Efficient biosynthesis of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol using a mutant short-chain dehydrogenase from Novosphingobium aromaticivorans

Article abstract of DOI:10.1016/j.tetlet.2020.151914

(R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol ((R)-CPEO) is an important chiral intermediate for antifungal drug synthesis. (R)-CPEO can be produced from 2-chloro-1-(2, 4-dichlorophenyl) ethanone (CPE) via a mutant short-chain dehydrogenase/reductase from Novosphingobium aromaticivorans (NaSDR). The Vmax of a mutant NaSDR-G145A/I199L toward CPE (6.32 U mg^?1) was greater than that of wild-type NaSDR (2.58 U mg^?). The Km of mutant NaSDR-G145A/I199L toward CPE (0.23 mM) was less than that of wild-type NaSDR (0.38 mM), indicating that the substrate affinity of mutant NaSDR-G145A/I199L was the greater of the two. Docking simulations were used to illustrate the mechanisms of the increased enzyme activity of NaSDR-G145A/I199L; these showed that NaSDR-G145A/I199L presented a more effective docking posture than that of the wild-type enzyme. Further, NaSDR-G145A/I199L and glucose dehydrogenase (GDH) were used to transform 120 g/L CPE into (R)-CPEO. After 6 h, the conversion rate and enantiomeric excess values were 99% and 99.95%, respectively. The present study provides a practical method for high substrate loading of (R)-CPEO for industrial-scale applications.

Full text of DOI:10.1016/j.tetlet.2020.151914

Tetrahedron Letters xxx (xxxx) xxx
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Tetrahedron Letters
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Efficient biosynthesis of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol
using a mutant short-chain dehydrogenase from Novosphingobium
aromaticivorans
Yafeng Zhou a,1, Qiaojun Zhao a,1, Shuyao Zhou a,1, Fandi Que , Guifang Li , Yunping Tang
a
a
a,b,
⇑
a
Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products, School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China
Zhejiang Changxing Pharmaceutical CO., LTD, Huzhou 313108, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
(R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol ((R)-CPEO) is an important chiral intermediate for antifun-
gal drug synthesis. (R)-CPEO can be produced from 2-chloro-1-(2, 4-dichlorophenyl) ethanone (CPE) via a
mutant short-chain dehydrogenase/reductase from Novosphingobium aromaticivorans (NaSDR). The Vmax
Received 15 February 2020
Revised 1 April 2020
Accepted 3 April 2020
Available online xxxx
À1
of a mutant NaSDR-G145A/I199L toward CPE (6.32 U mg ) was greater than that of wild-type NaSDR
À
(
2.58 U mg ). The Km of mutant NaSDR-G145A/I199L toward CPE (0.23 mM) was less than that of
wild-type NaSDR (0.38 mM), indicating that the substrate affinity of mutant NaSDR-G145A/I199L was
the greater of the two. Docking simulations were used to illustrate the mechanisms of the increased
enzyme activity of NaSDR-G145A/I199L; these showed that NaSDR-G145A/I199L presented a more effec-
tive docking posture than that of the wild-type enzyme. Further, NaSDR-G145A/I199L and glucose dehy-
drogenase (GDH) were used to transform 120 g/L CPE into (R)-CPEO. After 6 h, the conversion rate and
enantiomeric excess values were 99% and 99.95%, respectively. The present study provides a practical
method for high substrate loading of (R)-CPEO for industrial-scale applications.
Keywords:
Short-chain dehydrogenase/reductase
2
-Chloro-1-(2, 4-dichlorophenyl) ethenone
(R)-2-chloro-1-(2, 4-dichlorophenyl)
ethanol
Chiral drug intermediate
Antifungal drugs
Ó 2020 Elsevier Ltd. All rights reserved.
Introduction
methods have high selectivity, require mild conditions, and are
environmentally friendlier; thus, biological methods have
(
R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol ((R)-CPEO) is a key
attracted increasing attention for the asymmetric synthesis of chi-
ral drug intermediates [8–12]. Recently, reductases have been used
for the asymmetric reduction of pro-chiral ketones to their corre-
sponding chiral alcohols [13–16]. Many attempts have also been
made to convert 2-chloro-1-(2, 4-dichlorophenyl) ethanone (CPE)
into (R)-CPEO via reductase enzymes. For example, recombinant
ADH-T, ADH-CP, ADH-RS1 and ADH-A were screened for the asym-
metric reduction of CPE; these enzymes exhibited low enzymatic
activity toward CPE [2]. ADH-A was further chosen to scale-up of
the biosynthesis of (R)-CPEO. However, the substrate loading was
only 5.59 g/L in the reaction system, and was too low for industrial
application. Zhu et al [17] used recombinant PfADH from Pyrococ-
cus furiosus for the asymmetric reduction of 20 g/L CPE with glu-
cose dehydrogenase (GDH). After 24 h, (R)-CPEO was produced
with an enantiomeric excess (ee) value of 99%, and the final yield
was approximately 92%. Xu et al [6] used recombinant CgCR from
Candida glabrata for the asymmetric reduction of CPE (100 g/L);
the final yield was approximately 88% with an ee value >99%.
Recently, Shang et al [1] screened 50 enzymes from Scheffersomyces
stipites CBS 6045B for the production of (R)-CPEO and SsCR was
selected to catalyze the asymmetric hydrogenation of CPE. Based
chiral intermediate in the preparation of many antifungal drugs,
including (R)-miconazole, (R)-sertaconazole and (R)-econazole
1–3]. However, the effects of these racemates and optically-pure
enantiomers on microorganism survival are significantly different
2]. For example, (R)-miconazole was the most bioactive racemic
miconazole against the growth of a range of filamentous fungus
and yeasts [4], while (R)-sertaconazole bioactivity was approxi-
mately twice that of the racemic form against yeasts and fungi
[
[
[
5]. Furthermore, the use of (R)-miconazole or (R)-sertaconazol
reduces the risks of side effects and unspecific toxicities derived
from the non-active S-enantiomer.
(R)-CPEO can be produced by chemical and biological methods.
While most chemical methods are disadvantageous due to strict
reaction conditions and low stereoselectivity [6,7], biological
⇑
Correspondence author at: Zhejiang Provincial Engineering Technology Re-
search Center of Marine Biomedical Products, School of Food and Pharmacy,
Zhejiang Ocean University, Zhoushan 316022, China.
E-mail address: tangyunping1985@163.com (Y. Tang).
The authors shared first authorship.
1
https://doi.org/10.1016/j.tetlet.2020.151914
0
040-4039/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Y. Zhou, Q. Zhao, S. Zhou et al., Efficient biosynthesis of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol using a mutant short-chain
dehydrogenase from Novosphingobium aromaticivorans, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151914

2
Y. Zhou et al. / Tetrahedron Letters xxx (xxxx) xxx
on this catalytic system, 67.35 g/L CPE was converted into (R)-CPEO
with a 99% conversion rate and ee value >99%. However, the con-
centration of CPE mentioned above was not enough for indus-
trial-scale (R)-CPEO production. Therefore, more robust enzymes
for the production of high (R)-CPEO concentrations are necessary
for industrial-scale applications.
In our previous studies, a short-chain dehydrogenase/reductase
(
SDR) from Novosphingobium aromaticivorans (NaSDR) and its
mutant (NaSDR-G145A/I199L) have been used to prepare chiral
intermediates for (S)-fluoxetine [15] and orlistat [16] production.
In the present study, NaSDR and its variants were expressed and
used with GDH from B. subtilis to produce (R)-CPEO (Fig. 1). Recom-
binant NaSDR and its variants were purified to determine their
kinetic parameters, which were then compared. Docking simula-
tions were used to investigate the molecular mechanisms underly-
ing enzymes with greater activities. Finally, high concentrations of
Fig. 2. SDS-PAGE analysis of recombinant enzyme. M: Protein molecular weight
marker; Lane 1: Soluble proteins of NaSDR, Lane 2: Soluble proteins of NaSDR-
G145A), Lane 3: Soluble proteins of NaSDR-I199L, Lane 4: Soluble proteins of
NaSDR-G145A/I199L, Lane 5: Purified NaSDR; Lane 6: Purified NaSDR-G145A), Lane
(R)-CPEO were produced via the enantioselective reduction of CPE
120 g/L) by mutant NaSDR-G145A/I199L with GDH.
(
7: Purified NaSDR-I199L, Lane 8: Purified NaSDR-G145A/I199L. Kinetic parameters of
enzymes.
Results and discussion
Construction, expression, and purification of NaSDR and its variants
binding energy of the mutant was stronger, suggesting that the site
of the mutation was increased the catalytic efficiency of NaSDR.
Moreover, the distance between CPE and the active hydrogen of
NADH satisfied the conditions of the catalytic reaction, which
strengthens the reliability of molecular docking results [22].
Recombinant proteins are often expressed in insoluble inclusion
bodies or as soluble proteins in E. coli [18]. The modification of pro-
tein composition can lead to decreased soluble protein expression
[
19,20]. The constructed NaSDR and its variants were expressed,
and were purified by Ni-affinity chromatography. Crude and puri-
fied enzymes were analyzed by SDS-PAGE (Fig. 2). The bands on
SDS-PAGE gels indicate that the proteins ~28 kDa were NaSDR
and its variants, and there were no obvious differences in the levels
of NaSDR and its variants, suggesting that one or two amino acid
mutations did not affect the protein expression level of target
enzymes.
To evaluate the effect of mutations on enzyme activities toward
CPE, the kinetic parameters of wild-type NaSDR and its variants
were investigated against CPE (Table 1). The Vmax of NaSDR-
G145A and NaSDR-G145A/I199L were 2.33 and 2.45-fold greater,
respectively, than the Vmax of NaSDR. In addition, the Vmax of
NaSDR-I199L was little higher than the wild-type. Furthermore,
the NaSDR-G145A/I199L variant exhibited the highest Kcat/Km val-
ues against those of NaSDR, NaSDR-G145A, and NaSDR-I199L. So,
recombinant NaSDR-G145A/I199L was chosen for further study.
To investigate the mechanisms underlying greater enzyme
activity, the binding conformation of CPE with the substrate-bind-
ing cavities of NaSDR and NaSDR-G145A/I199L were modelled
Elucidating the mechanism of the enhanced activity
Docking simulations analysis showed that hydrophobic interac-
tions play a vital role in the recognition of CPE by NaSDR. The bind-
ing pattern was analyzed (Fig.4A-B) and the binding conformation
of CPE with NaSDR and with NaSDR-G145A/I199Lwere almost the
same. Both could theoretically catalyze the production of alcohol
products with R configuration, which is consistent with the cat-
alytic results. Hydrophobic residues, including Ile89, Val144,
Met195, Leu196, Val214, and Phe260, were involved in the
hydrophobic interaction. On one hand, these powerful hydropho-
bic amino acid residues provide a strong hydrophobic cavity for
Table 1
Kinetic parameters of NaSDR and its mutants towards 2-chloro-1-(2, 4-dichlorophe-
nyl) ethanone (CPE).
Enzyme
V
max
K
m
k
(s
cat
À1
k
cat/K
m
À1
(s ÁmMÀ1
À1
(
U mg
)
(mM)
)
)
NaSDR
2.58
6.01
3.28
6.32
0.38
0.30
0.33
0.23
2.00
3.97
2.72
3.51
5.26
13.23
8.24
(Fig. 3). CPE was bound in the strongly hydrophobic cavity of the
NaSDR-G145A
NaSDR-I199L
NaSDR-G145A/I199L
active site of the enzyme and formed strong hydrophobic interac-
tions with the surrounding [21]. The binding energies of CPE with
the substrate-binding cavities of NaSDR and NaSDR-G145A/I199L
were À5.28 kcal/mol and À5.69 kcal/mol, respectively. Thus, the
15.27
Docking simulations results of NaSDR and NaSDR-G145A/I199L.
Fig. 1. Schematic reduction of 2-chloro-1-(2, 4-dichlorophenyl) ethanone (CPE) into (R) À2-chloro-1-(2, 4-dichlorophenyl) ethanol ((R)-CPEO) with NaSDR-G145A/I199L
coupled with glucose dehydrogenase (GDH).
Please cite this article as: Y. Zhou, Q. Zhao, S. Zhou et al., Efficient biosynthesis of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol using a mutant short-chain
dehydrogenase from Novosphingobium aromaticivorans, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151914

Y. Zhou et al. / Tetrahedron Letters xxx (xxxx) xxx
3
Fig. 3. Illustration of the substrate-binding cavities of NaSDR (A) and NaSDR-G145A/I199L (B) with docked substrate 2-chloro-1-(2, 4-dichlorophenyl) ethanone (CPE). Red
stick represents CPE, yellow stick represents NADH, brown represents hydrophobic surface of protein, and blue represents hydrophilic surface of protein.
Fig. 4. Binding conformation and catalytic distance of 2-chloro-1-(2, 4-dichlorophenyl) ethanone (CPE) at the active site of NaSDR. (A) Binding conformation of CPE at the
active site of NaSDR. (B) Binding conformation of CPE at the active site of NaSDR-G145A/I199L. (C) Catalytic distance of CPE at the active site of NaSDR (a) and NaSDR-G145A/
I199L (b). Purple stick represents CPE, yellow stick represents NADH, blue dotted line represents the hydrogen bond.
the catalytic reduction of CPE by NADH, which is conducive to a
smooth catalytic reaction. On the other hand, Ser143 and Tyr156
are hydrogen bond donors that can form hydrogen bonds with
the carbonyl oxygen atoms of CPE. Based on molecular docking
analysis, CPE’s conformation at the enzyme active site is immobi-
lized, especially in the location of the carbonyl group, which can
easily be attacked by the reactive hydrogen atoms of NADH. This
binding pattern was observed for CPE with for both NaSDR and
NaSDR-G145A/I199L, and is consistent with the binding pattern
of most ketoreductases with their substrates [13,23,24].
Please cite this article as: Y. Zhou, Q. Zhao, S. Zhou et al., Efficient biosynthesis of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol using a mutant short-chain
dehydrogenase from Novosphingobium aromaticivorans, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151914

4
Y. Zhou et al. / Tetrahedron Letters xxx (xxxx) xxx
Table 2
reaction by using mutant NaSDR-G145A/I199L enzyme combined
with GDH. The final (R)-CPEO yield was approximately 92.4%. In
subsequent studies, a series of large-scale production and reaction
conditions optimization of this enzyme will be adopted to bolster
the potential for industrial-scale (R)-CPEO production.
Comparison of production of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol ((R)-CPEO)
using different enzyme.
Enzyme
Substrate
loading
Time
(h)
Yield
(%)
ee (%)
Reference
(g/L)
SsCR
CgCR
67.4
100
120.0
6.0
12.0
6.0
99
88
92.4
99 (R)
99 (R)
99.95 (R)
[1]
[6]
This study
Declaration of Competing Interest
NaSDR-G145A/I199L
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Substitutions of wild-type Gly145 with Ala increased the
hydrophobicity of the substrate-binding pocket of the mutant
NaSDR-G145A/I199L. Substitutions of Ile199 with Leu produced
almost no change, but the structural orientation of the side chain
of amino acid residues changed. The mutation resulted in a lower
probability collision of CPE with Met195, Leu196, and Leu199,
thereby increasing the affinity between CPE and the mutant
enzyme (+0.41 kcal/mol binding energy increase vs wild-type
enzyme) [25]. Based on the analysis above, it was concluded that
the NaSDR-G145A/I199L mutant could have a greater affinity for
CPE than NaSDR, and this conjecture was consistent with the
kinetic parameters. Moreover, to investigate the primary difference
in the catalytic efficiency between NaSDR and NaSDR-G145A/I199L
toward CPE, the catalytic distance was analyzed between the car-
bonyl carbon atom of CPE and reactive hydrogen atoms of NADH
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (grant No. 41806153), the Zhejiang
province postdoctoral program (211410), the Zhejiang Xin-miao
Talents Program (grant no. 2018R411052).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2020.151914.
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(
[
[
(
5
9
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2
0
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]D = À57.2 (c 1, CHCl
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), and the result was consistent with
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[
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with GDH. These features offer an excellent opportunity to
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Conclusions
In conclusion, 120 g/L of CPE was converted into (R)-CPEO with
the conversion rate of 99% and with the ee value of 99.95% after 6 h
Please cite this article as: Y. Zhou, Q. Zhao, S. Zhou et al., Efficient biosynthesis of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol using a mutant short-chain
dehydrogenase from Novosphingobium aromaticivorans, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151914