Stereochemical preference of Candida parapsilosis ATCC 7330 mediated deracemization: E- versus Z-aryl secondary alcohols

Article abstract of DOI:10.1016/j.tetasy.2012.09.014

The stereochemical preference of the biocatalyst, Candida parapsilosis ATCC 7330, was investigated with respect to the E/Z configuration in the deracemization and the asymmetric reduction of aryl secondary alcohols and prochiral ketones, respectively. The

Full text of DOI:10.1016/j.tetasy.2012.09.014

Tetrahedron: Asymmetry 23 (2012) 1360–1368
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Tetrahedron: Asymmetry
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Stereochemical preference of Candida parapsilosis ATCC 7330 mediated
deracemization: E- versus Z-aryl secondary alcohols
Thangavelu Saravanan a, Rajendran Selvakumar b, Mukesh Doble b, Anju Chadha a,c,
⇑
a Laboratory of Bioorganic Chemistry, Indian Institute of Technology Madras, Chennai 600 036, India
b Laboratory of Bioengineering and Drug Designing, Indian Institute of Technology Madras, Chennai 600 036, India
c National Centre for Catalysis Research, Indian Institute of Technology Madras, Chennai 600 036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2012
Accepted 19 September 2012
The stereochemical preference of the biocatalyst, Candida parapsilosis ATCC 7330, was investigated with
respect to the E/Z configuration in the deracemization and the asymmetric reduction of aryl secondary
alcohols and prochiral ketones, respectively. The biocatalyst preferred the E-isomers over Z-isomers as
substrates as evidenced from the experimental results of >99% ee and up to 86% isolated yield for E-sec-
ondary alcohols. The synthesis of enantiomerically pure E-4-phenylbut-3-ene-1,2-diol (ee >99%, isolated
yield 86%) by whole cell mediated deracemization is reported here for the first time. The geometric pref-
erence of the enzymes was confirmed by using the cell free extract of this biocatalyst. Mechanistic
insights using in silico studies showed that the E-isomers when located in the active site are favourably
placed with respect to the catalytic triad (Ser-Tyr-Lys) for hydride transfer from NADPH.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
nostoc mesenteroiles) in the reduction of trans- and cis-cinnamalde-
hydes.7 These enzymes react with the trans-isomers 7 to 647 times
Biocatalysis or enzyme-based synthetic catalysis challenges tra-
ditional synthetic methods for producing enantiomerically pure
secondary alcohols and pharmaceutical intermediates by effi-
ciently catalysing a chemical reaction with high enantio- and reg-
ioselectivity.1 Contrary to chemical synthesis, biocatalysis can be
carried out under mild conditions.2 In biocatalysis, both isolated
enzymes and whole cells are used as catalysts. Compared to iso-
lated enzymes, whole cell catalysts have the advantage of low cost,
eliminating cofactor addition and regeneration. In addition, en-
zymes in the cellular milieu are well protected and stable as com-
pared to pure (isolated) enzymes.3
The enantioselectivity of biocatalysts can be altered by various
methods, including substrate modification, optimizing the reaction
conditions, using enzyme specific inhibitors and also by medium
and solvent engineering.4 Slight modifications in the substrates
can lead to a switch in the selectivity of the biocatalyst. In addition
to chain length modification, functional group modification and
substituent in the phenyl ring, the size of the substrate can also
switch the selectivity of the biocatalyst.5 Attempts to understand
the enantioselectivity in particular the role of double bond geom-
etry (E/Z) selectivity have rarely been carried out.6
faster than with the cis-counterparts, thereby displaying remark-
able trans-specificity. The oxidative kinetic resolution of secondary
alcohols by lyophilized cells of Rhodococcus ruber DSM 44541
showed stereo-discrimination between the E- and Z-configured
secondary alcohols.8 The rac-E-3-penten-2-ol was resolved with
excellent enantioselectivity (E >100) whereas the corresponding
rac-Z-4-penten-2-ol was not oxidized. Similarly, Burgess and Jen-
nings reported the enantioselective esterification of unsaturated
alcohols mediated by a lipase prepared from Pseudomonas sp.,
which showed stereo-discrimination between E-and Z-4-phenyl-
but-3-en-2-ol.9 The E-isomer gave 50% conversion in 3 h with good
enantioselectivity (E >20) whereas the Z-isomer gave 57% conver-
sion in 64 h with poor enantioselectivity (E = 3). For over a decade,
reports from our laboratory showed that Candida parapsilosis ATCC
7330 is effective for the deracemization of secondary alcohols,10
the asymmetric reduction of aliphatic11 and aromatic prochiral ke-
tones12 and imines,13 and the resolution of N-acetylated amino es-
ters.14 This biocatalyst has also been used for the deracemization of
rac-E-aryl secondary alcohols to give enantiomerically pure enan-
tiomers with high ee (up to 99%) and isolated yields (up to 85%)
via stereoinversion.10 In order to better understand the stereospec-
ificity of oxidoreductase enzymes in C. parapsilosis ATCC 7330,
herein addresses the question of geometric preference using E/Z-
secondary alcohols as substrates for deracemization and attempts
to rationalize the experimentally observed results by in silico
studies.
Klibanov and Giannousis reported the geometric specificity of
three different dehydrogenases (from yeast, horse liver and Leuco-
⇑
Corresponding author. Tel.: +91 44 2257 4106; fax: +91 44 2257 4102.
E-mail address: anjuc@iitm.ac.in (A. Chadha).
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2012.09.014

T. Saravanan et al. / Tetrahedron: Asymmetry 23 (2012) 1360–1368
1361
Table 1
Deracemization of E-secondary alcohols using C. parapsilosis ATCC 7330
Entry
Product
Reaction time (h)
Isolated yield (%)
76
eea (%)
>99
½ ꢀ
a 2D5
OH
OH
OH
OH
OH
1a
2
+17.8 (c 1,MeOH)15a
+70.4 (c 1, CHCl3)15b
O
1b
1c
1d
1e
1
0.5
2
78
75
78
86
>99
>99
>99
>99
O
O
O
O
+115.2 (c 1,MeOH)15c
ꢁ6.8 (c 0.5,CHCl3)10c
+28.8 (c 1, CHCl3)16
O
OH
24
a
Enantiomeric excess was determined using chiral HPLC columns (Chiralcel OD-H and OJ-H, flow rate 1 ml/min).
tive dynamic kinetic asymmetric transformation of 3,4-epoxy-1-
butene into (2R)-3-butene-1,2-diol (ee = 85%).16 Cho and Shin syn-
thesized enantiomerically pure E-4-phenylbut-3-ene-1,2-diol in
98% ee via a multistep oxazaborolidine-catalysed borane reduction
of the corresponding b-keto sulfides.17 Enantiomerically pure E-
and Z-4-phenylbut-3-ene-1,2-diols were prepared by asymmetric
dihydroxylation and hydrolytic kinetic resolution of epoxides
2. Results and discussion
2.1. Deracemization of E- and Z-secondary alcohols
Earlier reports from our laboratory showed that E-secondary
alcohols were deracemized with good ee (up to >99%) using C. par-
apsilosis ATCC 7330 cells, which were harvested in the stationary
phase that is 40 h.10 Recently, culture conditions were re-opti-
mized for the asymmetric reduction of ethyl-4-chloro-3-oxobut-
anoate in which C. parapsilosis ATCC 7330 was harvested at the
mid exponential phase that is 14 h.11 Under the newly optimized
culture conditions, deracemization of rac-E-4-phenylbut-3-en-2-
ol resulted in the corresponding enantiomerically pure (R)-enan-
tiomer in excellent enantioselectivity (ee >99%) and isolated yield
(76%) (Table 1, entry 1a). The new culture conditions have the
advantages of reducing the harvesting time by more than half
(40 to 14 h) and doubling the substrate loading ability (from 0.97
to 2 mmol) compared to an earlier report.10e A variety of rac-E-sec-
ondary alcohols were used as substrates for deracemization to gen-
eralize the study.
The E-ethyl and E-methyl 2-hydroxy-4-phenylbut-3-enoates
were deracemized using whole cells of C. parapsilosis ATCC 7330
to give the (S)-enantiomer (ee >99%) with isolated yields of 78%
and 75%, respectively (Table 1, entries 1b and 1c). In addition,
rac-E-ethyl 3-hydroxy-5-phenylpent-4-enoate was deracemized
to the corresponding enantiomerically pure (S)-enantiomer with
excellent enantioselectivity (>99%). Notably, the isolated yield im-
proved from 28 to 78% compared to an earlier report (Table 1, entry
1d).10c In 14 h culture conditions, the formation of a by-product (3-
hydroxy-5-phenyl-pent-4-enoic acid) was not observed, unlike the
reaction with the 40 h culture conditions due to the action of a
hydrolase as described earlier.10c
using the chiral source 2,3-O-isopropylidene-D-glyceraldehyde
and (R)-(+)-2,2-dimethyl-1,3-dioxolane-4-carboxaldehyde.18 Aleu
et al., reported the Baker’s yeast mediated asymmetric reduction
of an acetoxy ketone to the corresponding 1-acetoxycarbinols,
which were further hydrolysed to 1,2-diols.19 Herein the deracemi-
zation of rac-E-4-phenylbut-3-ene-1,2-diol to the corresponding
enantiomerically pure (S)-enantiomer has been reported for the
first time with excellent ee (>99%) and isolated yield (86%) (Table 1,
entry 1e).
Deracemization under identical reaction conditions to those
used for rac-E-secondary alcohols was carried out with the coun-
terpart Z-isomers in order to answer the question of geometric
preference of this biocatalyst. The Z-secondary alcohols were not
deracemized, indicating that the biocatalyst C. parapsilosis ATCC
7330 had a geometric preference towards the E-isomer (Scheme 1).
Recently we reported the biocatalytic deracemization of alkyl-
2-hydroxy-4-arylbut-3-ynoates using the whole cells of C. parapsi-
losis ATCC 7330.10f The enantiomerically pure (S,Z)-ethyl-2-hydro-
xy-4-phenylbut-3-enoate 5a was prepared via hydrogenation of
the corresponding deracemized ethyl-2-hydroxy-4-phenylbut-3-
ynoate 4a using Lindlar’s catalyst20 with excellent enantiomeric
excess (>99%) and good yield (94%) (Scheme 2).
2.2. Asymmetric reduction of E- and Z-4-phenylbut-3-en-2-one
Biocatalyst C. parapsilosis ATCC 7330 mediated the deracemiza-
tion reaction by following a redox (stereoinversion) mechanism
that is a two-step one-pot process that consisted of the enantiose-
lective oxidation of one enantiomer to the keto intermediate, fol-
lowed by complementary enantioselective reduction of the keto
intermediate to a single enantiomer.10 In the above deracemization
Deracemization of rac-E-4-phenylbut-3-ene-1,2-diol was car-
ried out using whole cells of C. parapsilosis ATCC 7330. Although,
enantiomerically pure E-4-phenylbut-3-ene-1,2-diol has been syn-
thesized by chemical methods and biocatalytic methods, this is the
first report obtaining it by deracemization using a biocatalyst.
Cheeseman et al. reported the palladium-catalysed, enantioselec-

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T. Saravanan et al. / Tetrahedron: Asymmetry 23 (2012) 1360–1368
OH
HO
1a
2a
2b
2c
O
O
OH
HO
HO
O
O
O
1b
1c
O
OH
C. parapsilosis
ATCC 7330
O
(Water, 25°C)
O
OH
O
HO
HO
O
O
2d
2e
O
1d
1e
OH
OH
OH
ee = up to > 99%
Yield = up to 86
Scheme 1. Deracemization of E- and Z-4-phenylbut-3-en-2-ol derivatives.
OH
OH
HO
COOEt
C. parapsilosis
Pd/CaCO3, H2
ATCC 7330
COOEt
COOEt
quinoline
EtOH, 1h, rt
water, 3h
25°C
(S)
(S)
ee = >99%
ee = >99%
Yield = 94%
3a
Yield = 78%
[ ]D25 = +320.6 (c 1, CHCl3)
α
4a
5a
Scheme 2. Chemoenzymatic synthesis of (S,Z)-ethyl-2-hydroxy-4-phenylbut-3-enoate.
reactions, the stereochemical preference of the oxidizing enzyme
was studied using the E- and Z-secondary alcohols as substrates.
In order to study the selectivity of the reducing enzyme, E- and
Z-4-phenylbut-3-en-2-ones 6a and 7a were synthesized and used
as substrates for the asymmetric reduction.
deracemization of the E-isomer rac-1a gave (R,E)-4-phenylbut-3-
en-2-ol 1a with an ee of 98% and an isolated yield of 78% while
the reaction did not progress with the Z-isomer rac-2a. This con-
firmed that the geometric preference of the biocatalyst in the
deracemization is due to the enzymes and not the cell membrane.
The E-4-phenylbut-3-en-2-one 6a was reduced to (R,E)-4-phe-
nylbut-3-en-2-ol 6b with excellent ee (98%) and conversion
(96%). The counterpart Z-4-phenylbut-3-en-2-one 7a was synthe-
sized and subjected to asymmetric reduction under identical reac-
tion conditions. The Z-ketone 7a was not reduced, indicating that
the enzyme responsible for the asymmetric reduction also shows
a geometric preference towards the E-isomer (Scheme 3).
2.4. Mechanistic insights of the geometrical specificity
The geometrical preference of C. parapsilosis ATCC 7330 for the
asymmetric reduction was explained by modelling studies. The
crystal structure of C. parapsilosis carbonyl reductase (CPCR) depos-
ited in Protein Data Bank (PDB id: 3CTM)21a was not suitable for
docking, since the cofactor binding pocket of chain A was highly
collapsed and the catalytic site of chain B was completely rear-
ranged. Hence, a model of CPCR was generated by homology mod-
elling with the crystal structure of Mannitol Dehydrogenase
(MtDH), from Cladosporium herbarum (PDB id: 3GDF)21b showing
50% sequence identity and an intact cofactor binding pocket and
catalytic site. Since both 3CTM and 3GDF are short-chain dehydro-
genase/reductase (SDR) family proteins, the cofactor-binding do-
main of the model built was expected to fold into the classical
Rossmann-fold structure, a seven-strand parallel b-sheet flanked
2.3. Dismissing role of cell membrane in substrate geometric
preference
To ensure that the geometrical preference of the biocatalyst is
explicitly due to the selectivity of the enzymes (oxidoreductases)
and not due to the cell wall, which can influence the selectivity
during the uptake of substrates, deracemization of E- and Z-4-phe-
nylbut-3-en-2-ol was carried out using the cell free extract of C.
parapsilosis ATCC 7330. Under these conditions, enzymes directly
recruit the substrate without the cell wall barrier. As anticipated,
on both sides by
a-helices as found in all SDR family proteins

T. Saravanan et al. / Tetrahedron: Asymmetry 23 (2012) 1360–1368
1363
O
OH
(R)
6a
Conv = 96%
ee = 98%
C. parapsilosis
ATCC 7330
(water, 25°C)
6b
HO
O
(R)
7a
7b
Scheme 3. Asymmetric reduction of E- and Z-4-phenylbut-3-en-2-one.
Figure 1. Sequence alignment of CPCR with mannitol dehydrogenase (PDB code: 3GDF) with overall aligned score 50 using ClustalW 2.1. Notations: (⁄) indicates the
alignment contains identical amino acid residues in all sequences (or identical bases if protein sequences are aligned); (:) indicates the alignment contains different but highly
conserved (very similar) amino acids; (.) indicates the alignment contains different amino acids that are somewhat similar and () indicates that the alignment contains
dissimilar amino acids or gaps.
(Fig. 1).21 The active site includes the Ser-Tyr-Lys catalytic triad
which is located at the C-terminal end of the major parallel b-
sheet. This also harbours the coenzyme- and substrate-binding
pocket (Fig. 2).22 The Ser-Tyr-Lys catalytic triad forms a proton re-
lay system21c and the region is highly conserved which includes
the N-terminal TGxxxGxG motif as part of the nucleotide-binding
region.23 Based on the highly conserved cofactor-binding domain,
the putative enzyme-cofactor docking was performed with NADPH
and NADH (Fig. 3a and b). As reported by others,24 in both the low-
est-energy conformational ensembles, the modelled domain is
more favourable to NADPH (ꢁ28.01 kcal/mol) when compared to
NADH (ꢁ23.54 kcal/mol). The binding energy is lower with the for-
mer than with the latter. Hence, the CPCR-NADPH complex was
chosen for further docking with the E- and Z-isomers.
The docking of the co-enzyme (NADPH) with CPCR indicates
that Ser43, Ile46, Tyr65, Asn66, His68, Gly120, Thr122, Val143,
Tyr187 and Pro216 interact with it. Tyr187 is one among the three
amino acids in the catalytic residue. Either one of the hydrides
from the cofactor NADPH attacks the carbonyl carbon of the pro-
chiral ketones from the ‘Re’ or ‘Si’-face which leads to the formation
Figure 2. Structure of the modelled protein in apo form.

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T. Saravanan et al. / Tetrahedron: Asymmetry 23 (2012) 1360–1368
Figure 3. Structure of the modelled protein in holo form (a) NADPH bound (b) NADH bound.
Table 2
Interaction score between the substrate conformer with enzyme complex
Entry
Substrates
Scores (S)
Hydride transfer distance (Å)
Hydrogen bonding distance with TYR 187 (Å)
Absolute configuration
O
6a
ꢁ13.97
3.68
3.21
(R)
O
7a
8a
ꢁ10.26
ꢁ10.35
7.67
3.72
—
—
O
OH
3.70
(S)
9a
ꢁ8.52
7.89
—
—
OH
O
Figure 4a. Interaction of NADPH-C4 atom with E-4-phenylbut-3-en-2-one 6a indicating favourable ‘Si’ face attack.
of respective (S)- or (R)-alcohols.25 For docking studies, substrates
6a and 7a were also used in wet lab while substrates 8a and 9a (E-
and Z-1-hydroxy-4-phenylbut-3-en-2-one, respectively) were only
studied by in silico methods.
It was evident from the substrate docking studies, that both
6a and 8a interact with the catalytic residue (Tyr187, hydroxyl
group) and the NADPH-C4 atom is in close proximity to the sub-
strates (Table 2, 6a; 3.68 Å and 8a; 3.72 Å) favouring a ‘Si’ and
‘Re’ face attack respectively, (Figs. 4a and 4b). On the other hand,
neither 7a nor 9a is in close proximity to encounter attack from
the hydride on the C4 atom of the nicotinamide ring in the
cofactor (NADPH). The distances between the NADPH-C4 atom
to 7a and to 9a are 7.67 and 7.89 Å, respectively (Table 2). The
least London
D
G score26 for CPCR-NADPH-6a is ꢁ13.97 kcal/
mol which is lower in comparison to that of the CPCR-NADPH-
7a complex (ꢁ10.26 kcal/mol). The lower the score, the better

T. Saravanan et al. / Tetrahedron: Asymmetry 23 (2012) 1360–1368
1365
Figure 4b. Interaction of NADPH-C4 atom with E-1-hydroxy-4-phenylbut-3-en-2-one 8a indicating favourable ‘Re’ face attack.
the binding affinity is. Similarly, the energy of CPCR-8a-NAPDH
complex (ꢁ10.35 kcal/mol) is less than that of the CPCR-9a-
NADPH complex (ꢁ8.52 kcal/mol).
medium that contained 5 g/L peptic digest of animal tissue, 3 g/L
malt extract, 3 g/L yeast extract, 10 g/L dextrose and 20 g/L agar.
The entire chemicals for media preparation were purchased locally.
All chemicals used were of analytical grade and distilled prior to
use. TLC was carried out on Kieselger 60 F254 aluminium sheets
(Merck1.05554). Infrared spectra were recorded on a Shimadzu
IR 470 instrument. 1H and 13C NMR spectra were recorded in CDCl3
solution on a Bruker AV-400 and Bruker AVANCE III 500 MHz spec-
trometers. Chemical shifts are expressed in ppm values using TMS
as the internal standard. Mass spectra were recorded on a Q TOF
micro mass spectrometer. HPLC analysis was carried out on Jasco
PU-1580 liquid chromatogram with a PDA detector using Chiralcel
OD-H and OJ-H chiral columns (Daicel, 4.6⁄250 mm). Hexane/iso-
propanol was used as the mobile phase. Optical rotations were
determined on an Autopol-IV automatic polarimeter. The sub-
strates rac-1a-1e,10a,c,d ethyl-2-hydroxy-4-phenylbut-3-ynoate
3a27 and methyl-2-hydroxy-4-phenylbut-3-ynoate 3b27 were syn-
thesized and reported in earlier studies.
It should be noted that the cis-substrates 7a and 9a are away
from the cofactor (7.67 and 7.89 Å, respectively), making the hy-
dride attack difficult; therefore the product formation is not fa-
voured (Table 2). The favourable conformation of trans-substrates
6a and 8a brings them in close proximity to the CPCR-NADPH com-
plex to interact with the cofactor (3.68 and 3.72 Å, respectively)
and allows the formation of products. It should be noted that a ste-
reo-discrimination exists between substrates 6a and 8a. Although
the orientation of the substrate binding to the enzyme is the same
in both the cases, 6a results in the formation of the (R)-enantiomer
while 8a results in an (S)-enantiomer due to the difference in the
prioritization of the functional groups for assigning ‘Re’ and ‘Si’ face
(Figs. 4a and 4b). Thus, the geometric preference of C. parapsilosis
ATCC 7330 for the asymmetric reduction can be explained with
the help of docking studies.
4.2. Growth conditions for C. parapsilosis ATCC 733011
3. Conclusion
C. parapsilosis ATCC 7330 was grown in the yeast malt broth
medium (50 ml) in 250 ml Erlenmeyer flasks incubated at 25 °C,
200 rpm. The cells were harvested by centrifuging the 14th hour
culture broth at 10,000 rpm for 10 min at 4 °C and subsequent
washing with distilled water. The process was repeated twice,
and the wet cells were used for biotransformation.
The geometric preference in the C. parapsilosis ATCC 7330 med-
iated deracemization was studied using E- and Z-secondary alco-
hols. It was observed that E-isomers were preferred over the Z-
isomers. Enantiomerically pure E-secondary alcohols were synthe-
sized via deracemization with excellent enantioselectivity (> 99%)
and good isolated yields (up to 86%) using the 14 h culture of
whole cells of C. parapsilosis ATCC 7330. The enantiomerically pure
(S,E)-4-phenylbut-3-ene-1,2-diol (ee >99%, isolated yield 86%) was
synthesized by whole cell mediated deracemization and is re-
ported here for the first time. The geometric preference of C. par-
apsilosis ATCC 7330 was explained with the help of
computational studies. The enzyme was modelled using the X-
ray crystal structure of MtDH. The cofactor, NADPH, was initially
docked to this modelled enzyme and then various substrates were
docked to this complex. It was observed that the E-isomers were
favourably located in the active site cavity (6a; 3.68 Å and 8a;
3.72 Å) so that they could interact with the hydride of NADPH
and interact with the catalytic triad. The Z-isomers could not
achieve this proximity. Therefore, the E-isomers yielded the de-
sired products while the Z-isomers did not.
4.3. Typical procedure for the deracemization of rac-E- and Z-
secondary alcohols using the whole cells of C. parapsilosis ATCC
733010
To a 250 ml conical flask containing 50 g of harvested C. parapsi-
losis ATCC 7330 cells suspended in 100 ml of sterile distilled water
and 296 mg (2 mmol) of rac-E-4-phenylbut-3-en-2-ol rac-1a dis-
solved in 6 ml of ethanol as co-solvent were added. The reaction
was carried out in a water bath shaker at 200 rpm and 25 °C for
2 h. After incubation, the product formed was isolated using ethyl
acetate and the organic layer was dried over anhydrous sodium
sulfate. The solvent was removed by evaporation and enantiomeri-
cally pure (R,E)-4-phenylbut-3-en-2-ol 1a was obtained as a pale
yellow liquid after purification by silica gel column chromatogra-
phy using hexane/ethyl acetate (90:10) as the mobile phase eluent.
The ee was found to be >99%, as determined using HPLC. The yield
of the isolated product was 76%. Spectroscopic data were identical
to that reported in the literature.10d The rest of rac-E- and Z-sec-
ondary alcohols were used as substrates for the deracemization
under the same reaction conditions (Table 1). Control experiments
4. Experimental
4.1. General methods
C. parapsilosis ATCC 7330 was purchased from ATCC Manassas,
VA 201018, USA and maintained at 4 °C in the yeast malt agar

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T. Saravanan et al. / Tetrahedron: Asymmetry 23 (2012) 1360–1368
were done in parallel without the whole-cells under identical
conditions.
4.7.1. Structure validation
After building the protein 3D structure, in order to assess the
overall stereochemical quality of the modelled protein, Ramachan-
dran plot analysis was performed using the programme PRO-
CHECK.31 The ProSA32 test was applied to the final model in
order to check the energy criteria in comparison with the potential
mean force derived from a large set of known protein structures. To
assess the reliability of the modelled structure of CPCR, we further
calculated the root mean square deviation (RMSD); this was calcu-
lated by superimposing it on the template structure (3GDF) using
PYMOL. The RMSD value of the modelled CPCR protein based on
the approach was 0.074 Å.
4.4. Typical procedure for asymmetric reduction of E- and Z-4-
phenylbut-3-en-2-one using the whole cells of C. parapsilosis
ATCC 733011,12
To a 250 ml conical flask containing 50 g of harvested C. parapsi-
losis ATCC 7330 cells suspended in 100 ml of sterile distilled water
and 146 mg (1 mmol) of E-4-phenylbut-3-en-2-one 6a dissolved in
3 ml of 2-propanol as co-solvent were added. The reaction was car-
ried out in a water bath shaker at 200 rpm and 25 °C for 2 h. After
incubation, the product formed was isolated using ethyl acetate
and the organic layer was dried over anhydrous sodium sulfate.
The solvent was removed by evaporation and enantiomerically
pure (R,E)-4-phenylbut-3-en-2-ol 6b was obtained as a pale yellow
liquid after purification by silica gel column chromatography using
hexane/ethyl acetate (90:10) as a mobile phase eluent. The ee was
found to be >99%, as determined using HPLC. The yield of the iso-
lated product was 76%. Spectroscopic data were identical to those
reported in the literature.10d The Z-4-phenylbut-3-en-2-one 7a
was used as a substrate under the same conditions (Scheme 2).
Control experiments were done in parallel without the whole cells
under identical conditions.
4.7.2. Docking and simulation
The Molecular Operating Environment (MOE, version 2011.10)
software was used for all computations. MMFF94x force field33
was applied for energy minimization on CPCR and substrates.
The modelled CPCR enzyme structure was fixed whereas the co-
factor structure (NADPH) was assumed to be flexible during these
enzyme-cofactor dockings. While performing the docking with the
substrates, they were considered to be flexible but the enzyme-
cofactor complex was maintained rigid. The London
DG score
was calculated for all of the substrate-NADPH complexes in the
CPCR enzyme. This score estimates the free energy of binding of
a ligand for a given pose. The functional form of the energy term
is given below
X
X
X
4.5. Preparation of the cell free extract by cell disruption using a
sonicator28
G ¼ c þ Eflex
þ
CHBfHB
þ
CMfM
þ
DDi
hꢁbonds
mꢁlig
atomsi
Harvested cells (32 g of wet cells) of C. parapsilosis ATCC 7330
were re-suspended in 50 ml of lysis buffer (20 mM potassium
phosphate buffer, pH 6.8, 2 mM of PMSF). The suspension was then
homogenized on ice by ultrasonication (a pulse of 1 s on and 2 s off,
for 5 min, 40% amplitude). The homogenate was centrifuged at
10,000ꢂg for 15 min at 4 °C to pellet the undisrupted cells and deb-
ris. The supernatant was used as the cell free extract for further
experiments. Protein concentration (2.4 mg/ml) in the cell free ex-
tract was estimated by the Bradford method.
where, c represents the average gain/loss of rotational and transla-
tional entropies. Eflex is the energy due to the loss flexibility of the
Ligand (calculated from Ligand topology only, fHB measures geomet-
ric imperfections of hydrogen bonds and takes a value in (0,1), CHB is
the energy of an ideal hydrogen bond, fM measures geometric
imperfections of metal ligations and takes a value in (0,1), CM is
the energy of an ideal metal ligation and Di is the desolvation en-
ergy of atom i. Visualization, analysing and modelling were per-
formed using Discovery Studio Visualizer 2.5.5.34
4.8. Synthesis of the substrates
4.6. Deracemization rac-E- and Z-4-phenylbut-3-en-2-ol
reaction using cell free extract of C. parapsilosis ATCC 7330
The substrates E-4-phenylbut-3-en-2-one 6a,35 ethyl 3-hydro-
xy-5-phenylpent-4-ynoate 3c,36 4-phenylbut-3-yn-2-ol 3d37 and
4-phenylbut-3-yn-2-one 10a38 were synthesized as reported in
the literature.
To 40 ml of cell free extract, 40 mg (0.27 mmol) of substrate rac-
1a dissolved in 1 ml of DMSO was added. The reaction mixture was
incubated at 25 °C for 1 h at 200 rpm. After incubation, the product
formed was isolated using ethyl acetate and the organic layer was
dried over anhydrous sodium sulfate. The solvent was removed by
evaporation and enantiomerically pure (R,E)-4-phenylbut-3-en-2-
ol was obtained as a colourless liquid. The rac-Z-4-phenylbut-3-
en-2-ol rac-2a was also used as a substrate under identical reaction
conditions.
4.8.1. Synthesis of E-4-phenylbut-3-ene-1,2-diol rac-1e and 4-
phenylbut-3-yne-1,2-diol 3e27
The Ethyl-2-oxo-4-phenylbut-3-enoate (622 mg, 3 mmol) was
treated with ethanol (10 ml) in the presence of NaBH4 (228 mg,
6 mmol) and CeCl3.7H2O (1.6 g, 4.5 mmol) at room temperature.
The reaction was monitored by TLC and after completion, excess
alcohol was removed under vacuum. The reaction mixture was
quenched with dilute HCl and extracted with dichloromethane
(DCM). The organic layer was dried, concentrated, purified by sil-
ica-gel column chromatography and afforded 4-phenylbut-3-yne-
1,2-diol (320 mg, 64%). The same procedure was used for the syn-
thesis of 4-phenylbut-3-yne-1,2-diol.
4.7. Sequence alignment and homology modelling
The amino acid sequence of the carbonyl reductase was re-
trieved from the UniProtKB/TrEMBL database (Id: B2KJ46). NCBI’s
BLASTp was used to search against the protein data bank (PDB)
for similar sequence identity. Among the numerous homologous
sequences obtained as hits, mannitol dehydrogenase from Cladospo-
rium herbarum (PDB Code: 3GDF) with a resolution of 2.5 Å was
chosen as the template based on high sequence identity. The over-
all aligned score using ClustalW 2.1 was 50%.29 The resulting tem-
plate was used to model the 3D structure of C. parapsilosis carbonyl
reductase (CPCR). The homology modelling was carried out against
the chosen template using the Swiss Model.30
4.8.2. General procedure for the synthesis of Z-Secondary
alcohols rac-2a-2e and Z-4-phenylbut-3-en-2-one 7a20
Ethyl-2-hydroxy-4-phenylbut-3-ynoate (408 mg, 2 mmol) was
treated with Lindlar’s catalyst (180 mg) and quinoline (0.01 mmol)
in ethanol (6 ml) under a nitrogen atmosphere. The nitrogen was
then replaced by hydrogen gas and stirred at room temperature.
The reaction was monitored by TLC. After completion of reaction,

T. Saravanan et al. / Tetrahedron: Asymmetry 23 (2012) 1360–1368
1367
the reaction mixture was filtered through Celite and the filtrate
was concentrated. The residue was dissolved in ethyl acetate and
washed with dilute HCl and then water. The organic layer was
dried, concentrated and purified by silica gel column chromatogra-
phy (387 mg, 94%). The same procedure was used for the synthesis
of other substrates.
dd, J = 11.6 and 9.2 Hz), 6.59 (1H, d, J = 11. 6 Hz), 7.25–7.37 (5H,
m); 13C NMR (100 MHz, CDCl3): 14.3, 41.4, 61.0, 64.6, 127.6,
128.5, 128.9, 131.1, 132.1, 136.4, 172.5; IR (m
, cmꢁ1):3444, 3060,
2982, 2854, 2404, 1728, 1373, 1028, 758, 701, 669; HRMS: m/z,
Calculated Mass: 243.0997 [(M+Na)+], Found: 243.0990
[(M+Na)+]. The compound was resolved by HPLC analysis at
25 °C, using
a CHIRALCEL OD-H column (hexanes/2-propa-
4.9. Spectroscopic characterization of substrates
nol = 90:10, 1.0 mL/min; retention times 5.8 and 11.2 min).
4.9.1. E-4-Phenylbut-3-ene-1,2-diol16
4.9.9. Z-4-Phenylbut-3-en-2-ol38
1H NMR (500 MHz, CDCl3): d = 2.37 (1H, br s), 2.61 (1H, br s),
3.60 (1H, dd, J = 11.5 and 7.5 Hz), 3.75 (1H, dd, J = 11.5 and
3.5 Hz), 4.44 (1H, dd, J = 9.5 Hz and 6.5 Hz) 6.19 (1H, dd, J =
16 Hz and 6.5 Hz), 6.68 (d, 1H, J = 16), 7.23–7.27 (1H, m), 7.30–
7.33 (2H, m), 7.36–7.39 (2H, m); 13C NMR (125 MHz, CDCl3):
66.5, 73.2, 126.5, 127.7, 127.9, 128.6, 132.1, 136.3. The compound
was resolved by HPLC analysis at 25 °C, using a CHIRALCEL OD-H
column [hexanes/2-propanol = 90:10, 1.0 mL/min; retention times
13.0 (S), 14.8 min (R)].
1H NMR (500 MHz, CDCl3): d = 1.37 (3H, d, J = 6 Hz), 1.55 (1H,
bs) 4.76–4.82 (1H, m), 5.70 (1H, dd, J = 11.5 Hz and 9 Hz), 6.45
(1H, d, J = 11.5 Hz), 7.26–7.28 (3H, m), 7.33–7.36 (2H, m); 13C
NMR (125 MHz, CDCl3): 21.6, 64.2, 127.2, 128.3, 128.8, 130.1,
135.6, 136.2. The compound was resolved by HPLC analysis at
25 °C, using
a CHIRALCEL OJ-H column (hexanes/2-propa-
nol = 95:05, 1.0 mL/min; retention times 7.7 and 9.3 min).
4.9.10. Z-4-Phenylbut-3-ene-1,2-diol16
1H NMR (500 MHz, CDCl3): d = 2.58 (2H, br s), 3.59 (1H, dd,
J = 11.5 and 8 Hz), 3.71 (1H, dd, J = 11.5 and 3.5 Hz), 4.67–4.70
(1H,m) 5.64 (1H, dd, J = 11.5 Hz and 9 Hz), 6.63 (d, 1H, J = 11.5),
7.25–7.29 (3H, m), 7.32–7.36 (2H, m); 13C NMR (125 MHz, CDCl3):
66.2, 68.7, 127.5, 128.4, 128.6, 129.6, 133.2, 136.2. The compound
was resolved by HPLC analysis at 25 °C, using a CHIRALCEL OD-H
column (hexanes/2-propanol = 90:10, 1.0 mL/min; retention times
9.3 and 11.9 min).
4.9.2. E-4-Phenylbut-3-en-2-one35
1H NMR (400 MHz, CDCl3): d = 2.38 (3H, s), 6.71 (1H, d,
J = 16.4 Hz), 7.39–7.41 (3H, m), 7.51 (1H, d, J = 16.4 Hz), 7.54–
7.56 (2H, m); 13C NMR (100 MHz, CDCl3): 27.6, 127.2, 128.4,
129.1, 130.7, 134.5, 143.6, 198.6.
4.9.3. Ethyl 3-hydroxy-5-phenylpent-4-ynoate36
1H NMR (500 MHz, CDCl3): d = 1.29 (3H, t, J = 7.5 Hz), 2.83–2.84
(2H, m), 3.23(1H, d, J = 6 Hz), 4.21 (2H, q, J = 7 Hz), 4.99 (1H, dd,
J = 12 Hz and 6 Hz) 7.28–7.32 (3H, m), 7.41–7.43 (2H, J = 2H); 13C
NMR (125 MHz, CDCl3): 14.2, 42.0, 59.3, 61.0, 87.0, 88.0, 122.2,
128.2, 128.6, 131.7, 171.53.
4.9.11. Z-4-Phenylbut-3-en-2-one41
1H NMR (400 MHz, CDCl3): d = 2.14 (3H, s), 6.17 (1H, d,
J = 12.4 Hz), 6.89 (1H, d J = 12.4 Hz), 7.34–7.36 (3H, m), 7.46–7.48
(2H, m); 13C NMR (100 MHz, CDCl3): 30.8, 128.3, 128.5, 129.2,
129.4, 134.1, 140.1, 201.0.
4.9.4. 4-phenylbut-3-yn-2-ol37
1H NMR (500 MHz, CDCl3): d = 1.55 (3H, d, J = 6.5 Hz), 2.13 (1H,
d, J = 4.5) 4.73–4.78 (1H, m), 7.28–7.32 (3H, m), 7.41–7.44 (2H, m);
13C NMR (125 MHz, CDCl3): 24.3, 58.8, 84.0, 90.9, 122.6, 128.2,
128.4, 131.6.
Acknowledgments
Thangavelu Saravanan thanks the Indian Institute of Technol-
ogy (IIT) Madras and the University Grants Commission, India,
for the fellowship. We thank the Department of Biotechnology,
New Delhi, India for funding, Sophisticated Analytical Instrumen-
tation Facility (SAIF) and Department of Chemistry, IIT Madras
for NMR and mass analysis.
4.9.5. 4-Phenylbut-3-yn-2-one38
1H NMR (500 MHz, CDCl3): d = 2.45 (3H, s), 7.36–7.40 (2H, m),
7.43–7.47 (1H, m), 7.55–7. 58 (2H, m); 13C NMR (125 MHz, CDCl3):
32.7, 88.2, 90.3, 119.9, 128.6, 130.7, 133.0, 184.6.
References
4.9.6. Z-Ethyl 2-hydroxy-4-phenylbut-3-enoate39
1H NMR (500 MHz, CDCl3): d = 1.32 (3H, t, J = 7.5 Hz), 3.13 (1H,
d, J = 5 Hz), 4.27–4.31 (2H, m), 5.04 (1H, dd, J = 5 Hz and 0.5 Hz),
5.64 (1H, dd, J = 11 Hz and 9.5 Hz), 6.81 (1H, d, J = 11 Hz), 7.29–
7.32 (1H, m), 7.35–7.39 (2H, m), 7.44 (1H, d, J = 7.5 Hz); 13C NMR
(125 MHz, CDCl3): 14.1, 62.2, 67.3, 127.3, 127.7, 128.3, 128.9,
134.7, 135.7, 173.9; The compound was resolved by HPLC analysis
at 25 °C, using a CHIRALCEL OD-H column [hexanes/2-propa-
nol = 90:10, 1.0 mL/min; retention times 6.0 (R), 8.4 min (S)].
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