Enantioselective reduction of diaryl ketones catalyzed by a carbonyl reductase from Sporobolomyces salmonicolor and its mutant enzymes

Article abstract of DOI:10.1002/adsc.200900045

The carbonyl reductase from red yeast Sporobolomyces salmonicolor AKU4429 (SSCR) and its mutant enzymes effectively catalyzed the enantioselective reduction of diaryl ketones to give the corresponding chiral alcohols. Both conversion and enantioselectivity were dependent on the co-solvent in the reaction medium. Diaryl ketones with a para-substituent on one of the phenyl groups were reduced with high enantioselectivity (up to 99% ee), which is difficult to achieve using chemical methods such as chiral borane reduction, asymmetric hydrogenation or hydrosilylation. Mutation of SSCR at Q245 resulted in a higher amount of (S)-enantiomer in the products, and in the case of mutant Q245P with para-substituted diaryl ketones as substrate, this effect was so remarkable that the reduction enantiopreference was switched from (R) to (S). The present study provides valuable information about the catalytic properties of the carbonyl reductase SSCR toward the reduction of diaryl ketones, serving as basis for further engineering of this enzyme to develop efficient biocatalysts for highly enantiospecific reduction of diaryl ketones without high electronic dissymmetry or an ortho-substituent on one of the aryl groups.

Full text of DOI:10.1002/adsc.200900045

FULL PAPERS
DOI: 10.1002/adsc.200900045
Enantioselective Reduction of Diaryl Ketones Catalyzed by a
Carbonyl Reductase from Sporobolomyces salmonicolor and its
Mutant Enzymes
Hongmei Li,b Dunming Zhu,a,b,* Ling Hua,c and Edward R. Biehlb
a
State Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of
Sciences, Tianjin 300308, Peopleꢀs Republic of China
Fax : (+86)-22-2489-6830; phone: (+86)-22-2489-6830; e-mail: dzhu@smu.edu
Department of Chemistry, Southern Methodist University, Dallas, Texas 75275, USA
China Research Center, Genencor, A Danisco Division, Shanghai 200335, Peopleꢀs Republic of China
b
c
Received: January 21, 2009; Published online: March 4, 2009
Abstract: The carbonyl reductase from red yeast with para-substituted diaryl ketones as substrate, this
Sporobolomyces salmonicolor AKU4429 (SSCR) effect was so remarkable that the reduction enantio-
and its mutant enzymes effectively catalyzed the preference was switched from (R) to (S). The present
enantioselective reduction of diaryl ketones to give study provides valuable information about the cata-
the corresponding chiral alcohols. Both conversion lytic properties of the carbonyl reductase SSCR
and enantioselectivity were dependent on the co-sol- toward the reduction of diaryl ketones, serving as
vent in the reaction medium. Diaryl ketones with a basis for further engineering of this enzyme to devel-
para-substituent on one of the phenyl groups were op efficient biocatalysts for highly enantiospecific re-
reduced with high enantioselectivity (up to 99% ee), duction of diaryl ketones without high electronic dis-
which is difficult to achieve using chemical methods symmetry or an ortho-substituent on one of the aryl
such as chiral borane reduction, asymmetric hydro- groups.
genation or hydrosilylation. Mutation of SSCR at
Q245 resulted in a higher amount of (S)-enantiomer Keywords: bioreduction; diaryl ketones; enantiose-
in the products, and in the case of mutant Q245P lectivity; enzyme catalysis; oxidoreductase
Introduction
addition of additives such as polyethylene glycol ether
(PEG) are required to achieve synthetically useful re-
Chiral diarylmethanols are important intermediates sults, since achiral, non-catalytic background aryl ad-
for the synthesis of pharmaceutically interesting mole- dition often leads to a product of low enantiomeric
cules, such as (R)-neobenodine, (R)-orphenadrine, l- purity.[1,14]
cloperastine and (S)-carbinoxamine, inspiring consid-
An alternative approach to synthesize enantiomeri-
erable efforts to search for methods for the construc- cally enriched diarylmethanols is the reduction of
tion of the chiral diarylmethanol core moiety in high diaryl ketones,[1] which can be achieved chemically
optically purity.[1,2] As a result, a high number of pub- and biocatalytically. The asymmetric chemical reduc-
lications have appeared in the last 10 years or so to tions of diaryl ketones involve chiral borane reduc-
deal with the catalytic asymmetric addition of aryl nu- tion,[15–17] transition metal-catalyzed hydrogena-
cleophiles to aromatic aldehydes with the aim to pre- tion,[18–20] hydrogen transfer reduction[21], and hydrosi-
pare enantiomerically enriched diarylmethanols.[1,3–10] lylation.[22–24] Although these chemical methods have
The aryl nucleophiles are usually the expensive diphe- provided a useful access to diarylmethanol with high
nylzinc or arylzinc species formed in situ from aryl- enantiomeric purity in some cases, their common
boronic acids or triarylborane with excess of diethyl- drawback is the limited substrate range.[1,25] For exam-
zinc. Rhodium-, titanium- and copper-catalyzed enan- ple, the degree of enantiofacial discrimination in
tioselective aryl transfer reactions to aromatic alde- Coreyꢀs CBS reduction of diaryl ketones is deter-
hydes have also been reported and the corresponding mined by both electronic and steric effects, requiring
arylmetal species have been proposed to be the active that the ketones have an electron donor substituent
nucleophiles.[11–13] However, in many of the described on one aromatic ring and an acceptor group on the
processes, high catalyst loadings (10–20 mol%) and/or other,[26] or an ortho-substituent on one of the aryl
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Hongmei Li et al.
FULL PAPERS
groups.[15] For transition metal-catalyzed hydrogena- tive reduction of a wide range of ketones including
tion and hydrosilylation, the presence of an ortho-sub- 2,2-dimethylpropiophenone, 1-phenyl-1-pentanone
stituent on one of the aryl groups is necessary for and cyclopropyl(phenyl)methanone, showing that it is
AHCTUNGTRENNUNG
achieving high enantiocontrol in the products. The re- a useful biocatalyst for the reduction of sterically
duction of diaryl ketones with only a para- or meta- bulky ketones.[28,39] We envisioned that the carbonyl
substituent on one of the aryl groups affords the reductase SSCR might also take sterically bulky
diarylACHTUNGTRENNUNGmethanol in low enantiomeric purity (ee often diaryl ketones as substrates. Herein we report that a
being less than 50%).[18,19,22,24] Therefore, the highly variety of diaryl ketones were enantioselectively re-
enantioselective reduction of diaryl ketones without duced by this carbonyl reductase and its mutant
high electronic dissymmetry or an ortho-substituent enzyme Q245P.
on one of the aryl groups has proven to be a signifi-
cant challenge.[1]
Biocatalysts have been demonstrated to be highly Results and Discussion
enantioselective in the reduction of a wide range of
ketones including some bulky ones.[27–29] Enzymatic Given that chiral 4-chlorobenzhydrol (2b) and 4-
reduction offers a potentially complementary way of methylbenzhydrol (2c) are important precursors for
achieving highly enantioselective reduction of diaryl the synthesis of (S)-cetirizine hydrochloride and (R)-
ketones that are difficult with chemical catalysts.[30] In neobenodine, respectively, and that the enantioselec-
this context, a few reports dealing with the biocatalyt- tive reduction of such simple diaryl ketones with a
ic reduction of diaryl ketones have appeared. Ben- para-substituent on one of the aryl groups remains a
zoylpyridines were reduced to the corresponding opti- formidable task, 4-chlorobenzophenone (1b) and 4-
cally active phenylpyridylmethanols by whole-cell bio- methylbenzophenone (1c) were chosen as substrates
catalysts such as immobilized bakerꢀs yeast,[31,32] Rhi- and examined using SSCR as the catalyst in potassi-
zopus arrhizus,[33] Catharanthus roseus,[34] Nicotiana ta- um phosphate buffer with various organic solvents.
bacum[35] and Camellia sinensis cell cultures.[36] The organic solvent was added to increase the availa-
Preliminary results have shown that selected strains bility of the highly hydrophobic substrates in the reac-
from Hansenula nonfermentans, santamariae, ernobii, tion medium. Co-factor NADPH was regenerated
Rhodosporidium toruloides, Candida bombi, and C. with glucose dehydrogenase and d-glucose system, as
sorbophila reduced diaryl ketones with high selectivi- shown in Scheme 1. The reaction mixtures were
ty, producing the alcohol product with >95% ee, but shaken at room temperature for 24 h, and then ex-
the substrate was not specified and the conversion tracted with methyl tert-butyl ether. The extracts were
was less than 12%.[37] Debaryomyces marama enantio- dried over anhydrous sodium sulfate and subjected to
selectively reduced p-chlorobenzophenone to give a chiral HPLC analysis. The product alcohols (2) were
48% yield of the S-alcohol with 34% of ketone being identified by comparison with the authentic samples.
recovered.[38] Recently, the reductions of a series of The results are summarized in Table 1.
diaryl ketones were tested with commercially avail-
From Table 1, it can be seen that the carbonyl re-
able ketoreductases, and high enantioselectivity was ductase SSCR catalyzed the enantioselective reduc-
obtained in some cases.[30] Since the gene/protein se- tion of 4-chlorobenzophenone (1b) and 4-methylben-
quences or sources of most of the commercial en- zophenone (1c) to give the corresponding (R)-alcohol
zymes are not available, this provides limited informa- products. The co-solvent contained in the reaction
tion for the further search of more effective ketore- medium affected both conversion and enantioselectiv-
ductases for this formidable task. In our previous ity. For both substrates, the reaction in alcoholic sol-
studies, it has been found that a carbonyl reductase vents (isopropyl alcohol and methanol) gave highest
from red yeast Sporobolomyces salmonicolor conversion, while highest enantioselectivity was ob-
AKU4429 (SSCR) catalyzed the highly enantioselec- tained in buffer with THF. Since the addition of THF
Scheme 1. SSCR-catalyzed reduction of diaryl ketones with a glucose dehydrogenase/d-glucose NADPH regeneration
system.
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Enantioselective Reduction of Diaryl Ketones Catalyzed by a Carbonyl Reductase
FULL PAPERS
Table 1. Wild-type SSCR-catalyzed reduction of 4-chloro-
benzophenone (1b) and 4-methylbenzophenone (1c) in reac-
tion media with different co-solvents.
Solvent in buf-
fer[a]
1b
1c
Conv.
[%][b]
ee
Conv.
[%][b]
ee
[%][b]
[%][b]
No solvent
DMSO
2-PrOH
Methanol
Butyl acetate
MTBE
THF
THF (20%)
THF (30%)
47
97
93
95
14
46
62
11
0
50
70
74
78
62
58
88
45
98
>99
>99
11
52
67
9
82
80
80
84
Figure 1. Substituted benzophenones, benzoylpyridines, and
benzyl phenyl ketone.
[c]
–
78
92
product alcohols were identified by comparison with
the authentic samples, and their absolute configura-
tions were determined by comparing the HPLC elu-
tion sequence with the literature data using the same
type of chiral column,[6,7,17,40–44] and/or by comparison
with the enantio-merically enriched sample prepared
by literature methods.[30] The results are reported in
Table 2.
[c]
[c}
–
–
–
0
–
[a]
Potassium phosphate buffer (100 mM, pH 7.0) containing
10% (v/v) of organic solvent except where indicated oth-
erwise.
Determined by HPLC analysis, the product benzhydrols
were (R)-enantiomers.
Only (R)-enantiomer was observed by chiral HPLC anal-
ysis, since the low conversion might result in a trace of
(S)-enantiomer not being detected, the ee value was not
reported.
[b]
[c]
Table 2. Reduction of diaryl ketones catalyzed by wild-type
carbonyl reductase SSCR.
Ketone
THF
Methanol
Conv. [%][a] ee [%][a] Conv. [%][a] ee [%][a]
(10%) into the buffer increased the conversion and
the enantioselectivity, more THF was added into the
buffer in an attempt to further improve the conver-
sion while attaining a higher ee value, but lower con-
version or no reaction was observed, indicating the
enzyme was deactivated rapidly in these cases. Com-
pared to the asymmetric hydrogenation and hydrosily-
lation of 4-chlorobenzophenone and 4-methylbenzo-
phenone catalyzed by chiral metal catalysts, in which
the enantiomeric purity for p-chlorobenzhydrol was
0–47% ee and the best result for p-methylbenzhydrol
was 39% ee,[8,18,19,24] the carbonyl reductase SSCR-cat-
alyzed reduction exhibited much higher stereoselec-
tivity (up to 88 and 92% ee for 1b and 1c, respective-
ly), and even higher than the best results of the com-
mercially available ketoreductases (64 and 85% ee for
1b and 1c, respectively),[30] showing the promise of
this enzyme toward the reduction of sterically bulky
diaryl ketones.
1a (4-F)
1b (4-Cl)
1c (4-Me)
72
62
67
44 (R)
88 (R)
92 (R)
96 (R)
8 (S)
99
95
>99
95
>99
99
50 (R)
78 (R)
84 (R)
99 (R)
26 (S)
72 (R)
70 (R)
64 (R)
40 (S)
36 (R)
99 (R)
82 (R)
8 (R)
1d (4-MeO) 26
1e (2,6-F2)
1f (2-Cl)
1g (2-Me)
1h (3,4-Me2)
1i (3-NO2)
1j (3-NH2)
1k
49
44
8
74 (R)
–
–
[b] (R) 61
5
[b] (R) 58
33
50
>99
>99
9
48 (S)
56 (R)
99 (R)
78 (R)
28 (S)
81
>99
>99
>99
85
1l
1m
[a]
Determined by HPLC analysis.
[b]
Only one enantiomer was observed by chiral HPLC anal-
ysis, since the low conversion might result in a trace of
(S)-enantiomer not being detected, the ee value was not
reported.
To further explore the substrate scope of this car-
bonyl reductase, SSCR was applied to the reduction
Most diaryl ketones showed higher conversion in
of a series of diaryl ketones (Figure 1) in a potassium the medium with methanol as co-solvent. The enan-
phosphate buffer containing 10% (v/v) of either tioselectivity was also dependent on the co-solvent in
methanol or THF, since these two reaction media the buffer, although no trend was observed. The ee
showed the highest conversion or enantioselectivity. values of product alcohols for some ketones were
Co-factor NADPH was regenerated with the glucose higher with THF as co-solvent, while others exhibited
dehydrogenase and d-glucose system (Scheme 1). The higher enantioselectivity with methanol as co-solvent.
reaction mixtures were shaken at room temperature It seems that carbonyl reductase SSCR was more
for 24 h, and then extracted with methyl tert-butyl enantioselective toward the reduction of diaryl ke-
ether. The extracts were dried over anhydrous sodium tones with a para-substituent on one of phenyl groups
sulfate and subjected to chiral HPLC analysis. The than those with an ortho- or meta-substituent. This is
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Table 4. Reduction of diaryl ketones catalyzed by mutant
carbonyl reductase SSCR Q245P.
quite contrary to the observation in the chiral borane
reduction, asymmetric hydrogenation or hydrosilyla-
tion of diaryl ketones.[15,19,22,24] For the reduction of
para-substituted diaryl ketones, the enantioselectivity
was increased in the order of F<Cl<CH3 <CH3O,
which was in agreement with the increasing size and
electron-donating property of the para-substituent. It
is worth noting that 4-methoxybenzophenone was re-
duced to the corresponding (R)-alcohol with excellent
conversion (95%) and enantiomeric purity (99% ee)
in buffer containing 10% of methanol. 2-Benzoylpyri-
dine was completely converted to (R)-phenylpyridyl-
methanol in 99% ee with THF or methanol as co-sol-
vent.
Recently, three Q245 mutant enzymes (Q245P,
Q245L and Q245H) of carbonyl reductase SSCR
were found to invert the configuration of alcohol
products from the (R)-enantiomer to the (S)-enantio-
mer for the reduction of para-substituted acetophe-
nones.[45] It would be interesting to examine how
Ketone
THF
Methanol
Conv. [%][a] ee [%][a] Conv. [%][a] ee [%][a]
1a (4-F)
1b (4-Cl)
1c (4-Me)
1d (4-MeO) 70
1e (2,6-F2)
1f (2-Cl)
1g (2-Me)
1h (3,4-Me2) 40
1i (3-NO2)
68
88
79
24 (S)
74 (S)
52 (S)
76 (S)
70 (S)
12 (R)
20 (R)
36 (S)
10 (S)
32 (S)
90 (R)
74 (R)
12 (R)
>99
98
99
18 (S)
76 (S)
48 (S)
78 (S)
60 (S)
16 (R)
44 (R)
32 (S)
16 (S)
34 (S)
90 (R)
70 (R)
8 (S)
98
77
66
25
>99
>99
91
94
93
68
13
>99
>99
7
1j (3-NH2)
1k
1l
90
>99
>99
66
1m
[a]
Determined by HPLC analysis.
these mutants affect the reduction of diaryl ketones. phenyl groups, similar to the observation in the reduc-
Therefore, the catalytic properties of these mutant en- tion of para-substituted acetophenones.[45] The effect
zymes were studied using 4-methylbenzophenone (1c) on the enantioselectivity of those substrates with only
and 4-chlorobenzophenone (1b) as substrates an ortho- or meta-substituent was less significant, and
(Table 3). As indicated by the conversion, the three the increase in favour of forming the (S)-enantiomer
was not great enough to invert the enantiopreference
of the reduction, except for 3-aminobenzophenone.
A few chiral diaryl methanols such as (R)-4-methyl-
benzhydrol, (R)-4-methoxybenzhydrol, (R)-phenyl-(2-
pyridyl)methanol and (R)-phenyl-(3-pyridyl)methanol
were prepared in good yield and enantiomeric purity
using wild-type SSCR enzyme in potassium phosphate
buffer containing 10% (v/v) methanol (Table 5). This
provides a sustainable alternative method for the syn-
thesis of these pharmaceutically important com-
pounds.
Table 3. Reduction of 4-methylbenzophenone (1c) and 4-
chlorobenzophenone (1b) catalyzed by wild-type and
mutant SSCR enzymes.[a]
Enzyme
1c
1b
Conv [%][b] ee [%][b] Conv. [%][b] ee [%][b]
wild-type >99
84 (R)
48 (S)
46 (R)
46 (R)
95
98
>99
>99
78 (R)
76 (S)
28 (R)
22 (S)
Q245P
Q245L
Q245H
99
98
94
[a]
The reaction was carried out in potassium phosphate
buffer (100 mM, pH 7.0) with 10% (v/v) of methanol.
Determined by HPLC analysis.
Table 5. Preparation of a few diarylmethanols.
[b]
Diarylmethanol
Yield [%]
ee [%][a]
(R)-Methylbenzhydrol
(R)-Methoxybenzhydrol
(R)-Phenyl-(2-pyridyl)methanol
(R)-Phenyl-(3-pyridyl)methanol
89
87
81
84
84
99
99
82
mutant enzymes have comparable activity with wild-
type SSCR toward the reduction of the two tested
diaryl ketones. The ee values of alcohol products
showed that the ratio of (S)-enantiomer increased for
the mutant enzymes; thus single site mutation at
Q245 to P, L or H favours the formation of the (S)-
enantiomer compared to their wild-type counterpart.
[a]
Determined by chiral HPLC analysis.
This enantiopreference change was so significant for Conclusions
mutant Q245P that (S)-alcohols became the major
products. As such, the reduction of other diaryl ke- In summary, the enantioselective reduction of diaryl
tones were also investigated using Q245P mutant ketones catalyzed by carbonyl reductase SSCR and its
enzyme as the catalyst and THF or methanol as co- mutant enzymes was performed effectively in potassi-
solvent (Table 4).
um phosphate buffer containing an organic solvent
From Table 4 it can be seen that Q245P mutation (10% v/v) to give the corresponding enantiomerically
switched the enantiopreference for the reduction of enriched alcohols. Both conversion and enantioselec-
diaryl ketones with a para-substituent on one of tivity were dependent on the co-solvent in the buffer.
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Enantioselective Reduction of Diaryl Ketones Catalyzed by a Carbonyl Reductase
FULL PAPERS
Contrary to the results obtained by chemical methods General Procedure for Enzymatic Reduction of 4-
such as chiral borane reduction, asymmetric hydroge- Methylbenzophenone and 4-Methoxybenzophenone
nation or hydrosilylation, which exhibited low enan- (Preparative Scale)
tioselectivity (<47%) toward the reduction of diaryl
d-Glucose (20 gLÀ1), d-glucose dehydrogenase (2 gLÀ1),
ketones with a para-substituent on one of phenyl
groups, these diaryl ketones were reduced with high
enantioselectivity (up to 99% ee). The mutant en-
zymes of SSCR at Q245 showed a tendency in favour
of forming the (S)-enantiomer. Especially for mutant
Q245P with para-substituted diaryl ketone as a sub-
strate, the product enantiopreference is switched from
(R) to (S). The present study demonstrates that car-
bonyl reductase SSCR is a valuable biocatalyst
toward the reduction of diaryl ketones, and could
serve as a promising starting enzyme for further engi-
neering with the aim to development of efficient bio-
catalysts for highly enantiospecific reduction of diaryl
ketones without high electronic dissymmetry or an
ortho-substituent on one of the aryl groups, which re-
mains a formidable challenge in organic synthesis.
NADPH (1 gLÀ1), SSCR mutant enzyme (2 gLÀ1) were dis-
solved in potassium phosphate buffer (100 mM, pH 7.0), and
27 mL of the resulting solution were mixed with 3 mL of
diaryl ketone solution in methanol (0.1M). The reaction
mixture was shaken at room temperature for 24 h. The mix-
ture was saturated with sodium chloride and extracted with
methyl tert-butyl ether (3ꢂ30 mL). The organic extract was
dried over anhydrous sodium sulfate and removal of solvent
1
gave the product alcohols, which were characterized by H
and 13C NMR analysis.
(R)-4-Methylbenzhydrol (2c):[6] Yield: 52.3 mg (89%);
1H NMR (MeOH-d4): d=2.29 (s, 1H), 5.74 (s, 1H), 7.09 (m,
2H), 7.21 (m, 3H), 7.28 (m, 2H), 7.32 (m, 2H); 13C NMR
(MeOH-d4): d=21.1, 76.8, 127.6, 128.0, 129.2, 129.8, 137.9,
143.0, 145.8.
(R)-4-Methoxybenzhydrol (2d):[40] Yield: 55.3 mg (87%);
1H NMR (MeOH-d4): d=3.75 (s, 3H), 5.71 (s, 1H), 6.85 (m,
2H), 7.20–7.34 (m, H); 13C NMR (MeOH-d4): d=55.8, 76.5,
114.6, 127.5, 128.0, 129.0, 129.2, 138.0, 146.2, 160.5.
The procedures for the enzymatic reduction of 2-benzo-
pyridine and 3-benzopyridine were the same as described
above except that smaller amounts of enzymes and co-factor
were used: d-glucose dehydrogenase (0.2 gLÀ1), NADPH
(0.2 gLÀ1), SSCR enzyme (0.2 gLÀ1).
Experimental Section
The chiral HPLC analysis was performed on an Agilent
1200 high-performance liquid chromatography system with a
Chiracel OD-H chiral column. A mixture of hexane and iso-
propyl alcohol of different ratios was used as eluent
(hexane/isopropyl alcohol: 80/20 for 2j; 90/10 for 2l; 95/5
for 2b, 2c, 2f, 2g, 2h, 2i, and 2m; 98/2 for 2a, 2d, 2e and 2k).
All the ketone substrates were obtained from commercial
sources. The racemic alcohol standard samples were pre-
pared via the reduction of diaryl ketones with sodium boro-
hydride at room temperature. The optically active diaryl
methanol standard samples were prepared by the literature
method using commercially available enzymes.[30] The car-
bonyl reductase SSCR and its mutant enzymes were pre-
pared on a gram-scale as previously reported.[39,45]
(R)-Phenyl-(2-pyridyl)methanol (2k):[42] Yield: 44.5 mg
1
(81%); H NMR (MeOH-d4): d=5.79 (s, 1H), 7.2–7.4 (m,
6H), 7.62 (s, 1H), 7.80 (m, 1H), 8.42 (d, 2H); 13C NMR
(MeOH-d4): d=77.5, 122.2, 123.8, 127.9, 128.6, 129.4, 138.8,
149.1, 164.8.
(R)-Phenyl-(3-pyridyl)methanol (2l):[46] Yield: 46.3 mg
1
(84%); H NMR (MeOH-d4): d=5.84 (s, 1H), 7.3–7.4 (m,
6H), 7.81 (m, 1H), 8.39 (m, 1H), 9.54 (d, 1H); 13C NMR
(MeOH-d4): d=74.5, 125.0, 127.6, 128.7, 129.6, 136.5, 142.4,
144.6, 148.6.
General Procedure for Enzymatic Reduction of
Diaryl Ketones (1-mL scale)
Acknowledgements
d-Glucose (20 gLÀ1), d-glucose dehydrogenase (2 gLÀ1),
NADPH (1 gLÀ1), and SSCR or Q245 mutant enzymes
(2 gLÀ1) were dissolved in potassium phosphate buffer
(100 mM, pH 7.0), and 0.9 mL of the resulting solution was
mixed with 0.1 mL of diaryl ketone solution in the organic
solvent (0.1M). The reaction mixture was shaken for 24 h at
room temperature. The mixture was extracted with methyl
tert-butyl ether (1.0 mL). The organic extract was dried over
anhydrous sodium sulfate and subjected to chiral HPLC
analysis to determine the conversion and enantiomeric
excess. The absolute configurations of product alcohols were
identified by comparing the HPLC elution sequence with
the literature data using the same type of column,[6,7,17,40–44]
and/or by comparing with the enantiomerically enriched
sample prepared by literature methods.[30]
D.Z. thanks Chinese Academy of Sciences for support from
the Knowledge Innovation Program (Grant No. KSCX2-YW-
G-031).
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