Biocatalyzed asymmetric reduction of benzils to either benzoins or hydrobenzoins: pH dependent switch

Article abstract of DOI:10.1039/c5cy00158g

Enantiopure benzoins and hydrobenzoins are precursors of various pharmaceuticals and biologically active compounds. In addition, hydrobenzoins are precursors of chiral ligands and auxiliaries in stereoselective organic synthesis. Biocatalytic reduction of benzils is a straightforward approach to prepare these molecules. However, known methods are not selective and lead to formation of a mixture of benzoin and hydrobenzoin, requiring expensive separation procedures. Here, we describe an enzyme system Talaromyces flavus, which exhibited excellent pH dependent selectivity for the conversion of benzil to either benzoin or hydrobenzion in high ee. Thus, (S)-benzoin was the exclusive product at pH 5.0 (ee >99%), whereas at pH 7.0, (S,S)-hydrobenzoin (ee >99%, dl/meso 97 : 3) was the exclusive product. The observed pH dependent selectivity was shown to be due to the presence of multiple enzymes in Talaromyces flavus, which specifically accepted either benzil or benzoin as a substrate and exhibited different pH profiles of their activity. The biocatalyst efficiently reduced a variety of symmetrical and unsymmetrical benzils. Moreover, a 36.4 kDa benzoin reductase was purified, the N-terminal sequence of which did not show a significant similarity to any of the known reductase/dehydrogenase in the database. The protein therefore appears to be a novel reductase.

Full text of DOI:10.1039/c5cy00158g

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Biocatalyzed asymmetric reduction of benzils to
either benzoins or hydrobenzoins: pH dependent
switch†
Cite this: DOI: 10.1039/c5cy00158g
Mohan Pal,‡ Gautam Srivastava,§ Amar Nath Sharma, Suneet Kaur
*
and Ravinder S. Jolly
Enantiopure benzoins and hydrobenzoins are precursors of various pharmaceuticals and biologically active
compounds. In addition, hydrobenzoins are precursors of chiral ligands and auxiliaries in stereoselective
organic synthesis. Biocatalytic reduction of benzils is a straightforward approach to prepare these
molecules. However, known methods are not selective and lead to formation of a mixture of benzoin and
hydrobenzoin, requiring expensive separation procedures. Here, we describe an enzyme system
Talaromyces flavus, which exhibited excellent pH dependent selectivity for the conversion of benzil to
either benzoin or hydrobenzion in high ee. Thus, (S)-benzoin was the exclusive product at pH 5.0 (ee
>99%), whereas at pH 7.0, (S,S)-hydrobenzoin (ee >99%, dl/meso 97: 3) was the exclusive product. The
observed pH dependent selectivity was shown to be due to the presence of multiple enzymes in
Talaromyces flavus, which specifically accepted either benzil or benzoin as a substrate and exhibited
different pH profiles of their activity. The biocatalyst efficiently reduced a variety of symmetrical and
unsymmetrical benzils. Moreover, a 36.4 kDa benzoin reductase was purified, the N-terminal sequence of
which did not show a significant similarity to any of the known reductase/dehydrogenase in the database.
The protein therefore appears to be a novel reductase.
Received 1st February 2015,
Accepted 26th May 2015
DOI: 10.1039/c5cy00158g
www.rsc.org/catalysis
auxiliaries in stereoselective organic synthesis. Because of the
importance of α-hydroxyketones in organic synthesis and as
Introduction
Enantiopure benzoins (α-hydroxyketones) are important inter-
mediates for various pharmaceuticals, such as antifungal
agents, antitumor antibiotics (olivomycin A, chromomycin A3,
and epothilones),1 selective inhibitors of amyloid-β-protein
(for Alzheimer's disease treatment),2 farnesyltransferase
inhibitors kurasoin A and B,3 antidepressants (bupropion and
its metabolites),4 urease inhibitors5 and heterocycles.6 In
addition, these are very interesting molecules from a synthetic
chemistry point of view as these are precursors of amino alco-
hols and 1,2-diols, which in turn are precursors of various
biologically active compounds as well as chiral ligands and
intermediates of various bioactive molecules, many chemical
approaches have been developed for their synthesis. These
include α-hydroxylation of ketones and Sharpless asymmetric
dihydroxylation of the silyl enol ether of the corresponding
ketone,7 ketohydroxylation of olefins,8 asymmetric mono-
oxidation of 1,2-diols,9 oxidative kinetic resolution of racemic
α-hydroxyketones,10,11 organocatalytic strategies involving
asymmetric α-oxygenation of ketones in the presence of pro-
line or alanine12,13 and traditional benzoin condensation car-
ried out stereoselectively by means of optically active catalysts
such as chiral thiazolium and triazolium salts, in a biomi-
metic fashion.14,15 These reported chemical methods are suc-
cessful in producing a variety of molecules but suffer from
drawbacks, such as large number of steps, rare high ee, low
overall yields and too much waste.
Biocatalytic ketone reduction combined with economic
and environmentally friendly reaction conditions is an effec-
tive strategy to overcome these limitations.16–20 Penicillium
claviforme,21 Pichia glucozyma,22 Aspergillus oryzae, Fusarium
roseum,23 Bacillus cereus24 and Xanthomonas oryzae25 have
been used for the preparation of benzoins by asymmetric
reduction of benzils. Cryptococcus macerans,26 yeast strains,
Saccharomyces uvarum, Saccharomyces montanus27 and
Department of Chemistry, CSIR-Institute of Microbial Technology, Sector 39,
Chandigarh 160 036, India. E-mail: jolly@imtech.res.in; Fax: +91 0172 269 0585
† Electronic supplementary information (ESI) available: Fig. S1–3 showing
determination of 2a/3a ratios, dl/meso ratios and ee, Fig. S4 showing the
fractionation scheme for proteins of T. flavus, Fig. S5–7 showing the protein
purification profile on various columns and Table S1 showing the summary of
protein purification steps. Tables S2–4 showing HPLC traces of racemic and
biocatalyzed benzoins and hydrobenzoins. NMR images of all relevant
compounds. See DOI: 10.1039/c5cy00158g
‡ Present address: Department of Biochemistry and Molecular Biology,
Dalhousie University, Halifax, NS B3H 4R2, Canada. E-mail: mohanpal@dal.ca.
§ Present address: Department of Structural Biology, Weizmann Institute of
Science, Rehovot 76100, Israel.
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Rhizopus sp.28 and alcohol dehydrogenases from
Thermoanaerobacter sp., Lactobacillus brevis and Ralstonia
sp.29 have been used for enantio- and diastereo-selective syn-
thesis of hydrobenzoins.
In principle, chiral benzoins as well as chiral hydro-
benzoins are easily accessible via biocatalytic reduction of
the corresponding benzils. However, there is a dearth of bio-
catalysts which accept such bulky–bulky substrates.29 More-
over, known biocatalysts are not selective and bring about
the formation of a mixture of benzoin and hydrobenzoin,
requiring expensive separation procedures, which hampers
the acceptability of these biocatalysts for application in tech-
nical scale preparations.
Herein, we describe an enzyme system Talaromyces flavus,
which exhibited excellent pH dependent selectivity for the
conversion of benzil to either benzoin or hydrobenzion in
high ee. Thus, (S)-benzoin was the exclusive product at pH
5.0 (ee >99%), whereas at pH 7.0, (S,S)-hydrobenzoin (ee
>99%, dl/meso 97/3) was the exclusive product. We have dem-
onstrated that the pH dependent selectivity of T. flavus is due
to the presence of multiple enzymes in the system, which
specifically accepted either benzil or benzoin as a substrate
and exhibited different pH profiles of their activity. The bio-
catalyst T. flavus accepted a variety of symmetrical and
unsymmetrical benzils as substrates. Moreover, a 36.4 kDa
benzoin reductase was purified from T. flavus. The
N-terminal sequence obtained from the purified protein by
Edman degradation did not show a significant similarity to
any of the known reductases/dehydrogenases in the database.
The protein therefore appears to be a novel reductase.
sodium sulfate and solvents evaporated under reduced pres-
sure to yield a residue which was analysed by 1H NMR. All
the three strains were able to reduce 1a to a mixture of ben-
zoin (2a) and hydrobenzoin (3a) in varying ratios (Table 1).
1
The ratio of 2a/3a was determined by H NMR (Fig. S1, ESI†).
The single methine proton of pure rac-2a appeared as a bs
(broad singlet) at δ 5.9. Hydrobenzoin 3a prepared by sodium
borohydride reduction of 2a showed two bs at δ 4.7 and 4.8,
corresponding to two methine protons of the dl and meso
pairs. The ratio of 2a/3a of the biocatalyzed product in the
reaction mixture was determined from integral values of the
resonance peaks at δ 5.9, 4.7 and 4.8. The ee of 2a was deter-
mined by chiral HPLC using a Chiralcel OD-H column. Elu-
tion was carried out with hexane : isopropanol IJ90/10) at a
flow rate of 1 ml min−1. Detection was performed at λ254. In
contrast to two enantiomers of rac-2a eluted at 11.9 and 17.4
min, the biocatalyzed product showed the predominant pres-
ence of one enantiomer eluted at about 11.9 min (Fig. S2,
ESI†). Ee was calculated from the relative area under the two
peaks. T. flavus gave an ee of >99%, whereas Penicillium sp.
and A. alternata gave ees of 95% and 91%, respectively. The
absolute configuration of the T. flavus catalyzed product 2a
was assigned as (S) based on the comparison of the sign of
optical rotation ([α]2D5 = +115.2 (c 1.5, acetone)) with that in
the literature (lit. ref. 31, [α]2D5 = +115.0 (c 1.5, acetone)).
Accordingly, the enantiomer eluted at 11.9 min by HPLC of
(S)-2a corresponds to an (S)-enantiomer and that at 17.4 min
to an (R)-enantiomer.
Results and discussion
Strain selection
Enantiopure benzoins and hydrobenzoins are intermediates
for various bioactive molecules. They also serve as chiral
ligands in stereoselective organic synthesis. In our previous
report, we have described three biocatalysts Penicillium sp.
MTCC 10832, Alternaria alternata MTCC 10833 and
Talaromyces flavus MTCC 10834 for enantioselective reduc-
tion of methyl heteroaryl and aryl heteroaryl ketones to their
corresponding (S)-heteroaryl alcohols in >99% ee.30 We
tested these strains for enantioselective reduction of benzil
(1a). The fungal strains were grown as described in the exper-
imental section. The cells were isolated by centrifugation,
washed with phosphate buffer (0.2 M, pH 7.0) and
resuspended in the same buffer at a concentration of 100 g
L−1. Benzil (1a; 50 mg, 0.24 mmol) was added to a suspension
of 5 g of cells in 50 mL of phosphate buffer and the contents
were shaken at 200 rpm and 30 °C until complete consump-
tion of the substrate occurred. The progress of the reaction
was monitored by TLC using a commercially available stan-
dard sample of rac-benzoin (2a). At 100% conversion, cells
were removed from the reaction mixture by centrifugation
and the contents were extracted in ethyl acetate. The organic
layer was washed with saturated brine, dried over anhydrous
The dl/meso ratio of 3a was determined by HPLC and 1H
NMR. A sample of dl/meso-3a prepared by sodium borohy-
dride reduction of 2a on a chiral HPLC using a Chiralcel OJ
column was resolved into three peaks with retention times of
16.2, 18.8 and 22.6 min (Fig. S3, ESI†). The peaks at 16.2 and
18.8 min appeared in an almost 1 : 1 ratio and were assigned
to the dl pair of 3a. The peak eluting at 22.6 min was there-
fore assigned to meso-3a. The ratio of dl/meso in the borohy-
dride reaction was found to be 5 : 95, indicating the predomi-
nant formation of the meso product in the reaction. A similar
ratio was also obtained by comparison of integral values of
the resonance peaks at δ 4.84 and 4.72, corresponding to
methine protons of the meso and dl pairs, respectively, in the
1H NMR spectra of 3a obtained by borohydride reduction of
2a (Fig. S1, ESI†). Thus, the dl/meso ratio in all subsequent
studies was calculated by 1H NMR. In contrast to the predom-
inant formation of the meso product in borohydride reduc-
tion, the biocatalyzed reduction produced dl as the major
product. The ratios of dl/meso obtained from Penicillium sp.,
A. alternata and T. flavus were 65/35, 94/6 and 80/20, respec-
tively. The ee of the dl pair of 3a was determined by chiral
HPLC using a Chiralcel OJ column (Fig. S3, ESI†). T. flavus
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Table 1 Biocatalyzed reduction of benzil
(S)-2ab,c
(S,S)-3ab,c
dl/mesob
Conv.b
(%)
Time
(h)
2a :
Entry
Strain
3aa
Ee (%)
Ee (%)
1
2
3
Penicillium sp.
A. alternata
T. flavus
100
100
100
24
18
37
1 : 0.21
1 : 1.53
1 : 0.94
95
91
>99
65/35
94/6
80/20
93
85
99
a
b
The conversion, ratio of 2a : 3a and dl/meso ratio were determined by 1H NMR based on integral values of specified peaks. Ee of 2a and 3a
c
was determined by chiral HPLC on Chiralcel OD-H and OJ (Daicel, Japan) columns, respectively. Absolute configuration was assigned based
on comparison of the sign of optical rotation with that in the literature.
gave an ee of 99% while ees of 93% and 85% were obtained
in the case of Penicillium sp. and A. alternata, respectively.
The absolute configuration of T. flavus catalyzed 3a was
in high chemical purity. Demir et al. have reported an inter-
esting effect of pH on Rhizopus oryzae catalyzed reduction of
benzil/benzoin to give (R)-2a (>99% ee) at pH 6.5–8.5 and (S)-
2a (80% ee) at pH 4.2–5.0 in 5–6 days. Continuation of the
reaction for 21 days at pH 6.5–8.5 resulted in the formation
of 3a with a dl/meso ratio of 99/1 and an ee of >99%.28,34
Although a high ee was observed, this method suffered from
low yields and very long reaction time. Also, the reactions
were carried out with a growing culture, where media compo-
nents and secondary metabolites are likely to contaminate
the product. Moreover, no attempt was made to provide a
rationale for the effect of pH on the course of reaction.
assigned as 1S,2S based on the sign of optical rotation [α]D25
=
−91.0 (c 1.05, ethanol) [lit. ref. 32 and 33, [α]2D5 = +91.6 (c
1.05, ethanol), 99.9% ee (R,R)]. Thus, in the dl pair, the peak
eluting at 16.2 min corresponds to a (1S,2S) configuration.
Exclusive preparation of (S)-benzoin and (S,S)-hydrobenzoin
with T. flavus: pH dependent reduction of benzil
T. flavus was found to be the best biocatalyst for the enantio-
selective preparation of (S)-benzoin and (S,S)-hydrobenzoin
with >99% ee. However, it produced a mixture of 2a and 3a,
which limits its scope for the preparation of these molecules
In view of these observations, we studied the effect of pH
on T. flavus catalyzed asymmetric reduction of 1a. Biocata-
lytic reduction of 1a was carried out from pH 4.0–9.0 as
Table 2 pH dependent asymmetric reduction of benzil by Talaromyces flavus
Entry
pH
4.0
Time (h)
Conv.b (%)
2a : 3aa
eeb,c (%) (2a) (config)
dl : mesoa (3a)
Eeb,c (%) (3a) (config)
1
2
3
4
5
6
7
8
14
37
144
264
14
22
29
90
100
66
100
100
100
80
100
100
100
50
100
100
100
100
65
100
100
100
65
1 : 0
1 : 0
97 (S)
97 (S)
86 (S)
70 (S)
97 (S)
99 (S)
96 (S)
74 (S)
>99 (S)
>99 (S)
96 (S)
78 (S)
>99 (S)
>99 (S)
82 (S)
81 (S)
—
>99 (S)
>99 (S)
80 (S)
76 (S)
>99 (S)
>99 (S)
76 (S)
71 (S)
n.d.
n.d.
33/67
25/75
n.d.
n.d.
n.d.
33.3 (R,R)
45.9 (R,R)
n.d.
1 : 0.025
1 : 0.08
1 : 0
1 : 0.035
1 : 0.94
1 : 1.3
1 : 0.07
1 : 0.393
1 : 1.2
5.0
6.0
7.0
37
43/57
47/53
52/48
56/44
75/25
88/12
88/12
80/20
80/20
82/18
84/16
97/3
77/23
78/22
83/17
88/12
85/15
80/20
80/30
86/14
91 (S,S)
144
264
14
2.5 (S,S)
35 (R,R)
>99 (S,S)
>99 (S,S)
>99 (S,S)
97.2 (S,S)
>99 (S,S)
>99 (S,S)
>99 (S,S)
97.6 (S,S)
>99 (S,S)
>99 (S,S)
>99 (S,S)
96.2 (S,S)
63.7 (S,S)
>99 (S,S)
96 (S,S)
9
10
11
12
13
14
15
16
17d
18
19
20
21
22
23
24
25
37
144
264
14
1 : 1.6
1 : 0.392
1 : 0.94
1 : 1.76
1 : 2.14
0 : 1
1 : 0.174
1 : 0.441
1 : 1.07
1 : 1.26
1 : 0.25
1 : 0.51
1 : 0.95
1 : 1
37
144
264
18
14
37
144
264
14
37
144
264
8.0
9.0
98
100
100
93.7 (S,S)
78.2 (S,S)
a
b
The % conversion, ratio of 2a : 3a and dl/meso ratio were determined by 1H NMR based on integral values of specified peaks. Ee of 2a and
c
3a was determined by chiral HPLC on Chiralcel OD-H and OJ (Daicel, Japan) columns, respectively. Absolute configuration was assigned
based on comparison of the sign of optical rotation with that in the literature. Substrate concentration was 2 mM in this case. n.d. = not
d
detected.
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described in the experimental section using appropriate
buffers. Aliquots were drawn at 14, 37, 144 and 264 h and
analyzed for % conversion, product ratio and ee determina-
tion as described above for strain selection. The results are
summarized in Table 2. It was observed that (i) at pH 4.0–6.0,
(S)-2a was the major product in high ee at 100% conversion
(entries 4, 6, and 10; Table 2). (ii) The amount of 3a increased
with increasing pH. At pH 7.0, an almost 1 : 1 ratio of 2a to
3a was observed at 100% conversion in 37 h. While the ee of
(S)-2a was >99%, 3a was also produced in >99% ee (entry 14;
Table 2). (iii) A decrease in the ee of (S)-2a was observed at
pH 5.0 to 9.0 when the reaction was continued for longer
duration and (iv) at pH 4.0 and 5.0, not only a diol was
formed with a low ratio, but also a low dl/meso ratio was
obtained. Also, the diol was formed in low ee. The dl product
had an (R,R) configuration, whereas, at pH 6.0–8.0, diol 3a
was produced in high ee and had an (S,S) configuration.
In summary, pH 5.0 was found to be optimal for produc-
tion of (S)-2a (entry 6; Table 2). Similarly, pH 7.0 was found
to be optimal for production of (S,S)-3a (entry 15; Table 2).
Next, we reduced the substrate concentration from about 5
mM to 2 mM. We were pleased to note that at pH 7.0 and a
substrate concentration of 2 mM, (S,S)-3a was obtained as
the exclusive product in a dl/meso ratio of 97/3 and >99% ee
(entry 17; Table 2). Thus, depending on the pH and concen-
tration, either (S)-2a or (S,S)-3a can be selectively obtained in
high ee and high dl/meso ratio. In contrast to Rhizopus
oryzae, we did not observe pH dependent complete reversal
of enantioselectivity with T. flavus. However, we did observe a
drop in the ee of (S)-2a when the reaction was continued for
a longer period of 144–264 h. Optically pure (S)-2a did not
show any racemization at pH 4.0 to 9.0 in the absence of the
biocatalyst even after prolonged incubation for 264 h, indicat-
ing the presence of racemase activity in T. flavus.
appropriate buffers. The results are summarized in Table 3.
The following observations may be highlighted: (i) whereas
no reaction occurred at pH 4.0, the reaction at pH 5.0 was
very sluggish. (ii) At pH 6.0–9.0 and about 50% conversion,
(S)-2a was recovered in ~30% ee and 3a was obtained in a dl/
meso ratio of approximately 3/7 (entries 5, 8, 11, and 14;
Table 3). These results suggest that both (S)- and (R)-2a are
substrates for the diol reductase enzyme, but the (R)-enantio-
mer reacts faster than the (S)-enantiomer, which made partial
resolution of rac-2a possible at about 50% conversion. (iii) It
was interesting to note that after 24 h at pH 7.0–9.0, almost
complete conversion of 2a to 3a occurred to give a dl/meso
ratio of 3 : 7 (entries 9, 12, and 15; Table 3). However, upon
continuation of the reaction for 96 h, the ratio of dl/meso
changed to 1 : 1; the enantiomeric excess of the dl form
increased to 96–98% (entries 7, 10, 13, and 16; Table 3). Con-
tinuation of the reaction for longer times did not result in
any further change in the dl/meso ratio of 3a. These results
may be explained by the presence of oxidase activity, which
converted meso-3a to 2a which was re-reduced to (S,S)-
hydrobenzoin.
pH dependent selectivity of T. flavus due to the presence of
multiple dehydrogenases
T. flavus catalyzed bioreduction of 1a resulted in the forma-
tion of (S)-2a in >99% ee at pH 5.0 and (S,S)-3a in >99% ee
at pH 7.0. In principle, these results may be explained by
applying a single enzyme which has pH dependent substrate
specificity or by applying a two-enzyme system, each of which
has a different pH profile of activity. In addition to the above
pH dependent selectivity, conversion of meso-3a to dl-3a was
also observed, which could be explained based on the oxida-
tion of a hydroxyl group to the keto group to generate 2a as
the intermediate. Put together, these results indicate the
presence of more than one reductase in T. flavus, which are
We also studied the reduction of rac-2a with T. flavus as
the biocatalyst at various pH values in the range of 4.0–9.0 in
Table 3 pH dependent asymmetric reduction of rac-benzoin (2a) by Talaromyces flavus
Entry
pH
4
Time (h)
Conv.a (%)
Eeb,c (%) (residual (S)-2a)
dl : meso of 3aa
Eeb,c (%) ((S,S)-3a)
1
2
3
4
5
6
7
8
24
96
24
96
12
24
96
12
24
96
12
24
96
12
24
96
0
0
33
48
50
100
100
48
92
100
53
100
100
47
100
100
n.d.
n.d.
15
22
32
—
—
30
—
—
29
—
—
31
—
n.d.
n.d.
n.d.
n.d.
78.5
85
5
6
26/74
30/70
30/70
23/77
50/50
28/72
30/70
50/50
26/64
28/72
47/53
26/64
28/72
50/50
86
93.5
97.9
84.6
95.4
98.5
85.9
93.9
96.7
83.8
94.2
95.6
7
8
9
9
10
11
12
13
14
15
16
—
a
b
The % conversion and dl/meso ratio were determined by 1H NMR based on integral values of specified peaks. Ee of 2a and 3a was
c
determined by chiral HPLC on Chiralcel OD-H and OJ (Daicel, Japan) columns, respectively. Absolute configuration was assigned based on
comparison of the sign of optical rotation with that in the literature. n.d. = not detected.
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involved in the reduction of 1a. Thus, we fractionated pro-
teins of T. flavus in an attempt to isolate proteins with vari-
ous reductase activities.
after blotting the enzyme onto a polyvinylidene difluoride
(PVDF) membrane using a Trans-Blot cell. The obtained
N-terminal sequence, APTNQ_AYDHSV, was then searched
for homologous counterparts using the BLAST server at www.
ncbi.nlm.nih.gov. The retrieved hits for the N-terminal
sequence did not show significant homology to any of the
known benzil/benzoin reductases or dehydrogenases in the
database. Therefore, the protein appears to be a novel
reductase.
Similarly, the unbound protein fraction of the reactive red
step was also subjected to ion-exchange chromatography over
Q-sepharose followed by size-exclusion chromatography on a
Superdex S-200 column. The active fractions were pooled and
designated as fraction F2. However, a homogeneous prepara-
tion could not be obtained in this case as revealed by SDS-
PAGE of F2 run under reducing conditions (Fig. 1a). The
summary of fractionation of proteins of T. flavus is shown in
Fig. S4, ESI.†
The cell-free extract (F1), partially purified protein (F2)
and purified protein (F3) were studied for asymmetric reduc-
tion of 1a, rac-2a and (S)-2a at pH 5.0 and 7.0 as described in
the experimental section. The results are summarized in
Table 4. The presence of the following enzyme activities was
observed: (i) (S)-selective benzil reductase activity: fraction F2
converted 1a to (S)-2a at pH 5.0 as well as pH 7.0 (entries 6
and 7; Table 4). No trace of hydrobenzoin (3a) could be
detected in these reactions, which clearly shows that F2 is
devoid of any benzoin reductase activity. However, a lower ee
of 91% was obtained with F2 compared to 99% obtained with
whole cells at pH 5.0. The improved ee in whole cells could
be due to the conversion of (R)-2a to meso-3a by benzoin
reductase activity present in whole cells, but absent in frac-
tion F2; (ii) (R,R)-selective benzoin reductase activity: the pure
protein (F3) showed (R,R)-selectivity for reduction of 1a at pH
7.0 (entries 9 and 10; Table 4). F3 produced only (R,R)-3a and
no trace of (S,S)-3a or meso-3a could be detected either by
NMR or HPLC. However, the conversion rate for this enzyme
was very low compared to that for whole cells, requiring 24 h
for 25% conversion. Also, the (R,R)-selectivity was not strict,
because when (S)-2a was used as the substrate, (S,S)-3a was
produced in 53% ee at 50% conversion (entry 11; Table 4). F3
did not show any appreciable activity at pH 5.0; (iii) (S)-ben-
zoin to (R)-benzoin epimerase activity: when the pure protein
fraction F3 catalyzed conversion of (S)-2a (>99% ee) to 3a
was stopped at 50% conversion, the ee of the remaining (S)-
2a was found to be only 32% (entry 11; Table 4). This clearly
suggested that the pure protein in addition to benzoin reduc-
tase activity also has S-to-R epimerase activity. However, the
conversion rate for this activity was also very low; (iv) (S,S)-
selective benzoin reductase activity: the whole cell system of T.
flavus contains a very high (S,S)-selective benzoin reductase
activity at pH 7.0, but not at pH 5.0. However, this activity
could not be recovered from any of the fractions except F1
(entry 5; Table 4), which showed considerably lower activity
compared to the whole cell system, indicating that the
enzyme responsible for this activity is unstable outside the
The cell-free extract of T. flavus, designated as fraction F1
(see Fig. S4, ESI†) was obtained by disruption of cells in Dyno
Mill (KDL, Switzerland). T. flavus was mixed with glass beads
(0.5–0.75 μm) in a 1 : 1 ratio (wt/wt) and disruption was
conducted at 4 °C for 15 min followed by centrifugation at
20 000g for 20 min at 4 °C to remove cell debris. The cell-free
extract was subjected to ammonium sulfate fractionation,
maintaining the pH and temperature constant at 7.0 and 4
°C, respectively. Total activity was present in 40–80% ammo-
nium sulphate pellets. The protein was redissolved in phos-
phate buffer (10 mM, pH 8.0) and desalted using a Sephadex
G-25 column. The desalted protein was loaded into a reactive
red column. The column was washed with the same buffer
and the unbound protein fraction was assayed for activity.
The bound proteins were eluted with a 0–1.5 M NaCl gradient
and different fractions were assayed for activity. Both
unbound and bound fractions of proteins were found to be
active. The active fractions from the eluent were combined
and subjected to ion-exchange chromatography over
a
Q-sepharose column followed by size-exclusion chromatogra-
phy on a Superdex S-200 column. The active protein thus
obtained exhibited only one homogeneous band at ~40 kDa
in SDS-PAGE, run under the reducing conditions (Fig. 1b). It
was designated as fraction F3 (Fig. S4, ESI†). Approximately
20-fold purification was achieved in a 5-step purification pro-
cedure (see Table S1 and Fig. S5–7 in the ESI† for the sum-
mary of protein purification steps and purification profiles
on various columns).
The pure protein fraction (F3) showed a specific activity of
4.75 μmol per min per mg of protein. The purified protein
was subjected to MALDI analysis. The MALDI of the native
protein gave a molecular weight of ~36.4 kDa. Glycosylation
is a key feature of post-translational modification for protein
stability, secretion and activity in fungi.35 Accordingly, glyco-
sylation of the T. flavus enzyme reported herein is possible,
but its investigation was considered beyond the scope of this
study. The partial N-terminal amino acid sequence was
determined by using an automated Edman degradation pro-
cedure. The N-terminal amino acid sequence was determined
Fig.
1 SDS-PAGE of fractions F2 and F3 run under reducing
conditions: (a) fraction 2; lane 1: molecular wt. markers, lanes 2, 3 and
4: different fractions from the size exclusion chromatography step and
(b) fraction 3; lane 1: molecular wt. markers, lane 2: purified protein.
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Table 4 Asymmetric reduction of benzil (1a) and benzoin (2a) with protein fractions of Talaromyces flavus
Benzil (1a) as substrate
Conv.b Eec,d (%) (2a) % 3a
Benzoin (2a) as substratea
Eec,d (%) IJdl-3a) Conv.b Eec,d (%) (2a) % 3a
Eec,d (%) IJdl-3a)
(dl/meso)b (config)
Entry Fraction
pH (%)
(config)
(dl/meso)b
(config)
(%)
(config)
1
2
3
4
5
6
7
8
Whole cells 5.0
100
100
—
99 (S)
—
—
~3
—
48
28 (S)
n.d.
100 (S)
n.d.
11 (S)
n.d.
n.d.
26/74
50/50
80/20
n.d.
16/84
n.d.
n.d.
n.d.
99/1
100/0
100/0
75 (S,S)
97 (S,S)
>99
n.d.
86 (S,S)
n.d.
n.d.
n.d.
>99 (R,R)
>99 (R,R)
53 (S,S)
7.0
7.0
5.0
7.0
5.0
7.0
5.0
7.0
7.0
7.0
100 IJ97/3)
—
n.d.
7 IJ78/22)
n.d.
n.d.
>99 (S,S)
—
n.d.
80 (S,S)
n.d.
n.d.
100
80c
n.d.
15
n.d.
n.d.
n.d.
20
F1
F2
F3
20
40
50
50
<5
16
25
—
90 (S)
86 (S)
91 (S)
96 (S)
48 (S)
75 (S)
6 (R)
—
n.d.
n.d.
n.d.
9
10
11
20 (100 : 0) >99 (R,R)
33 (100 : 0) >99 (R,R)
—
36 (S)
25 (S)
32 (S)
50
—
50c
a
b
Benzoin was racemic except in entries 3 and 11 where (S)-benzoin was used as the substrate. The % conversion and dl/meso ratio were
determined by 1H NMR based on integral values of specified peaks. Ee of 2a and 3a was determined by chiral HPLC on Chiralcel OD-H and
OJ (Daicel, Japan) columns, respectively. Absolute configuration was assigned based on comparison of the sign of optical rotation with that in
c
d
the literature. n.d. = not detected.
cell, in spite of the presence of protease inhibitors in the
buffer at the time the cell-free extract was prepared from
whole cells. Several attempts to isolate the protein corre-
sponding to this activity were unsuccessful.
substituent (entry 6; Table 5). (ii) The substitution of electron
donating methoxy or electron withdrawing chloro groups
resulted in reduced enantioselectivity (entries 2 and 7;
Table 5). The loss of enantioselectivity was considerably
higher in the case of chloro substitution compared to
methoxy substitution. (iii) The ee in the case of methoxy sub-
stitution followed the order: 4-methoxy > 3-methoxy >
2-methoxy (entries 2–4; Table 5). It indicates that, in addition
to electronic effects, steric effects in the vicinity of the car-
bonyl group also play a role. Thus, the ee increased from
42% to 77% as the methoxy group was moved from the
2-position to the 3-position of the phenyl group. The ee was
82% when the methoxy group was moved to the 4-position.
(iv) An interesting observation was the reversal of stereo-
selectivity in the case of 2-chloro substitution (entry 7;
Table 5). Whereas unsubstituted benzil and methoxy or
methyl substituted benzil produced the corresponding ben-
zoin predominantly in the (S)-configuration, 2-chloro
substituted benzil produced the corresponding benzoin in
the (R)-configuration. (v) The substitution of the methyl
group at the 4-position of the phenyl group had a minimal
T. flavus catalyzed asymmetric monoreduction of various
symmetrical benzils (1a–1g)
In order to study the scope of T. flavus catalyzed mono-
reduction of benzils in general, various symmetrical benzils
1a–1g were synthesized and subjected to treatment with the
biocatalyst at pH 5.0 under the reaction conditions described
in the experimental section. Purification of the products was
carried out by flash chromatography over silica gel. The
desired products 2a–2g were obtained in 54–90% yield and
30–99% ee. The results are summarized in Table 5 (entries 1–
7). These results suggested that (i) the substitution of a phe-
nyl ring with a +I or −I substituent considerably slowed down
the reaction. Whereas benzil was reduced in 30 h, substituted
benzils typically required about 96 h for the reaction to go to
completion. No reduction occurred in the case of the 4-ethoxy
Table 5 Enantioselective reduction of symmetrical and unsymmetrical benzils to benzoins by Talaromyces flavus at pH 5.0
Entry
Substrate
Product
Eea (%) (config)
Yieldd (%)
Time (h)
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
4a
4b
4c
4d
4e
2a
2b
2c
2d
2e
2f
2g
5a
5b
5c
5d
5e
99 (S)b
42 (S)b
77 (S)b
82 (S)b
97 (S)b
n.r.
90
90
85
54
84
n.r.
62
91
88
84
80
85
30
96
96
96
96
n.r.
96
17
17
24
96
48
30 (R)b
89 (S)c
96 (S)c
>99 (S)c
96 (S)c
>99 (S)c
9
10
11
12
a
b
Ee was determined by chiral HPLC on a Chiralcel OD-H (Daicel, Japan) column. Absolute configuration was assigned based on comparison
of the sign of optical rotation with that in the literature. Configuration tentative. n.r. = no reaction. d Yield of isolated product.
c
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effect on the enantioselectivity of the biocatalyst and (vi) no
trace of hydrobenzoin was observed at 100% conversion with
any of the substrates under these reaction conditions.
various derivatives of benzil in order to study the effect of the
substituent on the conversion rate and ee of the product. T.
flavus catalyzed reduction of benzil derivatives (1a–1i) at pH
7.0 produced the corresponding 1,2-diols in 85–89% yield
(Table 6). The following observations are worth noting. (i)
The presence of a substituent at the 2- or 4-position of the
phenyl ring slowed down the reaction, whereas a substituent
at the 3-position had much less influence on the conversion
rate. No reaction occurred when a OCH3 group was present
at the 2-position of benzil (entry 2; Table 6), whereas the
2-chloro derivative required 168 h for the reaction to go to
completion (entry 7; Table 6). 4-Methoxy and 4-methyl deriva-
tives also required about 168 h for the reaction to go to com-
pletion (entries 4 and 5; Table 6). Surprisingly, no reaction
occurred with the 4-ethoxy substituted derivative (entry 6;
Table 6). (ii) The substitution of +I methoxy or −I chloro
groups resulted in a reduced dl/meso ratio. Whereas benzil
gave a dl/meso ratio of 97/3, the 3-methoxy and 4-methoxy
derivatives gave dl/meso ratios of 89/11 and 38/62, respectively
(entries 3 and 4; Table 6). The derivative with a chloro sub-
stituent at the 2-position gave a dl/meso ratio of 10/90 (entry
7; Table 6). (iii) The presence of a methoxy or methyl substit-
uent at the 4-position resulted in a decreased ee of 78–84%
(entries 4 and 5; Table 6), while with a 2-chloro substituent,
an ee of >99% was obtained (entry 7; Table 6).
T. flavus catalyzed enantio- and regio-selective mono-
reduction of various unsymmetrical benzils (4a–4e)
In order to study the scope of T. flavus for regioselective
reduction of benzils, various unsymmetrical benzil deriva-
tives 4a–4e were synthesized and subjected to treatment with
the biocatalyst at pH 5.0 under the reaction conditions
described in the experimental section. Purification of the
products was carried out by flash chromatography over silica
gel. The desired products 5a–5e were obtained in 80–91%
yield. The results are summarized in Table 5 (entries 8–12).
In summary, the results indicate that (i) in all the examples
studied, the carbonyl α to the unsubstituted phenyl group
was regioselectively reduced. (ii) Substitution had no effect
on the enantioselectivity as an ee of 96–99% was obtained in
all examples studied except when the methoxy group was
present at the 2-position of the phenyl ring, which gave an ee
of 89% (entry 8; Table 5). This is in contrast to symmetrical
benzils, where substitution had a detrimental effect on the ee
of the products. (iii) N-substituents were well tolerated as the
dimethylamino group at the 4-position of the phenyl ring
produced the desired product in >99% ee and 85% yield
(entry 12; Table 5). (iv) No hydrobenzoin was produced in any
of the examples studied even when the reactions were carried
out for longer times and (v) the ee and regioselectivity obtained
in the T. flavus catalyzed reaction for these examples were
higher compared to any report in the literature to date. In the
literature. Saccharomyces cerevisiae catalyzed regioselective
reduction of these substrates has been reported but the ee of
their products was not calculated.36 With Xanthomonas oryzae,
a mixture of both regioisomers was obtained.25
Conclusions
We have shown that by modulating the pH in T. flavus cata-
lyzed reduction of benzil, the reaction can be controlled to
obtain either (S)-benzoin (2a) or (S,S)-hydrobenzoin (3a) as
the exclusive product in high ee. Two prominent enzymes
involved in the pH dependent selectivity are (i) (S)-selective
benzil reductase, which is active at pH 5.0 and 7.0 and (ii) (S,
S)-selective benzoin reductase, which is active at pH 7.0, but
has no activity at pH 5.0. In addition, a novel dual-activity (R,
R)-selective benzoin reductase and benzil epimerase protein
was also purified. The (R,R)-selectivity of this enzyme was not
strict because it also converted (S)-benzoin to (S,S)-hydro-
benzoin. Benzoins were obtained in high ee when one or both
phenyl groups of benzil were unsubstituted. Except for 3a, all
other hydrobenzoins were obtained in moderate de. However,
considering the facts that (i) these bulky–bulky 1,2-diketones
are very tough substrates for enzymes and (ii) the ee and de
obtained in benzil reduction with T. flavus compare quite
favorably with those of any of the known biocatalysts in the
T. flavus catalyzed asymmetric reduction of various benzils
(1a–1i) to their corresponding 1,2-diols
We have already demonstrated that biocatalytic reduction of
benzils by Talaromyces flavus at pH 7.0 leads to the predomi-
nant formation of chiral 1,2-diols. We extended this study to
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Table 6 Enantio- and diastereo-selective reduction of 1,2-diketones to chiral 1,2-diols by Talaromyces flavus at pH 7.0
Entry
Substrate
Product
dl/mesoa
eeb,c (%) (config)
Yieldd,e (%)
Time (h)
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
1f
1g
1h
1i
3a
3b
3c
3d
3e
3f
3g
3h
3i
97/3
n.r.
>99 (S,S)
n.r.
n.d.
84 (S,S)
78.5 (S,S)
n.r.
>99 (S,S)
n.d.
86
n.r.
88
89
87
n.r.
85
88
56
n.r.
48
168
168
n.r.
168
48
89/11
38/62
67/33
n.r.
10/90
70/30
67/33
>99%IJS,S)
86
168
a
b
The dl/meso ratio was determined by 1H NMR based on integral values of specified peaks. Ee of the dl pair was determined by chiral HPLC
c
on a Chiralcel OJ (Daicel, Japan) column. Absolute configuration was assigned based on comparison of the sign of optical rotation with that
in the literature. d Combined yield for dl and meso. e Yield of isolated product. n.r. = no reaction, n.d. = not determined.
literature. Therefore, T. flavus expands and enriches the bio-
catalytic toolbox for asymmetric reduction of benzils.
12, 24 and 96 h for benzoin) and centrifuged (9000g, 4 °C,
15 min) to remove cells. The cell-free broth was then
extracted with ethyl acetate (3 × 10 ml), washed with brine
(1 × 5 mL) and dried over anhydrous sodium sulfate. Solvents
were removed under reduced pressure using a rotary evapora-
tor to leave a residue. The conversion, ratio of benzoin to
hydrobenzoin and dl/meso ratio of hydrobenzoin were calcu-
lated by comparison of integral values of the corresponding
peaks from 1H NMR and the ee of the products was deter-
mined by HPLC.
Experimental
General
Optical rotations were measured using a Rudolph Autopol IV
1
polarimeter. H NMR spectra were obtained at 300 MHz and
referenced to TMS (0.0 ppm) or the residual solvent peak
(CHCl3, 7.26 ppm). Chemical shifts were reported as parts
per million (ppm) using the δ scale. 13C NMR spectra were
recorded at 75 MHz and referenced to either TMS (0.0 ppm)
or the internal solvent (CDCl3, 77.0 ppm). Thin layer chroma-
tography (TLC) was performed on Merck silica gel DC
Alurolle Kieselgel 60F254 plates which were visualized under
UV lamp and/or with 0.25% w/v KMnO4 and 2% NaHCO3
solution in water. Flash column chromatography was carried
out using silica gel (200–400 mesh). Analytical HPLC analyses
were performed on a system equipped with a high pressure
gradient dual pump, an auto injector, a variable temperature
column compartment and a PDA detector. Ee was determined
by HPLC using one of the following systems: HPLC system 1
– column: 250 × 4.6 mm Chiralcel OD-H (Daicel, Japan),
detection: UV at 254 nm, elution: 10% 2-propanol in hexane
at a flow rate of 1 mL min−1, and temperature: 25 °C; HPLC
system 2 – column: 250 × 4.6 mm Chiralcel OJ (Daicel, Japan)
column, detection: 254 nm, elution: 10% 2-propanol in hex-
ane at a flow rate of 1 mL min−1, and temperature: 25 °C.
General procedure for enantioselective reduction of benzil
and benzoin by protein fractions of T. flavus
Enantioselective reduction of benzil, (S)-benzoin and
rac-benzoin by protein fractions from T. flavus was carried
out at pH 5.0 in 50 mM acetate buffer or pH 7.0 in 50 mM
phosphate buffer. A solution of NADP+ (784 nmol), glucose
dehydrogenases (14 units), glucose (0.5 mmol), the protein
fraction (8 units of dehydrogenase activity) and the substrate
(0.02 mmol) in 4 mL of appropriate buffer was energetically
mixed using a magnetic stirrer. After 16 h, the products were
extracted in ethyl acetate (3 × 4 ml) and dried over anhydrous
sodium sulfate. Solvents were removed under reduced pres-
sure to leave a residue. The conversion, ratio of benzoin/
hydrobenzoin and dl/meso ratio of hydrobenzoin were calcu-
lated as described in preceding section.
General procedure for microbial monoreduction of
symmetrical and unsymmetrical benzils by Talaromyces
flavus
General procedure for pH dependent asymmetric reduction
of benzil and benzoin by Talaromyces flavus
A 100 mL round bottomed flask was charged with cells (5 g,
wet weight) suspended in 50 ml of an appropriate 0.2 M
buffer (acetate buffer for pH 4.0 and 5.0; phosphate buffer
for pH 6.0–8.0; Tris-HCl buffer for pH 9). The substrate (50
mg, 0.24 mmol) was added to the cell suspension and the
mixture was shaken on an orbital shaker at 30 °C and 200
rpm. During the course of reaction, the pH of the reaction
mixture was maintained by addition of 1 N NaOH or 1 N
HCl. 10 ml of the sample from each reaction was taken after
different time intervals (14, 37, 144 and 264 h for benzil and
A 100 mL round bottomed flask was charged with cells (5 g,
wet weight) suspended in 50 mL of acetate buffer (0.2 M, pH
5.0). The substrate (0.24 mmol, 50–72 mg depending upon
the molecular mass of the substrate) was added and the mix-
ture was shaken on an orbital shaker at 30 °C and 200 rpm
till complete consumption of the starting material occurred.
The reaction mixture was then centrifuged (9000g, 4 °C, 15
min) to remove cells. The supernatant was extracted with
ethyl acetate (3 × 25 mL), washed with saturated brine (10
mL) and dried over anhydrous sodium sulfate. Solvents were
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removed under reduced pressure to leave a residue which
was purified by flash chromatography (silica, methanol/
chloroform 10 : 90). Ee was determined by using HPLC (sys-
tem 1). The yield, ee, NMR and HPLC data for the products
are given below.
CDCl3): δ = 75.5, 126.6, 127.4, 129.1, 129.4, 130.0, 130.1,
131.6, 132.4, 133.9, 134.9, 135.6, 200.9; HPLC data (HPLC
analysis, system 1): retention time = 11.9 min (minor) and
15.0 min (major).
IJS)-2-Hydroxy-1-IJ2-methoxyphenyl)-2-phenylethanone (5a).
1
IJS)-2-Hydroxy-1,2-diphenylethanone (2a). Yield: 45.2 mg
(90%); 99% ee; [α]2D5 = +115.2 (c 1.5, acetone) [lit. ref. 31, [α]2D5
Yield: 52.4 mg (91%); 89% ee. H NMR (300 MHz, CDCl3): δ =
3.79 (s, 3H), 4.64 (bs, 1H), 6.28 (s, 1H), 6.82–6.89 (m, 2H),
7.16–7.24 (m, 2H), 7.29–7.35 (m, 2H), 7.41–7.46 (m, 1H),
7.91–7.94 (m, 2H); 13C NMR (75 MHz, CDCl3): δ = 55.6, 71.1,
111.6, 121.3, 127.7, 127.8, 128.6, 128.8, 128.9, 129.2, 130.1,
133.7, 156.6, 199.6; HPLC data (HPLC analysis, system 1):
retention time = 19.4 min (major) and 21.7 min (minor). Con-
figuration was assigned tentatively as S based on the elution
order.
1
= +115.0 (c 1.5, acetone), >99% ee, (S,S)]. H NMR (300 MHz,
CDCl3): δ = 4.54 (bs, 1H), 5.95 (s, 1H), 7.23–7.34 (m, 5H),
7.36–7.41 (m, 2H), 7.49–7.54 (m, 1H), 7.89–7.92 (m, 2H); 13C
NMR (75 MHz, CDCl3): δ = 76.3, 127.8, 128.7, 129.2, 133.6,
134.0, 139.0, 199.0; HPLC data (HPLC analysis, system 1):
retention time = 11.9 min (major) and 17.4 min (minor).
IJS)-2-Hydroxy-1,2-bisIJ2-methoxyphenyl)ethanone
(2b).
Yield: 58.3 mg (90%); 42% ee; [α]2D5 = +52.0 (c 0.9, CHCl3) [lit.
IJS)-2-Hydroxy-1-IJ4-methoxyphenyl)-2-phenylethanone (5b).
ref. 37, [α]2D5 = −125.0 (c 0.9, CHCl3), >99% ee, (R)]. H NMR
Yield: 51 mg (88%); 96% ee. H NMR (300 MHz, CDCl3): δ =
1
1
(300 MHz, CDCl3): δ = 3.70 (s, 3H), 3.71 (s, 3H), 4.46 (bs, 1H),
6.09 (s, 1H), 6.73–6.77 (m, 2H), 6.80–6.85 (m, 1H), 6.89–6.94
(m, 1H), 7.12–7.20 (m, 2H), 7.32–7.38 (m, 1H), 7.65–7.69 (m,
1H); 13C NMR (75 MHz, CDCl3): δ = 55.2, 55.3, 75.9, 110.9,
111.2, 120.5, 125.3, 127.6, 129.6, 130.0, 130.7, 133.9, 157.3,
158.2, 201.7; HPLC data (HPLC analysis, system 1): retention
time = 21.7 min (minor) and 31.2 min (major).
3.81 (s, 3H), 4.64 (d, J = 5.16 Hz, 1H), 5.88 (d, J = 5.16 Hz,
1H), 6.86 (d, J = 8.9 Hz, 2H), 7.23–7.33 (m, 5H), 7.90 (d, J =
8.9 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ = 55.8, 75.8, 114.0,
126.3, 127.8, 128.5, 129.2, 131.7, 139.6, 164.2, 197.2; HPLC
data (HPLC analysis, system 1): retention time = 19.1 min
(major) and 21.2 min (minor). Configuration was assigned
tentatively as S based on the elution order.
IJS)-2-Hydroxy-1,2-bisIJ3-methoxyphenyl)ethanone
(2c).
IJS)-1-IJ4-Ethoxyphenyl)-2-hydroxy-2-phenylethanone
(5c).
1
Yield: 55 mg (85%); 77% ee; [α]2D5 = +119.0 (c 1.0, CH3OH)
Yield: 52 mg (84%); >99% ee. H NMR (300 MHz, CDCl3): δ =
1.36 (t, J = 6.87 Hz, 3H), 3.99 (q, J = 6.87 Hz, 2H), 5.90 (s,
1H), 6.82 (d, J = 8.9 Hz, 2H), 7.21–7.36 (m, 5H), 7.89 (d, J =
8.9 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ = 14.6, 63.9, 75.8,
114.4, 126.1, 127.8, 128.7, 129.1, 131.7, 139.7, 163.6, 197.3;
HPLC data (HPLC analysis, system 1): retention time = 15.7
min (major) and 16.3 min (minor). Configuration was assigned
tentatively as S based on the order of elution in HPLC.
[lit. ref. 37, [α]2D5 = −156.0 (c 1.0, CH3OH), >99% ee, (R)]. H
1
NMR (300 MHz, CDCl3): δ = 3.71 (s, 3H), 3.75 (s, 3H), 5.90 (s,
1H), 6.77–6.80 (m, 1H), 6.86–6.87 (m, 1H), 6.91–6.93 (m, 1H),
7.02–7.05 (m, 1H), 7.18–7.28 (m, 2H), 7.45–7.49 (m, 2H); 13C
NMR (75 MHz, CDCl3): δ = 55.3, 55.4, 76.3, 113.3, 113.5,
114.2, 120.2, 120.5, 121.8, 129.7, 130.2, 134.8, 140.5, 159.8,
160.2, 198.8; HPLC data (HPLC analysis, system 1): retention
time = 19.2 min (major) and 27.0 min (minor).
IJS)-2-Hydroxy-1-IJ2,3-dimethoxyphenyl)-2-phenylethanone
1
IJS)-2-Hydroxy-1,2-bisIJ4-methoxyphenyl)ethanone
(2d).
(5d). Yield: 52 mg (80%); 96% ee. H NMR (300 MHz, CDCl3):
Yield: 35 mg (54%); 82% ee; [α]2D5 = +73.8 (c 1.0, CH3OH) [lit.
δ = 3.77 (s, 3H), 3.78 (s, 3H), 4.71 (s, 1H), 5.86 (s, 1H), 6.70
(d, J = 8.2 Hz, 1H), 7.17–7.31 (m, 5H), 7.43–7.48 (m, 2H);
13C NMR (75 MHz, CDCl3): δ = 55.9, 56.0, 75.8, 110.1, 111.2,
124.3, 126.4, 127.7, 128.5, 129.1, 139.9, 149.0, 153.9, 197.2;
HPLC data (HPLC analysis, system 1): retention time = 35.3
min (major) and 41.9 min (minor). Configuration was assigned
tentatively as S based on the order of elution in HPLC.
IJS)-1-IJ4-IJDimethylamino)phenyl)-2-hydroxy-2-phenylethan-
one (5e). Yield: 52 mg (85%); >99% ee. 1H NMR (300 MHz,
CDCl3): δ = 3.01 (s, 6H), 4.80 (d, J = 5.85 Hz, 1H), 5.84 (d, J =
5.85 Hz, 1H), 6.56 (d, J = 8.9 Hz, 2H), 7.23–7.36 (m, 5H), 7.83
(d, J = 8.9 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ = 40.1, 75.3,
110.7, 120.8, 127.7, 128.2, 129.0, 131.6, 140.6, 153.8, 196.0;
HPLC data (HPLC analysis, system 1): retention time = 54.4
min (major) and 56.4 min (minor). Configuration was assigned
tentatively as S based on the order of elution in HPLC.
ref. 37, [α]2D5 = −90.4 (c 1.0, CH3OH), >99% ee, (R)]. H NMR
1
(300 MHz, CDCl3): δ = 3.74 (s, 3H), 3.81 (s, 3H), 5.84 (s, 1H),
6.82–6.86 (m, 4H), 7.24 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.6 Hz,
2H); 13C NMR (75 MHz, CDCl3): δ = 55.3, 55.5, 75.3, 114.0,
114.6, 126.4, 129.1, 131.6, 131.9, 159.7, 164.0, 197.4; HPLC
data (HPLC analysis, system 1): retention time = 42.0 min
(major) and 44.4 min (minor).
IJS)-2-Hydroxy-1,2-di-p-tolylethanone (2e). Yield: 48 mg
(84%); 97% ee; [α]2D5 = +144.6 (c 0.7, CH3OH) [lit. ref. 37, [α]D25
= −150 (c 0.7, CH3OH), >99% ee, (R)]. 1H NMR (300 MHz,
CDCl3): δ = 2.28 (s, 3H), 2.34 (s, 3H), 5.89 (s, 1H), 7.11 (d, J =
8.2 Hz, 2H), 7.16–7.23 (m, 4H), 7.82 (d, J = 8.2 Hz, 2H); 13C
NMR (75 MHz, CDCl3): δ = 21.2, 21.8, 76.8, 127.8, 129.4,
129.5, 129.9, 131.0, 136.4, 138.4, 145.0, 198.7; HPLC data
(HPLC analysis, system 1): retention time = 9.2 min (major)
and 11.6 min (minor).
IJR)-1,2-BisIJ2-chlorophenyl)-2-hydroxyethanone (2g). Yield:
41.5 mg (62%); 30% ee; [α]D25 = −14.1 (c 1.0, CHCl3) [lit. ref.
37, [α]2D5 = −46.0 (c 1.0, CHCl3), 97% ee, (R)]. 1H NMR (300
MHz, CDCl3): δ = 4.53 (bs, 1H), 6.34 (s, 1H), 7.11–7.17 (m,
2H), 7.18–7.25 (m, 3H), 7.27–7.32 (m, 3H); 13C NMR (75 MHz,
General procedure for microbial reduction of benzils to
hydrobenzoins by Talaromyces flavus
The procedure was the same as described above for the
monoreduction of benzils except that the phosphate buffer
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(0.2 M, pH 7.0) was used instead of the acetate buffer. Ee was
determined by using HPLC (system 2). The ee, NMR and
HPLC data for the products are given below.
imtech.res.in) with accession numbers MTCC 10832, MTCC
10833 and MTCC 10834, respectively.
IJ1S,2S)-1,2-Diphenylethane-1,2-diol ((S,S)-3a). >99% ee;
[α]2D5 = −91.0 (c 1.05, ethanol) [lit. ref. 32, [α]2D5 = +91.6 (c 1.05,
ethanol), 99.9% ee, (R,R)]. 1H NMR (300 MHz, CDCl3): δ =
4.72 (s, 2H), 7.11–7.15 (m, 4H), 7.21–7.25 (m, 6H) ppm; HPLC
data (HPLC analysis, system 2): retention time = 16.2 min (S,
S), 18.8 min (R,R) and 22.6 min (meso).
IJ1S,2S)-1,2-BisIJ3-methoxyphenyl)ethane-1,2-diol ((S,S)-3c).
Ee not determined. 1H NMR (300 MHz, CDCl3): δ = 3.71 (s,
6H), 4.68 (s, 2H), 6.71–6.80 (m, 5H), 7.12–7.26 (m, 3H) ppm.
IJ1S,2S)-1,2-BisIJ4-methoxyphenyl)ethane-1,2-diol ((S,S)-3d).
Growth conditions
Fungal strains were maintained as 25% glycerol stocks at −78
°C. A 100 mL Erlenmeyer flask was charged with 20 mL of
sterile PDB media (24 g L−1; pH 5.2–5.3). After inoculation
with fungi, the flask was incubated with shaking at 200 rpm
in an incubator shaker at 30 °C for 24 h. This was used to
inoculate a 2 L Erlenmeyer flask containing 400 mL of the
same media. The flask was then incubated at 30 °C with
shaking at 200 rpm on an orbital shaker for 72 h. Microbial
cells were isolated by centrifugation at 9000g for 15 min at 4
°C. Composition of PDB: potato infusions (200 g L−1) and
dextrose (3 g L−1).
84% ee; [α]2D5 = −99.1 (c 1.0, EtOH) [lit. ref. 38, [α]2D5
=
−118.3 (c 1.0, EtOH), 99% ee, (S,S)]. 1H NMR (300 MHz,
CDCl3): δ = 3.75 (s, 6H), 4.63 (s, 2H), 6.76 (d, J = 8.9 Hz, 4H),
7.0 (d, J = 8.9 Hz, 4H) ppm.
IJ1S,2S)-1,2-Di-p-tolylethane-1,2-diol ((S,S)-3e). 78.5% ee;
[α]2D5 = −89 (c 1.0, ethanol) [lit. ref. 38, [α]2D5 = −102.5 (c 1.0,
Preparation of cell-free extract
1
ethanol), 99% ee, (S,S)]. H NMR (300 MHz, CDCl3): δ = 2.29
T. flavus was grown in several 2 L flasks, as described in pre-
ceding section. Cells of T. flavus (75 g, wet weight) were
suspended in 200 mL of phosphate buffer (50 mM, pH 7.0)
containing 1 mM protease inhibitor cocktail (dithiothreitol,
phenylmethylsulfonyl fluoride and 1,10-phenanthroline) and
cooled to 4 °C. The suspension was disrupted by glass beads
(0.5–0.75 μm) in a 1 : 1 (wt/wt) ratio at 4 °C for 15 min in
Dyno Mill. The cell-free extract was obtained by centrifuga-
tion at 20 000g for 20 min at 4 °C
(s, 6H), 4.67 (s, 2H), 7.13 (d, J = 8.9 Hz, 4H), 7.19 (d, J = 8.9
Hz, 4H) ppm; HPLC data (HPLC analysis, system 2): retention
time = 12.2 min (S,S), 14.6 min (R,R) and 15.6 min (meso).
IJ1S,2S)-1,2-BisIJ2-chlorophenyl)ethane-1,2-diol
((S,S)-3g).
1
>99% ee. H NMR (300 MHz, CDCl3): δ = 5.36 (s, 2H), 7.16–
7.18 (m, 5H), 7.25–7.28 (m, 3H) ppm; HPLC data (HPLC anal-
ysis, system 2): retention time = 26.3 min (meso), 50.9 min (S,
S) and 57.6 min (R,R).
IJ1S,2S)-1,2-BisIJ3-chlorophenyl)ethane-1,2-diol ((S,S)-3h). Ee
1
not determined. H NMR (300 MHz, CDCl3): δ = 4.64 (s, 2H),
6.91 (d, J = 7.2 Hz, 2H), 7.12–7.26 (m, 6H) ppm.
Ammonium sulfate fractionation
IJ1S,2S)-1,2-BisIJ4-chlorophenyl)ethane-1,2-diol
((S,S)-3i).
>99% ee; [α]2D5 = −91.0 (c 1.0, CHCl3) [lit. ref. 38, [α]2D5 = +93
Solid ammonium sulfate was added in small portions to the
cell-free extract at 4 °C to 40% saturation. The pellet was
removed by centrifugation and the supernatant was brought
to 80% saturation with solid ammonium sulfate while
maintaining the temperature at 4 °C and the pH at 7.0. The
precipitated proteins were collected by centrifugation. The
pellet was re-dissolved in phosphate buffer (10 mM, pH 8.0)
and centrifuged to remove any precipitate.
1
(c 1.0, CHCl3), 99% ee, (R,R)]. H NMR (300 MHz, CDCl3): δ =
4.63 (s, 2H), 7.0 (d, J = 8.6 Hz, 4H), 7.21 (d, J = 8.6 Hz, 4H)
ppm; HPLC data (HPLC analysis, system 2): retention time =
44.9 min (S,S), 47.0 min (R,R) and 55.0 min (meso).
Materials and methods
Source of materials and microorganisms
Affinity chromatography
Symmetrical and unsymmetrical benzils were synthesized by
benzoin condensation followed by oxidation with pyridinium
chlorochromate.39 Culture media, TSB (tryptone soya broth)
and PDB (potato dextrose broth) were purchased from
HiMedia (Mumbai, India). Sephacryl S-200, bovine serum
albumin (BSA), β-mercaptoethanol, sodium dodecyl sulfate
(SDS), Reactive Red 120-agarose type 3000-CL, and molecular
weight standards were from Sigma Chemical Company, USA.
Q-Sepharose Fast Flow and Phenyl Sepharose CL-4B were
from Pharmacia (Freiburg, Germany). Penicillium sp.,
Alternaria alternata and Talaromyces flavus are our own iso-
lates and were described previously (Pal et al., 2012). These
have been deposited with Microbial Type Culture Collection
and Gene Bank (MTCC), IMTECH, Chandigarh (http://mtcc.
The supernatant from the above step was desalted using a
Sephadex G-25 column. This solution was applied on a Reac-
tive Red 120-agarose type 3000-CL column (2 × 13 cm) pre-
equilibrated with phosphate buffer (10 mM, pH 8.0). The col-
umn was washed with the same buffer until all unbound pro-
teins were completely removed. An increasing linear gradient
of NaCl (0–1.5 M) in phosphate buffer (10 mM, pH 6.5) at a
flow rate of 60 mL h−1 was used to elute the proteins. The
active fractions were pooled and concentrated by ultrafiltra-
tion (Millipore-Amicon Ultra-15 centrifugal filter units with a
10 kDa membrane). Desalting was carried out by repeated
dilution with Tris-HCl buffer (10 mM, pH 8.0) and the sam-
ple was finally concentrated to 1 mL volume.
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Ion-exchange chromatography
N-terminal sequencing and bioinformatics studies
The desalted and concentrated sample (1 mL) obtained from
Reactive Red chromatography was loaded into a Q-Sepharose
Fast Flow column (2 × 5 cm) pre-equilibrated with Tris-HCl
buffer (10 mM, pH 8.0). The column was then washed with
the same buffer and the enzyme was eluted with thirty col-
umn volumes of an increasing linear gradient of NaCl (0–0.5
M) in Tris-HCl buffer (10 mM, pH 8.0) at a flow rate of 60 mL
h−1. The active fractions were pooled and concentrated by
ultrafiltration (Millipore-Amicon Ultra-15 centrifugal filter
units with a 10 kDa membrane). Desalting was carried out by
repeated dilution with Tris-HCl buffer (10 mM, pH 8.0) and
the sample was concentrated to a final volume of 0.5 mL.
Purified protein was run on SDS-PAGE. The gel was washed
once with water and equilibrated with 10 mM CAPS
IJ3-IJcyclohexylamino)-1-propanesulfonic acid) buffer, pH 11.0
containing 0.037% IJw/v) SDS and 10% methanol for 15 min.
A polyvinylidene difluoride (PVDF) membrane of gel size was
cut and wetted with methanol for 1 min, rinsed once with
water and equilibrated with the above buffer for 15 min. Pro-
teins were electrophoretically transferred onto the PVDF
membrane at 300–350 mA for 1 h at 4 °C. The extent of trans-
fer was visualized by staining with 0.1% IJw/v) amido black (in
1% acetic acid) for 5 min. Destaining was carried out in 50%
IJv/v) methanol. The transferred protein of interest was
excised and washed once with 20% IJv/v) methanol for 1 h at
RT. Thereafter, cut-out PVDF bands were washed thoroughly
(10 times) with double distilled water to remove traces of
methanol. These PVDF bands were dried on Whatman sheets
and stored at 4 °C until analysis using an Applied Biosystems
476 A protein sequencer following the Edman degradation
Gel filtration chromatography
The desalted and concentrated sample (0.5 mL) obtained
from Q-Sepharose chromatography was loaded into
a
Sephacryl S-200 column (2 × 60 cm, 43 mL bed volume) previ-
ously equilibrated with Tris-HCl buffer (50 mM, pH 7.5)
containing 0.15 M NaCl at a flow rate of 30 mL h−1. The
active fractions were pooled and concentrated in an ultrafil-
tration cell using a 10 kDa membrane.
protocol.
The
obtained
N-terminal
sequence,
APTNQ_AYDHSV, was then searched for homologous coun-
terparts using the BLAST server at www.ncbi.nlm.nih.gov.
MALDI-TOF mass spectral analysis
Enzyme assay method
Purified enzyme (10 μM) and fresh matrix IJ3,5-dimethoxy-4-
hydroxycinnamic acid, sinapinic acid, 10 mM) solutions were
premixed in a small Eppendorf tube and applied directly to the
sample support (i.e., sample plate). The sample was allowed to
evaporate in air, irradiated with a nanosecond laser pulse and
analyzed using a MALDI-TOF Voyager DE-STR instrument.
The reductase activity was determined by monitoring the
decrease in absorbance of NADPH at 340 nm (molar absorp-
tion coefficient of 6800 M−1 cm−1) as described previously.40
The standard reaction mixture (1 mL) contained 0.1 mM
NADPH, 50 μl of the cell-free extract and 2.5 mM substrate.
One unit of enzyme activity was defined as the amount of
enzyme that catalyzed the oxidation of 1 μmol of NADPH per
minute under specified conditions. Protein concentration
was estimated by the Coomassie Brilliant Blue-G method of
Bradford using bovine serum albumin (BSA) as the
standard.41
Acknowledgements
This work was supported by
a Network Project grant
(NWP006) and a Supra Institutional Project grant (BSC 0211)
from the Council of Scientific and Industrial Research (CSIR),
New Delhi, India. We thank the Microbial Type Culture Col-
lection and Gene Bank (MTCC), IMTECH, Chandigarh for
preservation and supply of microbial cultures. MP, GS, ANS
and SK acknowledge the CSIR for awarding Senior Research
Fellowships. We acknowledge technical assistance provided
by Mr. Chander Prakash and Mr. Pradeep Patel.
Polyacrylamide gel electrophoresis (PAGE)
SDS-PAGE was performed in 12.5% IJw/v) gels according to
the procedure of Laemmli.42 Prior to loading into the gel, the
samples were heated in a boiling water bath for 5 min. The
discontinuous gel system usually had 5% stacking and 12.5%
resolving gels. Electrophoresis was carried out using Laemmli
buffer at a constant current of 15 mA first till the samples
entered the gel and then at 20 mA till completion. Upon com-
pletion of electrophoresis, the gel was immersed in 0.05%
Coomassie Blue R-250 in methanol : acetic acid : water (4 : 1 :
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