Development of a Practical Enzymatic Process for Preparation of (S)-2-Chloro-1-(3,4-difluorophenyl)ethanol

Article abstract of DOI:10.1021/acs.oprd.7b00230

(S)-2-Chloro-1-(3,4-difluorophenyl)ethanol (1) is a vital chiral intermediate for the synthesis of Ticagrelor - an effective treatment for acute coronary syndromes. A ketoreductase (KRED) KR-01 in our KRED library was screened to transform 2-chloro-1-(3,4

Full text of DOI:10.1021/acs.oprd.7b00230

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Full Paper
Development of a Practical Enzymatic Process for
Preparation of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol
Xiang Guo, Jia-Wei Tang, Jiang-Tao Yang, Guo-Wei Ni, Fu-Li Zhang, and Shao-Xin Chen
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00230 • Publication Date (Web): 14 Aug 2017
Downloaded from http://pubs.acs.org on August 14, 2017
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Development of a Practical Enzymatic Process for Preparation of (S)-
2
-chloro-1-(3,4-difluorophenyl)ethanol
1
1
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1
1
Xiang Guo , JiaꢀWei Tang , JiangꢀTao Yang , GuoꢀWei Ni , FuꢀLi Zhang* , Shaoꢀ
Xin Chen*
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Shanghai Institute of Pharmaceutical Industry, China State Institute of
Pharmaceutical Industry, 285 Gebaini Road, Pudong, Shanghai 201203, China and
2
Zhejiang University of Technology, 18 Chaowang Road, Xiacheng, Hangzhou
3
10014, China.
Email: zhangfuli1@sinopharm.com; sxzlb@263.net
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Abstract: (S)-2ꢀchloroꢀ1ꢀ(3,4ꢀdifluorophenyl)ethanol (1) is a vital chiral intermediate
for the synthesis of Ticagrelor—an effective treatment for acute coronary syndromes.
A ketoreductase (KRED) KRꢀ01 in our KRED library was screened out to transform
2
ꢀchloroꢀ1ꢀ(3,4ꢀdifluorophenyl)ethanone (2) into the chiral alcohol 1. During process
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optimization, the bioreduction procedure was performed at a substrate concentration
of 500 g/L, giving a near 100% conversion with >99.9% ee. The product 1 was
directly obtained by extraction and can be used for the synthesis of (1R, 2R)ꢀ2ꢀ(3,4ꢀ
difluorophenyl)cyclopropanecarboxylic acid ethyl ester (3) with a yield of 98%
and >99.9% de greatly simplifying the original process operation and reducing the
safety risk. This process is green and environmentally sound with high productivity of
biocatalysis and a spaceꢀtime yield of 145.8 mmol/L/h. It has an opportunity to be
very useful in industrial applications. Additional studies have indicated that KRꢀ01
can also be used to prepare (R)‑1ꢀ(3,5ꢀbis(trifluoromethyl)phenyl)ethanol (4) with a
substrate concentration of 500g/L.
Keywords: Enzymatic; Bioreduction; Asymmetric synthesis; Industrial application;
Keotreductase
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1
. Introduction
The synthesis of enantiopure alcohols has attracted significant attention due to
1
their broad versatility as key building blocks in the synthesis of pharmaceuticals. For
instance, (R)ꢀ3ꢀquinuclidinol is an important intermediate of Solifenacin (Scheme 1).
Other chiral alcohols are also key components in Atorvastatin, Ezetimibe and
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ꢀ4
Duloxetine.
Scheme 1. Examples of pharmaceuticals that contain chiral alcohol building blocks.
S)-2ꢀchloroꢀ1ꢀ(3,4ꢀdifluorophenyl)ethanol (1) is a valuable unit and is a vital
(
chiral intermediate for the synthesis of Ticagrelor that has been developed as an
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effective treatment for acute coronary syndromes (Scheme 2). The literatures show
5
,6
that 1 is generally synthesized though CBSꢀcatalyzed reduction system. Our team
previously found that boraneꢀTHF (tetrahydrofuran) catalyzed via (S)ꢀ2ꢀMeꢀCBS can
reduce the substrate 2ꢀchloroꢀ1ꢀ(3, 4ꢀdifluorophenyl)ethanone (2) to the target product
with 93ꢀ95% ee. Although chiral purity can be improved by recrystallization, the cost
of (S)ꢀ2ꢀMeꢀCBS and boraneꢀTHF makes the reaction inappropriate for industrial use.
In addition, harsh reaction conditions and tedious postꢀprocessing also limit this
method, implying that the synthesis of 1 remains to be further improved.
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Scheme 2. A schematic diagram of the synthesis of Ticagrelor.
Biocatalysis is a popular ‘green chemistry’ concept with environmentally
friendly reaction conditions and stringent stereoselectivity of enzymatic reactions. It is
7
increasingly considered to be an ideal alternative to current chemical methods.
Various biocatalysts are available, including ketoreductases (KREDs), that can be
used to prepare enantiopure alcohols with broad substrate specificity and high
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reactivity. There are several successful cases in which KREDs were used in the
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synthesis of chiral alcohols on an industrial scale.
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Recently, Wu et al. synthesized 1 via a mutant reductase ChKRED20. However,
the maximum substrate concentration for this KRED was 200 g/L with a spaceꢀtime
yield of 52.5 mmol/L/h. To establish a better process for bioreductivity of ketone 2,
we screened our KRED library to search for a KRED with higher substrate
concentration and excellent enantioselectivity. We noticed that the alcohol
dehydrogenase from Leifsonia sp. Strain S749 (KRꢀ01) had improved productivity
toward the chiral alcohol 1 and was suitable for industrial scale production. Besides, it
can be used to prepare enantiopure alcohols with other substrate. Herein, we report
the processes of screening, applying, and optimizing the KRꢀ01 in order to realize its
industrial application for the bioreduction of ketone 2 (Scheme 3).
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Scheme 3. Enzymatic reduction of ketone 2 with KRꢀ01.
2. Results and Discussion
2
.1. Screening of KREDs
To explore the potential biocatalyst for the asymmetric reduction of ketone 2 to
alcohol 1, 13 KREDs from our library were tested. Table 1 showed that almost all of
the KREDs were active toward substrate 2 except for KRꢀ04 and KRꢀ06. Thereinto,
KRꢀ09, KRꢀ05, KRꢀ07 and KRꢀ11 displayed relatively moderate reaction activity
(
>70%). Thus, KRꢀ01 and KRꢀ12 were selected due to their excellent conversion
>99%). Five of them showed the selectivity of (S)ꢀenantiomer, and KRꢀ01, KRꢀ09
(
and KRꢀ12 had high enantiomeric excess (>99%). Taking into account the conversion
and enantioselectivity, we selected KRꢀ01, KRꢀ09 and KRꢀ12 for further study.
Table 1. Screening of KREDs from our enzyme library
c
d
Entry
Enzyme Conversion (%)
Ee (%)
Configuration
a
1
2
3
4
5
6
7
8
KRꢀ01
>99
57.6
3.6
>99
S
R
b
KRꢀ02
KRꢀ03
>99
b
91.4
N.D.
>99
R
a
KRꢀ04
N.D.
82.7
N.D.
73.0
1.9
N.D.
R
b
KRꢀ05
a
KRꢀ06
N.D.
98.4
>99
N.D.
S
b
KRꢀ07
KRꢀ08
a
R
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KRꢀ09
KRꢀ10
KRꢀ11
KRꢀ12
KRꢀ13
85.8
65.7
75.1
>99
32.3
>99
>99
91.9
>99
96.8
S
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R
+
Reaction conditions: (a) 10g/L ketone 2, 20g/L wet cells, 0.5g/L NAD , 2eq IPA and
PBS buffer (100 mM , pH 7.0) in 10mL reaction system for 1.5h at 25 °C and 220
rpm. (b) 10g/L ketone 2, 20g/L wet cells, 0.2g glucose, 0.1g/L NADP and PBS
buffer (100 mM , pH 7.0) in 10mL reaction system for 1.5h at 25 °C and 220 rpm.
+
c
d
Measured by HPLC. Measured by chiral HPLC.
2.2. Further Optimization
2.2.1. Effect of substrate concentration
Substrate loading is key parameter for an industrial process for its influence on
1
volumetric productivity. The effect of substrate concentration (50 g/L – 500 g/L) on
conversion was investigated. As shown in Figure 1, we conclude that nearly
complete conversion was realized via KRꢀ01 and KRꢀ12 when the substrate
concentration was lower than 200 g/L. The substrate with KRꢀ09 was difficult to
convert. However, when the substrate loading was up to 400 g/L, the conversions
decreased to 88.4% and 80.2% by KRꢀ01 and KRꢀ12 respectively. There was no
yield increase even at longer reaction time. In conclusion, KRꢀ01 was selected due to
its better catalytic ability.
100
90
80
7
0
KR-01
KR-09
KR-12
60
50
50 100 150 200 250 300 400 500
Substrate Concentration (g/L)
Figure 1. The effect of substrate concentration on the bioreduction of ketone 2
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Reaction conditions: various concentration of ketone
NAD , 2eq IPA and PBS buffer (100 mM , pH 7.0) in 2mL reaction system for 24h at
2
, 20g/L wet cells and 0.5g/L
+
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5 °C and 220 rpm.
Considering the low conversion rate with high substrate concentration (500 g/L),
the process optimization of reaction conditions including coꢀsolvents, temperatures,
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pH, catalyst loading and NAD loading were subsequently performed.
2.2.2. Effect of co-solvents
In light of the poor solubility of ketone 2, six organic solvents were chosen to
improve the conversion rate in the waterꢀmediated reaction system. Figure 2 shows
that the addition of MeOH and IPA increased reaction activities; IPA was the best coꢀ
solvent giving an 18% improvement in conversion. Furthermore, IPA is a vital
parameter for the enzymatic reaction due to its role as a coꢀsubstrate for cofactor
11
regeneration. Thus, IPA was an optimal coꢀsolvent from this screening.
100
90
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50
Cosolvent
Figure 2. Comparison of the performance in different coꢀsolvents
+
Reaction conditions: 500g/L ketone
2, 100g/L wet cells and 0.5g/L NAD , 2eq IPA,
1
2
0% v/v coꢀsolvent and PBS buffer (100 mM , pH 7.0) in 2mL reaction system for
4h at 25 °C and 220 rpm.
On the other hand, organic solvents in the reaction system may equally change
the properties of the solvent including the polarity and dielectric constant. This would
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change the three dimensional structure of protein and influence the catalytic activity
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of the enzyme. Hence, we tried to confirm the optimum concentration by studying
different proportions of IPA so as to maximize the solubility of substrate and drive the
equilibrium to the desired product. As shown in Figure 3, the highest conversion was
achieved at proportion of 3.5 eq IPA, and the enzyme activity dropped gradually with
increasing levels of IPA. Therefore, 3.5 eq IPA was the optimal proportion that was
chosen into application.
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IPA (eq)
Figure 3. The effect of concentration of IPA on the activity of KRꢀ01
Reaction conditions: the proportions of IPA study was performed with 500g/L ketone
+
2
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, 100g/L wet cells and 0.5g/L NAD , 1~4.5eq IPA and PBS buffer (100 mM , pH
.0) in 2mL reaction system for 24h at 25 °C and 220 rpm.
2
.2.3. Effect of reaction temperature and pH
Reaction temperature and pH are the most critical influences on enzyme activity
in the KREDꢀmedicated reaction. Therefore, after an effective improvement in the
conversion rate with high substrate concentration, the parameters (namely reaction
temperature and pH) were subsequently explored to determine optimal reaction
conditions for industrialization.
Temperature significantly influences both the conversion rate of the reaction and
the stability of the protein. On the one hand an increase in temperature would lead to
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an increase reaction rate. On the other, high temperature may denature the protein
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leading to rapid enzyme inactivation. Thus, it is critical to find the appropriate
reaction temperature.
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As shown in Figure 4, KRꢀ01 showed relatively high reaction conversion at
temperatures ranging from 25°C to 45°C; a temperature of around 35°C was the
optimum temperature for the bioreduction. Additionally, we must consider the protein
stability as well. Thus, further study was to determine the thermal stability of KRꢀ01
at 25°C, 35°C, and 45°C. Figure 5 shows that the enzyme has better thermal stability
at 25°C and 35°C compared to 45°C. It maintained more than 90% residual activity
after storage at 25°C or 35°C for 12 h, but the initial activities reduced by 73% at
4
5°C after 12 h storage. Therefore, the reaction temperature was set to be 35°C to
balance reaction time and reaction activities.
100
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90
88
25
35
45
55
T (5 )
Figure 4. The effect of temperature on the activity of KRꢀ01
Reaction conditions: the temperature study was performed with 500g/L ketone
2
,
+
1
2
00g/L wet cells and 0.5g/L NAD , 3.5eq IPA and PBS buffer (100 mM , pH 7.0) in
mL reaction system for 24h at 220 rpm.
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1
h
2h
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12h
Time (h)
Figure 5. Activities of KRꢀ01 to thermal treatment at 25℃, 35℃and 45℃
b
respectively
a
b
The initial KRꢀ01 activity of the wet cells was defined as 100%. Reaction conditions:
the wet cells were incubated at temperatures of 25℃, 35℃, 45℃for different periods,
respectively.
The pH significantly affects the dissociation of the key amino acids at the active
center of the protein, so the choice of pH for enzymeꢀcatalyzed reaction cannot be
1
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neglected. The optimum value of pH was then explored in different buffer systems.
As shown in Figure 6, no significant changes in conversion were observed from pH 5
to 8—a relatively broad range. The highest conversion of alcohol
.0. Lower (<6) or higher (>6) pH led to a slight drop of the conversion. As a result,
pH 6.0 was selected as optimum condition.
1 was found at pH
6
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Figure 6. The effect of pH on the activity of KRꢀ01
Reaction conditions: the pH study was performed with 500g/L ketone
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, 100g/L wet
+
cells 0.5g/L NAD and 3.5eq IPA for 24h at 25 °C and 220 rpm in different PBS
buffer, including100mM sodium citrate (pH=5 or 6), 100mM potassium phosphate
(
pH=7) and 100mM Tris–HCl buffers (pH=8 or 9).
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.2.4. Effect of biocatalyst loading
Under these conditions, chiral alcohol
1 was achieved at near 100% conversion
in 10 h at 500 g/L substrate concentration. However, cost is important for a practical
and efficient biocatalysis route suitable for industry. Thus, biocatalyst and cofactor
loading were determined to minimize cost while maintaining complete conversion.
Increasing the biocatalyst loading is an efficient tool to increase the conversion
1
5
rate and percent conversion, but inevitably increasing catalyst consumption. Thus,
the influence of biocatalyst dose was studied to find relationship between maximum
conversion of substrate and utilization of enzyme. The additions of recombinant cells
ranging from 10 g/L to 100 g/L were tested. A plot of reaction conversion versus
biocatalyst loading was shown in Figure 7. Obviously, conversion steadily increased
with increasing biocatalyst loading until to 50 g (wet)/L cells, but slightly increased
when the cell amount was 100 g/L. Thus, to minimize the production cost, we reduced
the dose of biocatalyst loading to 50 g (wet)/L cells, which was sufficient to achieve
9
9% conversion of substrate to alcohol 1 within 18 h.
100
90
80
7
0
0
6
10
15
25
50
75
100
Biocatalyst Loading (g/L)
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Figure 7. The dosages of biocatalyst loading on the influence of conversion
Reaction conditions: the biocatalyst loading study was performed with 500g/L ketone
+
2
, 10~100g/L wet cells and 0.5g/L NAD , 3.5eq IPA and PBS buffer (100 mM , pH
6.0) in 2mL reaction system for 18h at 35 °C and 220 rpm.
+
10
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2.2.5. Effect of cofactor (NAD ) loading
+
In terms of production cost, the dosage of NAD was of equal importance for
1
6
KREDꢀmediated reaction. A series of experiments with different cofactor loading
+
were performed to investigate the effect of NAD concentration on reaction
conversion. As shown in Figure 8, the conversions were not significantly different
+
when NAD increased from 0.05 g/L to 0.5 g/L. In addition, the reaction could
+
+
progress without additional NAD due to the E. coli intracellular release of NAD , but
+
the reaction rate was low. Therefore, the addition of NAD was necessary at high
substrate concentration. Above all, when the production cost was considered, a
+
concentration of 0.05 g/L of NAD was applied.
100
0.50g/L of NAD+
8
0
0
.10g/L of NAD+
60
0.05g/L of NAD+
none of NAD+
40
20
0
0
.5
1
2
4
12 18 24 48
Time (h)
Figure 8. The effect of cofactor loading on the influence of conversion
Reaction conditions: the effect of different cofactor loading study was performed with
, 50g/L wet cells and 0~0.5g/L NAD , 3.5eq IPA and PBS buffer
100 mM , pH 6.0) in 2mL reaction system for 0.5~48h at 35 °C and 220 rpm.
+
5
(
00g/L ketone
2
2
.3. Scale up the reaction
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The reaction was carried out to 200 mL scale under the optimized conditions of
+
5
00 g/L substrate, 0.05 g/L NAD , 50 g/L biocatalyst, 3.5 eq IPA at pH 6.0 PBS
buffer (100 mM) and 35°C. The time course for the bioreduction of ketone
2 to
alcohol is shown in Figure 9. The conversion reached >99% within 18 h. After
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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1
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3
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2
3
4
5
6
7
8
9
0
completion of the reaction, the IPA and coꢀproduct of acetone were removed by
evaporation under reduced pressure, and the product was separated by extraction.
Upon isolation of desired product 1, the yield was 96% with a HPLC purity of 98.9%;
excellent stereoselectivity (ee>99.9%) was achieved without any further purification.
Overall, the production process of our system met the basic requirements of an
economically feasible process for industrialꢀscale production of optically pure alcohol
1
.
500
00
00
200
00
500
400
300
200
100
0
4
Product
3
Substrate
1
0
0
4
8
12
16
20
24
Time (h)
Figure 9. Time course for the bioreduction of ketone
Reaction conditions: 500g/L ketone
2
to alcohol 1
+
2
, 50g/L wet cells and 0.05g/L NAD , 3.5eq IPA
and PBS buffer (100 mM , pH 6.0) in 200mL reaction system for 24h at 35 °C and
20 rpm.
2
2
.4. Synthesis of (1R,2R)-2-(3,4-difluorophenyl)cyclopropanecarboxylic acid
ethyl ester (3)
Further studies found that chiral alcohol
1
can be obtained by extraction with
toluene from an enzymatic reaction system and directly converted to
3 with a yield of
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8% and >99.9% de. Compared with the current chemical synthetic method through
5
CBSꢀcatalyzed reduction system, our study allows the reaction to avoid using toxic
reagents and eliminates the cumbersome and dangerous quenching process. That is to
say, we reduced the environmental impact, improved operational safety and simplified
the original process operation. This study is of potential benefit for industrial
applications (Scheme 4).
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49
50
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55
56
57
58
59
60
Scheme 4. Comparison of the synthetic method of ester
3 from ketone 2
2.5. Synthesis of ( 1-(3,5-bis(trifluoromethyl)phenyl)ethanol (4)
R)‑
We were pleased to find that the selected KRꢀ01 could also be applied to the
preparation of alcohol which is a vital chiral intermediate for the synthesis of
Aprepitant. As shown in Table 2, the substrate 1ꢀ(3,5ꢀ
bis(trifluoromethyl)phenyl)ethanone ( ) can be converted almost completely in 500
g/L substrate loading with a spaceꢀtime yield of 108.4 mmol/L/h. Furthermore,
alcohol obtained by KRꢀ01 also showed high enantiomeric excess >99.9%. This
4
5
4
exciting finding further demonstrated that the KRꢀ01 shows potential for industrial
application.
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2
2
2
2
2
2
2
2
2
3
3
3
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3
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4
4
4
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5
5
5
5
5
5
5
5
5
6
a
Table 2. The preparation of diverse chiral alcohols using KRꢀ01
Substrate
Concentr
ation
Conv.
ee
(%)
Pharmace
Ketone
Product
b
c
Conf.
(
%)
utical
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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8
9
0
1
2
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4
5
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7
8
9
0
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2
3
4
5
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7
8
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0
1
2
3
4
5
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7
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9
0
5
00 g/L
>99
>98
>99.9
>99.9
S
Ticagrelor
5
00 g/L
R
Aprepitant
a
+
Reaction conditions: ketone
2 or 5 , 50g/L wet cells and 0.05g/L NAD , 3.5eq IPA
and PBS buffer (100 mM , pH 6.0) for 18h at 35 °C and 220 rpm Measured by HPLC.
b
c
Measured by chiral HPLC.
Thus far, the only bioreduction method for the synthesis of chiral alcohol
1 was
1
0
reported by Wu et al. using a mutant of enzyme ChKRED20. However, as shown in
Table 3, the maximum substrate concentration for Wu’s KRED can reach 200 g/L. In
contrast, our reaction system showed that the bioreduction process achieved nearly
complete conversion at a substrate concentration of 500 g/L with >99.9% ee giving a
2
.8ꢀfold increase in the spaceꢀtime yield (145.8 mmol/L/h). Meanwhile, a much
improved total turnover ratio (moles of product/moles of cofactor) of 34816 was
obtained for this cofactor. Furthermore, the cofactor loading was only a quarter of the
previous process. Lower costs are especially beneficial for industrial applications.
Above all, this is the best result for the bioreduction of
2 to date.
Table 3. Comparison of existing biocatalytic processes for the preparation of
1
ChKRED20
mutant L205A
Parameters
KRꢀ01
Solvent system
Cofactor (g/L)
25%Water/75%IPA
30%Water/70%IPA
0.05
2623.7 (500g/L)
2623.7
0.2
Concentration of ketone
Concentration of alcohol
2
1
(mM)
(mM)
1049.5 (200g/L)
1049.5
Reaction time (h)
Conversion (%)
20h
>99
18h
>99
145.8
34816
>99.9
This study
Space–time yield (mmol/L/h)
Turnover number (mol/mol)
Enantiomeric excess (%)
References
52.5
3481
>99
(ZhongꢀLiuWu et al. 2017)
3
. Conclusion
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A practical, efficient and environmental bioreductive process for the preparation
of the chiral alcohol
1 has been developed by using KRꢀ01. We have optimized the
reaction with KRꢀ01 to a higher degree than that achieved previously, making the
reaction more applicable for industrial use. Besides, this study simplifies the original
process operation and reduces the safety risk. We also found that KRꢀ01 can be used
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to prepare alcohol
4 with a 500 g/L substrate concentration.
4
. Experimental Section
4
.1. Materials and Methods
Ketone and were Energy Chemical (China), IPA was Sinopharm Chemical
®
2
5
Reagent Co.,Ltd. All other reagents were obtained commercially and used without
additional purification. All the KRED genes (the codonꢀoptimized open reading
frames encoding proteins) were synthesized by Bioligo (Shanghai). ClonExpress®
Entry One Step Cloning Kit used for molecular cloning was from Vazyme Biotech
(
Nanjin, China) and other enzymes from Takara (Dalian, China). BCA Protein Assay
Kit was purchased from cwbiotech (Beijin, China).
1
13
The H NMR and C NMR spectra were recorded using a Bruker Arance III 400
1
MHz spectrometer. The solvents used were CDCl
3
. The HꢀNMR chemical shift datas
were reported on δ (ppm) relative to internal standard tetramethylsilane (TMS) and
1
3
3
the CꢀNMR chemical shift datas were reported on δ (ppm) relative to CDCl . The
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
3
000 HPLC instrument.
4.2. Cloning and Expression
The KREDs used here are shown in Table 4. The codonꢀoptimized genes of KRꢀ
01, KRꢀ04, KRꢀ06, KRꢀ08, KRꢀ09, KRꢀ10, KRꢀ11, and KRꢀ13 were cloned into a
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2
2
2
2
2
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2
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pETꢀ22b(+) vector. A KRꢀ05 plasmid containing the KRꢀ05 and BmGDH (from
Bacillus megaterium) genes had a common T7 promoter, and an individual ribosomeꢀ
binding site (RBS) region was inserted into pETꢀ22b(+) by ClonExpress® Entry One
Step Cloning Kit. Other plasmids, namely KRꢀ02, KRꢀ03, KRꢀ07, and KRꢀ12, were
also performed by ClonExpress® Entry One Step Cloning Kit. All plasmids were
transformed into E. coli BL21 (DE3) cells for expression. The transformed cells
cultured at 37°C in 50 ml LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L
NaCl) containing 50 ug/mL kanamycin. When OD600 reached 0.6ꢀ0.8, 0.1 mM IPTG
was added in fermentation medium and cultivation was continued at 25°C overnight.
The cells were then harvested by centrifugation (4000 g, 4°C, 30 min) and stored at ꢀ
20 °C.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. KREDs used in this study
References/Genbank
Enzyme
Strain
Number
AB213459
17
KRꢀ01
KRꢀ02
KRꢀ03
KRꢀ04
KRꢀ05
KRꢀ06
KRꢀ07
KRꢀ08
KRꢀ09
KRꢀ10
KRꢀ11
KRꢀ12
KRꢀ13
Leifsonia sp. Strain S749
Exiguobacterium sp. F42
Streptomyces bingchenggensis
Microbacterium luteolum JCM 9174.
Chryseobacterium sp. CA49
Rhodococcus ruber
Debaryomyces hansenii CBS767
Corynebacterium sp. STꢀ10
Streptomyces cyaneogriseus
Lactobacillus kefir
WP043488748.1
AB733448
KC342003
WP017681535.1
XM459695
18
CP010849.1
19
Lactobacillus kefir
Lactobacillus kefir
Rhodococcus ruber DSM 44541
19
20
WP043801412.1
4
.3.
General
Protocol
for
the
synthesis
of
(S)-2-chloro-1-(3,4-
difluorophenyl)ethanol (1)
The 1g (500g/L) of substrate was dissolved in the solution of 1.4mL (3.5 eq) IPA
+
and 0.6mL PBS buffer (100 mM, pH 6.0). The 0.1mg (0.05g/L) of NAD and 0.1g
(
50g/L) of KRꢀ01 (wet cells) were added. The reaction was shaken at 220 rpm shaker
35°C) for 18h. Subsequently, the reaction was extracted with 1mL DCM (×3), and
(
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the organic layers was combined and dried over Na
determined by HPLC.
2 4
SO . The conversion and ee were
HPLC analysis was performed using a BDS C18 column (4.6×250 mm, 5 ꢁm,
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13
14
15
16
17
18
19
20
21
22
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49
50
51
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53
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59
60
Elite, Dalian), with a mobile phase of 60% water and 40% ACN (v/v). The substrate
and product were detected at 210 nm, and the retention time was 8.7 min (
1) and 13.4
min ( ).
2
Chiral HPLC was performed using a CHIRALPAK IC (4.6×250 mm, 5 ꢁm,
DAICEL, Shanghai) column with a mobile phase of 95% Hexꢀ0.1% TFA and 5% IPA.
The product was detected at 260 nm, and the retention times of enantiomers were 10.2
min (1) and 11.4 min, respectively..
4
.4. Scale-Up of the KRED Reaction for the synthesis of (S)-2-chloro-1-(3,4-
difluorophenyl)ethanol (1)
The reaction was performed in a 500 mL Erlenmeyer flask containing 60 mL
+
phosphate buffer (100 mM, pH 6.0), 140 mL IPA, 100 g ketone
2
, 0.01 g NAD , and
1
0 g wet cells. The reaction was carried out at 35°C and agitated by overhead stirring
for 18 h. The reaction mixture was then extracted with 200mL DCM (×3), and the
2 4
DCM layers were combined. The organic layers were dried over Na SO and
concentrated under reduced pressure, obtaining 97.0g oil (yield 96%). Spectral data:
1
H NMR (400 MHz, CDCl
3
) δ 7.24 (ddd, J = 10.6, 7.6, 2.2 Hz, 1H), 7.20 – 7.12 (m,
1
H), 7.12 – 7.07 (m, 1H), 4.90 – 4.83 (m, 1H), 3.71 (dd, J = 11.3, 3.5 Hz, 1H), 3.59
13
(
3
dd, J = 11.3, 8.5 Hz, 1H), 2.80 (d, J = 3.3 Hz, 1H). C NMR (101 MHz, CDCl ) δ
1
51.68 (dd, J = 26.1, 12.6 Hz), 149.20 (dd, J = 26.0, 12.6 Hz), 137.24 – 136.93 (m),
22.26 (dd, J = 6.5, 3.7 Hz), 117.58 (d, J = 17.5 Hz), 115.37 (d, J = 18.2 Hz), 73.00 (d,
1
2
5
+
J = 1.3 Hz), 50.64 (s). [α]589 +31.325 (c 1.0, CH
3
OH). HRMS m/z [M] calcd for
8 7 2
C H F ClO: 192.0153, found: 192.0157
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.5. Synthesis of (1
R,2R)-2-(3,4-difluorophenyl)cyclopropanecarboxylic acid
ethyl ester (3)
The tꢀBuONa (169.4g, 3.5eq) and toluene (500mL) were charged into a reaction
vessel. Subsequently, triethyl phosphonoacetate (282.3g, 2.5eq) was added to the
mixture with stirring during which the internal temperature should be controlled
below 20°C. After finishing, the temperature was set to 40°C. The organic layer that
was extracted from enzymatic process by toluene (600mL), contained about 97g
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
alcohol
1 (1eq), was added dropwise to the mixture with keeping the internal
temperature below 45°C. After addition, stirring was continued for 24h at 60°C. After
2
cooling to room temperature, the mixture was washed with H O, and the organic layer
was dried over Na
2
SO
4
and concentrated under reduced pressure to give the product 3
1
as oil (111.4g, yield 98%). Spectral data: H NMR (400 MHz, CDCl
3
) δ 7.05 (dt, J =
1
0.1, 8.4 Hz, 1H), 6.89 (ddd, J = 11.3, 7.4, 2.2 Hz, 1H), 6.86 – 6.81 (m, 1H), 4.17 (q,
J = 7.1 Hz, 2H), 2.52 – 2.43 (m, 1H), 1.84 (ddd, J = 8.6, 5.3, 4.2 Hz, 1H), 1.62 – 1.58
13
(m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 1.26 – 1.21 (m, 1H). C NMR (101 MHz, CDCl
3
) δ
1
1
1
2
73.03 (s), 151.63 (d, J = 12.8 Hz), 150.45 (d, J = 12.6 Hz), 149.16 (d, J = 12.8 Hz),
48.00 (d, J = 12.7 Hz), 137.30 (dd, J = 5.8, 3.7 Hz), 122.45 (dd, J = 6.1, 3.5 Hz),
17.28 (d, J = 17.3 Hz), 115.27 (d, J = 17.7 Hz), 61.00 (s), 25.26 (d, J = 1.4 Hz),
2
0
+
3
4.22 (s), 16.99 (s), 14.33 (s). [α]589 ꢀ226.553 (c 0.9, CH OH). HRMS m/z [M] calcd
for C H F O : 226.0805, found: 226.0802
12 12
2
2
HPLC analysis was performed using a phenomenex Gemini 5u C18 110A
column (4.6×250 mm, 5 ꢁm). The mobile phases consisting of A (0.1%TFAꢀH O)
2
and B (0.1%TFAꢀACN) were used with the gradient mode. The substrate and product
were detected at 260 nm. Initial gradient starts with 20% of B and at 15 min it was set
to 80%. The ratio had been set to 20% at 35.1 min which continued up to 40 min. The
retention time was 16.8 min (3) and 12.3 min (1).
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The chiral HPLC was performed using a CHIRALPAK ICꢀ3 (4.6×250 mm, 5
ꢁ
m, DAICEL, Shanghai) column with a mobile phase of 99% Hexꢀ1% IPA. The
product was detected at 260nm, and the retention times of diastereoisomers were 9.6
min ( ) and 10.5 min, respectively.
3
10
11
12
13
14
15
16
17
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4
.6. Synthesis of (R)‑1-(3,5-bis(trifluoromethyl)phenyl)ethanol (4)
The reaction was performed in a 500 mL Erlenmeyer flask containing 60 mL
+
phosphate buffer (100 mM, pH 6.0), 140 mL IPA, 100 g ketone
5, 0.01 g NAD , and
1
0 g wet cells. The reaction was carried out at 35°C and agitated by overhead stirring
for 18 h. The reaction mixture was then extracted with 200mL DCM (×3), and the
DCM layers were combined. The organic layers were dried over Na SO and
2
4
concentrated under reduced pressure, obtaining 95.7g offꢀwhite solid (yield 95%).
1
Spectral data: H NMR (400 MHz, CDCl
3
) δ 7.84 (s, 2H), 7.79 (s, 1H), 5.04 (qd, J =
13
6
.5, 4.0 Hz, 1H), 2.08 (d, J = 3.8 Hz, 1H), 1.55 (d, J = 6.5 Hz, 3H). C NMR (101
3
MHz, CDCl ) δ 148.36 (s), 132.43ꢀ31.44 (q), 127.58 (s), 125.75 (d, J = 2.6 Hz),
1
24.87 (s), 122.16 (s), 121.46 (dt, J = 7.7, 3.8 Hz), 119.46 (s), 69.44 (s), 25.52 (s). [α]
2
5
5
+
89 +23.377 (c 1.0, DMF). HRMS m/z [M] calcd for C10
H F O: 258.0479, found:
8 6
2
58.0471.
HPLC analysis was performed using a BDS C18 column (4.6×250 mm, 5 ꢁm,
Elite, Dalian). The mobile phases consisting of A (H O) and B (ACN) were used with
2
the gradient mode. The substrate and product were detected at 210 nm. Initial gradient
starts with 10% of B and at 20 min it was set to 80%. The ratio had been set to 10% at
3
0 min which continued up to 36 min. The retention time was 22.8 min (
5) and 22.2
min ( ).
4
The chiral HPLC was performed using a CHIRALPAK IBꢀ3 (4.6×250 mm, 3
ꢁ
m, DAICEL, Shanghai) column with a mobile phase of 97% Hexꢀ0.1% TFA and 3%
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IPA. The product was detected at 260nm, and the retention times of enantiomers were
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.7 min and 8.3 min (
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Asssociated Content
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Supporting Information. H NMR, C NMR, HRMS spectrogram, specific rotation
and chiral HPLC chromatogram of compounds 1, 3, 4
.
Acknowledgments
We are grateful to China State Institute of Pharmaceutical Industry for financial
support.
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