Separation of enantiopure m-substituted 1-phenylethanols in high space-time yield using Bacillus subtilis esterase

Article abstract of DOI:10.1039/c3ra41999a

A recombinant Bacillus subtilis esterase (BsE) expressed in E. coli was found to exhibit excellent enantioselectivity (E was always greater than 100) towards m-substituted 1-phenylethanol acetates in the enantioselective hydrolysis reaction. An explanation for the high enantioselectivity observed towards these substrates was provided by molecular modeling. Moreover, the BsE also showed strong tolerance towards a high concentration of m-substituted 1-phenylethanol acetates (up to 1 M). Based on these excellent catalytic properties of BsE, a kind of m-substituted 1-phenylethanols, (R)-1-(3-chlorophenyl)ethanol, was efficiently synthesized in space-time yield of 920 g per L per day and 97% ee, indicating that the BsE was considered as a potentially ideal and promising biocatalyst for large-scale production of optically active m-substituted 1-phenylethanols. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra41999a

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Separation of enantiopure m-substituted
1-phenylethanols in high space-time yield using
Cite this: RSC Adv., 2013, 3, 20446
Bacillus subtilis esterase†
a
a
a
b
Gao-Wei Zheng, Xu-Yun Liu, Zhi-Jun Zhang, Ping Tian, Guo-Qiang Linb
and Jian-He Xu
*
a
*
A recombinant Bacillus subtilis esterase (BsE) expressed in E. coli was found to exhibit excellent
enantioselectivity (E was always greater than 100) towards m-substituted 1-phenylethanol acetates in
the enantioselective hydrolysis reaction. An explanation for the high enantioselectivity observed
towards these substrates was provided by molecular modeling. Moreover, the BsE also showed strong
tolerance towards a high concentration of m-substituted 1-phenylethanol acetates (up to 1 M). Based
on these excellent catalytic properties of BsE, a kind of m-substituted 1-phenylethanols, (R)-1-(3-
chlorophenyl)ethanol, was efficiently synthesized in space-time yield of 920 g per L per day and 97% ee,
indicating that the BsE was considered as a potentially ideal and promising biocatalyst for large-scale
production of optically active m-substituted 1-phenylethanols.
Received 23rd April 2013
Accepted 21st August 2013
DOI: 10.1039/c3ra41999a
www.rsc.org/advances
more serious. Recently, scientists have shown great interest in
biocatalysis because of its lower energy consumption, envi-
Introduction
Optically active m-substituted 1-phenylethanols are frequently
used as important and valuable intermediates for the
production of pharmaceuticals and other ne chemicals.1 For
example, enantiopure 1-(3-chlorophenyl)ethanol (6a) was
recently employed for the synthesis of DP1 (a receptor for
prostaglandin D2) antagonists, which has been used for
treating the side effects of niacin-induced ushing.2 In addi-
tion, (R)-1-(3-methoxyphenyl)ethanol (16a) was also used as a
precursor for the preparation of (S)-rivastigmine, which is the
rst drug approved by FDA for the treatment of mild to
moderate dementia associated with Alzheimer's and Parkin-
son's disease.3
In the past, some physical or chemical methods have been
developed for the production of m-substituted 1-phenyl-
ethanols. For example, a supramolecular chiral host consist-
ing of N-(2-naphthoyl)-L-aspartic acid and meso-1,2-
diphenylethylenediamine was used for enantioseparation of 1-
arylethanols, giving 96% ee and 100% inclusion ratio.4
However, environmental pollution and energy pinch arising
from physical or chemical methods are becoming more and
ronmental friendliness, shorter synthetic routes and avoid-
ance of functional group protection. Esterases (EC 3.1.1.1) and
lipases (EC 3.1.1.3) as powerful and promising biocatalysts
have been applied in a variety of pharmaceutically useful
synthetic reactions because of their excellent activity and
robustness. More importantly, they could smoothly catalyze
reactions without addition of expensive cofactors and could be
reused aer immobilization, which makes them commercially
available at competitive prices.5–7 Several lipases and esterases
have been utilized for the preparation of optically pure m-
substituted 1-phenylethanols. For instance, Candida antarctica
lipase B (CALB) has been used to prepare (R)-1-(3-methox-
yphenyl)ethanol (16a) with excellent enantiomeric excess
(>96%).3 Although a few kinetic resolution processes have
been developed for preparing optically active m-substituted 1-
phenylethanols, most of them provided the low space-time
yields, which has limited their practical application. There-
fore, efficient synthesis process is still of great interest to
achieve economic feasibility and competitiveness for the large-
scale preparation of enantiopure m-substituted 1-
phenylethanols.
We recently cloned and expressed a p-nitrobenzyl esterase
(BsE) from Bacillus subtilis ECU0554.8,9 The BsE protein
exhibited extremely high hydrolytic activity (as high as 45 kU per
mg protein) and excellent enantioselectivity towards l-menthol.
Herein, we found that the BsE also showed excellent enantio-
selectivity (E > 100) towards m-substituted 1-phenylethanol
acetates in the enantioselective hydrolysis of a series of acety-
lated rac-aryl alcohols. A molecular modelling was performed to
aLaboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of
Bioreactor Engineering, East China University of Science and Technology, 130
Meilong Road, Shanghai 200237, P.R. China. E-mail: jianhexu@ecust.edu.cn; Fax:
+86-21-6425-0840
bKey Laboratory of Synthetic Chemistry of Natural Substances, Shanghai Institute of
Organic Chemistry, Chinese Academy of Science, 345 Lingling Road, Shanghai
200032, P.R. China. E-mail: tianping@sioc.ac.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra41999a
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obtain a better understanding of the signicant differences
observed in enantioselectivity towards different substrates.
Furthermore, the study showed that the BsE also exhibited
strong tolerance against high substrate concentrations. Finally,
a BsE-catalyzed hydrolysis process with high space-time yield
for synthesis of optically activity m-substituted 1-phenyl-
ethanols was developed.
Results and discussion
To learn the substrate specicities of BsE, analytical-scale
kinetic resolutions were carried out for a variety of racemic
alcohol acetates (1–13) using BsE (Scheme 1). The results
revealed that the activity and enantioselectivity of BsE differed
Scheme
2
Enantioselective hydrolysis of m-substituted 1-phenylethanol
acetates with BsE.
Table 2 Enantioselective hydrolysis of m-substituted 1-phenylethanol acetates
14–18 using BsE at an analytical scale
Substrate
Time (min)
eesa (%)
eepa (%)
Conv.b (%)
Eb
14
15
16
17
18
45
90
45
180
45
92
84
97
91
96
96 (R)
99 (R)
99 (R)
97 (R)
93 (R)
49
46
50
48
51
>100
>100
>100
>100
>100
a
Determined by chiral GC. b Conversion and E were calculated from ees
and eep.
considerably depending on the structure of substrate (Table 1).
An increase in the carbon-chain length (2) and the introduction
of an electron-withdrawing group (3) at the R2 substituent led to
a signicant reduction in the enantioselectivity (E) from 56 to 21
and 8, respectively. Besides, the introduction of electron-with-
drawing and electron-donating groups at the R1 substituent in
the o- (4 and 5) and p-positions (7–9) resulted in poor enantio-
selectivity. However, interestingly, the substrate bearing a
chloro-substituent in the m-position (6) was hydrolyzed with an
excellent enantioselectivity (E > 100). Based on this preliminary
and unexpected result, it was envisaged that the enzyme might
also exhibit excellent enantioselectivity towards other m-
substituted substrates.
Scheme 1 Enantioselective hydrolysis of racemic alcohol acetates 1–13 by BsE.
Table 1 Enzymatic resolution of chiral alcohol acetates 1–13 by BsE at an
analytical scale
Substrate
Time (min)
eesa (%)
eepa (%)
Conv.b (%)
Eb
1
2
3
4
5
6
7
8
60
90
60
60
60
91
81
60
50
79
95
74
39
84
73
79
24
—
89 (R)
74 (R)
63 (R)
70 (R)
57 (R)
98 (R)
71 (R)
37 (R)
75 (R)
59 (R)
85 (R)
21 (R)
—
51
48
47
41
58
49
51
51
52
55
48
53
n.d.c
56
21
8
9
8
>100
13
3
19
8
Table 3 Results of BsE-catalyzed hydrolysis of m-substituent 1-phenylethanol
acetates 6 and 14–18 at preparative scale
Acetate (%)
Alcohol (%)
60
a
a
Substrate Time (min) ees
Yieldb eep
Yieldb Conv.c (%)
120
180
60
60
60
6
360
240
360
200
420
180
94
92
93
97
94
94
40
45
44
42
42
40
98
97
99
99
96
94
42
45
40
38
36
41
49
49
48
49
49
50
9
14
15
16
17
18
10
11
12
13
30
2
720
720
—
a
Enantiomeric excess (% ee) was determined by chiral HPLC or GC.
Conversion and E value were calculated from ees and eep. n.d.: not
b
c
a
Determined by chiral GC. b Isolated yield. c Conversion was calculated
determined.
from ees and eep.
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The results showed that the BsE exhibited not only excellent
enantioselectivity, but also strong tolerance towards all of the
m-substituted substrates tested.
To obtain a better understanding of the signicant differ-
ences observed in enantioselectivities of BsE towards
substrates with and without substituent in the m-position,
protein modeling was performed using faster reacting enan-
tiomers (R)-1 and (R)-16 as examples. The modeling showed
that the substituent in the m-position of the substrate mole-
cule stretched into a hydrophobic pocket consisting of Ile-270,
Met-358, Leu-362 and Phe-363, and the hydrophobic interac-
tion between the substituent and the hydrophobic pocket
could result in better binding of the substrate in the active site.
This suggestion was conrmed by the distances between the
nitrogen atom in the oxyanion hole and the carbonyl oxygen
atom of the substrates. In the case of (R)-16, the distance was
˚
found to be 3.0 A, whereas, the distance in the case of (R)-1 was
˚
3.5 A, (Fig. 1), suggesting that BsE bound (R)-16 more tightly
than (R)-1. The enhanced exibility of the substrates without
any substituent at the m-position in the active site could
provide an explanation for the lower enantioselectivities
observed towards these substrates.
To investigate the feasibility of using BsE for the large-scale
preparation of m-substituted 1-phenylethanols, substrate 6 was
selected for further investigation. Pleasingly, a relatively high
space-time yield (307 g per L per day) was achieved in
conjunction with a nearly complete conversion (49%) within 6 h
(Table 4, entry 1) for the hydrolysis of 199 g Lꢀ1 of substrate 6
(1.0 M). Further increases in the substrate loading to 397 g Lꢀ1
(Table 4, entry 2) and 596 g Lꢀ1 (Table 4, entry 3) resulted in
excellent conversion (50%) and extremely high space-time
yields of 1-(3-chlorophenyl)ethanol (6a) (as high as 940 g per L
per day), which markedly exceeded the threshold of industrially
feasible space-time yield (500 g per L per day).10 The BsE derived
from B. subtilis ECU0554 was further demonstrated to be a
robust biocatalyst with excellent tolerance towards high
substrate loading; enabling the efficient preparation of optically
active m-substituted 1-phenylethanols as well as their esters.
Next, the biohydrolytic reaction with the highest space-time
yield (Table 4, entry 3) was scaled up to 100 mL to evaluate the
feasibility of this bioprocess. As a result, the hydrolysis of
substrate 6 (59.6 g) over a 6 h period provided the desired
product with a conversion of 49% and an enantiomeric excess of
97% (Scheme 3). Finally, following an extraction and standard
work-up, 19.6 g of (R)-1-(3-chlorophenyl)ethanol was isolated
Fig. 1 Comparison of (R)-1 (a) and (R)-16 (b) placed in the active site of BsE. The
models suggested that (R)-16 binds more tightly in the active site than (R)-1 likely
because of the substituent in the m-position, which stretches into a hydrophobic
pocket thereby resulting in better binding of the substrate. The greater flexibility
of substrates without any substituent at the m-position in the active site explains
their lower enantioselectivities.
To conrm this hypothesis, a series of racemic 1-phenyl-
ethanol acetates that were substituted in the m-position with
different electron-withdrawing or electron-donating groups,
were subjected to biocatalytic hydrolysis (Scheme 2,
compounds 6 and 14–18). As expected, the BsE displayed
excellent enantioselectivities (E > 100) towards all of the m-
substituted substrates tested (Table 2). The subsequent
preparative-scale hydrolysis of high concentration of 6 and 14–
18 (up to 1 M) was also smoothly catalyzed by BsE (Table 3).
Table 4 Enantioselective hydrolysis of substrate 6 at high loads with BsEa
Substrate
Space-time yield
(g per L per day)
Entry
(g Lꢀ1
)
(M)
Enzyme (kU)
Time (h)
eesb (%)
eepb (%)
Conv.c (%)
1
2
3
199
397
596
1.0
2.0
3.0
47
94
141
6
6
6
94
94
95
98
97
97
49
50
50
307
626
940
a
Reaction conditions: substrate (2.0–6.0 g, 10–30 mmol), enzyme (47–141 kU), EtOH (10%, 1 mL), phosphate buffer (100 mM, pH 8.0, 9 mL), 30 ꢁC.
Determined by chiral GC analysis. Conversion was calculated from ees and eep.
b
c
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Conclusion
Enantioselective hydrolysis of racemic alcohol acetates by the
p-nitrobenzyl esterase (BsE) from Bacillus subtilis ECU0554 is
noteworthy for its excellent enantioselectivity (E > 100) and
strong tolerance against high substrate loading for all of the
m-substituted 1-phenylethanol acetates tested. This enantio-
selective hydrolysis using the BsE also provided an easy access
to m-substituted 1-phenylethanols with high enantio-purity and
the extremely high space-time yield.
Scheme 3 BsE-catalyzed enantioselective hydrolysis of 1-(3-chlorophenyl)ethyl
acetate with BsE at 59.6 g/100 mL scale.
(42% yield), representing an outstanding space-time yield of
920 g per L per day. The results suggest that the reaction could
be potentially used as a competitive process for the large-scale
production of enantiopure m-substituted 1-phenylethanols.
Acknowledgements
We are very grateful to Prof. Romas Kazlauskas at University of
Minnesota for his critical reading and fruitful revision of the
text and constructive suggestions on the protein modelling.
Thanks to the nancial supports from National Natural
Science Foundation of China (no. 31200050 & 21276082),
Ministry of Science and Technology, P.R. China (no.
Materials and methods
Molecular modeling
Molecular modeling was performed with Maestro (version 9.2,
Schrodinger, New York, NY, USA) using OPLS-2005 force eld11
starting with the X-ray crystal structure of Bacillus subtilis
esterase (PDB le ID: 1QE3).12 The dielectric constant was set to
1 to mimic the solvation effects of water. Hydrogen atoms were
added and His-399 was protonated. The geometry of structure
was optimized in a stepwise manner. Initially, the geometries of
the hydrogen atoms on water were optimized followed by the
hydrogen atoms on the protein. The whole protein with water
molecules was then optimized followed by water, whole protein
and the bound substrate. Each step in the optimization of the
geometry was conducted using the PolaRibiere conjugated
2011AA02A210
& 2011CB710800), and the Fundamental
Research Funds for the Central Universities, Ministry of
Education, P.R. China.
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This journal is ª The Royal Society of Chemistry 2013
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