Efficient Synthesis of (R)-2-Chloro-1-(2,4-dichlorophenyl)ethanol with a Ketoreductase from Scheffersomyces stipitis CBS 6045

Article abstract of DOI:10.1002/adsc.201601003

By enzyme screening, a ketoreductase cloned from Scheffersomyces stipitis CBS 6045 and named SsCR was identified that could catalyze the asymmetric hydrogenation of a variety of aromatic ketones. SsCR exhibited a specific activity of 65 U mg^?1p

Full text of DOI:10.1002/adsc.201601003

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DOI: 10.1002/adsc.201601003
Efficient Synthesis of (R)-2-Chloro-1-(2,4-dichlorophenyl)ethanol
with a Ketoreductase from Scheffersomyces stipitis CBS 6045
Yue-Peng Shang,a Qi Chen,a Xu-Dong Kong,a Yu-Jun Zhang,a Jian-He Xu,a
and Hui-Lei Yua,*
a
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East
China University of Science and Technology, 130 Meilong Road, Shanghai 200237, Peopleꢀs Republic of China
Fax : (+86)-21-6425-0840; e-mail: huileiyu@ecust.edu.cn
Received: September 27, 2016; Revised: November 22, 2016; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201601003.
Abstract: By enzyme screening, a ketoreductase
cloned from Scheffersomyces stipitis CBS 6045 and
named SsCR was identified that could catalyze the
asymmetric hydrogenation of a variety of aromatic
ketones. SsCR exhibited a specific activity of
65 UmgÀ1 protein and excellent enantioselectivity
(99.9% ee) towards the hydrophobic substrate 2-
chloro-1-(2,4-dichlorophenyl)ethanone, which is an
intermediate in the synthesis of common antifungal
agents such as miconazole, econazole and sertaco-
nazole. The kinetic parameter kcat/Km was 4.51ꢁ
103 sÀ1 mMÀ1, showing the great catalytic efficiency
of SsCR towards this substrate. Molecular dynamic
simulation results shed light on the higher substrate
binding free energy change for this substrate rela-
tive to other substrates. Based on the good catalytic
properties of SsCR, (R)-2-chloro-1-(2,4-dichloro-
phenyl)ethanol could be obtained with a space-time
yield (STY) of up to 268 gLÀ1 dÀ1 without any addi-
tional cofactor required in the reductive reaction
process. On scaling up the bioreaction, the (R)-alco-
hol was isolated with 88.2% yield and 99.9% ee.
The environmental factor (E factor) of this reaction
was 7.25 when process water was excluded.
agents, such as miconazole, econazole and sertacona-
zole.[2] Based on new reports from the BCC research,
the global market for human antifungal therapeutics
may reach $13.9 billion by 2018. Miconazole is one of
the most important basic medications on the World
Health Organizationꢀs list of essential medicines.[3]
The demand for this kind of antifungal agent requires
industrial production on a massive scale.
Various kinds of catalysts are exploited during the
process of asymmetric transfer hydrogenation. These
include chiral oxazaborolidine catalysts, chiral metal
catalysts and biocatalysts. The substrate spectrum of
chiral oxazaborolidine catalysts is broad,[4] but the
enantioselectivity of the products is related to com-
plex ligands.[5] Compounds based on transition metals
such as rhodium, ruthenium and iridium are the most
popular catalysts in the hydrogenation of ketones.
However, transition metals need additional chelating
functional groups for high enantioselectivities, and the
high cost of transition metals is a limitation to their
industrial use.
Compared with chemical catalysts, the principal ad-
vantages of biocatalysts are their unsurpassed selec-
tivity and environmental friendliness.[6] Lipases are ki-
netically suitable for the synthesis of various chiral al-
cohols,[7] but are limited by a 50% theoretical yield.
Reductases have been exploited and applied to real-
ize the asymmetric hydrogenation of ketones. Bakerꢀs
yeast cells were used for the asymmetric synthesis of
(S)-2-chloro-1-(2,4-dichlorophenyl)ethanol, but the
requirement for lyophilized cells was up to
120 gLÀ1.[8] Kluyveromyces thermotolerans carbonyl
Keywords: biocatalysis; bioreduction; (R)-2-chloro-
1-(2,4-dichlorophenyl)ethanol; cofactor recycle; re-
ductase
The asymmetric hydrogenation of prochiral substrates reductase (KtCR) was investigated to synthesize this
is considered as an important method to prepare opti- (S)-product and had a specific activity of approxi-
cally active compounds. Chiral alcohols are vital mately 1.2 UmgÀ1 protein.[9] To produce (R)-2-chloro-
building blocks for drug synthesis as a result of the 1-(2,4-dichlorophenyl)ethanol, PFADH was cloned
different activities of enantiomers in pharmaceutical from Pyrococcus furiosus.[10] The ee value was up to
applications.[1]
ACHTUNGTRENNUNG
(R)-2-Chloro-1-(2,4-dichlorophenyl)- 99%, but the space-time yield (STY) was only
ethanol is applied in the synthesis of many antifungal 20 gLÀ1 dÀ1 in preparations at the 100 mL scale. To
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find more efficient biocatalysts, many alcohol dehy-
drogenases, such as ADH-T, ADH-PR2, ADH-CP,
ADH-A, ADH-RS1 and ADH-LB, were screened by
Gotor et al.[2b] Among these enzymes, ADH-T
showed a relatively high catalytic efficiency and enan-
tioselectivity. However, the substrate loading was only
To fully determine the catalytic properties of SsCR
6.7 gLÀ1 in the bioreduction system. Low substrate towards the substrate 10a, the reaction system was op-
loading and STY markedly hindered its industrial timized in terms of catalyst dosage, substrate concen-
scale application.
tration and NADP+ addition. All of the reaction opti-
Herein, we have exploited a novel ketoreductase mizations were carried out in aqueous solution con-
with high specific activity toward various halogenated taining 10% v/v DMSO to increase the solubility of
aromatic ketones. To identify an enzyme catalyzing the hydrophobic substrate 10a.
the reduction of 2-chloro-1-(2,4-dichlorophenyl)etha-
A preliminary experiment was conducted with
none (10a), 50 enzymes were screened for their activi- 100 mM substrate in 10 mL mixture [PBS 100 mM,
ty (Supporting Information, Table S1). After expres- pH 6.5, 2.5 gLÀ1 SsCR-expressing lyophilized cells,
sion in Escherichia coli, cell-free extracts containing 0 mM NADP+, 10 KULÀ1 d-glucose dehydrogenase
recombinant protein were applied to measure the ac- from Bacillus megaterium (BmGDH)]. The substrate
tivity spectrophotometrically. Sixteen reductases were was transformed by only 59% after 12 h. When
identified with catalytic activity towards 10a 0.5 mM NADP+ was added to accelerate the catalytic
(Figure 1). On the basis of the best enantioselectivity rate, SsCR could completely catalyze conversion of
the substrate to the corresponding (R)-product within
2 h (Table 1, entry 2). Next, the substrate loading was
increased to 300 mM, but the conversion was only
63% (Table 1, entry 3). It was 97% after 12 h with
5.0 gLÀ1 catalyst loading (Table 1, entry 4). It was nec-
essary to increase the dosage of SsCR to 9.5 gLÀ1 to
realize complete transformation; the substrate conver-
sion reached up to 99% in 6 h (Table 1, entry 5). For
cost reasons, it is preferable not to add extra NADP+
(Table 1, entry 6), but under these conditions the con-
version of substrate reached only 87% after 12 h.
From this series of optimization experiments, we con-
cluded that the coenzyme was very important for ef-
fective operation of the catalyst. The endogeneous
content of NADP+ was reported as 1.86 mmolgÀ1 in
lyophilized E. coli cells.[11] Taking into account the
Figure 1. The screening results of cloned enzymes based on
amount of NADP+ in lyophilized E. coli, we in-
the enantioselectivity and specific activity of cell-free ex-
tract.
creased the total dosage of SsCR to 12.5 gLÀ1 lyophi-
lized cells without adding any extra coenzyme. Now
the substrate was transformed completely with 99%
and activity, the protein cloned from Scheffersomyces conversion and the TTN of the catalyst was estimated
stipitis CBS 6045, named SsCR was selected for fur- to be about 1.26ꢁ104. Analogous to substrate 10a,
ther study.
conversion of substrate 11a was also transformed into
Table 1. Asymmetric reduction of substrate 10a with recombined E. coli cell harboring SsCR.
Substrate
Entry Ketone
[mM]
SsCR
NADP+
[mM]
Time
[h]
Conv.
[%][a]
ee
STY
TTN
[gLÀ1]
[%][a]
[gLÀ1 dÀ1]
1
2
3
4
5
6
7
100
100
300
300
300
300
300
2.5
2.5
2.5
5.0
9.5
9.5
12.5
0
12
2
12
12
6
59
99
63
97
99
87
99
>99
>99
>99
>99
>99
>99
>99
63.4
269
113
130
268
117
268
1.24ꢁ104
2.10ꢁ104
3.97ꢁ104
3.15ꢁ104
1.66ꢁ104
1.44ꢁ104
1.26ꢁ104
0.5
0.5
0.5
0.5
0
12
6
0
[a]
Determined by GC analysis.
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the corresponding (R)-product effectively at a gram fold higher than that of KtCR.[9] SsCR could catalyze
scale. For 300 mM substrate 11a, 12.5 gLÀ1 SsCR substrate 10a with a specific activity of up to
could complete the transformation within 3 h. These 65.0 UmgÀ1, 54-fold higher than that of KtCR. If the
results showed that SsCR was an efficient catalyst to substituents on the benzene ring were changed to flu-
realize the reduction of halogenated aromatic ketones orine, SsCR showed a highest specific activity of
and had a good application potential.
253 UmgÀ1 towards substrate 11a (see Figure 2).
To explore the catalytic properties of SsCR in Meanwhile, the enantioselectivity for substrates 10a
depth, the specific activities toward a wide range of and 11a reached the highly desirable level of 99%.
aromatic ketones (Figure 2) were detected using pro-
The kinetic parameters of purified SsCR toward
tein purified by nickel affinity chromatography. These several halogenated aromatic ketone substrates were
substrates contained different substituents on the measured in the enzyme assay (Table 2). The Km of
phenyl ring and adjacent to the carbonyl group. The
specific activities towards all the a-position substitut-
ed ketones (6a–11a) were higher than that toward
acetophenone with distinct magnitudes. SsCR showed
significantly higher specific activities toward sub-
strates 10a and 11a, which had additional halo-sub-
stituents on both the para-position of the phenyl ring
and the a-position of the carbonyl ketone, compared
Table 2. Kinetic parameters of SsCR for several aromatic
ketones.
Substrate Km [mM]
kcat [sÀ1] kcat/Km [sÀ1
mMÀ1]
1a
6a
2.15Æ0.20
3.85Æ0.1 1.79
28.3Æ1.0 64.3
0.440Æ0.060
with its activity toward 3a, whose chloro-substituent 10a
was at the phenyl ortho-position. Many reductases,
2.44ꢁ10À2 Æ0.80ꢁ10À2 110Æ11 4.51ꢁ103
7.19ꢁ10À3 Æ0.40ꢁ10À3 191Æ0.8 2.65ꢁ104
11a
for example, a short-chain alcohol dehydrogenase
YMR226c from Saccharomyces cerevisiae,[12] showed
poor catalytic efficiency toward acetophenone analogs SsCR for 10a was merely 2.44ꢁ10À2 mM. This indicat-
with different groups only on the phenyl ring. These ed that the binding affinity was high for this substrate.
results showed that substrates with electron-withdraw- kcat/Km is a very important parameter to evaluate the
ing groups at the a-position of the carbonyl ketone catalytic efficiency of an enzyme.[13] For SsCR with
could be catalyzed more efficiently by SsCR. The ac- substrate 10a kcat/Km reached 4.51ꢁ103 sÀ1 mMÀ1, indi-
tivity of SsCR toward 8a was up to 124 UmgÀ1, nine- cating that the catalysis was efficient. Substrate 11a
had a similar chemical structure to 10a, and its kcat/Km
was 2.65ꢁ104 sÀ1 mmÀ1. These values were higher than
those observed for substrates 1a and 6a. Based on the
kinetic parameters determined for the four substrates
(Table 2), the halogenated substituent groups and
their position on the substrate dramatically affected
the substrate binding and catalytic efficiency of SsCR.
To shed new light on the substrate binding mode,
the crystal structure of SsCR was resolved at 2.3 ꢃ
(PDB: 5GMO). Meanwhile, we performed all-atom
MD simulations of SsCR in complex with three differ-
ent substrates using NAMD v.2.9 (Figure 3).[14] Ac-
cording to the binding energy results estimated using
Molecular Mechanics–Poisson Boltzmann (General-
ized Born)/surface area (MM-PB(GB)/SA) meth-
ods,[15] the calculated that the binding free energy of
substrate 1a was À9.03Æ1.65 kcalmolÀ1, which in-
creased to À14.29Æ2.79 kcalmolÀ1 and À19.67Æ
2.57 kcalmolÀ1 for substrates 6a and 10a, respectively.
The overall trend of the calculated binding free ener-
gies was consistent with the experimentally deter-
mined Km values.
Based on the optimized conditions (Table 1,
entry 7), the reaction volume was scaled up 100-fold
to 1 L. Substrate (67 g) was transformed in a thermo-
statted stirred-tank reactor at 308C. After 16 h, the
conversion of substrate was up to 99%, with 99.9%
ee. The reaction mixture was extracted twice with
Figure 2. The specific activity (U mgÀ1 protein, upper value)
and enantionselectivity (% ee, lower value) of SsCR towards
various aromatic ketones (1a–11a). The specific activity was
measured with purified enzyme based on the oxidation of
NADPH at 340 nm. The ee values were determined by GC
[Supporting Information, Table S2].
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Figure 3. Comparison of different substrate binding modes using MM-PB/SA.
ethyl acetate (EtOAc) followed by distillation. Satu- smoothly with 88.2% isolated yield and an E factor of
rated sodium chloride solution was used to wash the 7.25 (excluding water). Our findings indicate that
mixture to remove water and DMSO. Finally, 59.6 g SsCR has a significant potential as a catalyst for in-
product were collected representing an 88.2% yield. dustrial production of halogenated alcohols.
The asymmetric reduction reaction of the highly con-
centrated hydrophobic substrate was easily realized
by SsCR because of its high specific activity and low Experimental Section
Km. Notably, because no additional cofactor had to be
added, the cost of the reaction was reduced.
Screening of Ketoreductases
According to the principles of green chemistry, this
reductase-based process was greener than transition
metal-catalyzed reduction (involving heavy metals)
and kinetic resolution (50% maximum yield).[16] The
environmental factor (E factor) of this reduction pro-
cess was shown in the Supporting Information, Table
S3. Solvent (EtOAc [92.3% recovery rate] and
DMSO) losses (68%) were the highest contributor to
the E factor. The glucose was transformed into
sodium gluconate, which constituted 18% of the
waste. As a co-substrate for cofactor regeneration,
glucose was cost effective. It was also renewable and
sodium gluconate produced could be further uti-
lized.[16] If water was excluded, the E factor was 7.25.
The E factor for whole reduction process was 27.4 if
water was included.
To identify an enzyme sufficiently catalytic toward substrate
10a and its analogs, a reductase kit in our lab was screened.
The enzyme activity was measured in cell-free extracts after
cloning and expression, based on the measurement of the
absorbance of NADPH. The enantioselectivity was deter-
mined by GC after reduction reaction in aliquots of 1 mL.
The GC detection method is described in the Supporting In-
formation.
Bioreduction of 2-Chloro-1-(2,4-dichlorophenyl)-
ethanone with SsCR
To explore the catalytic efficiency of SsCR toward substrate
10a, several reduction reactions were performed on the
gram scale. The reaction mixture was as follows: substrate
10a (100 mM, 300 mM), d-glucose (1.5 substrate equiva-
lents), SsCR (2.5 gLÀ1, 5 gLÀ1, 9.5 gLÀ1, 12.5 gLÀ1 lyophi-
lized cells), GDH (1 SsCR activity unit equivalent to lyophi-
In summary, a novel reductase from S. stipitis CBS
6045, SsCR, was identified and had an extremely high lized crude enzyme), NADP+ (0 mM, 0.5 mM), PBS
(100 mM, pH 6.5) and DMSO (10% v/v). These reactions
were stirred at 200 rpm and 308C until the reaction stopped.
To maintain stable activity of SsCR, the reaction pH was au-
totitrated to 6.5 using 1M NaOH. The mixtures were ex-
tracted using ethyl acetate and dried with anhydrous sodium
sulfate, after which the reaction products were analyzed by
GC. All of the reactions were optimized in a 10 mL reactor.
The final product was identified by NMR. H NMR (CDCl3,
400 Hz): d=2.74 (d, J=3.6 Hz, 1H), 3.52 (dd, J=11.2,
8.4 Hz, 1H), 3.88 (dd, J=11.2, 2.8 Hz, 1H), 5.27 (m, 1H),
specific activity toward various halogenated aromatic
ketones, especially 2-chloro-1-(2,4-dichlorophenyl)-
ACHTUNGTRENNUNGethanone and its analogs. A series of reaction optimi-
zations was performed to evaluate the catalytic prop-
erties of SsCR. Without the addition of extra
NADP+, SsCR could transform 300 mM 2-chloro-1-
(2,4-dichlorophenyl)ethanone completely within 6 h.
Under these conditions, the STY was up to
268 gLÀ1 dÀ1 and the TTN was 1.26ꢁ104. When the re-
1
action was scaled up to 1 L, the process performed 7.31 (dd, J=8.4, 2.0 Hz, 1H), 7.38 (d, J=2.0 Hz, 1H), 7.58
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(d. J=8.4 Hz, 1H). These data corresponds to those in
ref.[2b] 13C NMR (CDCl3, 100 MHz): d=49.1, 70.3, 127.6,
128.5, 129.3, 132.4, 134.6, 135.9. [a]2D7: À57 (c 1.0, CHCl3).
(No. 222201514039), Shanghai Science and Technology Pro-
gram (No. 15JC1400403) and SKLBE (No. 2060204). We
thank Prof. Jia-Hai Zhou for his kind advice on crystal data
analyses. We are grateful for access to beamline BL17U1 at
Shanghai Synchrotron Radiation Facility and thank the
beamline staff for technical support.
Substrate Profile and Enantioselectivity
The specific activity of SsCR toward various aromatic ke-
tones was measured with purified protein (Supporting Infor-
mation, Figure S1). The substrate ketones were diluted with
DMSO to 2 mM. The purified enzyme was diluted to differ-
ent concentrations with PBS (100 mM, pH 6.5) based on the
reasonable absorbance change of NADPH at 340 nm. The
enantioselectivity of different substrates was determined in
a 400 mL reaction mixture. In these reactions, 10 mM sub-
strate, 15 mM glucose, 0.1 mM NADP+, 10 mg SsCR-ex-
pressing lyophilized cells and 10 mg BmGDH lyophilized
crude enzyme were mixed to react at 308C with stirring at
1000 rpm. After 24 h, the reaction mixture was extracted
with ethyl acetate. The enantioselectivity of the product was
determined by GC (Supporting Information, Table S4).
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X-Ray Structure of SsCR and MD Analysis
A crystal of SsCR was obtained at 188C in the optimal crys-
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COMMUNICATIONS
Efficient Synthesis of (R)-2-Chloro-1-(2,4-
dichlorophenyl)ethanol with a Ketoreductase from
Scheffersomyces stipitis CBS 6045
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Adv. Synth. Catal. 2017, 359, 1 – 7
Yue-Peng Shang, Qi Chen, Xu-Dong Kong, Yu-Jun Zhang,
Jian-He Xu, Hui-Lei Yu*
Adv. Synth. Catal. 0000, 000, 0 – 0
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ꢂ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!