Stereoselective synthesis of aryl γ,δ-unsaturated β-hydroxyesters by ketoreductases

Article abstract of DOI:10.1016/j.molcatb.2013.09.003

The biocatalytic reduction of aryl γ,δ-unsaturated-β- ketoesters was evaluated utilizing 24 different commercially available ketoreductases. In all cases, both (R) and (S)-enantiomers of γ,δ-unsaturated β-hydroxyesters were synthesized by one or more keto

Full text of DOI:10.1016/j.molcatb.2013.09.003

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 264–269
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Stereoselective synthesis of aryl ꢀ,ı-unsaturated ˇ-hydroxyesters
by ketoreductases
∗
Zhipeng Dai, Kate Guillemette, Thomas K. Green
Department of Chemistry and Biochemistry, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks 99775, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 May 2013
Received in revised form 16 August 2013
Accepted 2 September 2013
Available online 9 September 2013
The biocatalytic reduction of aryl ꢀ,ı-unsaturated-ˇ-ketoesters was evaluated utilizing 24 different
commercially available ketoreductases. In all cases, both (R) and (S)-enantiomers of ꢀ,ı-unsaturated
ˇ-hydroxyesters were synthesized by one or more ketoreductases in excellent optical purity and yields.
©
2013 Elsevier B.V. All rights reserved.
Keywords:
Ketoreductases
Enantioselectivity
Ketoesters
Enzymes
1
. Introduction
to control. Recently, Ma et al. published a Ru-catalyzed hydro-
genation of ꢀ-halo-ꢀ,ı-unsaturated-ˇ-ketoesters in up to 97%
ee [27], where halogen at the ꢀ-position is needed to protect
the ꢀ,ı-carbon–carbon double bond with further organometallic
transformation required to remove the vinyl halide moiety. The
enantioselectivity of chiral oxazaborolidines catalysts are sensi-
tive to moisture and temperature and must be conducted under
strictly anhydrous conditions and typically at low temperature
Enantiomerically pure ꢀ,ı-unsaturated-ˇ-hydroxyesters and
their derivatives are important chiral building blocks for synthesis
of many biologically active compounds, pharmaceutical products
and their intermediates, such as (−)-CP -Disorazole C [1], turna-
2
1
gainolides A and B [2], epothilone [3] and Seimatopolide A [4]. Many
traditional methods have been addressed and provide pathways to
make ˇ-hydroxyesters asymmetrically, for example, (1) enantio-
selective Mukaiyama aldol addition between aldehyde and methyl
◦
(−20 C and below). Also, the use of BH ·THF (or BH ·Me S) is
3
3
2
often incompatible with carbon–carbon double bonds. Although
these chemicalmethodsusing chiral auxiliariesare frequentlyused,
other limitations remain in many cases; the incompatibility of cat-
alytic conditions with a number of functionalities [28–30] and the
catalytic behavior (enantiomeric purity and productivity) being
sensitive to even slight modifications in the substances due to the
change of steric and electronic properties.
(
ethyl) acetate O-silyl enolates with a chiral Ti(IV) complexes as
catalyst [5–7], (2) enantioselective ruthenium-catalyzed hydro-
genation of saturated ˛-ketoesters [8], ˇ-ketoesters [9–18] and
˛
catalyzed reduction of prochiral ketones, cyclic and acyclic ˛, and
ˇ-enones [21–25].
,ˇ-unsaturated carbonyls [19,20], and (3) chiral oxazaborolidine-
All of these methods, while potentially providing high enan-
tioselectivity, have disadvantages. The Mukaiyama aldol methods
often require an expensive chiral ligand (e.g. BINAP) and mul-
tiple steps to make the catalyst [7]. While ruthenium-catalyzed
hydrogenation has been shown to selectively reduce the carbonyl
of ˇ-ketoesters chemoselectively in the presence of a nonconju-
gated carbon–carbon double bond [10,26], we and others [27] have
found that partial hydrogenation of the double bond of conju-
gated ꢀ,ı-unsaturated-ˇ-ketoesters often occurs, and is difficult
With the advantages of high enantioselectivity, broader sub-
strate acceptance, mild and environmental friendly reaction
conditions, tolerance of organic solvents and easy separation, bio-
catalysts using whole cells and isolated enzymes have received
increasing interest toward the production of optically pure ˇ-
hydroxyesters and their derivatives [31–36]. For instances, the
asymmetric reduction of ˛- and/or ˇ-ketoesters was evaluated
by means of whole cells of Candida parapsilosis ATCC 7330 (on
the reduction alkyl 2-oxo-4-arylbutanoates) [37], the recombi-
nant Escherichia coli (for the synthesis of 3-hydroxybutyrate) [38],
Chlorella strains (toward the reduction of ˛-ketoesters) [39], yeast
(reduction of ˛- and ˇ-ketoesters) [34,40], carbonyl reductase from
Candila magnolia [33], nicotinamide-dependent ketoreductases
∗ Corresponding author. Tel.: +1 9074741559; fax: +1 9074745640.
E-mail address: tkgreen@alaska.edu (T.K. Green).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.09.003

Z. Dai et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 264–269
265
(
reduction of ˇ-ketoesters with alkyl substituents at ˛- or ˇ-
2.3. Synthesis of ꢀ,ı-unsaturated ˇ-keto ethyl esters 2A–2F
position) [41,42], and NADPH-dependent ketoreductases (toward
the reduction of ˛-alkyl-ˇ-ketoesters) [43]. What is more, Candida
parapsilosis ATCC 7330-induced deracemisation of unsaturated aryl
ˇ-hydroxyesters to a single enantiomer has also been reported
(E)-Ethyl 4-(diethoxyphosphinyl)-3-oxobutanoate (2.234 g,
6.6 mmol), prepared according to the literature [47], in 20 mL
anhydrous THF was reacted with 2.0 equiv. of n-BuLi (6.0 mL,
◦
[
44,45].
In this paper, we describe the synthesis of a series of aryl
,ı-unsaturated-ˇ-hydroxyesters from aryl ꢀ,ı-unsaturated-ˇ-
13.2 mmol, 2.2 M in hexanes) at 0 C. After gas formation ceased,
strirring was continued for 1 h at room temperature. One equiv-
alent of aldehyde (6.0 mmol) was added over 10 min, and the
reaction mixture was stirred at room temperature for another
2.5 h. After completion, the reaction was quenched by adding
ꢀ
ketoesters via the action of 24 isolated NAD(P)H dependent
ketoreductases from Codexis. Either enantiomer of ꢀ,ı-unsaturated
ˇ-hydroxyesters can be synthesized with high enantioselectivity
15 mL of saturated NH Cl. The reaction mixture was concentrated
4
◦
(
>99% ee) and chemoselectivity (no olefin reduction) in good to
under vacuum at 50–60 C. The residue was extracted by CH Cl
2
2
excellent yield in one step by one or more enzymes. Additionally,
conversions and purifications were achieved economically, safely
and readily under the enzymatic reaction condition used.
(3× 15 mL), the combined organic extracts were washed with satu-
rated NaCl (2× 15 mL), and then dried with anhydrous Na SO . The
2
4
◦
organic solvent was removed under vacuum at 30 C. Product was
isolated by chromatography on silica gel using 10:1 CH Cl /EtOAc
2
2
1
13
and verified by H and C NMR spectroscopy.
2
. Experimental
2.4. Synthesis of racemic ꢀ,ı-unsaturated ˇ-hydroxyesters 3A–3F
2.1. Materials
In a round bottom flask ˇ-ketoester (1 mmol) was dissolved in
mL ethanol. In portion, cautiously and intermittently, 0.4 equiv. of
All chemicals were purchased from Sigma–Aldrich Chemical
5
Company unless otherwise noted. Spectroscopy grade chloroform
was used for all optical rotation measurements. Cyclodextrins
used for capillary electrophoresis, heptakis (2,3-di-O-methyl-6-
O-sulfobutyl) cyclomaltoheptaose, sodium salt (NaSBDM-ˇ-CD)
and heptakis (2,3-di-O-ethyl-6-O-sulfopropyl) cyclomaltohep-
taose potassium salt (KSPDE-ˇ-CD), were synthesized according
to procedures reported in the literature [46]. Codex® KRED
screening kit was purchased from Codexis, Inc. Both reconstituted
KRED Recycle Mix N (containing 250 mM potassium phosphate,
NaBH4 (15 mg, 0.4 mmol) was added and the mixture was stirred
for 30 min at room temperature. The reaction mixture was con-
centrated under vacuum at 60 C. The product was isolated by
◦
chromatography on silica gel using 10:1 CH Cl /EtOAc and verified
2
2
1
13
by H and C NMR spectroscopy.
2.5. Stereoselectivity of enzymatic formation of ˇ-hydroxyesters
3A–3F using NADH system (enzyme 1–5)
2
mM magnesium sulfate, 1.1 mM NADP+, 1.1 mM NAD+, 80 mM
Into a solution of ˇ-ketoester (25 ␮mol) in 50 ␮L methanol, was
d-glucose, 10 U/mL glucose dehydrogenase, pH 7.0) and recon-
stituted KRED Recycle Mix P (containing 125 mM potassium
phosphate, 1.25 mM magnesium sulfate, 1.0 mM NADP+, pH 7.0)
are available from Codexis, Inc. Aluminum coated silica gel WF254s
plates were used to monitor reactions products and flash chro-
matography eluents. Column chromatography was performed with
added KRED Mix N (57.4 mg in 1.0 mL deionized H O) and 1.0 mg
2
◦
ketoreductase. The mixture was shaken at 32 ± 1 C. After 24 h, the
reaction was extracted by EtOAc (2× 1 mL). The combined organic
extract was dried over anhydrous Na SO and was subjected to
2
4
1
chiral HPLC or CE analysis, and H NMR spectroscopy.
®
silica gel SiliaFlash P60 (40–60 ␮m, 230–400 mesh).
2
.6. Stereoselectivity of enzymatic formation of ˇ-hydroxyesters
3A–3F using NADPH system (enzyme 6–24)
2.2. Instrument
Into a solution of ˇ-ketoester (25 ␮mol) in 400 ␮L isopropanol
and 50 ␮L methanol, was added KRED Mix P (29.1 mg in 1.0 mL
1H and 13C NMR spectra were recorded in CDCl solution with
3
a Varian 300 MHz instrument. Chemical shifts are reported in ppm
relative to TMS as internal standard. HPLC was performed on Agi-
lent 1100 series with isocratic pump and UV-visible detector. A
deionized H O) and 1.0 mg ketoreductase. The mixture was stirred
2
◦
at 32 ± 1 C. After 24 h, the reaction was extracted by EtOAc (2×
1 mL). The combined organic extract was dried over anhydrous
®
Na SO4 and was subjected to chiral HPLC or CE analysis, and 1
Phenomenex Lux 3 ␮ cellulose-1 column (50 mm × 4.60 mm) was
H
2
◦
used for the chiral separation at 23 C. The mobile phase consisted
NMR spectroscopy. For ˇ-ketoesters 2A–2E except 2D, the reac-
tions were scaled up by a factor of 20 or 100 using enzymes that
yielded high conversions and ee, and all products were isolated by
chromatography on silica gel using 10:1 CH Cl /EtOAc and verified
of hexanes and isopropanol in the ratio of 90:10, and flow rate
of 0.5 mL/min. Optical rotations were measured on Krüss P3000
polarimeter operating at the sodium D line 589 nm and reported
2
2
2
5
3
89
1
13
as follows: [˛] , concentration (g/100 mL), and solvent. Capil-
by H and C NMR spectroscopy.
lary electrophoresis was performed with an Agilent 3D Capillary
Electrophoresis System using bare fused silica capillary (purchased
from Polymicro Technologies, L.L.C.) (50 ␮m i.d., 32.5 cm total
length, 24.0 cm to detector) under reverse polarity (−10 or −15 kV),
and detection was by UV absorbance at 254 nm. Prior to first use,
the capillary was primed for 2 min with 1 M NaOH solution and then
for 2 min with 0.1 M NaOH solution. For each use, the capillary was
preconditioned for 1 min using background electrolyte (BGE). BGE
was either 5.0 mM NaSBDM-ˇ-CD or 5.0 mM KSPDE-ˇ-CD as chiral
selector in 25 mM tris buffer, pH 2.5. Samples (diluted 10 fold into
2.7. Synthesis of MTPA ester of ꢀ,ı-unsaturated ˇ-hydroxyesters
[48]
Into a solution of 64 ␮mol ˇ-hydroxyester (racemic or made
by enzyme 8 KRED-P1-B05 or enzyme 23 KRED-P3-G09) and dry
pyridine (16 ␮L, 200 ␮mol, 3.1 equiv.) in 1.0 mL anhydrous CH Cl ,
2
2
was added the R-(−)-MTPA-Cl (23 ␮L, 120 ␮mol, 1.9 equiv.). The
reaction mixture was stirred at ambient temperature till the
reaction was completed as monitored by TLC plate (eluent:
10:1 = CH Cl :EtOAc) (usually 3 h). After completion, the reaction
9
0:10 v/v deionized water/acetone solvent) were injected into the
2
2
capillary under 50.0 mbar pressures for 3 s. At the end of each run,
the capillary was post-conditioned with 0.1 M NaOH solution for
was quenched by 2 mL H O, extracted by EtOAc (2× 5 mL). The
2
combined organic layer were dried over anhydrous Na SO and
2
4
1
min and then with deionized water for 1 min.
removed under vacuo. The product MTPA ester of ꢀ,ı-unsaturated

2
66
Z. Dai et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 264–269
Scheme 1. Synthesis of ꢀ,ı-unsaturated-ˇ-ketoesters from appropriate aldehydes and 4-(diethoxyphosphinyl)-3-oxobutanoate in the presence of two equivalents of n-BuLi.
ˇ-hydroxyesters was isolated by chromatography on silica gel
using 10:1 CH Cl /EtOAc. (S)-MTPA ester spectra are included in
Supporting Information.
reaction condition. A significant by-product was obtained because
␣-hydrogen of phenylacetaldehyde is activated by the phenyl and
formyl groups and easily deprotonated under basic conditions, fol-
lowed by intermolecular aldol condensation.
2
2
3
. Results and discussion
3.2. Stereoselectivity of enzymatic formation of ˇ-hydroxyesters
3.1. Preparation of ꢀ,ı-unsaturated ˇ-keto ethyl esters 2A–2F
3A–3F
By optimizing previously reported procedures [49–53], synthe-
The five para-substituted phenyl (2A–E) and one benzyl (2F)
sis of ꢀ,ı-unsaturated ˇ-keto ethyl esters 2A–F, shown in Scheme 1,
was accomplished via the condensation of appropriate aldehydes
with pretreated 4-(diethoxyphosphinyl)-3-oxobutanoate [47] by
two equivalents of n-BuLi at room temperature. No Z-olefins were
ꢀ,ı-unsaturated ˇ-ketoesters were selected as substrates to eval-
uate the stereoselectivity of twenty-four isolated ketoreductases
listed in Table 1 from the Codex® ketoreductase screening kit which
includes five wild type KREDs 1–5 and nineteen engineered KREDs
6–24. All KREDs reactions used NADPH recycle system except
enzyme 4 (KRED-NADH-101) and 5 (KRED-NADH-110) which used
NADH instead of NADPH, as shown in Scheme 2. For engineered
KREDs 6–24 which have high tolerance to high concentration
1
detected by H NMR (E-olefins have a coupling constant with a
1
value of 15 Hz in H NMR spectrometry). The condensation afforded
yields of 75–90% for 2A–E, but the ketoester 2F was formed in only
1
5% yield when phenylacetaldehyde 1F was used under the same
Table 1
Stereocontrolled reduction of ꢀ,ı-unsaturated ˇ-ketoesters to ꢀ,ı-unsaturated ˇ-hydroxyesters by 24 Codex® ketoreductases.
KRED
3A
3B
3C
3D
3E
3F
3F’
e.e.a,b
%
Conv.c (%) e.e.a,b
%
Conv. c (%) e.e.b
%
Conv.c (%) e.e.a,b
%
Conv.c (%) e.e.a
%
Conv.c (%) e.e. a
%
Conv.c (%) e.e. a
%
Conv.c (%)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
101
119
130
>99
>99
−>99
92
94
84
w
94
–
>99
>99
−>99
−>99
>99
–
90
>99
84
45
–
95
–
>99
>99
>99
–
>99
96
>99
–
–
67
96
84
w
75
–
90
79
13
28
–
>99
77
95
85
–
66
–
>99
>99
40
<3
–
>99
>99
−>99
<3
>99
–
>99
>99
72
>99
–
54
–
96
25
>99
–
–
–
>99
–
62
24
6
<3
13
–
86
92
<3
<3
–
<3
–
96
90
96
–
>99
>99
49
87
74
–
8
–
87
>99
–
–
–
–
–
>99
>99
98
–
–
–
>99
–
–
91
96
−85
−>99
>99
–
98
82
52
38
–
–
48
>99
>99
>99
–
–
–
>99
>99
81
50
>99
–
97
89
<3
<3
–
–
–
–
–
–
–
8
90
–68
−76
–
–
–
–
–
–
–
3
11
<3
<3
–
–
<3
3
2
6
–
–
<3
–
–
94
−93
–
>99
–
>99
>99
68
50
–
40
–
>99
>99
>99
80
92
82
>99
–
–
−>99
NADH-101 −>99
–
NADH-110
P1-A04
P1-B02
P1-B05
P1-B10
P1-B12
P1-C01
P1-H08
P1-H10
P2-B02
P2-C02
P2-C11
P2-D03
P2-D11
P2-D12
P2-G03
P2-H07
P3-B03
P3-G09
P3-H12
>99
–
84
>99
53
49
90
98
–
>99
>99
>99
94
96
95
>99
–
–
>99
–
54
>99
–
–
–
–
–
>99
>99
>99
–
–
–
>99
–
–
88
>99
28
31
<3
<3
–
>99
93
96
<3
33
<3
94
–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
8
–
<3
–
–
–
<3
97
98
94
–
–
–
>99
–
–
14
95
>99
>99
–
>99
>99
93
–
10
<3
97
–
>99
>99
>99
<3
<3
<3
>99
–
–
–
81
–
–
–
−46
>99
–
–
–
–
–
–
15
8
–
38
<3
–
>99
20
–
–
9
31
−>99
−>99
−>99
−>99
73
43
−>99
40
15
−>99
43
62
−>99
−>99
−>99
−>99
−>99
−>99
−90
21
a
The percent ee was measured by chiral HPLC. The positive ee value indicates that (R)-enantiomer is the major product, while negative ee value indicates that (S)-
1
enantiomer is the major product. In each case, the racemic mixture was prepared for calibration. Absolute configuration of stereogenic C-3 was identified by H NMR analysis
of corresponding MTPA ester [48].
b
The percent ee was determined by CE using charged ␤-CD as a chiral selector.
c
The conversion was obtained by 1H NMR integration using d-chloroform as a solvent. When <3% conversion is reported, the presence of byproducts made the measurement
1
uncertain, although a small amount of product is detected. If no value is given, the product was not detected by either HPLC, CE, H NMR spectroscopy or TLC.

Z. Dai et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 264–269
267
Scheme 2. Enzymatic reduction of ꢀ,ı-unsaturated ˇ-ketoesters with NAD(P)H-dependent Codex® ketoreductases.
of isopropanol (IPA), IPA was used to assist in dissolving poor
water soluble substances and served to recycle NADPH cofactor
from NADP+, and the reconstituted KRED recycle Mix P serviced
as the reaction medium. However, for wild type KREDs 1–5, the
reconstituted KRED recycle Mix N containing d-glucose/glucose
dehydrogenase (GDH) and NAD(P)+ was used to provide a cofactor
recycle system. In each case, methanol was used as co-solvent to
enhance the solubility of ˇ-ketoesters, but the amount of co-solvent
was no more than 5% of the total volume of reaction solution as
recommended in the protocol provided by Codexis.
Table 2
Isolated yields of ꢀ,ı-unsaturated-ˇ-hydroxyesters 3 using ketoreductases which provide both good yield and high enantioselectivity.
eea (%)
[˛]5
89
23
#
Substance
KRED
Isolated yield (%)
OH
OH
O
O
◦
◦
3
A
OEt
P2-B02
90
>99
+14 (c 0.57, CHCl3) lit. +13.6 [5]
130
70
79
75
−>99
>99
−16◦ (c 0.34, CHCl3) lit. −2.6◦ [44], −6.8◦ [45]
OEt
O
OH
◦
3
B
OEt
P1-B05
130
+20 (c 0.10, CHCl3)
OH
O
OEt
OEt
−>99
−15◦ (c 0.33, CHCl3)
OH
O
◦
3
C
P2-C11
P3-G09
85
81
>99
+15 (c 0.13, CHCl3)
Cl
Cl
OH
O
OEt
−16◦ (c 0.16, CHCl3)
−>99
OH
OH
O
O
◦
3
E
OEt
OEt
P1-B05
130
77
64
>99
+20 (c 0.10, CHCl3)
−>99
−11◦ (c 0.55, CHCl3)
a
The percent ee was measured by chiral HPLC.

2
68
Z. Dai et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 264–269
Scheme 3. Enzymatic reduction of 2F to 3F and rearranged product 3F’.
®
In the protocol provided by Codex , 1–4 mg of KREDs was rec-
(R)-3A with over 99% ee and 83–99% conversion. Impressively, the
H NMR spectra of several crude products (e.g. those from enzyme
8) showed no impurities, and were essentially identical to the spec-
trum of the “purified” product.
For ˇ-ketoesters 2A–E except 2D, the reactions were scaled up
by a factor of 20 or 100 using chosen ketoreductases, as shown in
Table 2. The product ꢀ,ı-unsaturated ˇ-hydroxyesters 3 were iso-
lated by chromatography on silica gel using 10:1 CH2Cl2/EtOAc.
Isolated yields were slightly lower compared to NMR yields in
Table 1. The same optical purity was obtained in all scaled reac-
tions, compared to the one obtained from crude product as shown
in Table 1.
1
ommended to reduce 1 mmol of substance. Different from that
in the protocol, in our experiment 40 mg/mmol ratio of KREDs
relative to unsaturated ˇ-ketoesters was used to perform all the
microscale reactions (using 25 ␮mol of unsaturated ˇ-ketoesters)
◦
shown in Table 1. All reactions were carried at 30–32 C. Approx-
imate yields for the microscale reactions were determined by
integration of the vinyl hydrogen regions of the 1H NMR spec-
tra of the crude mixtures, which also served to establish whether
substantial byproducts formed and whether the double bond was
reduced. There was no evidence of double bond reduction in
any reaction. When conversion of ˇ-ketoesters was greater than
3
%, only the presence of unreacted ˇ-ketoesters and product ˇ-
In the case of ˇ-ketoester 2F, 1,3-hydrogen rearrangement
occurred to a small extent when some ketoreductases were used,
which produced not only ethyl (4E)-3-hydroxy-6-phenylhex-4-
enoate 3F, but also ethyl (5E)-3-hydroxy-6-phenylhex-5-enoate
3F’, as shown in Scheme 3. In the rearrangement process, the hydro-
gen was transferred without formation of the Z-isomer. Evidence
of 1,3-hydrogen rearrangement comes from the HPLC ultraviolet
absorption spectra of 3F and 3F’ which show different absorption
maxima (ꢁmax) for 3F and 3F’. Indeed, a ꢁmax at 250 nm is observed
in the UV spectrum of 3F’ while a ꢁmax of 206 nm is the spec-
trum of 3F (Fig. 34s and 35s). Further conclusive evidence for allylic
hydroxyester was observed in the spectrum (See example spectra
in Fig. 43s).
To determine the absolute configuration of ˇ-hydroxyesters, the
reductions of ˇ-ketoesters with enzyme 8 and 23, which provide
>99% ee of R and S isomers, respectively, were scaled by a factor
of 10 (0.25 mmol) by using the same relative ratio of enzyme to ˇ-
ketoesters as the microscale reaction. The Mosher ester method
was then used to determine the absolute configuration of each
enantiopure ˇ-hydroxyester from enzyme 8 or 23 by compari-
son with the racemic mixture (Fig. 38s–42s) [48]. Optical rotations
of both enantiomers of aryl ꢀ,ı-unsaturated ˇ-hydroxyesters 3
except 3D were also measured (Table 2). Comparing the signs of
measured values of 3A with those of known literature values, the
absolute configurations of ˇ-hydroxyesters were further confirmed
1
rearrangement was obtained by examination of the H NMR spectra
of the crude products (Fig. 33s).
The pathway for the formation of 3F’ is not clear. Double bond
migration of 2F may occur prior to enzymatic reduction to 3F’.
Alternatively, 2F may be enzymatically reduced to 3F, followed by
isomerization to 3F’. Current literature is lacking on the mechanistic
aspects of this process and further studies will be required. Nev-
ertheless, product (R)-3F can be obtained in pure form with high
ee and conversion (Enzymes 5 and 20) but further optimization is
required for obtaining pure (S)-3F with high yield and conversion.
[
5,44]. The percent enantiomeric excess (% ee) was determined
by either chiral HPLC or chiral capillary electrophoresis using a
charged cyclodextrin as chiral selector (Fig. 44s–48s and Fig. 49s).
The data reported in Table 1 present the results of the reduc-
tion of ꢀ,ı-unsaturated ˇ-ketoesters under the action of 24 KREDs
1
–24. With exceptions of eight enzymes (P1-A04, P1-C01, P1-
H08, P1-H10, P2-D03, P2-D12, P2-H07 and P2-D11) which have
no (or very weak) effects on the conversion of ˇ-ketoesters and
two enzymes (P1-B10 and P1-B12) which provide low to medium
enantiomeric excess of the desired ˇ-hydroxyesters, most enzymes
showed activity and excellent stereoselectivity for the reduction
of ˇ-ketoesters 2A–2F toward ˇ-hydroxyesters 3A–3F. We do not
discern any trends, either in terms of ee or conversion, as the
substituent on the aromatic rings changes from weakly electron-
donating (2B and 2E, alkyl) to strongly electron-withdrawing (2D,
nitro).
Both (R) and (S) enantiomers of each ˇ-hydroxyester were
produced impressively in optically pure form (>99% ee) by a num-
ber of KREDs. (S)-enantiomers ˇ-hydroxyesters were made from
four enzymes (130, NADH-101, P3-G09 and P3-H12) while (R)-
enantiomers can be accessed using nine enzymes (101, 119, P1-B02,
P1-B05, P2-B02, P2-C02, P2-C11, P2-D11 and P2-G03). For each
ˇ-ketoesters, at least one enzyme could be used to catalyze the for-
mation of the corresponding (S)-enantiomer and several enzymes
could be used to produce the corresponding (R)-enantiomer, both
with excellent stereoselectivity and good conversion. For example,
in the formation of (S)-3A, three out of four enzymes (NADH-101,
P3-G09 and P3-H12) had low conversion with over 99% enan-
tiomeric excess. However, enzyme 3 (130) could catalyze formation
of (S)-3A with both excellent stereoselectivity (>99%) and conver-
sion (84%). Seven out of nine enzymes catalyzed the formation of
4
. Conclusion
We have developed an efficient enzymatic method utiliz-
ing isolated NAD(P)H dependent ketoreductases for the direct
and stereoselective synthesis of either enantiomer of aryl ꢀ,ı-
unsaturated ˇ-hydroxyesters in excellent optical purity (>99%)
with good to excellent conversion for at least one ketoreductase.
The scaled reaction with chosen enzyme shows that the isolated
yields are very close to the conversion monitored by NMR spec-
troscopy and with same optical purity (>99%). These results suggest
that Codexis ketoreductases offer potential for large scale synthesis
of either enantiomer of aryl ꢀ,ı-unsaturated-ˇ-hydroxyesters.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2
013.09.003.
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