Stereoselective enzymatic synthesis of chiral alcohols with the use of a carbonyl reductase from Candida magnoliae with anti-prelog enantioselectivity

Article abstract of DOI:10.1021/jo0603328

In our effort to search for carbonyl reductases with anti-Prelog enantioselectivity, the activity and enantioselectivity of a carbonyl reductase from Candida magnoliae have been examined with various ketones of diverse structures. This carbonyl reductase catalyzed the reduction of a series of ketones, α-and β-ketoesters, to anti-Prelog configurated alcohols in excellent optical purity. The usefulness of this carbonyl reductase has been demonstrated by synthesis of several chiral alcohol intermediates of pharmaceutical importance.

Full text of DOI:10.1021/jo0603328

Stereoselective Enzymatic Synthesis of Chiral Alcohols with the Use
of a Carbonyl Reductase from Candida magnoliae with Anti-Prelog
Enantioselectivity
Dunming Zhu, Yan Yang, and Ling Hua*
Department of Chemistry, Southern Methodist UniVersity, Dallas, Texas 75275
lhua@smu.edu
ReceiVed February 16, 2006
In our effort to search for carbonyl reductases with anti-Prelog enantioselectivity, the activity and
enantioselectivity of a carbonyl reductase from Candida magnoliae have been examined with various
ketones of diverse structures. This carbonyl reductase catalyzed the reduction of a series of ketones, R-
and â-ketoesters, to anti-Prelog configurated alcohols in excellent optical purity. The usefulness of this
carbonyl reductase has been demonstrated by synthesis of several chiral alcohol intermediates of
pharmaceutical importance.
Introduction
have been made to develop biocatalytic ketone reduction
processes, and many examples with isolated carbonyl reductases
have been described.8-12 The stereoselectivity of carbonyl
reductase-catalyzed ketone reduction is usually dependent on
the steric situation of substrates, and may be predicted with
“Prelog’s rule”.13,14 The majority of known carbonyl reductases
used for the enantioselective ketone reduction follow Prelog’s
rule as shown in eq 1.10,13
Enantiometrically pure alcohols including R- and â-hydroxy-
esters are important and valuable intermediates in the synthesis
of pharmaceuticals and other fine chemicals. A variety of
synthetic methods have been developed to obtain optically pure
alcohols, R- and â-hydroxy acids, and their derivatives. Among
these methods, a straightforward approach is the reduction of
prochiral ketones to chiral alcohols. In this context, a variety
of chiral metal complexes have been developed as catalysts in
the asymmetric ketone reductions.1-3 However, in many cases
difficulties remain in the process operation, and obtaining
sufficient enantiomeric purity and productivity.2,3 In addition,
residual metal in the products originated from the metal catalyst
presents another challenge because of ever more stringent
regulatory restrictions on the level of metals allowed in
pharmaceutical products.4 An alternative to the chemical asym-
metric reduction processes is the biocatalytic transformation with
isolated enzymes or whole-cell microorganisms that offers
advantages such as mild and environmentally benign reaction
conditions, high chemo-, regio-, and stereoselectivity, and being
void of residual metal in the products.5-7 Therefore, great efforts
Because of the equal importance of both enantiomers, there
is a great demand for the carbonyl reductases with anti-Prelog
(5) Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Curr. Opin. Chem.
Biol. 2004, 8, 120-126.
(6) Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T. Tetrahedron:
Asymmetry 2003, 14, 2659-2681.
(7) Patel, R. N. Curr. Opin. Biotechnol. 2001, 12, 587-604.
(8) Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am.
Chem. Soc. 2004, 126, 12827-12832.
(9) Zhu, D.; Mukherjee, C.; Rozzell, J. D.; Kambourakis, S.; Hua, L.
Tetrahedron 2006, 62, 901-905.
(1) Ohkuma, T.; Noyori, R. Compr. Asymmetric Catal., Suppl. 2004, 1,
1-41.
(10) Zhu, D.; Rios, B. E.; Rozzell, J. D.; Hua, L. Tetrahedron: Asymmetry
2005, 16, 1541-1546.
(2) Noyori, R.; Okhuma, T. Angew. Chem., Int. Ed. 2001, 40, 40-73.
(3) Wills, M.; Hannedouche, J. Curr. Opin. Drug DiscoVery DeV. 2002,
5, 881-891.
(11) Kaluzna, I. A.; Feske, B. D.; Wittayanan, W.; Ghiviriga, I.; Stewart,
J. D. J. Org. Chem. 2005, 70, 342-345.
(12) Inoue, K.; Makino, Y.; Itoh, N. Tetrahedron: Asymmetry 2005, 16,
2539-2549.
(4) Thayer, A. Chem. Eng. News 2005, 83, 55-58.
10.1021/jo0603328 CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/04/2006
4202
J. Org. Chem. 2006, 71, 4202-4205

Enzymatic Synthesis of Chiral Alcohols
CHART 1
stereopreference, which lead to the formation of anti-Prelog
configurated alcohols. Recently a carbonyl reductase gene from
Candida magnoliae was cloned and the encoded protein was
found to catalyze the reduction of ethyl 4-chloro-3-oxobutanoate
to give ethyl (S)-4-chloro-3-hydroxybutanoate in optically pure
form.15-18 Since the chloromethyl group is smaller in size than
CH2COOC2H5, but has higher priority in the CIP rules for R/S
configuration assignment, we reasoned that the carbonyl reduc-
tase from Candida magnoliae (CMCR) might not obey Prelog’s
rule in the ketone reduction to afford anti-Prelog configurated
alcohols. To initiate our effort to search for carbonyl reductases
with anti-Prelog enantioselectivity, the activity and enantiose-
lectivity of CMCR have been evaluated toward the reduction
of various ketones including R- and â-ketoesters (Chart 1). This
carbonyl reductase indeed reduced a diversity of ketones to anti-
Prelog configurated alcohols in excellent enantiomeric purity.
To explore the synthetic potential of this carbonyl reductase, it
was then applied to the synthesis of several pharmaceutically
important chiral alcohol intermediates.
TABLE 1. Specific Activity and Enantioselectivity of Carbonyl
Reductase from Candida magnoliae toward the Reductions of r- or
â-Ketoesters
R-/â-ketoester
specific activitya
ee (%)b
1a (CH3)
1b (C2H5)
100
682
1545
209
6614
1000
118
>99 (R)c
>99 (R)
>99 (S)
>99 (S)
97 (S)
1c (CH(CH3)2)
1d (CF3)
1e (CH2Cl)
2a (CH(CH3)2)
2b (C(CH3)3)
>99 (R)
99 (R)
a The unit of specific activity was nmol‚min-1‚mg-1
.
b The ee value was
measured by chiral GC analysis. c The absolute configuration of product
alcohol was determined by comparing the retention time with that of
standard samples.
R- or â-ketoesters and the catalytic activity was dependent on
the substrate structure. It was interesting that the specific activity
of CMCR for reduction of â-ketoesters increased as the alkyl
group became larger (1a f 1b f 1c). The chloro substituent
at the 4-position of ethyl 3-oxo-butyrate also greatly enhanced
the enzyme activity. In contrast to â-ketoesters, CMCR showed
less activity for the reduction of R-ketoester (2b) with a bulky
tert-butyl group than ethyl 3-methyl-2-oxo-butyrate (2a).
Table 2 showed that the carbonyl reductase (CMCR) was
active for a series of acetophenone derivatives. The substituents
at the para position of acetophenone derivatives greatly affected
the enzyme catalytic activity. The electron-withdrawing sub-
stituents enhanced the enzyme activity (3b, 3c, 3d, and 3h),
while the substrates with an electron-donating substituent were
less active than the unsubstituted acetophenone (3e, 3f, and 3g).
The acetophenone derivatives with a substituent at the ortho or
meta position were much less active (data not shown). R-Chlo-
roacetophenone (3i) showed much higher activity than ace-
tophenone (3a) and was an excellent substrate for the carbonyl
reductase from Candida magnoliae. The aliphatic ketones were
also good substrates for this carbonyl reductase, and chain length
exerted some effect on the enzyme activity as shown in Table
3.
Results and Discussion
The carbonyl reductase gene from Candida magnoliae
(Genbank Accession No. AB036927) was overexpressed in E.
coli and the encoded protein was purified and lyophilized by
following the literature procedure.15 This lyophilized enzyme
was stable for months at 4 °C without significant loss of activity.
The specific activity of the carbonyl reductase from Candida
magnoliae (CMCR) toward the reduction of ketones in Chart 1
was determined by spectrophotometrically measuring the oxida-
tion of NADPH at 340 nm at room temperature. Activity assay
with the control cell-free extract, which was obtained by
expression of pET15b vector without CMCR gene in E. coli
Rosetta2(DE3)pLysS strain, did not show any activity toward
all the substrates tested. The specific activities for different
ketone substrates are presented in Tables 1-3.
From Table 1 it can be seen that the carbonyl reductase from
Candida magnoliae efficiently catalyzed reduction of the tested
(13) Faber, K. Biotransformation in Organic Chemistry; Springer-
Verlag: Berlin, Germany, 2004.
(14) Prelog, V. Pure Appl. Chem. 1964, 9, 119-30.
(15) Yasohara, Y.; Kizaki, N.; Hasegawa, J.; Wada, M.; Kataoka, M.;
Shimizu, S. Biosci., Biotechnol., Biochem. 2000, 64, 1430-1436.
(16) Wada, M.; Kataoka, M.; Kawabata, H.; Yasohara, Y.; Kizaki, N.;
Hasegawa, J.; Shimizu, S. Biosci., Biotechnol., Biochem. 1998, 62, 280-
285.
(17) Yasohara, Y.; Kizaki, N.; Hasegawa, J.; Wada, M.; Kataoka, M.;
Shimizu, S. Tetrahedron: Asymmetry 2001, 12, 1713-1718.
(18) CMCR has been recently mutated to be NADH-dependent, see:
Morikawa, S.; Nakai, T.; Yasohara, Y.; Nanba, H.; Kizaki, N.; Hasegawa,
J. Biosci., Biotechnol., Biochem. 2005, 69, 544-552.
The enantioselectivity of the carbonyl reductase from Candida
magnoliae toward the reduction of ketones in Chart 1 was
studied by using a NADPH regeneration system consisting of
D-glucose dehydrogenase (GDH) and D-glucose (Scheme 1).19
(19) Eguchi, T.; Kuge, Y.; Inoue, K.; Yoshikawa, N.; Mochida, K.;
Uwajima, T. Biosci., Biotechnol., Biochem. 1992, 56, 701-3.
J. Org. Chem, Vol. 71, No. 11, 2006 4203

Zhu et al.
TABLE 2. Specific Activity and Enantioselectivity of Carbonyl
Reductase from Candida magnoliae toward the Reduction of Aryl
Ketones
TABLE 4. Isolated Yields, Enantiomeric Excess, and Optical
Rotation of the Product Alcohols in the Preparative Scale
a
ketones
yield (%)
ee (%)
[R]22
D
2a
2b
3b
3h
3i
4b
4d
4e
87
91
89
94
92
92
95
90
>99 (R)
99 (R)
-9.5
-30.3
+45.2
+29.3
+49.5
-9.4
>99 (R)
>99 (R)
>99 (S)
>99 (R)
>99 (R)
>99 (R)
ketone (X, R)
specific activitya
ee (%)b
+15.7b
-8.8
3a (H, H)
3b (H, F)
3c (H, Cl)
3d (H, Br)
3e (H, CH3)
3f (H, OCH3)
3g (H, C(CH3)3)
3h (H, CF3)
3i (Cl, H)
65
864
455
682
59
18
10
1136
818
99 (R)c
>99 (R)
>99 (R)
>99 (R)
99 (R)
>99 (R)
99 (R)
>99 (R)
>99 (S)
a [R]22 was measured in CHCl3 (c 1). b [R]22 was measured as 1-(1-
adamantyl)ethyl acetate.
D
D
to achieve such high enantioselectivty in the aliphatic ketone
reduction by metal-catalyzed hydrogen transfer or hydrogena-
tion.22,23 This indicated that the carbonyl reductase from Candida
magnoliae was indeed an oxidoreductase with excellent anti-
Prelog enantioselectivity for a diversity of ketones.
a The unit of specific activity was nmol‚min-1‚mg-1
.
b The ee value was
measured by chiral GC analysis. c The absolute configuration of product
alcohol was determined by comparing the retention time with that of
standard samples, or by the sign of optical rotation.
Many chiral alcohols are important intermediates in the
synthesis of pharmaceuticals and agrichemicals. For example,
optically active 2-hydroxy-3-methylbutyrate is an important
chiral synthon in the preparation of a potent, selective, and cell-
penetrable inhibitor of caspase 3.24 (R)-2-Hydroxy-3,3-dimeth-
ylbutyrate is a key component P3 of thrombin inhibitor identified
by Merck.25 Chiral 2-chloro-1-phenylethanol is a key synthon
for the preparation of a large group of anti-depressants and
potential cocaine-abuse therapeutic agents.26 Optically pure
1-(4′-flurophenyl)ethanol and 1-(4′-trifluromethylphenyl)ethanol
have been used to synthesize m2 muscarinic antagonists and
the modulators of the CCR-5 chemokine receptor for treating
patients with HIV.27-29 To further explore the potential ap-
plication of CMCR in the synthesis of these pharmaceutucally
important chiral alcohols, the reduction of ethyl 3-methyl-2-
oxo-butanoate (2a), ethyl 3,3-dimethyl-2-oxo-butanoate (2b),
R-chloroacetophenone (3i), 4′-fluoroacetophenone (3b), 4′-
trifluoromethylacetophenone (3h) ,and aliphatic ketones (4b, 4d,
and 4e) was carried out in a preparative scale. The isolated
yields, enantiomeric excess, and optical rotation of the product
alcohols are summarized in Table 4. From Table 4 it can be
seen that these chiral alcohols were obtained in essentially
optically pure form in excellent yields. This demonstrated the
TABLE 3. Specific Activity and Enantioselectivity of Carbonyl
Reductase from Candida magnoliae toward the Reduction of
Aliphatic Ketones
ketone (R, R′)
specific activitya
ee (%)b
4a (CH3, n-C5H11)
4b (CH3, n-C6H13)
4c (CH3, n-C7H15)
4d (CH3, 1-adamantyl)
4e (C2H5, n-C5H11)
200
95
73
18
>99 (R)c
>99 (R)
>99 (R)
>99 (R)
>99 (R)
15
a The unit of specific activity was nmol‚min-1‚mg-1
.
b The ee value was
measured by chiral GC analysis. c The absolute configuration of product
alcohol was determined by comparing the retention time with that of
standard samples, or by the sign of optical rotation.
SCHEME 1
(22) Schlatter, A.; Kundu, M. K.; Woggon, W.-D. Angew. Chem., Int.
Ed. 2004, 43, 6731-6734.
(23) Reetz, M. T.; Li, X. J. Am. Chem. Soc. 2006, 128, 1044-1045.
(24) Mellon, C.; Aspiotis, R.; Black, C. W.; Bayly, C. I.; Grimm, E. L.;
Giroux, A.; Han, Y.; Isabel, E.; McKay, D. J.; Nicholson, D. W.; Rasper,
D. M.; Roy, S.; Tam, J.; Thornberry, N. A.; Vaillancourt, J. P.; Xan-
thoudakis, S.; Zamboni, R. Bioorg. Med. Chem. Lett. 2005, 15, 3886-
3890.
(25) Nelson, T. D.; LeBlond, C. R.; Frantz, D. E.; Matty, L.; Mitten, J.
V.; Weaver, D. G.; Moore, J. C.; Kim, J. M.; Boyd, R.; Kim, P.-Y.;
Gbewonyo, K.; Brower, M.; Sturr, M.; McLaughlin, K.; McMasters, D.
R.; Kress, M. H.; McNamara, J. M.; Dolling, U. H. J. Org. Chem. 2004,
69, 3620-3627.
(26) Hsin, L.-W.; Dersch, C. M.; Baumann, M. H.; Stafford, D.; Glowa,
J. R.; Rothman, R. B.; Jacobson, A. E.; Rice, K. C. J. Med. Chem. 2002,
45, 1321-1329.
(27) Harriman, G. C. B.; Carson, K. G.; Flynn, D. L.; Solomon, M. E.;
Song, Y.; Trivedi, B. K.; Roth, B. D.; Kolz, C. N.; Pham, L.; Sun, K.-L.;
Millennium Pharmaceuticals, Inc., USA., Warner-Lambert Company, Ap-
plication WO, 2002, 307 pp.
(28) McKittrick, B. A.; Guo, G.; Zhu, Z.; Ye, Y. Schering Corporation,
USA, Application WO, 2002, 26 pp.
The enantiomeric excess (ee) values of the product alcohols
were determined by chiral GC analysis. The absolute config-
uration of product alcohols was assigned by comparing either
the retention time with standard sample or the specific rotation
with reported data. The standard samples were from either
commercial sources or the preparation in our previous stud-
ies.9,10,20,21 The results are summarized in Tables 1-3. It can
be seen that the carbonyl reductase from Candida magnoliae
efficiently catalyzed the reduction of various ketones including
R- and â-ketoesters to give anti-Prelog configurated alcohols
in excellent optical purity (g97% ee). More importantly,
aliphatic ketones were also reduced to the corresponding
alcohols in optically pure form (Table 3). It is usually difficult
(20) Zhu, D.; Mukherjee, C.; Hua, L. Tetrahedron: Asymmetry 2005,
16, 3275-3278.
(21) Uray, G.; Stampfer, W.; Fabian, W. M. F. J. Chromatogr., A 2003,
992, 151-157.
(29) Tagat, J. R.; Steensma, R. W.; McCombie, S. W.; Nazareno, D.
V.; Lin, S.-I.; Neustadt, B. R.; Cox, K.; Xu, S.; Wojcik, L.; Murray, M. G.;
Vantuno, N.; Baroudy, B. M.; Strizki, J. M. J. Med. Chem. 2001, 44, 3343-
3346.
4204 J. Org. Chem., Vol. 71, No. 11, 2006

Enzymatic Synthesis of Chiral Alcohols
determined by spectrophotometrically measuring the oxidation of
NADPH at 340 nm (ꢀ ) 6.22 mM-1 cm-1) in the presence of an
excess amount of ketones. The activity was measured at room
temperature in a 96-well plate, in which each well contained ketone
(6.25 mM) and NADPH (0.25 mM) in potassium phosphate buffer
(100 mM, pH 6.5, 180 µL). The reaction was initiated by the
addition of the carbonyl reductase (20 µL solution containing 2-40
µg of enzyme). The specific activity was defined as the number of
nanomoles of NADPH converted in 1 min by 1 mg of enzyme
(nmol‚min-1‚mg-1).
Enantioselectivity of the Reduction of Ketones Catalyzed by
the Carbonyl Reductase from Candida magnoliae (CMCR). The
enantioselectivity of the enzymatic reduction of ketones was studied
with an NADPH recycle system.19 The general procedure was as
follows: D-glucose (4 mg), D-glucose dehydrogenase (0.5 mg),
NADPH (0.5 mg), the carbonyl reductase (CMCR, 0.5 mg), and
the ketone solution in DMSO (50 µL, 0.25 M) were mixed in a
potassium phosphate buffer (1 mL, 100 mM, pH 6.5) and the
mixture was shaken overnight at room temperature. The mixture
was extracted with methyl tert-butyl ether (1 mL). The organic
extract was dried over anhydrous sodium sulfate and subjected to
chiral GC analysis to determine the enantiomeric excess.21 The
absolute configuration of product alcohols was identified by
comparing the chiral GC data with the standard samples, or by
comparing the specific rotation of the product alcohols with the
literature data.
usefulness of the carbonyl reductase from Candida magnoliae
as an enzyme catalyst in the synthesis of chiral alcohol
intermediates.
In conclusion, the carbonyl reductase from Candida magno-
liae (CMCR) was an efficient enzyme catalyst for the enantio-
selective reduction of a diversity of ketones including aliphatic,
aromatic ketones, R- and â-ketoesters. More importantly, this
carbonyl reductase showed anti-Prelog enantioselectivity, which
was complementary to most of the known ketoreductases, and
would find more applications in the synthesis of chiral alcohol
intermediates of pharmaceutical and agricultural interest.
Experimental Section
Gene Expression and Purification of the Carbonyl Reductase
from Candida magnoliae (CMCR). The carbonyl reductase gene
from Candida magnoliae (Genbank Accession No. AB036927) was
cloned by gene assembly techniques.30 Ten oligonucleotides ranging
from 100 to 120 nucleotides were designed on the basis of the
nucleotide sequence of the C. magnoliae carbonyl reductase gene,15
the open reading frame of which is composed of 852 nuclotides
(284 amino acid residues). The Nco I and Bam HI sites were franked
to the open reading frame for easy cloning into expression vector
pET15b (Novagen). The plasmid DNA containing CMCR gene was
transformed into the E. coli Rosetta2(DE3)pLysS strain. Overnight
culture was diluted into fresh LB medium containing ampicillin
(100 µg/mL) and chloramphenicol (34 µg/mL) and incubated at
37 °C until the optical density reached 0.6 at 595 nm. The
expression was induced by addition of IPTG to 0.5 mM and the
culture was incubated at 30 °C for another 6 h. Cells were harvested
by centrifugation at 4100 rpm at 4 °C for 30 min. The cell pellet
was resuspended in 100 mM potassium phosphate buffer (pH 6.5)
and the cells were disrupted by EmulsiFlex-C5 Homogenizer. The
cell-free extract was mixed with an equal volume of PEI solution
(0.25% polyethyleneimine MW 40-60 K, 6% NaCl, 100 mM
Borax, pH 7.4) to remove lipids.31 The supernatant was precipitated
with 25% ammonium sulfate and the precipitate was discarded.
The remaining supernatant was precipitated with 55% ammonium
sulfate. The resulting precipitate was collected after centrifugation
and dissolved in potassium phosphate buffer (10 mM, pH 7.0, 2
mM 2-mercaptoethanol). The lysate was desalted by gel filtration
into potassium phosphate buffer (10 mM, pH 7.0, 2 mM 2-mer-
captoethanol) and lyophilized to afford the CMCR enzyme as white
powder with a protein content of 86% measured with Bradford
assay. The expression vector pET15b without the CMCR gene was
also expressed in Rosetta2(DE3)pLysS. The cell-free extract was
purified by the same procedure and used as a control in the activity
assay.
Preparative Procedures. The preparative synthesis was carried
out as follows: D-Glucose (1.0 g), d-glucose dehydrogenase (10
mg), NADPH (10 mg), and ketoreductase (10 mg) were mixed in
a potassium phosphate buffer (50 mL, 100 mM, pH 6.5). To the
mixture was added a ketone solution (500 mg in 2.0 mL of DMSO).
The mixture was stirred at room temperature and the pH was
controlled at 6.5-6.6 with addition of 0.5 M NaOH solution until
conversion was complete (usually overnight). The mixture was
extracted with ether or methyl tert-butyl ether. The organic extract
was dried over anhydrous sodium sulfate and removal of the solvent
1
gave product alcohol, which was identified by comparison of H
and 13C NMR with literature data.12,25,32,33 The absolute configura-
tions of the product alcohols were determined by comparison of
the specific rotation data with those in the literature.12,32,34,35
Supporting Information Available: General methods and chrial
GC chromatograms of all product alcohols and their racemates.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO0603328
(31) Milburn, P.; Bonnerjea, J.; Hoare, M.; Dunnill, P. Enzyme Microb.
Technol. 1990, 12, 527-32.
(32) Ma, Y.; Liu, H.; Chen, L.; Cui, X.; Zhu, J.; Deng, J. Org. Lett.
2003, 5, 2103-2106.
Activity Assay of the Carbonyl Reductase from Candida
magnoliae (CMCR). The activity of the carbonyl reductase from
Candida magnoliae toward the reduction of ketones in Chart 1 was
(33) Li, X.; Wu, X.; Chen, W.; Hancock, F. E.; King, F.; Xiao, J. Org.
Lett. 2004, 6, 3321-3324.
(34) Brown, H. C.; Cho, B. T.; Park, W. S. J. Org. Chem. 1988, 53,
1231-8.
(30) Lin, Y.; Cheng, G.; Wang, X.; Clark, T. G. Gene 2002, 288, 85-
94.
(35) Nakamura, K.; Matsuda, T. J. Org. Chem. 1998, 63, 8957-8964.
J. Org. Chem, Vol. 71, No. 11, 2006 4205