Diastereoselective reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4- fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to Ezetimibe by the whole cell catalyst Rhodococcus fascians MO22

Article abstract of DOI:10.1016/j.molcatb.2010.09.006

The asymmetric microbial reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4- fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)-propyl]-4(S) -(4-hydroxyphenyl)azetidin-2-one (Ezetimibe) by Rhodococcus fascians MO22 is described. The catalytic capability of the microorganism for reduction has been examined also with protected ketone, an intermediate from chemical synthesis of Ezetimibe. Various parameters of the bioreduction have been optimized: the strain converted 94.8% of ketone and 63% of protected ketone into Ezetimibe with the same de of 99.9%. In the later case, two chemical steps are replaced with a single biotransformation.

Full text of DOI:10.1016/j.molcatb.2010.09.006

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 266–270
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Diastereoselective reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-
fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to Ezetimibe by
the whole cell catalyst Rhodococcus fascians MO22
Eva Kyslíkováa, Peter Babiakb, Helena Maresˇováa, Andrea Palyzováa, Josef Hájícˇekb, Pavel Kyslíka,∗
a Laboratory of Enzyme Technology, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Vídenˇská 1083, 142 20 Prague 4, Czech Republic
b Zentiva Group, k.s., U kabelovny 130, 102 37 Prague 10, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2010
Received in revised form 31 August 2010
Accepted 15 September 2010
Available online 21 September 2010
The asymmetric microbial reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)-propyl]-
4(S)-(4-hydroxyphenyl)azetidin-2-one to 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)-
propyl]-4(S)-
(4-hydroxyphenyl)azetidin-2-one (Ezetimibe) by Rhodococcus fascians MO22 is described. The cat-
alytic capability of the microorganism for reduction has been examined also with protected ketone, an
intermediate from chemical synthesis of Ezetimibe. Various parameters of the bioreduction have been
optimized: the strain converted 94.8% of ketone and 63% of protected ketone into Ezetimibe with the
same de of 99.9%. In the later case, two chemical steps are replaced with a single biotransformation.
© 2010 Elsevier B.V. All rights reserved.
Keywords:
Biotransformation
Rhodococcus fascians MO22
Ezetimibe
Reduction
1. Introduction
reactions catalyzed by enzymes over chemical syntheses are that
enzymatic reactions are often highly stereoselective, regioselective
Ezetimibe 2 is 2-azetidione that selectively and potently inhibits
the adsorption of biliary and dietary cholesterol and related plant
sterols from the small intestine without affecting the adsorption of
fat-soluble vitamins, triglycerides or bile acids. The chiral alcohol
Ezetimibe 2 is an effective option for treating patients with primary
hypercholesterolemia, reducing the risk of coronary heart disease
[1,2]. A deprotection and reduction of the respective ketone to alco-
hol belong among key steps of synthesis of Ezetimibe 2. The usual
methods employed in the preparation of optically active alcohols
are asymmetric reductions of ketones by chemical [3,4] or chemo-
enzymatic syntheses [5,6]. Several reviews deal with the use of
enzymes in organic syntheses of the drugs [7–11] in which the chi-
rality is a key factor in their efficiency. For pharmaceutical industry,
the production of a single stereoisomer of the chiral molecule is of
great importance.
and the biocatalysis is a process that is environment friendly [13].
In this paper, we describe the biotransformation of 1-(4-fluo-
rophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)-propyl]-4(S)-(4-hyd-
roxyphenyl)azetidin-2-one 1 or its carboxybenzyl-protected form
3
to 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)-
propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one 2 by the nature
isolate Rhodococcus fascians MO22 [14] with high Ezetimibe 2 yield
and excellent diastereoselectivity (Scheme 1). The alternative
enzyme-based method for the last step or two steps of synthesis
of Ezetimibe 2 was described.
2. Experimental
2.1. Chemicals and general methods
Recently a number of microbial strains having ketone reductase
(KR) activity have been reported and exploited for the stereoselec-
tive reduction of ketones. KRs show a broad substrate range and
an excellent stereodifferentiation. However, specific catalyst must
be developed for each substrate as these compounds are not nat-
ural substrates for the microbial enzymes [12]. The advantages of
A ketone 1, Ezetimibe 2, protected ketone 3, and protected
Ezetimibe 4 were synthesized in the Development Department,
Zentiva Group, k.s., Czech Republic.
Media components were purchased from Hi-media and Difco
and solvents were obtained from Merck and Chromasolv.
Thin layer chromatography (TLC) was made on plastic sheets
Silica gel 60 F254 from Merck and the detection was done
under UV (ꢀ = 254 nm). Elution system, the mixture of ethylac-
etate:hexane = 1:1 was used; rf for ketone 1 = 0.5 and rf for alcohol
2 = 0.3.
∗
Corresponding author. Tel.: +420 241 062 777; fax: +420 241 727 021.
E-mail address: kyslik@biomed.cas.cz (P. Kyslík).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.09.006

E. Kyslíková et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 266–270
267
OH
OH
O
OH
Microbial Reduction
N
N
F
F
O
O
Ketone 1
Alcohol 2
F
F
OCbz
OH
OH
O
O
OH
+
N
N
F
F
N
O
F
O
O
Ketone 1
F
F
Alcohol 2
Protected Ketone 3
F
+
OCbz
OH
N
F
O
Protected Ezetimibe 4
F
Scheme 1. Preparation of Ezetimibe 2. Cbz is a carboxybenzyl protecting group.
HPLC analysis was made on Dionex Summit with PDA detec-
tor. Column Luna C18 5 ␮m was used for determination of the
degree of conversion and column Chiralpak AD-H (Daicel) for de
determination.
To determine quantitatively the percentage of conversion of
the substrate 1 to the product 2, the reaction mixtures contain-
ing the product 2 were also analyzed by HPLC. Mobile phase was
0.02 M KH2PO4 buffer (pH 3.0):acetonitrile = 3:7, isocratic elution
with flow rate of 1.3 mL/min, column temperature of 20 ◦C, detec-
tion spectrophotometer ꢀ = 220 nm, injection volume of 10 ␮L, and
run time 6 min. The retention time of 2 and 1 was 3.13 and 4.07 min,
respectively.
In the case of isolates capable of reduction and exhibiting more
than 4% conversion of the substrate, the diastereomeric excess
of Ezetimibe 2 was determined. Mobile phase used was hex-
ane:ethanol = 1:1 + 0.5 mL/L of diethylamine. The isocratic elution
was performed as follows: the flow rate of 1 mL/min, column tem-
perature of 40 ◦C, injection volume of 10 ␮L, run time 12 min, and
the reactants were detected at the wavelength ꢀ = 275 nm. The
retention time of 2 and R-alcohol was 4.7 and 6.6 min, respectively
(Fig. 1).
For isolation of Ezetimibe 2 after biotransformation of ketone
1, 30 mL of 20% cell suspension was saturated with NaCl and
the reaction mixture was treated with ethylacetate (4 × 15 mL).
Ethylacetate fractions were collected, dried out with Na2SO4 and
evaporated on a rotavap. The Ezetimibe 2 and ketone 1 were puri-
fied by flash chromatography on Silica gel 60 from Fluka (elution
system ethylacetate:hexane = 1:1).
2.2. Microorganisms
Microorganisms were obtained from the Culture Collection of
Industrial Microorganisms, the Research Institute of Food Indus-
try, Prague, and the Collection of Microorganisms of the Laboratory
of Enzyme Technology, the Institute of Microbiology ASCR, v.v.i.,
Prague. Microorganisms were maintained as frozen glycerol stock
cultures at −70 ◦C.
2.3. Culture conditions and screening procedures
Samples of the cell suspensions used in biotransformations were
prepared by batch cultivations in 50-mL shake flasks containing
Fig. 1. Example of chromatogram from biotransformation. The retention time of Ezetimibe 2 was 4.7 min and 9.4 min for ketone 1. A negligible peak around 6.6 min was
R-alcohol.

268
E. Kyslíková et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 266–270
Table 1
15 mL of the culture medium. The cultures of bacteria and yeasts
were grown in LB medium (1.0% tryptone, 0.5% yeast extract, 1.0%
sodium chloride, pH 7.0) and YPD medium (1.0% yeast extract, 1.0%
peptone, 1.0% glucose, pH 5.5), respectively. The value of pH in a
medium was adjusted before sterilization. One vial of the frozen
stock culture of each strain was used as inoculum and cultures
were performed on an orbital shaker (200 rpm) for 48 h and at
28 ◦C. In the course of a cultivation or microbial reduction, pH was
not controlled. To prepare the cell suspensions for biotransforma-
tions, a biomass was harvested from the cultures by centrifugation
(10 000 × g, 15 min, 10 ◦C), washed with 50 mM potassium phos-
phate buffer (pH 7.0, buffer A), frozen overnight at −20 ◦C, and used
for the reduction of 1 after resuspending in a required volume of
the buffer A.
Screening for microorganisms capable of the diastereoselective
reduction of the ketone 1 was performed as follows: the volume
of 5 mL of 10% suspension of cells (cell wet weight/v) in buffer A
was added into 25 mL Erlen-Mayer flasks. The reactions were per-
formed at 200 rpm, 28 ◦C and initiated by the addition of 0.1 mL
of solution of 1 in ethanol (25 mg/mL) so that the final concentra-
tion of 1 equaled 0.5 g/L. After 24 h, a sample of the reaction mixture
(0.5 mL) was removed and extracted for 15 min with 0.5 mL of ethy-
lacetate. The sample was centrifuged (10 000 × g, 5 min) and the
organic layer was used for the detection of 1 and 2 by TLC.
Asymmetric microbial reduction of ketone 1 to Ezetimibe 2.
Microorganism
Yield (%)
de of 2 (%)
Saccharomyces cerevisiae No. 3
Saccharomyces cerevisiae No. 4
Saccharomyces cerevisiae No. 13
Torulopsis azyma CCY 26592
Candida tropicalis RIFIS 216
Rhodococcus sp. No.654
8.6
8.4
4.8
4.3
4.7
99.9
99.8
95.8
n.d.
n.d.
99.2
99.9
24.3
41.5
Rhodococcus fascians MO22
Yield is expressed as a percentage (w/w) of the ketone converted to Ezetimibe, n.d.:
not determined.
mate at concentrations 10, 50 and 100 g/L. 10% (cell wet weight/v)
suspension of cells was used in these experiments. The following
parameters optimized in this work were used in the conversion
experiments with cofactor regeneration: pH 7.0 and temperature
of 30 ◦C.
3. Results and discussion
A number of yeasts and bacteria [15–18] were reported as
ketoreductase positive microorganisms. To detect the useful bio-
catalyst for the production of Ezetimibe 2 with high conversion and
de, a number of microorganisms have to be analyzed. As a total, we
screened 230 microbial strains for the asymmetric reduction of the
ketone 1 to alcohol 2. Among them, eight yeast and nine bacterial
strains were detected and isolated as KR positive microorganisms.
The conversion and de of the product 2 were determined by HPLC
with cell suspensions of the strains exhibiting at least a conver-
sion of 4% of the ketone into the product. The results are shown
in Table 1. The highest conversion of 41.5% and de of 99.9% were
achieved with the strain R. fascians MO22 (collection of microor-
ganisms of the Laboratory of Enzyme Technology) that was isolated
from the population of microorganisms growing in a biofilter of the
exhaust gas installed to remove pollutants the styrene and acetone.
The growth of R. fascians MO22 in a stirred bioreactor in the
medium R and bioconversion of the ketone 1 by culture samples
withdrawn at regular intervals from the bioreactor were conducted
to study the effect of the culture growth phase on the bioconver-
sion (Fig. 2). The culture growth was completed in 44 h when the
culture reached the stationary phase of growth. The bioconver-
sion increases in the course of the exponential phase of growth
and reaches the maximum of 45% in the 22nd h of cultivation. The
same conversion was maintained for the period of time of 22 h in
the course of which the growth rate of the culture ceased. Later
on, the fast decrease of the bioconversion was observed, but cells
2.4. Optimization of conditions for biotransformation of 1 by cells
of R. fascians MO22
2.4.1. The influence of the growth phase of a culture
The microbial strain R. fascians MO22 was grown in a 10 L stirred
bioreactor with a working volume of 8.0 L. The culture medium R
contained 0.2% yeast extract, 0.3% beef extract, 0.5% bacteriological
peptone, 0.7% tryptone, and 1.0% sodium chloride (pH 7.2).
Two-stage inoculum (F1 and F2) was prepared. Inoculum F1:
a vial of the glycerol stock culture was inoculated into 100 mL of
medium R in a 500 mL flask. The cultivation was carried out on the
orbital shaker (200 rpm) for 24 h at 28 ◦C. 5 mL of the culture F1 was
used to inoculate 100 mL of the medium R, and the inoculum F2 (2
flasks) was incubated for 34 h at 28 ◦C.
The bioreactor containing 8 L of the medium R was inoculated
with 200 mL of the F2 inoculum and the batch culture was con-
ducted under the following conditions: cultivation temperature of
28 ◦C, stirring frequency of 500 rpm, aeration rate of 10 L/min, and
cultivation time 58 h. During fermentation, the cells were harvested
periodically from 2 mL samples of the culture by centrifugation
(10 000 rpm, 15 min, 10 ◦C), washed with buffer A, and kept at
−20 ◦C. To evaluate the bioconversion ability of cells, the frozen
biomass in a vial was resuspended in 1 mL of buffer A and the reac-
tion was started by addition of the ethanol solution of ketone 1
(final concentration 0.5 g/L). The vials were shaken (200 rpm, 28 ◦C)
for 4 h and the sample of the reaction mixture was removed and
processed as described earlier. The organic layer was used for the
determination of concentrations of 1 and 2 by HPLC.
4.5
50
45
40
35
30
25
20
15
10
5
4
3.5
3
2.5
2
2.4.2. The effect of reaction pH, temperature and cofactor
regeneration
1.5
1
To find pH optimum for the bioreduction, the reaction was car-
ried out at the pH ranging from 2 to 10, using 0.1 M buffers as
follows: the citrate buffer for the pH range from 2 to 6, the phos-
phate buffer for the pH 7 and 8, and the Tris–HCl buffer for the
pH 9 and 10. The temperature optimum was determined by incu-
bation of the resting cell suspension in buffer A at temperature
ranging from 25 ◦C to 40 ◦C. To improve the efficiency of the micro-
bial reduction, the effect of routinely used substrates for cofactor
regeneration was studied: the conversion of 1 was performed in the
buffer A supplemented with glucose, glycerol, isopropanol, and for-
0.5
0
0
0
20
40
60
Fermentation time (h)
Fig. 2. The time-course of the biomass concentration (ꢀ: cell dry weight in g/L) in
a batch culture of the strain R. fascians MO22 performed in a stirred bioreactor and
the growth phase-dependent capability of the cells for diastereoselective reduction
of ketone 1 to Ezetimibe 2 (ꢁ: conversion in %).

E. Kyslíková et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 266–270
269
Table 2
The effect of pH on the microbial reduction of ketone 1 to Ezetimibe 2 by cell
suspension of R. fascians MO22.
pH
2
3
4
5
6
7
8
9
10
Conversion (%) 4.8 47.9 86.0 90.0 95.6 97.0 82.1 83.1 83.1
Conditions of biotransformation: T = 30 ◦C, t = 4 h, cells were resuspended in the
buffer of a given pH value.
exhibited high diastereoselectivity: de of 2 was 99.9% in the course
of the whole cultivation.
The effects of the temperature and pH on the asymmetric micro-
bial reduction of the ketone 1 to Ezetimibe 2 were evaluated using
10% cell suspension of R. fascians MO22 (cell wet weight/v) and
0.1 g/L of 1 in a buffer in the absence of a substrate for cofactor
regeneration. An effect of pH of the reaction mixture on the biore-
duction was followed at pH value ranging from 2 to 10.
It is evident (Table 2) that the process of biotransformation
reaches similar degree of conversion at the pH range from 4 to 10.
The pH optimum for the conversion lies between the pH values 6
and 7. A distinct decrease of the conversion occurs on the acidic
side of the range of pH values.
Fig. 4. Concentration effect of the glucose, isopropanol, formate and glycerol as
substrates for the regeneration of the cofactor on the conversion of ketone 1 (0.1 g/L)
to Ezetimibe 2 by cell suspension of R. fascians MO22 (pH 7.0, 25 ◦C). Conversion was
determined after 1 and 4 h of biotransformation.
The effect of the reaction temperature on the conversion process
was followed at temperatures ranging from 25 ◦C to 40 ◦C (Fig. 3).
The optimum temperature for the bioreduction of the ketone 1
corresponded to 30 ◦C. The percentage of conversion at other tem-
peratures was markedly reduced: the conversion was reduced by
50% and 70% at the temperature of 25 ◦C and 35 ◦C, respectively.
In all cases, the maximum conversion was reached in 4 h. We pre-
sume that any internal source of energy has been depleted after
4 h of the reaction and no regeneration of cofactor occurred in a
reaction buffer.
could reduce the rate of conversion at this concentration (a lower
percentage reached after 1 h).
Deprotection of Cbz protecting group of 3 is a chemical reac-
tion preceding microbial diastereoselective reduction of 1 to 2.
Bioreduction of 3 to 2 was determined with 10% or 20% (cell wet
weight/v) suspension of the biomass withdrawn from the biore-
actor culture of the strain R. fascians MO22 at the 22nd h of the
cultivation. Optimal conditions for the biotransformation of 1 to 2
(buffer A supplemented with 50 g/L of glucose, 30 ◦C) were applied
andthe protectedketone3 was usedatconcentrations ranging from
0.1 to 4.0 g/L. The suspensions were able to transform 3 directly into
Ezetimibe 2 with conversion of 63%, with a few percents of 1 and
protected Ezetimibe 4 (Table 3).
Biotransformation of 1 to 2 was evaluated under identical
conditions as with protected ketone 3 (Table 4). The maximum
conversion of 90 and 95% in 24 h was reached with 10% and 20%
suspension of cells, respectively. As regards the productivity of the
process, the highest value was obtained with 20% cell suspension
at 0.5 g/L of ketone 1 within the 1st h of reaction: 175 mg/L h.
After 24 h, the reduction of 1 gives higher conversions at lower
concentrations of the substrate (0.1–1.0 g/L) while the biotransfor-
mation of 3 gives higher conversions at concentration range from
1.0 to 4.0 g/L.
At temperatures up to 30 ◦C, de was 99.9% but the parameter
decreased to 86.4% at temperature of 35 ◦C. Since biotransforma-
tions were performed with whole cells (not with the purified
enzyme) we cannot exclude the presence of alternative KR with
lower stereoselectivity which may be more stable or more active
at higher temperatures.
Percentage of conversion can be affected by the depletion of
the cofactor NADH or NADPH. The effect of addition of glucose,
glycerol, isopropanol, and formate, substrates routinely used for
the regeneration of cofactors of KRs, is shown in Fig. 4.
The glucose had a marked positive effect on the percentage of
the conversion of 1 at the concentration of 50 g/L: in comparison
to the control experiment, the percentage increased by 17% after
4 h. The effect of glycerol on the percentage of the conversion of 1
was positive in the whole concentration range: in comparison to
the control experiment, the percentage increased by 22% after 4 h
even at the concentration of 100 g/L.
To isolate the product 2 and determine the yield of isolation,
30 mL of 20% suspension of cells was shaken for 48 h at optimal
A similar effect was observed if isopropanol (50 g/L) or formate
(10 g/L) was added to the reaction buffer. While the formate seems
to be a good candidate for regeneration of the cofactor, isopropanol
Table 3
Biotransformation of protected ketone 3 used at different concentrations by 10% or
20% cell suspension of R. fascians MO22 at 30 ◦C.
Cell suspension
Protected
ketone 3 (g/L)
Conversion after 24/48 h (%)
100
90
80
25°C
Ezetimibe 2
Ketone 1
Protected
Ezetimibe 4
70
60
50
40
30
20
10
0
30°C
35°C
40°C
0.1
0.5
1
2
4
0.1
0.5
1
2
4
44/56
42/49
29/41
27/39
24/29
53/63
44/44
39/43
36/38
28/37
2/0
4/3
16/4
19/6
21/6
1/5
3/6
5/9
12/10
19/11
0/0
4/0
7/0
8/3
8/5
0/0
4/2
9/3
11/3
10/4
10%
20%
0
5
10
15
20
25
Bioconversion time (h)
Fig. 3. The effect of temperature on the microbial reduction of ketone 1 (0.1 g/L) to
Ezetimibe 2 by cell suspension of R. fascians MO22. The time course of the processes
was studied in buffer A.

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E. Kyslíková et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 266–270
Table 4
The use of the extraction step and protocol for isolation
of Ezetimibe 2 at industrial scale requires further optimization
because the yield of 2 (68%) is low when compared to conversion
of 84%.
Biotransformation of protected ketone 3 could replace one more
synthetic step, usually a deprotection by hydrogenation on a pala-
dium catalyst, and can avoid Ezetimibe 2 contamination with the
heavy metal. Maximum conversion of 63% in 48 h was reached with
20% suspension of cells.
The timecourse ofthe biotransformation ofketone 1 usedat different concentrations
to Ezetimibe 2 by 10% or 20% cell suspension of R. fascians MO22 at 30 ◦C.
Cell suspension
10%
Ketone 1 (g/L)
Conversion (%)
1 h
4 h
24 h
48 h
0.1
0.5
1
2
4
0.1
0.5
1
2
4
62.0
28.6
2.5
88.5
41.2
7.3
89.8
58.4
24.4
23.5
17.8
94.8
84.4
44.8
37.3
26.3
86.5
82.1
41.1
36.7
23.5
90.0
92.1
50.2
47.2
33.4
1.9
6.2
1.1
3.9
20%
72.3
35.0
7.3
3.6
2.6
93.9
62.6
20.5
11.4
8.6
Acknowledgments
This work was supported by Zentiva Group, k.s. and by the insti-
tutional research concept No. AV0Z50200510. We thank Jaroslav
Marsˇálek for technical assistance for bioreactor cultures, Lukásˇ
Placˇek and Vladislav Kubelka for HPLC analysis.
conditions for the biotransformation of 1 to 2 (buffer A supple-
mented with 1.5 g of glucose, 15 mg of ketone 1, 30 ◦C). The reaction
mixture was processed and the reactants were purified by flash
chromatography from 330 mg of brown tar obtained after evapo-
ration. The conversion reached 84%, yield of isolated Ezetimibe 2
was 68% (10.2 mg) and recovery of ketone 1 equaled 14% (2.1 mg).
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The maximum conversion of 95% in 24 h was reached with 20%
suspension of cells. The highest productivity of 175 mg/L h was
obtained with 20% cell suspension at 0.5 g/L of ketone 1 after 60 min
of the reaction (Table 4).
To our knowledge, this is the first report on achievement of
excellent de and a high degree of conversion of 1 to Ezetimibe 2
using a whole cell catalyst.