White solid, m.p. 128 ◦C, 57% yield.
1H NMR (500 MHz, CD3OD) (major/minor) ␦ 2.42/2.32 (s/s, 3H),
7.34/7.15 (d/d, 2H, J = 8/8.5 Hz), 7.99–8.00/7.51–7.53 (m/m, 2H)
(major:minor = 3:0.26).
IR(KBr, cm−1) 3402, 3194, 1684, 1629, 1605, 1431, 1409.
HRMS: Calcd. for C9H9NO2Na 186.0531 (M+Na)+; found, 186.0540.
2-(4-Methoxyphenyl)-2-oxoacetamide 3c [53]
White solid, m.p. 150 ◦C, 48% yield.
1H NMR (500 MHz, CD3OD) ␦ 3.89 (s, 3H), 7.03–7.06 (m, 2H),
8.09–8.12 (m, 2H).
The spectral data of R-2a, R-2g and R-2h were in accordance
with the lit. value [5]. Compound R-2i is known in its racemic form
[42] but the corresponding optically pure form is not known. The
(R)-enantiomer of R-2i and R-2j is reported here.
13C NMR (125 MHz, CD3OD) ␦ 56.2, 115.1, 127.2, 133.8, 166.4,
169.6, 189.7.
IR(KBr, cm−1) 3451, 3221, 2385, 1693, 1648, 1597, 1566, 1508.
HRMS: Calcd. for C9H9NO3Na 202.0480 (M+Na+); found, 202.0471.
2-(4-Chlorophenyl)-2-oxoacetamide 3d
(R)-N-benzyl-2-(4-chlorophenyl)-2-hydroxyacetamide R-2g
[˛]D24 = −11.2 (c 0.5, CHCl3) for 24% ee {Lit. [˛]D25 = −57.2 (c 1.0,
CHCl3) for 97% ee [5]}.
White solid, m.p. 124 ◦C, 40% yield.
1H NMR (500 MHz, CD3OD) (major/minor)
␦ 4.58 (s, 1H),
(R)-N-benzyl-2-hydroxy-2-(4-methoxyphenyl)acetamide R-
2h
7.53–7.56/7.33–7.36 (m/m, 2H), 8.10–8.12/7.61–7.64 (m/m, 2H)
(major:minor = 2:1).
[˛]D24 = −6.6 (c 0.5, CH2Cl2) for 16% ee {Lit. [˛]D25 = −47.0 (c 1.0,
CH2Cl2) for 77% ee [5]}.
IR(KBr, cm−1) 3433, 3286, 3203, 1711, 1661, 1584, 1483, 1393.
EI/MS 183.5160.
(R)-N-benzyl-2-hydroxy-4-phenylbutanamide R-2i
White solid, m.p. 88 ◦C, [˛]D25 = −1.4 (c 1, CHCl3) for 16% ee.
1H NMR (500 MHz, CDCl3) ␦ 1.93–2.01 (m, 1H), 2.15–2.22 (m,
1H), 2.76 (t, 2H, J = 7 Hz), 2.86 (d, 1H, J = 5 Hz), 4.14–4.17 (m, 1H),
4.40–4.48 (m, 2H), 6.83 (bs, 1H), 7.17–7.34 (m, 10H).
2-(4-Bromophenyl)-2-oxoacetamide 3e
White solid, m.p. 122–124 ◦C, 50% yield.
1H NMR (500 MHz, CD3OD)(major/minor) ␦ 7.72/7.50 (d/d, 2H,
J = 8/8 Hz), 8.03/7.56 (d/d, 2H, J = 8/8 Hz) (major:minor = 2:1.3).

348
S. Stella, A. Chadha / Catalysis Today 198 (2012) 345–352
13C NMR (125 MHz, CDCl3) ␦ 31.3, 36.4, 43.1, 71.5, 126.1, 127.6,
100
90
Conversion(%)
ee(%)
127.7, 128.47, 128.48, 128.7, 137.9, 141.1, 173.7.
IR(KBr, cm−1) 3256, 1617, 1533, 1454, 1328.
80
(R)-N-benzyl-2-hydroxy-3-methylbutanamide R-2j
White solid, m.p. 64 ◦C, [˛]D28 = +23.0 (c 1, CHCl3) for 75% ee {Lit.
[˛]D23 = −20.8 (c 3.3, CHCl3) for >99% ee of S enantiomer [55]}.
1H NMR (500 MHz, CDCl3) ␦ 0.86 (d, 3H, J = 7 Hz), 1.02 (d, 3H,
J = 7 Hz), 2.16–2.22 (m, 1H), 4.02 (d, 1H, J = 3 Hz), 4.41–4.51 (m, 2H),
7.26–7.35 (m, 5H).
70
60
50
40
30
20
10
0
13C NMR (125 MHz, CDCl3) ␦ 15.5, 19.1, 31.9, 43.2, 76.4,
127.5,127.8, 128.7, 138.0, 173.3.
IR(KBr, cm−1) 3273, 2695, 1619, 1536, 1457, 1340.
0
12
24
36
48
60
72
84
2.8. Biocatalytic reduction of primary ˛-keto amides 3a–3e using
whole cells of C. parapsilosis ATCC 7330
Reaction time (h)
Fig. 1. Time course study of biocatalytic reduction of N-benzyl-2-oxo-2-
phenylacetamide 1a.
6.4 g wet cells of C. parapsilosis ATCC 7330 was suspended in
16 ml of potassium phosphate buffer (pH 7.2, 10 mM). 5 mmol glu-
cose and 15 mg benzoyl formamide 3a (0.1 mmol) dissolved in
0.5 ml acetonitrile were added into the cell suspension. The reaction
mixture was incubated in a shaker at 200 rpm, 25 ◦C. The reduction
was monitored by HPLC and the conversion was determined by C
18 column using acetonitrile and water mixture (85:15). The reac-
tion was done in triplicates. Though high conversion of the product
(R)-2-hydroxy-2-phenylacetamide R-4a was obtained, extraction
of the product using ethylacetate from the reaction mixture gave
low yields as the primary ␣-hydroxy amides are partially soluble
in water. Therefore, after the reaction was complete, the reaction
mass was centrifuged to remove the cell pellet and the supernatant
was taken and lyophilized to remove the water to get the crude
product. Further, it was purified by column chromatography using
ethylacetate and methanol mixture (9:1) to get the product in 95%
yield. The ee of R-4a (85%) was determined by Phenomenox, Lux-2
chiral column using hexane:isopropanol mixture (3:1). The spe-
cific rotation of the (R)-hydroxy amide R-4a {[˛]D25 = −53.5 (c 1,
acetone) for 85% ee} was compared with the literature value{Lit.
[˛]D25 = −73.0 (c 1.2, acetone) for >99% ee [56]} to assign the abso-
lute configuration. It was found to be ‘R’. The same procedure was
followed for the reduction of remaining primary ␣-keto amides.
The spectral data of R-4a was in accordance with the lit. value
[48]. For new compounds (R-4b and R-4c) and for those compounds
(R-4d and R-4e) complete characterizations not given in the liter-
ature are given below.
13C NMR (125 MHz, CD3OD) ␦ 74.6, 129.4, 129.5, 134.8, 140.6,
178.1.
IR(KBr, cm−1) 3405, 3266, 1644, 1594, 1489, 1407.
HRMS: Calcd. for C8H8NO2NaCl 208.0141(M+Na)+; found,
208.0150.
(R)-2-(4-bromophenyl)-2-hydroxyacetamide R-4e.
White solid, m.p. 140 ◦C, 12% ee, [˛]D28 = −5.2 (c 1, H2O) for 12%
ee {Lit.[˛]D20 = −70.6 (c 0.11, H2O) for ee 99% [57]}.
1H NMR (500 MHz, CD3OD) ␦ 4.98 (s, 1H), 7.39–7.41(d, 2H,
J = 8.5 Hz), 7.49–7.50 (d, 2H, J = 8.5 Hz).
13C NMR (125 MHz, CD3OD) ␦ 74.6, 122.8, 129.8, 132.4, 141.0,
178.1.
IR(KBr, cm−1) 3389, 3256, 1639, 1598, 1489, 1402.
HRMS: Calcd. for C8H8NO2BrNa 251.9636 (M+Na)+; found,
251.9628.
3. Results and discussion
3.1. Reduction of secondary ˛-keto amides using whole cells of C.
parapsilosis ATCC 7330
(R)-2-hydroxy-2-p-tolylacetamide R-4b
White solid, m.p. 130 ◦C, [˛]D28 = −41.6 (c 2, acetone) for 94% ee.
1H NMR (500 MHz, CD3OD) ␦ 2.32 (s, 3H), 4.96 (s, 1H), 7.15–7.16
(d, 2H, J = 8 Hz), 7.33–7.35 (d, 2H, J = 8 Hz).
Initially, N-benzyl-2-oxo-2-phenylacetamide 1a was reduced
using the resting cells of C. parapsilosis ATCC 7330 in ethanol (5%,
v/v) as the cosolvent and glucose (1 mmol) as the cosubstrate
at 25 ◦C. The reduction was monitored by HPLC at regular time
intervals and the maximum conversion (53%) and ee (59%) of the
(R)-N-benzyl-2-hydroxy-2-phenylacetamide R-2a was obtained at
24th hour. Further increase in time did not show any significant
increase in conversion and ee of the (R)-␣-hydroxy amide R-2a
(Fig. 1).
The viability of the cells during the bioreduction of 1a was mea-
sured by cell counting method and a viability loss of 12% (78%
cells are viable) was observed at 24 h. So, a fresh biomass (4 g)
was added to the biotransformation at 24 h and the reduction was
continued. At the end of another 24 h, the conversion of R-2a was
increased to 72% and the ee was 58%. Further, when the amount
of the cosubstrate (5 mmol) was increased, instead of addition of
excess biomass, good conversion (84%) of the (R)-hydroxy amide
R-2a was obtained at 24 h and the ee remained the same (58%). The
use of excess amount of the glucose enhanced the conversion of the
product R-2a. Hence, for the subsequent reactions 5 mmol glucose
and 24 h reaction time were used.
13C NMR (125 MHz, CD3OD) ␦ 21.2, 75.3, 127.9, 130.0, 138.7,
139.0, 178.8.
IR(KBr, cm−1) 3245, 1679, 1511, 1416.
HRMS: Calcd. for C9H11NO2Na 188.0687 (M+Na)+; found,
188.0694.
(R)-2-hydroxy-2-(4-methoxyphenyl)acetamide R-4c
White solid, m.p. 142 ◦C, [˛]D28 = −53.4 (c 1, acetone) for 70% ee.
1H NMR (500 MHz, CD3OD) ␦ 3.78 (s, 3H) 4.95 (s, 1H), 6.89–6.91
(m, 2H), 7.35–7.38 (m, 2H).
13C NMR (125 MHz, CD3OD) ␦ 55.7, 75.0, 114.8, 129.3, 133.8,
161.1, 178.9.
IR(KBr, cm−1) 3389, 3206, 1651, 1609, 1510, 1462.
HRMS: Calcd. for C9H11NO3Na 204.0637 (M+Na)+; found,
204.0642.
(R)-2-(4-chlorophenyl)-2-hydroxyacetamide R-4d
White solid, m.p. 110 ◦C, [˛]D28 = −9.4 (c 2, acetone) for 20% ee.
1H NMR (500 MHz, CD3OD) ␦ 4.99 (s, 1H), 7.33–7.35 (m, 2H),
7.45–7.47 (m, 2H).

S. Stella, A. Chadha / Catalysis Today 198 (2012) 345–352
349
Table 1
Biocatalytic reduction of various N-aryl/alkyl ␣-keto amides using whole cells of C. parapsilosis ATCC 7330.
Entry
1
R
(R)-␣-hydroxy amide
Conversion (%)a
ee (%)b
–CH2Ph
1a
87
68
16
68
64
69
71
R-2a
2
3
4
5
-Ph
1b
79
29
34
34
11
R-2b
R-2c
R-2d
R-2e
R-2f
–CH3
1c
–CH2CH3
1d
–(CH2)2CH3
1e
6
–(CH2)4−
1f
a
Conversion was determined by HPLC using C18 column.
ee was determined by chiral HPLC using Daicel OJH and ODH columns.
b
3.2. Effect of cosolvents on the biocatalytic reduction
Considering the conversion (87%) and ee (68%), acetonitrile was
used as the cosolvent for further reactions.
Organic solvents have been reportedly used to improve enan-
tioselectivity in biocatalytic reductions [58–61]. Since the ee of the
(R)-hydroxy amide R-2a was moderate (59%), solvent engineering
was done to increase the ee of R-2a. Various water miscible (CH3CN,
EtOH, MeOH, IPA, THF, DMSO and acetone), immiscible organic
solvents (MTBE, EtOAc) and ionic liquids ([BMIM]OAc, [BMIM]BF4,
[BMIM]Br) were examined as cosolvents (3.1%, v/v) for the biotrans-
formation of 1a. Conversion of the hydroxy amide R-2a improved in
ionic liquids and water miscible organic solvents (60–89%) as com-
pared to the water immiscible organic solvents like ethylacetate
(20%) and tert-butylmethylether (65%), possibly due to less avail-
ability of substrates to the enzymes for the reduction to occur in
water immiscible organic solvents. However, the ee of the hydroxy
amide R-2a was moderate (50–68%) in all the solvents investigated.
3.3. Inhibitor studies on the biocatalytic reduction
The moderate ee (68%) of the ␣-hydroxy amide R-2a sug-
gests that there are atleast two alcohol dehydrogenases present in
the whole cells which compete for the same substrate and yield
products of opposite enantioselectivity. Inhibition of yeast alco-
hol dehydrogenase [62,63] was therefore carried out to inhibit
one of the enzymes selectively. Addition of the inhibitors 2-chloro
ethanol, sodium azide, methylvinylketone and acetamide (5 mM)
to the biocatalytic reduction of 1a marginally decreased both the
conversion (66–79%) and ee (55–67%) of R-2a which indicates that
both the enzymes involved in the reduction were inhibited. For
further reactions no inhibitor was added.

350
S. Stella, A. Chadha / Catalysis Today 198 (2012) 345–352
Table 2
Biocatalytic reduction of various N-benzyl ␣-keto amides using whole cells of Candida parapsilosis ATCC 7330.
Entry
1
R
(R)-␣-hydroxy amide
Conversion (%)a
Yield (%)
71
ee (%)b
Ph–
87
68
1a
R-2a
2
p-Cl-Ph–
1g
52
34
33
26
24
16
R-2g
R-2h
3
4
p-OMe-Ph–
1h
Ph-(CH2)2−
97
94
86
16
75
1i
R-2i
5
(CH3)2CH–
58c
1j
R-2j
a
Conversion was determined by HPLC using C18 column.
ee was determined by chiral HPLC using Daicel OJH and ODH columns.
The recovery of the product was low compared to its conversion due to the partial solubility of the product in water.
b
c
3.4. Effect of temperature on the biocatalytic reduction
␣-keto amide 1b. The optical purity of (R)-N-phenyl ␣-hydroxy
amide R-2b was low (ee 16%), possibly due to steric reasons. Of all
the N-substituted ␣-keto amides investigated for the biocatalytic
reduction, the model substrate N-benzyl ␣-keto amide 1a afforded
good conversion 87% and ee 68% of the hydroxy amide R-2a as
compared to the other N-substituted (alkyl/aryl) ␣-keto amides.
Further, the effect of temperature on the enantioselectivity
of the bioreduction of 1a was studied at various temperatures
between 20 and 60 ◦C. At 20 ◦C the product R-2a was obtained in
80% conversion and 48% ee. In the temperature range from 25 to
35 ◦C, the biotransformation was not significantly affected and gave
64–68% ee and 86–87% conversion of the product R-2a. Increasing
the temperature above 35 ◦C resulted in a sharp decrease in both
ee (54–47%) and conversion (79–12%) of the product. The reaction
was continued at 25 ◦C for further reactions.
3.6. Biocatalytic reduction of various N-benzyl ˛-keto amides
Various N-benzyl ␣-keto amides were synthesized and used as
the substrate for the biocatalytic reduction (Table 2). Substituents
on the phenyl ring had profound influence on the stereoselectivity
of the reaction. Electron withdrawing and donating substituents
in the phenyl ring such as p-Cl and p-OMe groups dramatically
decreased the ee (24 and 16%, Table 2, entries 2 and 3) and con-
version (52 and 34%) of the corresponding (R)-␣-hydroxy amides.
When the phenyl ring was separated by two methylene groups
from the keto group (1i), good conversion of the product R-2i (97%)
was obtained but ee was decreased to 16%. Alkyl substitution (iso-
propyl group) in place of phenyl ring (1j) increased the ee (75%) as
well as conversion (94%) of the (R)-hydroxy amide R-2j (Table 2,
entry 5). Notably, alkyl substitution at the amide nitrogen (1c–1f)
3.5. Effect of N-alkyl/aryl substitution on the amide nitrogen of
˛-keto amides in the biocatalytic reduction
Under the optimized conditions, various secondary N-aryl and
N-alkyl ␣-keto amides were used as substrates for the biocatalytic
reduction. Reduction of N-aryl ␣-keto amides 1a and 1b (Table 1,
entries 1 and 2) gave good conversion (79–87%) of the correspond-
ing (R)-hydroxy amides as compared to N-alkyl ␣-keto amides
1c–1f (11–34%) at 24 h. However, ee of all (R)-hydroxy amides
was moderate (64–71%) except for the bioreduction of N-phenyl

S. Stella, A. Chadha / Catalysis Today 198 (2012) 345–352
351
Table 3
Biocatalytic reduction of primary ␣-keto amides using whole cells of Candida parapsilosis ATCC 7330.
Entry
1
R
Time
4
(R)-␣-hydroxy amide
Conversion (%)a
Yield (%)
95
ee(%)b
H
3a
99
85
94
R-4a
2
CH3
5
97
74
3b
R-4b
R-4c
R-4d
R-4e
3
4
OCH3
3c
12
88
99
99
76
97
96
70
20
12
Cl
3d
4
4
5
Br
3e
a
Conversion was determined by HPLC using C18 column.
ee was determined by chiral HPLC using Phenomenox Lux-2 chiral column.
b
did not improve the ee and conversion of the corresponding prod-
ucts (Table 1, entries 3–6) but the alkyl substitution near to the
carbonyl group (1j) increased the ee and conversion of the product
(Table 2, entry 5).
dramatically decreased the ee of the corresponding products R-4d
and R-4e (20 and 12%, Table 3, entries 4 and 5).
4. Conclusions
In the present study, the less explored biocatalytic reduction of
various ␣-keto amides was investigated. The biocatalytic reduction
of both primary and secondary ␣-keto amides using whole cells of
C. parapsilosis ATCC 7330 gave the corresponding (R)-␣-hydroxy
amides. The various parameters for the reduction of N-benzyl-2-
oxo-2-phenylacetamide were optimized to get the maximum ee
(68%) and conversion (87%) of the (R)-␣-hydroxy amide. Primary
␣-keto amides gave better results than secondary ␣-keto amides
i.e. in 4–12 h; the former gave up to 94% ee of (R)-␣-hydroxy amide.
3.7. Biocatalytic reduction of primary ˛-keto amides to
(R)-mandelamide derivatives
Unlike secondary ␣-keto amides, primary ␣-keto amides were
reduced quickly and the reduction was complete in 4–12 h. In sec-
ondary ␣-keto amides, the N-alkyl/aryl substitution at the amide
nitrogen makes it more electron rich which in turn makes the keto
carbonyl group less reactive for the reduction. The absence of sub-
stitution at the amide nitrogen in the primary ␣-keto amides leads
to fast reduction. Benzoyl formamide 3a was reduced using the
whole cells of C. parapsilosis ATCC 7330 to (R)-mandelamide R-4a in
4 h with 95% yield and 85% ee (Table 3, entry 1). The electron donat-
ing substituent p-CH3 group in the phenyl ring (3b) increased the
ee (94%) of the (R)-hydroxy amide R-4b. p-OMe substituted keto
amide 3c gave the (R)-hydroxy amide 4c in 70% ee and 76% yield.
Further, the halogen substituents (p–Cl and p-Br) in the phenyl ring
Acknowledgements
One of the authors [S. Stella] thanks the Council of Scientific and
Industrial research (CSIR, India) for the fellowship. We thank the
Sophisticated Analytical Instrumentation Facility (SAIF, IIT Madras)
for the NMR analysis.

352
S. Stella, A. Chadha / Catalysis Today 198 (2012) 345–352
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