Enzymatic enantioselective reduction of α-ketoesters by a thermostable 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis

Article abstract of DOI:10.1016/j.tet.2006.02.041

A thermostable 7α-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was cloned and over-expressed in E. coli, and its substrate specificity and stereoselectivity toward reduction of various ketones were examined. This alcohol dehydrogenase was active toward a series of aromatic and bulky aliphatic α-ketoesters. The substituents at the phenyl ring of aromatic α-ketoesters greatly affected the activity, but their effects on enantioselectivity were minimal. The synthetic application of this enzyme was then demonstrated through the preparation of a few α-hydroxy carboxylic acid esters of pharmaceutical interest.

Full text of DOI:10.1016/j.tet.2006.02.041

Tetrahedron 62 (2006) 4535–4539
Enzymatic enantioselective reduction of a-ketoesters by
a thermostable 7a-hydroxysteroid dehydrogenase from
Bacteroides fragilis
*
Dunming Zhu, Jennifer Elisabeth Stearns, Monica Ramirez and Ling Hua
Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA
Received 28 December 2005; revised 10 February 2006; accepted 15 February 2006
Available online 10 March 2006
Abstract—A thermostable 7a-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was cloned and over-
expressed in E. coli, and its substrate specificity and stereoselectivity toward reduction of various ketones were examined. This alcohol
dehydrogenase was active toward a series of aromatic and bulky aliphatic a-ketoesters. The substituents at the phenyl ring of aromatic
a-ketoesters greatly affected the activity, but their effects on enantioselectivity were minimal. The synthetic application of this enzyme was
then demonstrated through the preparation of a few a-hydroxy carboxylic acid esters of pharmaceutical interest.
q 2006 Elsevier Ltd. All rights reserved.
1. Introduction
made to develop enzyme catalysts for the enantioselective
reduction of ketones and varied levels of success have been
achieved.14–16 However, most research has been focused
on the enantioselective reduction of aryl ketones and
b-ketoesters.17–20 Studies on enzymatic reduction of
a-ketoesters have been only scarcely reported.14,21,22
Especially biocatalytic reduction of aromatic a-ketoesters
has been very limited and much less successful than that of
their small counterparts.23 Hydroxysteroid dehydrogenases
normally reduce 3,7,12-oxo group of sterically bulky
steroids in vivo.24 These dehydrogenases belong to short-
chain dehydrogenase family and their synthetic application
has been largely unexplored.25 Recently, a thermostable
7a-hydroxysteroid dehydrogenase from Bacteroides
fragilis ATCC 25285 has been cloned,26 and it reduces
sterically demanding native substrate 7-keto-lithocholic
acid to cheno-deoxycholic acid in gastrointestinal tract
(Scheme 1).
Optically pure a-hydroxy carboxylic acids and their
derivatives are important intermediates in the synthesis of
pharmaceuticals and other fine chemicals. Many
approaches have been developed to obtain enantiomeri-
cally-enriched a-hydroxy carboxylic acid esters.1 This
includes (dynamic) kinetic resolution of racemic
a-hydroxy esters,2,3 hydrolysis of optically pure cyano-
hydrin, which in turn can be obtained via several
asymmetric synthetic methods.4–6 Another straightforward
method to enantiomerically pure a-hydroxy carboxylic
acid esters is the asymmetrical reduction of prochiral
a-ketoesters that could be performed either chemically7–10
or enzymatically.1,11–13 Because of environmentally benign
reaction conditions and unparallel selectivity, biocatalytic
reduction has attracted more and more attention from both
academia and industry. Recently, great efforts have been
COOH
COOH
7α−HSDH
NADH
H
H
H
H
O
H
H
HO
HO
OH
H
H
Scheme 1.
Keywords: a-Ketoesters; Dehydrogenase; Enzyme.
*
Corresponding author. Tel.: C1 214 768 1609; fax: C1 214 768 4089; e-mail: lhua@smu.edu
0040–4020/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.02.041

4536
D. Zhu et al. / Tetrahedron 62 (2006) 4535–4539
We reasoned that this enzyme might be useful in the
reduction of bulky ketones such as aromatic a-ketoesters.
Therefore, we have cloned and over-expressed this
7a-hydroxysteroid dehydrogease in E. coli and examined
its substrate specificity and stereoselectivity toward
reduction of various ketones including aromatic and
aliphatic a-ketoesters. This NADH-dependent alcohol
dehydrogenase was found to be active toward a series of
Table 1. The activity and enantioselectivity of 7a-hydroxysteroid dehydrogenase toward various a-ketoesters
Entry
a-Ketoester
Relative activitya
Productb
ee (%)c
O
OH
O
O
1
100
98
O
O
O
OH
O
O
2
3
354
421
O99
O
O
O
OH
O
O
95
O
O
F
Cl
F
Cl
O
OH
O
O
4
5
6
7
209
163
85
99
99
99
99
O
O
O
OH
O
O
O
O
Br
Br
O
OH
O
O
O
O
H3C
H3C
O
OH
O
O
788
O
O
NC
F
NC
F
O
OH
O
O
8
9
364
172
98
99
O
O
F
F
O
OH
Cl
Cl
O
Cl
Cl
O
O
O
O
OH
O
O
O
10
11
12
22
63
97
O99
O99
O
O
O
OH
O
O
O
O
OH
O
O
823
O
O
a The relative activity for methyl benzoylformate was defined as 100.
b The absolute configuration was determined by comparison with authentic standards, or the reported optical rotation.
c The ee value was determined by chiral HPLC or GC analysis.

D. Zhu et al. / Tetrahedron 62 (2006) 4535–4539
4537
aromatic a-ketoesters and aliphatic a-ketoester with bulky
O
groups such as tert-butyl and cyclohexyl groups. The
substituents at the phenyl ring of aromatic a-ketoesters
greatly affected the enzyme activity, but exerted less effect
on enantioselectivity of the reduction. This enzyme was
then applied to the synthesis of a few pharmaceutical
important a-hydroxy carboxylic acid esters.
R
R
R'
O
NADH
NAD+
CO2
FDH
HCOO–
O
7-HSDH
R'
O
O
OH
R
2. Results and discussion
Scheme 2. Reduction of a-ketoesters catalyzed by 7a-hydroxysteroid
dehydrogenase from B. fragilis.
The 7a-hydroxysteroid dehydrogenase gene from B. fragilis
ATCC 25285 was cloned and expressed in E. coli and the
recombinant enzyme was purified sequentially by heat
treatment, PEI treatment and fractional ammonium sulfate
precipitation of cell-free extract (see Section 4). The
obtained 7a-hydroxysteroid dehydrogenase was assayed
for activity toward various ketones by spectrophotometri-
cally measuring the oxidation of NADH at 340 nm at room
temperature. The results are presented in Table 1. Surpris-
ingly, while this dehydrogenase showed almost no activity
toward a series of acetophenone derivatives and b-keto-
esters (data not shown), it was very active for the reduction
of aromatic and bulky aliphatic a-ketoesters using NADH as
co-factor. This suggested that a carboxylic group adjacent to
the carbonyl group might be necessary for being the
substrate of 7a-hydroxysteroid dehydrogenase from
B. fragilis ATCC 25285. From Table 1 it can be seen that
the substituent at para-position of phenyl ring of aromatic
a-ketoesters greatly affected the activity. For example, the
fluoro- and cyano-group at para-position showed higher
activity than ethyl benzoylformate, while chloro-, bromo-
and methyl-substituents decreased the activity. The ester
group also exerted some effects on the enzyme activity with
ethyl ester being more active than methyl counterpart.
Among the tested aliphatic a-ketoesters, ethyl 2-cyclo-
hexyl-2-oxoacetate showed highest activity. Interestingly,
this alcohol dehydrogenase was more active for the
reduction of ethyl 3,3-dimethyl-2-oxo-butyrate than that of
less bulky ethyl 3-methyl-2-oxo-butyrate, and showed
almost no activity for the reduction of ethyl pyruvate (data
not shown). Thus 7a-hydroxysteroid dehydrogenase from
B. fragilis ATCC 25285 took more sterically demanding
carbonyl compounds as substrates. This is probably due to
the spacious active site cavity for its native substrate 7-keto-
lithocholic acid. The steric crowdness of the bulky non-
native substrates might fit better to the enzyme’s active site
for the hydride transfer from NADH to the carbonyl group
of substrates, while the less bulky substrates failed to
accomplish this hydride transfer.
Many of the a-hydroxy carboxylic acid esters listed in
Table 1 are important intermediates in the synthesis of many
pharmaceuticals. 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 3.27 (R)-2-Hydroxy-3,3-dimethyl-
butyrate is a key component P3 of thrombin inhibitor
identified by Merck.28 Optically pure 3,5-difluoromandelate
has recently been used to synthesize amino alcohol
dipeptides designed to inhibit b-amyloid peptide (Ab)
formation, which is related to Azheimer’s desease.29,30 The
results in Table 1 showed that 7a-hydroxysteroid dehydro-
genase from B. fragilis ATCC 25285 had synthetic potential
for the preparation of these a-hydroxyesters in optically
pure form. Therefore, the reductions of ethyl 3-methyl-2-
oxobutyrate, ethyl 3,3-dimethyl-2-oxobutyrate and ethyl
(3,5-diflurophenyl)-glyoxylate were performed in 1 mmol
scale. Ethyl (R)-2-hydroxy-3-methylbutyrate, ethyl (R)-2-
hydroxy-3,3-dimethylbutyrate and ethyl (R)-2-hydroxy-2-
(3,5-diflurophenyl)acetate were indeed obtained in isolated
yields of 85–94%. The enantiomeric purities of the product
a-hydroxyesters were from 97 up to O99%.
3. Conclusion
A thermostable recombinant 7a-hydroxysteroid dehydro-
genase from B. fragilis ATCC 25285 was produced by over-
expression of the 7-HSDH gene in E. coli. This alcohol
dehydrogenase catalyzed the reduction of aromatic and
sterically demanding aliphatic a-ketoesters to the corre-
sponding a-hydroxyesters in essentially optically pure form.
The synthetic application of 7a-hydroxysteroid dehydro-
genase was then demonstrated by the preparation of ethyl
(R)-2-hydroxy-3-methylbutyrate, ethyl (R)-2-hydroxy-3,3-
dimethylbutyrate and ethyl (R)-2-hydroxy-2-(3,5-
diflurophenyl)acetate.
The enantioselectivity for reduction of various aromatic and
aliphatic a-ketoesters catalyzed by 7a-hydroxysteroid
dehydrogenase from B. fragilis was evaluated using
NADH as co-factor, which was regenerated with a recycling
system comprising formate dehydrogenase and sodium
formate (Scheme 2). The results are summarized in Table 1.
From the results it can be seen that both aromatic and
aliphatic a-ketoesters were reduced to the (R)-enantiomer of
the corresponding a-hydroxy carboxylic acid esters in high
enantioselectivity with up to O99% ee. The substituent on
phenyl ring of aromatic a-ketoesters had minimal effect on
the enzyme enantioselectivity.
4. Experimental
The chiral HPLC analysis was performed on an Agilent
1100 series high-performance liquid chromatography
system with (S,S)-Whelk-O 1 column (25 cm!4.6 mm,
Regis Technologies Inc.). 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 CP-Chirasil-Dex CB
chiral capillary column (25 m!0.25 mm) (Table 2). The
7a-hydroxysteroid dehydrogenase activities toward the
reduction of a-ketoesters (Table 1) were assayed using

4538
D. Zhu et al. / Tetrahedron 62 (2006) 4535–4539
Table 2. Details of chiral HPLC and GC analysis
a-Hydroxyester
potassium phosphate buffer (10 mM, pH 7.0), and then
lyophilized as powder.
Methoda
Retention
time (min)
4.2. Activity assay of 7a-hydroxysteroid dehydrogenase
tR
tS
Methyl 2-hydroxy-2-phenylacetate
Ethyl 2-hydroxy-2-phenylacetate
A
A
A
A
A
A
A
B
A
C
D
E
10.2 11.0
9.8 10.7
The activity of 7a-hydroxysteroid dehydrogenase toward
the reduction of a-ketoesters (Table 1) was determined by
spectrophotometrically measuring the oxidation of NADH
at 340 nm (3Z6.22 mMK1 cmK1) in the presence of excess
a-ketoesters. The activity was measured at room tempera-
ture in 96-well plate, in which each well contained
a-ketoester (6.25 mM), NADH (0.25 mM) in potassium
phosphate buffer (100 mM, pH 7.0, 190 ml). The reaction
was initiated by the addition of 7a-hydroxysteroid
dehydrogenase (10 ml solution containing 18 mg of
enzyme). The specific activity was defined as the number
of micromolar of NADH converted in 1 min by 1 mg
of enzyme (mmol minK1 mgK1). The specific activity for
methyl benzoylformate was 0.32 mmol minK1 mgK1 and its
relative activity was defined as 100.
Ethyl 2-hydroxy-2-(4-fluorophenyl)acetate
Ethyl 2-hydroxy-2-(4-chlorophenyl)acetate
Ethyl 2-hydroxy-2-(4-bromophenyl)acetate
Ethyl 2-hydroxy-2-(4-methylphenyl)acetate
Ethyl 2-hydroxy-2-(4-cyanophenyl)acetate
Ethyl 2-hydroxy-2-(3,5-diflurophenyl)acetate
Ethyl 2-hydroxy-2-(3,4-dichlorophenyl)acetate
Ethyl 2-hydroxy-3-methylbutyrate
8.4
8.9
8.9
9.5
9.4 10.0
11.6 12.3
21.6 22.6
9.2
8.8
9.7
9.6
22.0 22.3
11.2 11.8
15.6 15.9
Ethyl 2-hydroxy-3,3-dimethylbutyrate
Ethyl 2-hydroxy-2-cyclohexylacetate
a (A) HPLC, flow rate 1.0 ml/min, hexane/isopropanol (0.1% HOAc)Z
95:5; (B) HPLC, flow rate 1.0 ml/min, hexane/isopropanol (0.1%
HOAc)Z99:1; (C) GC, 60 8C for 2 min, 1 8C/min to 90 8C, 90 8C for
5 min; (D) GC, 80 8C for 2 min, 1 8C/min to 110 8C, 110 8C for 5 min; (E)
GC, 90 8C for 2 min, 1 8C/min to 120 8C, 120 8C for 5 min, a-hydroxyl
group was acylated as trifluoroacetate.
SpectraMax M2 microplate reader (Molecular Devices).
Methyl phenylglyoxylate, ethyl phenylglyoxylate, ethyl (4-
cyanophenyl)glyoxylate, ethyl (3,4-dichlorophenyl)glyoxy-
late, ethyl (3,5-difluorophenyl)glyoxylate, and ethyl
3-methyl-2-oxobutyrate were purchased from Aldrich or
Acros. All the other a-ketoesters were prepared by Friedel–
Crafts acylation of substituted benzene with ethyl oxalyl
chloride in the presence of anhydrous AlCl3,31 or reaction of
diethyl oxalate with the corresponding Grignard reagents.10
The racemic a-hydroxyester standards were prepared by
reduction of a-ketoesters with sodium borohydride. Methyl
and ethyl (S)-mandelate were purchased from Aldrich. (R)
or (S) enantiomers of other a-hydroxyesters were prepared
by following the literature methods.28,32,33
4.3. Enantioselectivity of reduction of a-ketoesters
catalyzed by 7a-hydroxysteroid dehydrogenase
The enantioselectivity of the enzymatic reduction of
a-ketoesters was studied using an NADH recycle system.
The general procedure was as follows: sodium formate
(3.4 mg), formate dehydrogenase (0.4 mg), NADH
(0.4 mg), 7a-hydroxysteroid dehydrogenase (0.2 mg) and
a-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 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 was subjected to chiral HPLC or GC analysis to
determine the enantiomeric excess. The absolute configur-
ation of product a-hydroxyesters was identified by
comparing the chiral HPLC or GC data with the standard
samples, or by comparing the optical rotation of the product
alcohols with the literature data.
4.1. Gene expression and purification of 7a-hydroxy-
steroid dehydrogenase
Plasmid pBPC-1 (from James P. Coleman) containing
7-HSDH gene from B. fragilis ATCC 25285 was used as
template for PCR amplification. The PCR fragment was
cloned into pTXB1 expression vector at the Nde I and BamH
I sites to give JS2.2 and the cloned 7-HSDH gene was
confirmed by DNA sequencing. The plasmid JS2.2 was
transformed into Rosetta2(DE3)pLysS for expression.
Overnight culture (20 ml) was diluted into 1 l of LB
media containing 100 mg/ml of ampicillin and 34 mg/ml
of chloramphenicol and propagated until OD595 reached
0.6–1.0 at 37 8C. The cells were then induced with 0.1 mM
of IPTG and continuing grown at 30 8C for 5 h. The cells
were harvested and lysed in 10 mM of potassium phosphate
(pH 7.0) by homogenizer. The cell-free extract was heat-
treated in a water-bath for 30 min at 55–60 8C and
centrifuged at 20,000g for 30 min. The heat-treated lysate
was then mixed with equal volume of PEI solution (0.25%
polyethyleneimine MW 40–60 K, 6% NaCl, 100 mM
Borax, pH 7.4) to remove lipids.34 The PEI-treated
supernatant was precipitated with 45% ammonium sulfate.
The resulting precipitate was collected after centrifugation
and dissolved in potassium phosphate buffer (10 mM,
pH 7.0). The lysate was dialysed by gel filtration into
4.4. Preparation of ethyl (R)-2-hydroxy-3-methylbuty-
rate, ethyl (R)-2-hydroxy-3,3-dimethylbutyrate, and
ethyl (R)-2-hydroxy-2-(3,5-difluorophenyl)acetate
Sodium formate (4 mmol), formate dehydrogenase (40 mg),
NADH (10 mg), 7a-hydroxysteroid dehydrogenase (10 mg)
and ethyl 3-dimethyl-2-oxo-butyrate (1 mmol) were mixed
in a potassium phosphate buffer (50 ml, 100 mM, pH 7.0)
and the mixture was stirred at room temperature. After 24 h,
GC analysis indicated that reduction was complete. The
reaction mixture was extracted with ethyl ether (30 ml!2).
The organic extract was dried over anhydrous Na2SO4 and
removal of the solvent gave ethyl (R)-2-hydroxy-3-
1
methylbutyrate as clear oil (124 mg, 85% yield). H and
13C NMR (CDCl3) were in accordance with literature
data.35 [a]2D2 K9.4 (c 1.0, CHCl3); lit.35 [a]2D5 K10.5 (c 0.5,
CHCl3).
Similar procedures were followed for the preparation of
other two a-hydroxyesters. Ethyl (R)-2-hydroxy-3,3-
1
dimethylbutyrate (145 mg, 91% yield). H and 13C NMR
(CDCl3) were in accordance with literature data.28 [a]D22

D. Zhu et al. / Tetrahedron 62 (2006) 4535–4539
4539
K31.3 (c 1.0, CHCl3); lit.36 [a]D22 C27.7 (c 3.4, CHCl3) for
(S)-enantiomer. Ethyl (R)-2-hydroxy-2-(3,5-difluoro-
phenyl)acetate (203 mg, 94% yield). H NMR (400 MHz,
17. Zhu, D.; Mukherjee, C.; Hua, L. Tetrahedron: Asymmetry
2005, 16, 3275–3278.
1
18. Zhu, D.; Rios, B. E.; Rozzell, J. D.; Hua, L. Tetrahedron:
Asymmetry 2005, 16, 1541–1546.
CDCl3) d ppm 1.27 (t, 3H, JZ7.2 Hz), 3.58 (s, 1H),
4.21–4.34 (m, 2H), 5.15 (s, 1H), 6.78 (m, 1H), 7.03 (m, 2H).
13C NMR (100.6 MHz, CDCl3) d ppm 14.4, 63.2, 72.2,
104.1 (t, 2JC–FZ25 Hz), 109.8 (d, 2JC–FZ19 Hz), 109.9 (d,
19. Kaluzna, I. A.; Feske, B. D.; Wittayanan, W.; Ghiviriga, I.;
Stewart, J. D. J. Org. Chem. 2005, 70, 342–345.
20. Zhu, D.; Mukherjee, C.; Rozzell, J. D.; Kambourakis, S.; Hua,
L. Tetrahedron 2006, 62, 901–905.
1
2JC–FZ19 Hz), 142.5, 163.3 (d, JC–FZ247 Hz), 163.4
(d, 1JC–FZ247 Hz), 173.0. [a]D22 K81.2 (c 1.0, CHCl3).
21. Ishihara, K.; Yamaguchi, H.; Omori, T.; Uemura, T.;
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University for providing us the plasmid of 7a-hydroxy-
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Southern Methodist University for start-up support.
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