A recombinant ketoreductase tool-box. Assessing the substrate selectivity and stereoselectivity toward the reduction of β-ketoesters

Article abstract of DOI:10.1016/j.tet.2005.10.044

The substrate selectivity and stereoselectivity of a series of ketoreductases were evaluated toward the reduction of two sets of β-ketoesters. Both the structural variety at β-position and the substituent at α-position greatly affected the activity and stereoselectivity of these ketoreductases. For the first set of β-ketoesters, at least one ketoreductase was found that catalyzed the formation of either (d) or (l) enantiomer of β-hydroxyesters from each substrate with high optical purity, with the only exception of ethyl (d)-3-hydroxy-3-phenylpropionate. For the second set of β-ketoesters with α-substituents, the situation is more complex. More commonly, a ketoreductase was found that formed one of the four diastereomers in optically pure form, with only a few cases in which enzymes could be found that formed two or more of the diastereomers in high optical purity. The continued development of new, more diverse ketoreductases will create the capability to produce a wider range of single diastereomers of 2-substituted-3-hydroxy acids and their derivatives.

Full text of DOI:10.1016/j.tet.2005.10.044

Tetrahedron 62 (2006) 901–905
A recombinant ketoreductase tool-box. Assessing the substrate
selectivity and stereoselectivity toward the reduction
of b-ketoesters
Dunming Zhu,a Chandrani Mukherjee,a J. David Rozzell,b Spiros Kambourakisb and Ling Huaa,
*
aDepartment of Chemistry, Southern Methodist University, Dallas, TX 75275, USA
bBioCatalytics, Inc., Pasadena, CA 91106, USA
Received 22 August 2005; revised 12 October 2005; accepted 14 October 2005
Available online 7 November 2005
Abstract—The substrate selectivity and stereoselectivity of a series of ketoreductases were evaluated toward the reduction of two sets of
b-ketoesters. Both the structural variety at b-position and the substituent at a-position greatly affected the activity and stereoselectivity of
these ketoreductases. For the first set of b-ketoesters, at least one ketoreductase was found that catalyzed the formation of either (D) or (L)
enantiomer of b-hydroxyesters from each substrate with high optical purity, with the only exception of ethyl (D)-3-hydroxy-
3-phenylpropionate. For the second set of b-ketoesters with a-substituents, the situation is more complex. More commonly, a ketoreductase
was found that formed one of the four diastereomers in optically pure form, with only a few cases in which enzymes could be found that
formed two or more of the diastereomers in high optical purity. The continued development of new, more diverse ketoreductases will create
the capability to produce a wider range of single diastereomers of 2-substituted-3-hydroxy acids and their derivatives.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Since optically pure b-hydroxy carboxylic acids and their
derivatives are key building blocks in the synthesis of
bioactive compounds,6 many efforts have been made to
develop effective methods for their synthesis in enantio-
merically pure form.7 Enzymatic reduction of b-ketoesters
catalyzed by ketoreductases represents an attractive
approach to enantiomerically pure b-hydroxy carboxylic
acids and their derivatives. The b-ketoester starting
materials are readily available in many cases, and the
environmentally friendly reaction conditions are easily
introduced in an industrial setting and can be scaled to
commercial volumes.3a,8 In this study, we report on the
substrate selectivity and stereoselectivity of a recombinant
ketoreductase tool-box by evaluating the reduction of a
series of diverse b-ketoesters. These studies represent a
systematic look at enzyme-catalyzed stereoselective ketone
reduction, which will serve as a useful guideline for
developing enzymatic processes for the production of
optically pure b-hydroxy carboxylic acid esters and
also guide the needs for future development of new
ketoreductase enzymes.
With the advantages of environmentally benign reaction
conditions, broad reaction scope, and high stereo- and
regioselectivity, biocatalytic reductions of prochiral ketones
offer significant potential in the synthesis of optically pure
alcohols.1 A biocatalytic reduction can be carried out using
either whole cell systems2 or isolated ketoreductases.3 Since
a whole cell may contain more than one ketoreductases,
frequently with opposing stereoselectivities, not all whole-
cell-mediated ketone reductions provide product chiral
alcohols in high optical purity.4 A straightforward approach
to solve this problem is to carry out the reduction with an
isolated ketoreductase in an in vitro reaction system.
However, until recently the application of isolated keto-
reductases to ketone reduction has been hampered by their
limited availability.3 In this context, we have developed an
‘easy-to-use’ ketoreductase tool-box consisting of more
than 30 recombinant ketoreductases KRED101–131 by
genome mining and protein engineering, and have shown
that these isolated recombinant ketoreductase enzymes
efficiently catalyze the enantioselective reduction of a
variety of substituted aryl ketones to optically pure aryl
alcohols.5
2. Results and discussion
Keywords: Ketoreductase; Stereoselectivity; b-Ketoesters.
*
Two sets of b-ketoesters have been chosen as substrates
to evaluate the activity and stereoselectivity of the
Corresponding author. Tel.: C1 214 768 1609; fax: C1 214 768 4089;
e-mail: lhua@smu.edu
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.10.044

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D. Zhu et al. / Tetrahedron 62 (2006) 901–905
ketoreductase tool-box, which comprises 31 recombinant
ketoreductase enzymes KRED101–131. The first set of
b-ketoesters 1–8 have diverse structure at the b-position,
while the second ones 9–15 possess different substituents at
the a-position as shown in Figure 1. The activity and
stereoselectivity of the ketoreductases toward these two sets
of b-ketoesters will be presented separately.
The activities of the ketoreductases toward the reduction of
b-ketoesters 1–8 were determined by spectrophotometri-
cally measuring the oxidation of NADPH at 340 nm at room
temperature. The relative activity of KRED101 in the
reduction of ethyl 3-oxobutyrate 1 was defined as 100. The
enantioselectivity of the ketoreductase-catalyzed reduction
of b-ketoesters 1–8 were studied using an NADPH recycle
system as shown in Scheme 1.
O
O
O
O
The selected data of the relative activity and enantioselec-
tivity for the reduction of b-ketoesters 1–8 are presented
together in Table 1. From Table 1 it can be seen that most of
the ketoreductases effectively catalyzed the reduction of
ethyl 3-oxobutyrate 1. When the chain length of the
b-ketoesters increases or become branched (from 1/2/
3/4/5), three major trends were observed for the
ketoreductase activities. For example, the activity of
KRED108 followed a descending order from 1/2/3/
4/5 as shown in Figure 2. Several other ketoreductases,
KRED102,103,106, and KRED107, followed the same
decreasing order. This may be due to the alkyl group at
b-position becoming more bulky from 1 to 5. An interesting
trend was observed for KRED112 and KRED118, which
showed an overall decrease in activity from 1/2/3/
4/5, but with an unexpected increase in activity for
substrate 3 (KRED112 is shown in Fig. 2 as an example).
KRED114,121,123, and KRED130 did not show significant
activity change across the series of b-ketoesters 1–5
(KRED114 is shown in Fig. 2 as an example). Interestingly,
KRED101 showed the highest activity toward the reduction
of ethyl 4,4-dimethyl-3-oxo-pentanoate 5. For the reduction
of ethyl 4-chloro-3-oxo-butyrate 6 and ethyl 4,4,4-trifluoro-
3-oxo-butyrate 8, most of the ketoreductases were efficient
catalyst, while only a few ketoreductases were effective in
the reduction of ethyl benzoylacetate 7.
R
O
O
R'
R' =
R =
1
CH3
9
CH3
2
3
4
5
6
7
8
CH2CH3
CH2CH2CH3
CH(CH3)2
C(CH3)3
CH2Cl
10
11
12
13
CH2CH3
CH(CH3)2
CH2C6H5
Cl
C6H5
CF3
O
O
O
O
O
O
14
Figure 1. b-Ketoesters 1–15.
15
O
O
R
R
O
O
NADPH
NADP+
D-Gluconic acid
GDH
D-Glucose
R'
OH
KRED
O
R'
Scheme 1. Reduction of b-ketoesters catalyzed by ketoreductases with
NADPH recycle system.
Table 1. The relative activity and enantioselectivity of the ketoreductases toward the reduction of b-ketoesters 1–8
O
O
OH
O
OH O
+
R
OC2H5
R
OC2H5
R
OC2H5
D
L
KRED
1 (CH3)
37a (100)b
2 (CH2CH3)
3 (CH2CH2CH3)
4 (CH(CH3)2)
5 (C(CH3)3)
6 (CH2Cl)
7 (C6H5)
8 (CF3)
101
102
103
106
107
108
110
112
113
114
118
121
123
128
130
131
K65 (154)
O99 (16)
O99 (13)
O99 (24)
KO99 (21)
O99 (114)
—
75 (120)
c
—
K99 (170)
—
—
KO99 (277)
—
—
KO99 (394)
99 (226)
99 (311)
K61 (94)
—
—
K88 (71)
87 (9)
96 (9)
O99 (51)
O99 (97)
O99 (67)
KO99 (60)
O99 (354)
O99 (13)
84 (361)
89 (318)
37 (43)
O99 (630)
K23 (30)
K22 (17)
96 (200)
—
—
—
—
97 (353)
—
98 (4)
KO99 (7)
95 (34)
—
KO99 (3)
—
—
K89 (127)
K87 (79)
K73 (45)
O99 (369)
KO99 (20)
K97 (41)
65 (16)
KO99 (1)
—
—
KO99 (29)
K99 (29)
K99 (54)
99 (13)
KO99 (71)
KO99 (17)
—
KO99 (271)
98 (406)
K8 (14)
K9 (516)
K89 (24)
K63 (263)
97 (396)
K82 (164)
K93 (174)
79 (137)
K8 (294)
K94 (404)
K54 (3)
66 (29)
99 (10)
K68 (93)
K67 (60)
73 (174)
K77 (136)
80 (189)
—
K97 (6)
83 (77)
—
K75 (237)
K65 (203)
K46 (27)
91 (224)
K91 (49)
K89 (29)
64 (63)
23 (210)
52 (60)
96 (355)
97 (296)
K6 (61)
O99 (617)
K72 (46)
K62 (51)
O99 (149)
K88 (20)
K98 (41)
K56 (51)
O99 (516)
K90 (47)
K78 (27)
98 (223)
0 (14)
—
—
—
70 (20)
K52 (16)
K99 (16)
KO99 (16)
KO99 (7)
—
K90 (81)
35 (337)
K91 (34)
a ee%, the positive ee value indicates that L-enantiomer is the major product, while the negative ee value indicates that D-configuration is the major enantiomer.
b The initial reaction rate was measured by the procedure described in the Section 4, the relative activity of KRED101 in the reduction of ethyl 3-oxobutyrate 1
was defined as 100 and its specific activity was 88 nmol minK1 mgK1
.
c The conversion of the reduction was less than 20% after 24 h, the ee value and relative activity was thus not reported.

D. Zhu et al. / Tetrahedron 62 (2006) 901–905
903
400
350
300
250
200
150
100
50
0
1
2
3
4
5
Substrate
Figure 2. Three major patterns for the structural effect on the activity of
ketoreductases toward the reduction of b-ketoesters (1–5). KRED108
(diamond), KRED112 (square), and KRED114 (triangle).
Reduction of the first set of b-ketoesters produced two
enantiomers (L and D) of b-hydroxyesters, since the enzyme
delivered the hydride either from the re- or si-face of the
prochiral ketone. The enantioselectivity of the enzymatic
reduction was clearly dependent on the structure of the
b-ketoesters. In some cases, structure of the b-ketoesters
also affected the stereochemical preference, that the enzyme
delivered the hydride preferentially at the re- or si-side of
the carbonyl group. For example, KRED101-catalyzed
reductions of b-ketoesters 1 and 3 gave (L)-enantiomer as
the major product, resulting from the hydrogen transfer
occurring at the re-face of the carbonyl group; while
(D)-enantiomer was obtained as the major product in the
KRED101-catalyzed reduction of b-ketoesters 2, 4, and 5,
indicating that the hydrogen transfer occurred at the si-face
of the carbonyl group. Similar structure-induced inversion
of configuration was observed for KRED112,113,114, and
KRED130. From the results, it can be found that both
enantiomers of b-hydroxyesters were obtained in high
optical purity by one or more ketoreductases for each of the
tested b-ketoesters, the only exception was ethyl (D)-3-
hydroxy-3-phenylpropionate. The breadth of scope offered
by ketoreductases demonstrates that such a group of
enzymes can serve as a useful tool-box for the synthesis
of optically pure b-hydroxyesters.
The selected data for the relative activity and stereo-
selectivity of the ketoreductase-catalyzed reduction of
a-substituted-b-ketoesters 9–15 are presented in Table 2.
When the size of the alkyl group at the a-position of ethyl
3-oxo-butyrate increased (from 9/10/11), the activity of
most ketoreductases showed a decreasing trend. The benzyl
group at the a-position 12 had an unexpectedly diverse
effect on the enzyme activity when compared with the
introduction of an a-methyl group 9. Most of the
ketoreductases showed high activity toward the reduction
of ethyl 2-chloro-3-oxo-butyrate 13. Both ethyl 2-oxo-
cyclopentanecarbonate 14 and ethyl 2-oxo-cyclohexane-
carbonate 15 were good substrates for most of the tested
ketoreductases, although the activities toward these two
substrates were significantly varied.
The reduction of the second set of b-ketoesters generated
two chiral carbon atoms, with the possible formation of four
diastereomers (Table 2). In many cases, a single

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D. Zhu et al. / Tetrahedron 62 (2006) 901–905
diastereomer was obtained with very high diastereoselec-
tivity. Mechanistically, the high diastereoselectivity results
from the acidity of the a-proton in the b-ketoester, allowing
the epimerization of the a-carbon of the b-ketoester starting
material, but not the b-hydroxyester product, under the
reaction conditions. Especially for ethyl 2-chloro-3-oxo-
butyrate 13, almost every ketoreductase showed high
diastereoselectivity. In most cases, diastereomer A was
obtained. A few exceptions were KRED102- and
KRED103-catalyzed reduction of ethyl 2-isopropyl-
3-oxo-butyrate 11, in which diastereomer C was obtained
with selectivity greater than 96%. Although the other
diastereomers were also obtained as the major product in
other cases, the diastereoselectivity was not as high, ranging
from 40–95% in most cases. This indicates that while this
current group of ketoreductases show high stereoselectivity
and good diversity in terms of the substrate acceptance
toward the reduction of 2-substituted-3-ketoesters, further
efforts are required to develop ketoreductases with high and
diverse diastereoselectivities, that is, for the production of
diastereomers B, C, and D.
340 nm (3Z6.22 mMK1 cmK1) in the presence of excess
b-ketoesters. The activity was measured at room tempera-
ture in 96-well plate, in which each well contained
b-ketoester (6.25 mM), NADPH (0.25 mM) in potassium
phosphate buffer (100 mM, pH 7.0, 180 ml). The reaction
was initiated by the addition of the ketoreductase
(20 ml solution containing 2.5–100 mg of enzyme). The
specific activity was defined as the number of nmol of
NADPH converted in one minute by 1 mg of enzyme
(nmol minK1 mgK1).
4.2. Stereoselectivity of the enzymatic reduction
of b-ketoesters
The stereoselectivity of the enzymatic reduction of
b-ketoesters was studied using an NADPH recycle system.
The general procedure was as follows: D-glucose (4 mg),
D-glucose dehydrogenase (0.5 mg), NADPH (0.5 mg),
ketoreductase (0.5 mg) and b-ketoester solution in DMSO
(50 ml, 0.25 M) were mixed in a potassium phosphate buffer
(1 ml, 100 mM, pH 7.0) and the mixture was shaken at
25 8C overnight. The mixture was extracted with methyl
tert-butyl ether (1 ml). The organic extract was dried over
anhydrous sodium sulfate and was subjected to chiral GC
analysis. The absolute configuration of ethyl 3-hydroxy-
butyrate, ethyl 3-hydroxypentanoate were determined by
comparison of the chiral GC data with that in the
literature.19 The absolute configuration of other b-hydroxy-
esters were identified by comparing the chiral GC data with
the standard samples. The racemate and enantiomers or
diastereomers of the b-hydroxyesters were prepared by
sodium borohydride reduction or following the literature
methods: ethyl 3-hydroxyhexanoate,9 ethyl 3-hydroxy-
3. Conclusion
The substrate selectivity and stereoselectivity of a series of
ketoreductases have been studied toward the reduction of
two sets of b-ketoesters. Both the structural variety at
b-position and the substituent at a-position greatly affect the
activity and stereoselectivity of these reductions. For the
synthetic purposes, the ability to prepare each stereoisomer
of b-hydroxyester building blocks in homochiral form is
important. For the first set of b-ketoesters, our ketoreductase
collection has been demonstrated to be a useful tool-box for
this goal. For the second set of b-ketoesters with
a-substituent, one of the four diastereomers was produced
in optically pure form, although diastereomer A was
obtained in most cases. We are continuing efforts to develop
new ketoreductases with differing diastereoselectivity in the
reduction of a-substituted-b-ketoesters.
4-methylpentanoate,10
ethyl
4-chloro-3-hydroxy-
butyrate,9a,11 ethyl 3-hydroxy-3-phenylpropionate,12 ethyl
3-hydroxy-4,4,4-trifluorobutyrate13 ethyl 3-hydroxy-
2-methylbutyrate,9a,14
ethyl
2-ethyl-3-hydroxy-
butyrate,9a,14a ethyl 2-isopropyl-3-hydroxybutyrate,14a,15
ethyl 2-benzyl-3-hydroxybutyrate,16 ethyl 2-chloro-3-
hydroxybutyrate,17 ethyl 2-hydroxycyclopentanecarboxy-
late18 and ethyl 2-hydroxycyclohexanecarboxylate.18
4. Experimental
4.3. Preparative scale reduction of ethyl 4,4-dimethyl-
3-oxo-pentanoate and absolute configuration determi-
nation of ethyl 4,4-dimethyl-3-hydroxy-pentanoate
The chiral GC analysis was performed on a Hewlett Packard
5890 series II plus gas chromatograph equipped with
autosampler, EPC, split/splitless injector, FID detector and
25 m!0.25 mm CP-Chirasil-Dex CB chiral capillary
column. The ketoreductase activities toward the reduction
of b-ketoesters (Tables 1 and 2) were assayed using
SpectraMax M2 microplate reader (Molecular Devices).
All the ketoreductases were purified recombinant enzymes,
which were developed by genome mining and protein
engineering, and are commercially available from Bio-
Catalytics, Inc. All the b-ketoesters were purchased from
Aldrich and the b-hydroxyester standards were prepared by
following the literature procedures.9–18
D-Glucose (400 mg), D-glucose dehydrogenase (5 mg),
NADPH (5 mg), KRED101 (5 mg) and ethyl
4,4-dimethyl-3-oxo-pentanoate (200 mg) were mixed in a
potassium phosphate buffer (50 ml, 100 mM, pH 7.0) and
the mixture was stirred at room temperature with pH being
controlled by 0.5 M sodium hydroxide solution. After 24 h,
GC analysis indicated that reduction was complete. The
reaction mixture was extracted with MTBE (30 ml!2).
The organic extract was dried over anhydrous Na2SO4 and
removal of the solvent gave product b-hydroxyester as
1
clear oil (185 mg, 92% yield). H and 13C NMR (CDCl3)
are in accordance with literature data.20 The ester was then
hydrolyzed in H2O with 1.5 equiv of NaOH solution
4.1. Activity assay of the ketoreductases
1
to give (D)-4,4-dimethyl-3-hydroxy-pentanoic acid. H and
The activities of the ketoreductases toward the reduction of
b-ketoesters (Tables 1 and 2) were determined by spectro-
photometrically measuring the oxidation of NADPH at
13C NMR (CDCl3) are in accordance with literature data.21
[a]2D2 K53.4 (c 1.0, CHCl3); lit.21 [a]2D5 53.0 (c 1.0,
CHCl3) for L-enantiomer.

D. Zhu et al. / Tetrahedron 62 (2006) 901–905
905
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