Whole cell application of Lactobacillus paracasei BD101 to produce enantiomerically pure (S)-cyclohexyl(phenyl)methanol

Article abstract of DOI:10.1002/chir.23048

In this study, a total of 10 bacterial strains were screened for their ability to reduce cyclohexyl(phenyl)methanone 1 to its corresponding alcohol. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful biocatalyst to redu

Full text of DOI:10.1002/chir.23048

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Received: 2 November 2018
DOI: 10.1002/chir.23048
Revised: 12 December 2018
Accepted: 16 December 2018
R E G U L A R A R T I C L E
Whole cell application of Lactobacillus paracasei BD101 to
produce enantiomerically pure (S)‐cyclohexyl(phenyl)
methanol
Engin Şahin
| Hüseyin Serencam | Enes Dertli
Department of Food Engineering, Faculty
of Engineering, Bayburt University,
Bayburt, Turkey
Abstract
In this study, a total of 10 bacterial strains were screened for their ability to
reduce cyclohexyl(phenyl)methanone 1 to its corresponding alcohol. Among
these strains, Lactobacillus paracasei BD101 was found to be the most
successful biocatalyst to reduce the ketones to the corresponding alcohols.
The reaction conditions were systematically optimized for the reducing agent
L paracasei BD101, which showed high enantioselectivity and conversion
for the bioreduction. The preparative scale asymmetric reduction
of cyclohexyl(phenyl)methanone (1) by L paracasei BD101 gave (S)‐
cyclohexyl(phenyl)methanol (2) with 92% yield and >99% enantiomeric
excess. The preparative scale study was carried out, and a total of 5.602 g
of (S)‐cyclohexyl(phenyl)methanol in high enantiomerically pure form
(>99% enantiomeric excess) was produced. L paracasei BD101 has been
shown to be an important biocatalyst in asymmetric reduction of bulky sub-
strates. This study demonstrates the first example of the effective synthesis of
(S)‐cyclohexyl(phenyl)methanol by the L paracasei BD101 as a biocatalyst in
preparative scale.
Correspondence
Engin Şahin, Department of Food
Engineering, Faculty of Engineering,
Bayburt University, Bayburt 69000,
Turkey.
Email: esahin@bayburt.edu.tr
KEYWORDS
asymmetric reduction, biocatalyst, biotransformations, chiral aryl methanols, enantioselectivity
1 | INTRODUCTION
enantioselective formation of carbinols is one of the most
essential strategies in modern organic chemistry as they
Chirality is a key factor in the safety of many drugs, and
therefore, the synthesis of enantiopure drugs has become
increasingly important issue in the pharmaceutical indus-
try.1 The interaction of chiral compounds with chiral envi-
ronments may result in biologically different enantiomers
that may have deteriorative effects. The modern pharma-
ceutical industry demands for the obtaining of biologically
active chiral compounds in their enantiomerically pure
forms for the production of drugs and other chemicals
found in biological systems. So the enantiomeric purity
of the synthesized product is of great importance.2,3 The
are commonly used as intermediates for the synthesis of
drugs and other industrially important compounds.4,5
Heteroaryl methanols can be used for the production of
pharmaceutically significant molecules that might have
antihistaminic, anesthetic, diuretic, antidepressive, antiar-
rhythmic, and anticholinergic roles.6 Thus, chiral carbi-
nols can act as precursors of many biologically and
pharmacologically relevant compounds,7 and important
examples of these compounds are (R)‐orphenadrine,8 (S)‐
phenyl(pyridin‐2yl)methanol,9 and carbinoxamine10
(Figure 1). Generally, production of these compounds is
Chirality. 2019;1–8.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

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ŞAHIN ET AL.
FIGURE 1 Chiral carbinol‐based drugs
carried out by classic synthetic procedures that use
catalysts such as metal hydrides, but the product obtained
in these reactions ate in their non‐enantiomerically
form. Although classic chemical methods for the
enantioselective synthesis of chiral carbinols have been
comprehensively developed, the synthesis of some of these
compounds is still extremely complicated, toxic, and
expensive.11 Biocatalytic synthesis is a very successful
technique as a green chemistry application for preparing
chiral compounds. However, enantioselective reduction
of aryl heteroaryl ketones in the presence of biocatalyst is
still challenging.12,13 Diaryl ketones are usually known as
“hard‐to‐reduce” substances for biocatalyst owing to their
big steric obstacle and similar of two aromatic groups.14,15
Biocatalysts have many superiorities compared with clas-
sic chemical catalysts. Chemical catalysts might produce
toxic waste and a large range of by‐products, whereas
biocatalysts are biodegradable products and provide a
clean and environmentally friendly process to carry out
these reactions under mild reaction conditions with high
selectivity for the substrate.16 Therefore, highly selective
and environmentally friendly biocatalytic process to syn-
thesis optically active carbinols is the utilization of isolated
enzymes and whole cell microorganisms.17 Whole cell
biocatalysts compared with the pure enzyme are advanta-
geous as they are usually cheap and more stable. Whole
cells contain various dehydrogenases that are able to
accept a wide range of unnatural substrates. All enzymes
and cofactors are well protected within their natural
cellular environments. Moreover, the use of whole cell
biocatalysts avoids enzyme purification and cofactor addi-
tion.18-20 Enantiomerically pure carbinols are useful build-
ing blocks for the synthesis of complex molecules as the
alcohol functional group can be easily converted, without
racemization, into other functional groups.21-24
involving reduction of this compound using biocatalyst,
and in these examples, the corresponding chiral secondary
alcohols were obtained by low enantiomeric excess (ee) and
conversion. Ema and coworkers obtained (S)‐2 with 59% ee
by the lipase enzyme catalyst.35 In the literature, it has been
reported that cyclohexyl(phenyl)methanone is reduced to
(R)‐2 by 75% ee and 55% conversion with yeast Candida
magnoliae as biocatalyst.36 The use of [α]‐branched aro-
matic ketones as a biocatalyst for whole cell or pure
enzymes as a biocatalyst has been reported in the literature
in which the asymmetric reductions are generally of low
selectivity or low conversion.1 Recently, we performed
asymmetric reduction of acetophenones and piperonyl
methyl ketone by high enantioselectivity and conversion
using Lactobacillus paracasei BD101 as biocatalyst.37,38
In this study, we report the asymmetric reduc-
tion of cyclohexyl(phenyl)methanone 1 to the (S)‐
cyclohexyl(phenyl)methanol 2 by L paracasei BD101 with
>99% ee and 92% yields. Moreover, to the best of our
knowledge, this is the first report on asymmetric reduc-
tion of cyclohexyl(phenyl)methanone 1 using biocatalyst
in the enantiomerically pure form, high yield, and scale.
Scale‐up bioreduction of cyclohexyl(phenyl)methanone 1
was reduced to the (S)‐cyclohexyl(phenyl)methanol 2 in
excellent yield and enantiopure form using L paracasei
BD101 as a biocatalyst. The effects of the reaction condi-
tions in terms of pH, temperature, and agitation speed on
the yield, conversion, and ee were also optimized for the
bioreduction reaction.
2 | MATERIALS AND METHODS
2.1 | General
Sales of single enantiomeric drug products are
expanding at an alarming rate every year in the world.
Therefore, enormous efforts have been made in recent
years to detect enantioselective routes to enantiomerically
pure carbinols. Enantiopure diarylmethanols are impor-
tant structural scaffolds in the synthesis of pharmaceuticals
and bioactive compounds.25-30 There are a number of
studies in the literature that contain cyclohexyl(phenyl)
methanone 1 in high enantiomeric selectivity using chem-
ical catalysts31-34; however, there are very limited studies
All chemicals and all solvents were purchased from Sigma‐
Aldrich (purity of >99%). The growth mediums for bacterial
growth were purchased from Merck. Reactions were moni-
tored by thin‐layer chromatography (TLC) performed by
using plates (aluminum, silica gel 60 F254 Merck, 0.25 mm)
and hexane to ethyl acetate (4:1, v/v) as eluent. For analysis
purpose, a small fraction of the product was prepared by pre-
parative TLC. Purification of (S)‐cyclohexyl(phenyl)metha-
nol was performed by column chromatography filled with
silica gel (0.063‐0.2 mm), and the product was eluted with

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ŞAHIN ET AL.
3
a mixture of hexane to ethyl acetate (90:10, v/v). High‐
performance liquid chromatography (HPLC) analysis was
performed on an Agilent 1260 systems equipped with a
UV and chiral detector. The racemic 2 was obtained by
reducing the 1 with NaBH4 in methanol at room tempera-
ture (RT). Optical rotation was measured with a Belling-
(Table 1, entries 1–7) and Turkish sourdough (Table 1,
entries 8–10).40 Bacterial strains were propagated from
their glycerol stocks by inoculation to 10‐mL MRS broth
medium followed by overnight growth at 30°C. From
these cultures, exponentially grown bacterial cells were
inoculated to 1‐L MRS broth medium at 10% concentra-
tion and incubated 2 days at 30°C under aerobic condi-
tions. Following the growth of the bacteria in the flasks,
bacterial cells were centrifuged, and following the wash
process, whole cells were obtained freeze‐drying
(Labconco, TR) and stored at RT.
1
ham + Stanley, ADP220, 589 nm spectropolarimeter. H
and 13C nuclear magnetic resonance (NMR) spectra were
recorded on Bruker 400 MHz spectrometer in CDCl3. (S)‐
cyclohexyl(phenyl)methanol39: White solid, m.p.: 63‐65°C,
1H NMR (400 MHz, CDCl3) δ = 7.36‐7.28 (m, 5H), 4.39 (d,
J = 7.2 Hz, 1H), 2.01‐1.61 (m, 6H), 1.42‐0.94 (m, 6H); 13C
NMR (100 MHz, CDCl3) δ = 143.6, 128.2, 127.4, 126.6,
79.4, 45.0, 29.3, 28.8, 26.4, 26.1, 26.0; [α]D25 = −39.5 (c 0.4,
CHCl3) >99% ee; HPLC, Chiralcel OD column, hexane/i‐
PrOH, 95:5, flow rate of 0.5 mL/min, 254 nm, tR (S)
21.3 minutes. HPLC condition of 1: Chiralcel OD column,
n‐hexane/i‐PrOH, 95:5, flow rate of 0.5 mL/min, 254 nm,
10.7 minutes (Supporting Information).
2.3 | General procedure for the
asymmetric reduction of
cyclohexyl(phenyl)methanone
Forty‐milligram dry bacterial strain was added to 100‐mL
MRS broth and stirred on an orbital shaker at 150 rpm
and 30°C. After 2 hours, pH was adjusted 6 and shaken
2
hours, followed by the addition of 1‐mmol
cyclohexyl(phenyl)methanone 1 directly to the medium,
and incubated on a shaker (150 rpm) at 30°C for 48 hours.
At the end of the incubation period, the cells were sepa-
rated by centrifugation at 6000g for 5 minutes at 4°C
and the supernatant was saturated with NaCl and then
2.2 | Bacterial strains and culture
conditions
The microorganism used in this study was isolated
previously from boza, a cereal‐based fermented beverage
TABLE 1 Screening of bacteria strains for the bioreduction of 1
Entry
Microbial straina
Conversion, %b
Yield, %c
ee, %d,e (S)
1
Lactobacillus plantarum BY14
Lactobacillus fermentum BY35
Enterococcus faecium BY48
Lactobacillus paracasei BD28
Lactobacillus paracasei BD101
Lactobacillus paracasei BD71
Lactobacillus paracasei BD87
Leuconostoc mesenteroides N6
Weissella paramesenteroides N7
Weissella cibaria N9
61
58
54
64
74
62
54
70
21
10
54
51
47
54
70
56
48
64
15
5
52
63
48
65
81
72
15
51
62
45
2
3
4
5
6
7
8
9
10
Abbreviations: ee, enantiomeric excess; HPLC, high‐performance liquid chromatography. Reaction condition: substrate 1 mmol/100 mL; temperature, 25°C;
time, 24 h; 100 rpm.
aComparison of the best microbial strain.
bThe conversions were determined by HPLC.
cIsolated yield.
dDetermined by HPLC using Chiralcel OD column.
eAbsolute configurations were assigned by comparison of the sign of optical rotations relative to the values in the literature.

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4
ŞAHIN ET AL.
extracted with dichloromethane. Dichloromethane
extracts were combined and dried over anhydrous
Na2SO4. After removal of the solvent under reduced pres-
sure, the crude product was identified by NMR analysis
followed by the purification of this product by column
chromatography on silica gels using hexane to ethyl ace-
tate as eluents (90:10). The absolute configuration was
determined by sign of specific rotation. The conversion
was determined by chromatography on a chiral OD col-
umn on HPLC (n‐hexane/i‐PrOH, 95:5, flow rate of
0.5 mL/min, 254 nm) after filtering the crude product
with a column containing small silica gel and comparing
the alcohol peaks with the ketone peaks. The ee of the
products was determined by HPLC analysis using chiral
OD column.
obtained when whole cell of L paracasei BD101 was used.
The bioreduction was taken place with high conversion
and produced 2 at 81% ee with this strain (Table 1, entry
5). One of the primary requirements of a microbial
biotransformation is the optimization of the reaction con-
ditions.41 Therefore, we decided to determine the perfor-
mance of the microorganism under optimized reaction
conditions. To detect the optimal reaction conditions for
the asymmetric reduction of 1 with L paracasei BD101,
pH, temperature, incubation period, and agitation speed
were examined. The first parameter that was investigated
was the effect of pH on the production of 2 from 1.
Table 2 shows the results for the effect for the pH range
from 4.0 to 7.0 on the product's ee and bioconversion of
1. The reactions were carried out for 24 hours using
1 mmol of 1 in an orbital shaker at 25°C at 100 rpm.
The pH performs a key role in the biocatalytic reactions
when using whole cells due to the fact that it affects the
activity and enantioselectivity of the enzymes involved
in the reaction, alters the ionic state of substrates,
products, and enzymes involved in the reaction, and
affects the binding of enzyme's active site to substrates,
especially for several isoenzymes with different
enantioselectivities. As can be seen from Table 2, the
bioreduction of 1 was importantly affected by effects of
pH. The results indicate that ketone 1 could be reduced
to alcohol 2 at pH 6.0 (Table 2, entry 4). Above or below
these values, low ee and mild conversion were observed.
Probably, the three‐dimensional structure of the enzyme
changes as the pH changes, and it can be said that con-
version and ee have changed as a result of the interaction
of the enzyme and, also, the solubility of the substrate
may have affected the selectivity of the biocatalyst.37
2.4 | Scale‐up asymmetric reduction of
cyclohexyl(phenyl)methanone
The 400‐mg dry L paracasei BD101 was inoculated into 1‐L
sterilized fresh MRS broth as working volume in 5‐L
Erlenmeyer flask under sterile conditions and stirred on
an orbital shaker at 30°C, 150 rpm for 2 hours. After 2 hours,
pH was adjusted to 6 and the flask was shaken 2 hours
followed by the addition of 32 mmol cyclohexyl(phenyl)
methanone 1 to the medium and the reaction mixture was
incubated at 30°C, for 56 hours under agitation at 150
rpm. At the end of the incubation period, the cells were
separated by centrifugation at 6000g for 5 minutes at 4°C
and the supernatant was saturated with NaCl and then
extracted with dichloromethane. Dichloromethane extracts
were combined and dried over anhydrous Na2SO4. After
removal of the solvent under reduced pressure, the crude
product was purified on a silica gel column and eluted using
hexane to ethyl acetate (10:1, v/v) to obtain the product.
TABLE 2 Effects of different pHs on the reduction of 1 by
Lactobacillus paracasei BD101
Entry
pH
Conversions, %a
ee, %b,c (S)
3 | RESULTS AND DISCUSSION
1
2
3
4
5
6
7
4.5
5
67
76
71
78
70
64
20
51
59
65
84
79
10
5
Ten different bacterial strains were evaluated for the
reduction of 1 to 2. The reactions for screening process
were carried out in 200‐mL Erlenmeyer flask containing
100 mL of fresh culture medium (MRS broth). The micro-
bial reduction was performed by suspending the dry cells
(40 mg) in a 100‐mL fresh medium; then substrate 1
(1 mmol, 188.27 mg) was added directly, and the mixture
was incubated on conditions of 100 rpm and 25°C for
24 hours. The bioreduction progress was monitored
TLC, and a chiral HPLC column was used for the deter-
mination of the ee of product 2 and conversion of 1 to
2. As can be seen in Table 1, the bacterial strains used
in this study reduced 1 to 2 at levels of 5% to 70% ee.
The best result for the asymmetric bioreduction was
5.5
6
6.5
7
7.5
Abbreviations: ee, enantiomeric excess; HPLC, high‐performance liquid
chromatography.
Substrate 1 mmol.
aThe conversion was determined by HPLC.
bDetermined by HPLC using Chiralcel OD column.
cAbsolute configurations were assigned by comparison of the sign of optical
rotations relative to the values in the literature.