Development of an Enzymatic Process for the Synthesis of (S)-2-Chloro-1-(2,4-dichlorophenyl) Ethanol

Article abstract of DOI:10.1021/acs.oprd.9b00037

(S)-2-Chloro-1-(2,4-dichlorophenyl) ethanol (3) is a chiral intermediate in the synthesis of luliconazole ((R)-E-1). Here, we report a novel biopreparation of 3 by bioreduction of 2-chloro-1-(2,4-dichlorophenyl) ethanone (2) using recombinant Escherichia

Full text of DOI:10.1021/acs.oprd.9b00037

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Full Paper
Development of an enzymatic process for the
synthesis of (S)-2-chloro-1-(2,4-dichlorophenyl) ethanol
Teng-Yun Wei, Jia-Wei Tang, Guo-Wei Ni, Hong-Yi Wang, Dong Yi, Fu-Li Zhang, and Shao-Xin Chen
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00037 • Publication Date (Web): 16 Aug 2019
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Development of an enzymatic process for the synthesis of (S)-2-chloro-1-(2,4-
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dichlorophenyl) ethanol
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Teng-Yun Wei1#, Jia-Wei Tang1#, Guo-Wei Ni1, Hong-Yi Wang1, Dong Yi2*, Fu-Li
Zhang1*, Shao-Xin Chen1*
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1Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical
Industry, 285 Gebaini Road, Pudong, Shanghai 201203, China
2 Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, Felix-
Hausdorff-Str.4, D-17487 Greifswald, Germany
Email: Prof. Dr. S.-X. Chen sxzlb@263.net, Prof. Dr. F.-L. Zhang
zhangfuli1@sinopharm.com, Dr. D. Yi charlesdyi@yahoo.com
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TOC figure
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ABSTRACT
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(S)-2-chloro-1-(2,4-dichlorophenyl) ethanol (3) is a chiral intermediate for synthesis of
Luliconazole ((R)-E-1). Here, we report a novel biopreparation of 3 by bioreduction of
2-chloro-1-(2,4-dichlorophenyl) ethanone (2) using recombinant Escherichia coli
expressing LK08, a ketoreductase mutant from Lactobacillus kefiri, as biocatalyst. The
reaction conditions for the biotransformation including pH, temperature, concentration
of isopropanol and NADP+, as well as the amount of recombinant E. coli cells, were
optimized to improve the process productivity. When the enzymatic process was carried
out on a 300 g scale under the optimized conditions, the ketone 2 was fully converted
to chiral alcohol 3 with a product ee value of >99%. Furthermore, the 3 was isolated
and used to chemically synthesize Luliconazole with 38% yield and 99% ee. This study
presents an efficient and cost-effective chemoenzymatic process for the production of
(R)-E-1.
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Keywords: (S)-2-chloro-1-(2,4-dichlorophenyl) ethanol, Luliconazole, ketoreductase,
biocatalysis, Lactobacillus kefiri
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1. INTRODUCTION
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Luliconazole, chemically known as (2E)-2-[(4R)-4-(2,4-dichlorophenyl)-1,3-dithiolan-
2-ylidene] (1H-imidazol-1-yl) acetonitrile), was approved and indicated by U. S. Food
and Drug Administration (FDA) in 2013 for the topical treatment of interdigital tinea
pedis, tinea cruris, and tinea corporis caused by the organisms Trichophyton rubrum
and Epidermophyton floccosum.1
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(S)-2-Chloro-1-(2,4-dichlorophenyl) ethanol (3) is a key intermediate in the
preparation of Luliconazole (Scheme 1, step 2 and step 3). Therefore, the production of
intermediate 3 with a high chiral purity is a key step in the synthesis route of
Luliconazole.2 Recently, three synthetic methods for obtaining the intermediate 3 have
been reported, including chemical reduction, enzymatic resolution, and bioreduction.
For the chemical reduction of 2, the BH3-Corey-Bakshi-Shibata catalyst (BH3-CBS)
was used to transform ketone 2 to alcohol 3 with ee 98%.3 However, controlling the
chiral purity of Luliconazole using this method becomes more difficult. Besides, the
high cost of BH3-CBS and requirement of strict reaction condition under anhydrous and
oxygen-free conditions also limit its industrial application. The search for biocatalytic
methods for the synthesis of Luliconazole has attracted increasing attention owing to
the various advantages afforded by biocatalysis, such as high stereoselectivity and
environment friendliness.4 Recently, a method that involves chemoenzymatic synthesis
mediated by lipases has been reported.5 The ee value of the product 3 can be up to 99%,
however the conversion is 50% as the process is a kinetic resolution of a racemate,
which leads a waste of substrate if without a recycling step. In addition, the synthesis
of the substrate 2 still requires chemical steps including reduction and acetylation.
Therefore, this enzymatic resolution approach is limited by the complicated process
involved and the resulting low yield.
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Scheme 1. A schematic diagram of the synthesis of Luliconazole.
Ketoreductases (KREDs) catalyze the asymmetric reduction of ketones to chiral
alcohols in the presence of the cofactor NAD(P)H.6 With the increasing use of KREDs,
an increasing number of chiral intermediates are being synthesized by KREDs instead
of chemical catalysts.7 For example, Guo et al. reported an enzymatic process to
synthesize (S)-2-chloro-1-(3,4-difluorophenyl) ethanol, the chiral intermediate of
Ticagrelor by KRED KR01 from Leifsonia sp. strain S749.8 Moreover, three KREDs,
i.e. KtCR from Kluyveromyces thermotolerans, DhCR from Debaryomyces hansenii,
and CgKR1 from Candida glabrata, were reported for their abilities to reduce 2 to 3
enantioselectivity with >99% ee. However, these three KREDs can only accept the
substrate at low concentrations (less than 45 g/L) with unsatisfying conversion rates
(38%–89%), which restricts their industrial application.9 Therefore, it is necessary to
search for novel KREDs with high enantioselectivity towards 2 at a higher substrate
concentration.
In this study, we report a novel KRED mutant, LK08 from L. kefiri, which can reduce
ketone 2 to chiral alcohol 3 at a high substrate concentration with excellent activity and
enantioselectivity (Scheme 1). An efficient bioreduction process for production of
chiral alcohol 3 was established and further used for the synthesis of Luliconazole.
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2. RESULTS AND DISCUSSION
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2.1. Screening of KREDs for bioreduction of 2.
In order to obtain a suitable KRED for the enzymatic synthesis of 3, we screened the
enzyme library preserved in our laboratory and found seven recombinant KREDs
showed enzyme activity to the substrate 2. For preparing the biocatalysts, these seven
KRED genes were inserted into pET-28a(+) vector and over-expressed in E. coli
BL21(DE3), respectively. These KREDs in the form of whole recombinant E. coli cells
were used for the conversion of 2 to 3. As shown in Table 1, these KREDs displayed
different activity and enantioselectivity towards 2. Three enzymes, KR02, KR05 and
KR08, generated (R)-[2-chloro-1-(2,4-dichlorophenyl)] ethanol with >99% ee. Only
LK08 was able to reduce 2 to 3 with high (S)-enantioselectivity and conversion.
Therefore, LK08 was chosen for further studies.
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Table 1. Screening of KREDs for bioreduction of 2.
Enzyme
KR01a
KR02b
KR05b
KR07b
KR08a
LKCRa
LK08a
Strain
Genbank access
AB213459
WP_023468191.1
KC342003
XM_459695
AB190261
AY267012.1
-
Conversion (%)c
ee (%)d
66 (S)
Leifsonia sp.S749
1.4
15.4
16.6
8.7
Exiguobacterium sp.F42
Chryseobacterium sp.CA49
Debaryomyces hansenii CBS767
Corynebacterium sp. ST-10
Lactobacillus kefiri
>99 (R)
>99 (R)
71 (S)
0.6
>99 (R)
90 (S)
17.7
99.9
Lactobacillus kefiri
>99 (S)
aReaction conditions: 10 g/L ketone 2, 10 g/L wet cells, 30% isopropanol (v/v),
phosphate buffer solution (PBS) (100 mM, pH 7.0) in 2 mL reaction system for 1.5 h
at 25 °C and 220 rpm. bReaction conditions: 10 g/L ketone 2, 10 g/L wet cells, 20 g/L
glucose, PBS buffer (100 mM, pH 7.0) in 2 mL reaction system for 1.5 h at 25 °C and
220 rpm. cMeasured by HPLC. dMeasured by chiral HPLC.
LK08 is a random mutant of LKCR. The protein sequence alignment of LK08 and
LKCR shows that LK08 contains mutations at 5 positions: E67D, A94T, F147L, L199H,
and A202L (Figure 1a). In order to figure out which amino acids play the key role in
improving enzyme activity or/and stereoselectivity, we performed single-site mutation
on these positions. As shown in Table 2, the mutation of L199H resulted in a significant
increase in stereoselectivity, while the other mutations did not improve the
stereoselectivity. Both the mutants L199H and A202L have an effect on the increase in
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activity. For the mutation site with positive action, we made a combination mutation
for L199H and A202L, but the results showed that although the activity was
significantly increased, which was the same as LK08, the stereoselectivity was not
improved. We suspect that the increase in activity and stereoselectivity of LK08 is the
result of several sites working together.
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Table 2. Comparison of different mutant of LKCR for bioreduction of 2.
Enzyme
LKCR
mutation position
Conversion (%)
9.15 ± 0.64
99 ± 1.13
ee (%)
93 (S)
>99 (S)
89 (S)
71 (S)
82 (S)
>99 (S)
93 (S)
LK08
E67D, A94T, F147L, L199H, A202L
LKCR-1
LKCR-2
LKCR-3
LKCR-4
LKCR-5
E67D
3.7 ± 1.13
A94T
5.7 ± 0.14
F147L
L199H
A202L
7.7 ± 1.56
76.9 ± 1.80
87.6 ± 1.20
LKCR-6
L199H, A202L
99 ± 0.92
92 (S)
Reaction conditions: 10 g/L ketone 2, 10 g/L wet cells, 30% isopropanol (v/v),
phosphate buffer solution (PBS) (100 mM, pH 7.0) in 2 mL reaction system for 1.5 h
at 25 °C and 220 rpm.
To evaluate the effects of these mutations to the substrate tolerance and
stereoselectivity of LK08, we simulated the protein structure of LK08 using the webtool
SWISS-MODEL (https://swissmodel.expasy.org/) with the crystal structure of LKCR
(PDB entry 4RF2) as template.6 The obtained structure was aligned to the crystal
structure of an alcohol dehydrogenase from Lactobacillus brevis (PDB entry 1ZK4)
which contains an acetophenone ligand, to identify the substrate binding pocket of
LK08. The structure of 2 was aligned to that of acetophenone. An energy minimization
of the whole protein structure with 2 was carried out by YASARA software. The final
simulated structure is shown in Figure 1b. In this simulated activity pocket, it is clearly
shown that the residue 94T likely forms a hydrogen bond with the methylene chlorine
together with Y156. 199H and Y190 could offer hydrogen linkages to the para- and
ortho-chlorine on the benzyl ring of 2, respectively. 202L might offer a better non-polar
pocket then its wild-type to accept the benzyl ring of 2. As a result, the ketone 2 is
trapped in the active pocket, which can explain the great improvement of both activity
and stereoselectivity. E67D and F147L locate out of substrate binding pocket but might
still have effect to altering the corresponding secondary structure of the active pocket.
However, according to the activity screening result described above, single-site
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mutation at these two sites did not present any improvement to both conversion rate
and stereoselectivity. Therefore, these two mutations have no contribution to the
improved substrate binding. In conclusion, the mutation in the active pocket create
additional hydrogen bonds and non-polar interaction between the amino acid residues
and substrate which stabilize the binding of 2 in the activity pocket, thus NADPH can
attract 2 on the re-side of carbonyl. Therefore, the mutant LK08 displays high activity
towards 2 with excellent S-enantioselectivity.
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Figure 1. (a) Alignment of LK08 and LKCR protein sequence. (b) Model of the
acceptor binding pocket based on the X-ray crystal structure of LKCR (PDB entry
4RF2). The dotted red line indicates the potential hydrogen bonds for substrate binding.
Graphic was created with PyMOL
2.2. Optimization of pH and temperature for bioproduction of chiral alcohol 3.
The effect of pH on LK08 activity was measured at pH values ranging from 3.0 to 9.0.
As shown in Figure 2a, the enzymatic activity decreased when the pH was above 7.0
or below 5.0, and no significant changes were observed from pH 5.0 to 7.0. The LK08
showed maximum activity at pH 6.0.
(c)
(b)
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Time (h)
18
pH
T (°C)
Figure 2. (a) Effects of pH on the activity of LK08. Reaction conditions: the reaction
was performed with 10 g/L ketone 2, 10 g/L wet cells, 30% isopropanol (v/v) in 2 mL
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reaction system for 1 h at 25 °C and 220 rpm in different pH buffer solutions, including
100 mM sodium citrate (pH 3, 4, or 5), 100 mM potassium phosphate (pH 6 or 7), and
100 mM Tris-HCl buffers (pH 8 or 9). (b) Effects of temperature on the activity of
LK08. Reaction conditions: the temperature study was performed with 10 g/L ketone
2, 10 g/L wet cells, 30% isopropanol (v/v), and PBS buffer (100 mM, pH 6.0) in 2 mL
reaction system for 1 h at 220 rpm. (c) Effect of temperature on enzyme stability.
aThe initial LK08 activity of the wet cells was defined as 100%. Reaction conditions:
the wet cells were incubated at temperatures of 25°C, 35°C, and 45°C for different
periods.
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To test the effect of temperature on LK08 activity, reactions were performed at
various temperatures (25°C–55°C). As shown in Figure 2b, temperature greatly
influenced on the enzymatic activity. The optimum temperature for LK08 activity
towards substrate 2 was 35°C. When the temperature was increased from 25 to 35, the
LK08 activity increased from 70% to 98% and it stayed relatively high (>92%) at
temperatures ranging from 35°C to 45°C, however, it sharply decreased when the
temperature was above 45°C. In addition, the influence of temperature on the stability
of enzyme activity should be considered.10 The LK08 was kept at temperature of 25°C,
35°C, 45°C for 18 h, respectively. The results are shown in Figure 2c. We found that
LK08 is sensitive to higher temperatures. When the temperature was up to 45°C, the
activity of LK08 dropped to 6% after 12 h and no activity was observed after 18 h.
However, activities of 85% and 95% were observed at 35°C and 25°C for 18 h,
respectively. Considering the activity and stability, 35°C was selected as the optimum
temperature for the further enzymatic reactions.
2.3. Optimization of the reaction parameters for bioproduction of chiral alcohol
3.
A competitive enzymatic process must exhibit a high volumetric productivity mainly
decided by the enzyme reactivity as well as the high substrate loading.11 To investigate
the effect of substrate concentration, the biotransformation was carried out at 100–300
g/L of 2 with E. coli/pET-28a(+)-LK08 wet cells. As shown in Figure 3, 100 g/L of 2
could be completely converted to 3 with a high conversion of 99%, and the conversion
decreased to 68% when the substrate concentration was 300 g/L. To enhance the
conversion efficiency at high substrate concentrations, optimization of crucial reaction
parameters was further performed.
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Figure 3. Effect of ketone 2 concentration on conversion by E. coli/pET-28a(+)-LK08
wet cells. Reaction conditions: various concentrations of substrate, 300 g/L wet cells
(The wet cells volume is not counted in the reaction volume in the paper), 30%
isopropanol (v/v), PBS buffer (100 mM, pH 6.0) in 2 mL reaction system for 24 hours
at 35°C and 220 rpm.
2.3.1. Optimization of concentration of isopropanol for bioproduction of chiral
alcohol 3.
Cofactor regeneration is an essential step for bioreduction reaction.12 Isopropanol has
been well used as a substrate oxidized to acetone by LK08 for in situ regeneration of
NADPH from NADP+.13 For a reversible reaction, driving into the desired direction is
necessary. Thus, excess isopropanol is required to drive the equilibrium into the
forward reaction. On the other hand, the substrate 2 is saturated in the aqueous reaction
system when the concentration is more than 100 g/L. Isopropanol in high concentration
can take a role in improving the solubility of the substrate. As shown in Figure 4, there
was an obvious increase in conversion when the concentration of isopropanol increased
from 20% to 50%. The conversion increased to 98% when 70% isopropanol was added.
In addition, considering that the catalyst may be denaturated or inhibited by the high
concentration of isopropanol, we chose 70% isopropanol for the reaction, which is the
lowest isopropanol concentration that can achieve the highest reaction rate.
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20 30 40 50 60 70 80 90
Isopropanol (%)
Figure 4. Effect of isopropanol concentration on conversion by E. coli/pET-28a (+)-
LK08 wet cells. Reaction conditions: 300 g/L ketone 2, 300 g/L wet cells, PBS buffer
(100 mM, pH 6.0), and various concentrations of isopropanol in 2 mL reaction system
for 24 hour at 35°C and 220 rpm.
2.3.2. Optimization of biocatalyst loading for bioproduction of chiral alcohol 3.
Increasing the biocatalyst loading can contribute to the improvement of conversion and
conversion rate but inevitably increases catalyst consumption and complicates the post-
treatment process.14 In general, under the premise of a near 100% conversion, the
biocatalyst loading will be reduced as much as possible. Thus, the time curve of
biocatalyst loading ranging from 100 g/L to 300 g/L was studied. As shown in Figure
5, as the biocatalyst loading increases, the rate of conversion increases. When the
biocatalyst loading is 100 g/L, the final conversion is only 80%. Increasing the catalyst
concentration to 300 g/L allowed conversion to reach >99% within 28 h. Taking into
account the reaction time and the usage of biocatalyst, we finally chose 300 g/L wet
cells as the optimized concentration.
100
80
60
100 g/L wet cells
200 g/L wet cells
40
300 g/L wet cells
20
0
400 g/L wet cells
4
8
12 16 20 24 28 32 36 40
Time (h)
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Figure 5. Effect of biocatalyst loading on conversion by E.coli/pET-28a (+)-LK08
wet cells. Reaction conditions: 300 g/L ketone 2, 70% isopropanol (v/v), PBS buffer
(100 mM, pH 6.0), and various concentrations of wet cells in 2 mL reaction system at
35°C and 220 rpm.
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2.3.3. Optimization of the concentration of NADP+ for bioproduction of chiral
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alcohol 3.
In the bioreduction process mediated by ketoreductases, NADPH is essential as a
hydrogen donor.15 Noey et al. reported that NADPH is the cofactor of LKCR from L.
kefir.6 Compared with the purified enzyme as a catalyst, NADPH in cells can be used
to provide hydrogen for the reaction by wet cells catalysis. For this reason, the
conversion efficiency was still high without the addition of NADP+ in the previous
condition optimization. However, considering the fact that sufficient amount of
NADPH can increase the reaction rate, the concentration of NADP+ is optimized. As
shown in Figure 6, compared with the absence of NADP+, the addition of NADP+ has
an impact on the reaction rate and caused a decrease in the time required for the
complete reaction. When 0.1 g/L NADP+ was added, the conversion reached >99%
within 20 h.
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0 g/L NADP+
0.01 g/L NADP+
0.05 g/L NADP+
0.1 g/L NADP+
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0.5 g/L NADP+
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Time (h)
Figure 6. Effect of NADP+ concentration on conversion by E. coli/pET-28a (+)-LK08
wet cells. Reaction conditions: 300 g/L ketone 2, 70% isopropanol (v/v), PBS buffer
(100 mM, pH 6.0), 300 g/L concentration of wet cells and various concentrations of
NADP+ in 2 mL reaction system at 35°C and 220 rpm.
2.4. Scale-up of the reaction.
The reaction was carried out on a 300 g scale under the optimized conditions of 300
g/L substrate 2, 300 g/L wet cells (approximately 16.7 g/L of LK08, Figure S3), 0.1
g/L NADP+, and 70% (v/v) IPA at pH 6.0 PBS buffer (100 mM), and 35 °C. The time
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curve for the bioreduction of ketone 2 to alcohol 3 is shown in Figure 7. The conversion
reached >99% within 30 h. After completion of the reaction, the isopropanol and by-
product of acetone were removed by evaporation under reduced pressure, and the
product was separated by extraction. Upon isolation of desired product 3, the yield was
94.3% with a HPLC purity of 99%. Excellent enantioselectivity (>99% ee) was
achieved without any further purification. Overall, the production process of our system
met the basic requirements of an economically viable process for industrial-scale
production of optically pure alcohol 3.
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Figure 7. Time curve for the bioreduction of ketone 2 to alcohol 3.
Reaction conditions: 300 g ketone 2, 700 mL isopropanol, 0.1 g NADP+ and PBS buffer
(100 mM, pH 6.0) were added to 1 L in a 3 L reaction vessel, 300 g of wet cells was
added to the reaction system and the reaction was carried out at 35°C and 220 rpm.
2.5. Synthesis of Luliconazole
The chiral alcohol 3 was prepared under the optimized conditions described above with
a conversion of >99% and product ee of >99%. After crystallization in heptane, the
final chemical purity of 3 reached 99%. Then, alcohol 3 was treated with
methanesulphonyl chloride to obtain (S)-2-chloro-1-(2,4-dichlorophenyl)-2-
methanesulfonate (4) in a 93.6% yield (Scheme 1). Finally, 4 was subjected to react
with 1-cyanomethylimidazole (5) in the presence of CS2 and KOH in DMSO. In this
case, a mixture of (R)-E-1 and (R)-Z-1 was obtained in a ratio of 65:35 (Figure S1). It
could be purified through salt formation with hydrochloric acid in acetone to elevate
the ratio of (R)-E-1 to (R)-Z-1 to 99.5:0.5 (Figure S2). Recrystallization in acetone was
carried out to afford the highly pure (R)-E-1 without traces of (R)-Z-1. (R)-E-1
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hydrochloride salt was then transformed to Luliconazole with a chiral purity of 99%.
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3. CONCLUSION
In this study, we developed a novel approach involving a chemoenzymatic process
mediated by a ketoreductase to synthesize Luliconazole. The LK08, a mutant of LKCR,
was discovered to have an excellent enantioselectivity for ketone 2. We optimized the
reaction conditions with LK08 and successfully realized the preparation of 3 on a 300
g scale with >99% conversion and >99% ee. This study presents an efficient and cost-
effective chemoenzymatic process for the production of (R)-E-1, which can be a
potential biosynthetic step for the industrial-scale preparation of Luliconazole.
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4. EXPERIMENTAL SECTION
4.1. Materials and methods.
Ketone 2 was purchased from Energy Chemical (China). Glucose and isopropanol were
purchased from Sinopharm Chemical Reagent Co, Ltd (China). The other chemicals
were obtained commercially in analytical grade. The ClonExpress Entry One Step
cloning kit used for molecular cloning was purchased from Vazyme Biotech (Nanjing,
China). The other enzymes were purchased from Takara (Dalian, China).
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The H NMR and C NMR spectra were recorded using a Bruker Arance III 400
MHz spectrometer with CDCl3 solvent. The 1H NMR chemical shift data were reported
on δ (ppm) relative to tetramethylsilane as the internal standard, and the 13C NMR
chemical shift data were reported on δ (ppm) relative to CDCl3.
The enzymatic reaction was monitored by HPLC, and the ee was determined by chiral
HPLC using the conditions listed below. The HPLC analyses were recorded on a
Dionex UItiMate 3000 HPLC instrument.
4.2. Cloning and expression.
The genes of KR01, KR02, KR05, KR07, KR08, LKCR and LK08 were cloned into a
pET-28a(+) vector using the ClonExpress Entry One Step cloning kit. The resulting
plasmids were transformed into E. coli BL21 (DE3) cells which was cultured at 37°C
in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) containing 50 μg/mL
kanamycin. When the optical density at 600 nm of the culture reached 0.6-0.8, 0.1 mM
IPTG was added to the fermentation medium. The cultivation was continued at 25°C
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overnight. The cells were then harvested by centrifugation (4000×g, 4°C, 30 min) and
stored at −20°C.
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The single-site mutations of LKCR were performed by overlap extension PCR.
Primers were listed in Table S1.
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4.3. General protocol for the synthesis of 3.
The reaction mixture (2 mL) was prepared by dissolving substrate 2 (0.6 g), wet cells
of LK08 (0.6 g), and 70% isopropanol (v/v) in potassium phosphate buffer (100 mM,
pH 6.0). The reaction solution was shaken at 220 rpm (35°C) for 24 h. Subsequently,
the reaction was extracted with CH2Cl2 (3 × 1 mL). The organic layers were combined
and dried over anhydrous Na2SO4. The conversion and ee value were determined by
HPLC.
The product alcohol 3 and substrate ketone 2 in the reaction solution was analyzed
using a BDS C18 column (4.6 × 250 mm, 5 μm, Elite, Dalian), with a mobile phase of
50% water and 50% acetonitrile (v/v) at 210 nm. The retention times were 10.7 min (3)
and 15.4 min (2).
Chiral HPLC was performed using a CHIRALPAK IA (4.6 × 250 mm, 5 μm,
DAICEL, Shanghai) column with a mobile phase of 90% hexane (0.1% trifluoroacetic
acid + 0.1% diethylamine) and 10% isopropanol. The product was detected at 260 nm.
The retention times of enantiomers were 5.9 min and 6.7 min (3).
4.4. Scale-up of the KRED reaction for the synthesis of (S)-2-chloro-1-(2,4-
dichlorophenyl) ethanol (3).
The reaction was performed on a 300 g scale. 700 mL of isopropanol, 300 g of substrate
ketone 2, 0.1 g NADP+ and phosphate buffer (100 mM, pH 6.0) were added to 1 L in a
3 L reaction vessel, then, 300 g of wet cells was added to the reaction system. The
reaction was carried out at 35 °C and was agitated by overhead stirring for 30 h.
Subsequently, the isopropanol was removed by evaporation under reduced pressure.
The reaction mixture was extracted with CH2Cl2 (3 × 1 L), then centrifuged to remove
the proteins, and the CH2Cl2 layers were combined. The organic layers were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. Finally, 285.4 g crude
product was obtained as a pale yellow solid with the yield of 94.3% (HPLC purity 99%,
ee >99%). [α]20 589= + 57.3° (c 2.50 in CHCl3). Spectral data: 1H NMR (600 MHz,
CDCl3) δ 7.58 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.31 (dd, J = 8.4, 2.1 Hz,
1H), 5.26 (dt, J = 8.4, 3.2 Hz, 1H), 3.87 (dd, J = 11.3, 2.8 Hz, 1H), 3.52 (dd, J = 11.3,
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8.5 Hz, 1H), 2.81 (d, J = 3.6 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 136.00, 134.75,
132.60, 129.44, 128.68, 127.72, 70.41, 49.31. Anal. calcd for C8H7Cl3O: C 42.61, H
3.13, Cl 47.17; Found C 42.69, H 3.26, Cl 47.22.
4.5. Synthesis of Luliconazole [(R)-E-1].
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Alcohol 3 (30 g, 133 mmol) was dissolved in CH2Cl2 (300 mL). Triethylamine (27 g,
267 mmol) and methanesulphonyl chloride (23.1 g, 202 mmol) were added to the
mixture with stirring at 0 °C for 1 h, and then the mixture was stirred for further 6 h at
room temperature. After that, distilled water (200 mL) was added which separated the
organic phase, then the water phase was extracted with CH2Cl2 (2 × 100 mL). Then,
the combined organic phases were washed with saturated aqueous NaHCO3 solution
(200 mL). The organic phase was dried with anhydrous Na2SO4 and filtered. The
solvent was evaporated under reduced pressure to give 4 (0.607 g, 2.0 mmol), a yellow
solid (37.9 g, 93.6%) of chiral purity of 99% ee and without purification for the next
step.
CS2 was added to the solution of 1-cyanomethylimidazole (5) (8 g, 74.7 mmol) and
KOH (8.4 g, 149.7 mmol) in DMSO (80 mL). The mixture was stirred for 2 h at room
temperature. Then the solution of 4 (11.3 g, 37.2 mmol) in DMSO (20 mL) was added
slowly at 4°C and the mixture was stirred for 24 h at room temperature. After that, cold
water (100 mL) was added and the mixture was stirred for 30 min. The mixture was
extracted with ethyl acetate (2 × 100 mL). The organic phase was washed with water
(2 × 100 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced
pressure to get the crude product which was dissolved in ethyl acetate (60 mL) and
salified by using 2N HCl in ethyl acetate (20 mL) to produce Luliconazole
hydrochloride. Then it was recrystallized in acetone (50 mL) to collect the product.
After that, it was dissociated with saturated aqueous solution of NaHCO3 in H2O (40
mL) and extracted with CH2Cl2 (40 mL). The aqueous phase was then extracted with
CH2Cl2 (2 × 20 mL). Finally, the combined the organic phase was dried and
concentrated to get Luliconazole (5.0 g, 38%), 99% for HPLC purity and 99% of ee
value. Spectral data: 1H NMR (600 MHz, CDCl3) δ 7.63 (s, 1H), 7.60 (d, J = 8.4 Hz,
1H), 7.49 (d, J = 2.1 Hz, 1H), 7.35 (dd, J = 8.4, 2.1 Hz, 1H), 7.18 (s, 1H), 7.06 (t, J =
1.2 Hz, 1H), 5.70 (dd, J = 7.3, 5.3 Hz, 1H), 3.89 (dd, J = 12.1, 5.2 Hz, 1H), 3.66 (dd, J
= 12.0, 7.4 Hz, 1H).13C NMR (151 MHz, CDCl3) δ 164.55, 137.06, 135.83, 134.19,
131.31, 130.37, 130.20, 128.99, 127.98, 119.12, 114.65, 93.81, 55.06, 44.19. HRMS
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calcd. for C14H10Cl2N3S2 [M+H], 353.9688, found 353.9697, C14H9NaCl2N3S2 [M+Na]
375.9507, found 375.9515.
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HPLC analysis was performed using an Agilent X-bridge-C18 (4.6 × 150 mm, 3.5
µm). The mobile phases consisting of A (0.1% HCOOH and 99.9% H2O) and B
(acetonitrile) were used with the gradient mode. The substrate and product were
detected at 210 nm. The initial gradient starts with 5% of B and at 15 min it was set to
75%. The ratio had been set to 20% at 35.1 min which continued up to 30 min. The
retention time was 14.6 min [(R)-E-1] and 13.9 min [(R)-Z-1].
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The chiral HPLC was performed using a Chiralcel OD-3 (4.6 × 250 mm, 5 µm)
column with a mobile phase of 70% Hexane and 30% Ethanol. The product was
detected at 298 nm, and the retention times of diastereoisomers were 12.8 min[(R)-E-
1] and 20.3 min.
Associated Content
Supporting Information. Figure S1, Figure S2 and spectral data of compounds 1, 3,
4.
Author Contributions
# These authors contributed equally to this work.
Acknowledgments
This project was sponsored by Program of Shanghai Technology Research Leader.
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