Green synthesis of chiral aromatic alcohols with Lactobacillus kefiri P2 as a novel biocatalyst

Article abstract of DOI:10.1080/00397911.2020.1729809

Biocatalytic reduction is a very important field of research in synthetic organic chemistry. Herein, three different Lactic Acid Bacteria (LAB) strains were evaluated for their bioreduction potential using acetophenone as a model substrate. Among these strains, Lactobacillus kefiri P2 strain was determined as the best asymmetric reduction biocatalyst. Reaction optimization parameters such as reaction time, temperature, agitation speed and pH were systematically optimized using Lactobacillus kefiri P2 strain and model substrate acetophenone. Under these optimized reaction conditions, secondary chiral alcohols were obtained by bioreduction of various prochiral ketones with results up to 99% enantiomeric excess. In addition, the steric and electronic effects of substituents on enantioselectivity and conversion were evaluated. It has been shown that Lactobacillus kefiri P2 biocatalyst was an effective catalyst for asymmetric reduction. This method provides an environmentally friendly method for the synthesis of optically pure alcohols and an alternative approach to chemical catalysts.

Full text of DOI:10.1080/00397911.2020.1729809

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Green synthesis of chiral aromatic alcohols with
Lactobacillus kefiri P2 as a novel biocatalyst
Yasemin Baydaş, Enes Dertli & Engin Şahin
To cite this article: Yasemin Baydaş, Enes Dertli & Engin Şahin (2020): Green synthesis of chiral
aromatic alcohols with Lactobacillusꢀkefiri P2 as a novel biocatalyst, Synthetic Communications,
DOI: 10.1080/00397911.2020.1729809
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https://doi.org/10.1080/00397911.2020.1729809
Green synthesis of chiral aromatic alcohols with
Lactobacillus kefiri P2 as a novel biocatalyst
Yasemin Baydas¸a, Enes Dertlia, and Engin S¸ahinb
b
aFaculty of Engineering, Department of Food Engineering, Bayburt University, Bayburt, Turkey; Faculty
of Health Sciences, Department of Nutrition and Dietetics, Bayburt University, Bayburt, Turkey
ABSTRACT
ARTICLE HISTORY
Received 3 February 2020
Biocatalytic reduction is a very important field of research in syn-
thetic organic chemistry. Herein, three different Lactic Acid Bacteria
(LAB) strains were evaluated for their bioreduction potential using
KEYWORDS
acetophenone as
a
model substrate. Among these strains,
Asymmetric synthesis;
aromatic ketones;
bioreduction; Lactobacillus
kefiri; whole cell
Lactobacillus kefiri P2 strain was determined as the best asymmetric
reduction biocatalyst. Reaction optimization parameters such as reac-
tion time, temperature, agitation speed and pH were systematically
optimized using Lactobacillus kefiri P2 strain and model substrate
acetophenone. Under these optimized reaction conditions, secondary
chiral alcohols were obtained by bioreduction of various prochiral
ketones with results up to 99% enantiomeric excess. In addition, the
steric and electronic effects of substituents on enantioselectivity and
conversion were evaluated. It has been shown that Lactobacillus kefiri
P2 biocatalyst was an effective catalyst for asymmetric reduction.
This method provides an environmentally friendly method for the
synthesis of optically pure alcohols and an alternative approach to
chemical catalysts.
GRAPHICAL ABSTRACT
Introduction
Chiral aromatic secondary alcohols are very important due to their widespread use for
the production of intermediates in pharmaceutical chemistry. These chiral secondary
aromatic alcohols are widely used as leading molecules in the organic chemistry and
pharmaceutical industry due to their stable structure. Biocatalytic reaction is a highly
significant moiety of synthetic organic chemistry for the synthesis of drugs or biological
CONTACT Engin S¸ahin
esahin@bayburt.edu.tr
Faculty of Health Sciences, Department of Nutrition and Dietetics,
Bayburt University, Bayburt 6900, Turkey.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2020 Taylor & Francis Group, LLC

2
Y. BAYDAŞ ET AL.
Figure 1. Examples of drugs derivatives from chiral secondary alcohols.
active molecules.[1] Chirality has become indispensable for the asymmetric organic syn-
thesis, as it has crucial roles for the drug interaction in the body. Hence, the investiga-
tion of reactions related to the synthesis of chiral molecules is of significant attention.[2]
Synthesis of enantiomerically pure chiral molecules is one of the significant targets of
[3–5]
drug industry
as many drugs are composed of enantiomers that might have diverse
biological activities due to the presence of the chiral center, even the physical and
chemical properties of the molecules are similar. It is known that chiral molecules pro-
duced by biocatalytic reduction constitute 20% of total chiral alcohol precursors in
industrial products.[6] Synthesis of only one enantiomers of some chiral molecules is
one of the key aims of drug industry as the great number of the drugs in the market
are formed from at least bearing one chiral center and optically pure molecules.[7–10]
Chiral secondary alcohols are usually used in the production of drugs and precursor
compounds with high enantiomeric purity.[11] For instance, Prozac[12] which is used in
the treatment of depression, isoproterenol[13] which is used for the therapy of bradycar-
dia and neobenodine[14] which displays anticholinergic action and antihistaminic effects
can be produced from optically pure secondary alcohols (Figure 1).
These optically pure molecules can be synthesized with several methods and biocata-
lytic method is one of the most chosen method among them due to its several benefits
compared to chemical methods.[15–17] Asymmetric ligands, which is used in the reduc-
tion reactions, form toxic and a large amount of undesirable products while biocatalyst
provide environmentally friendly and clean method to perform reactions under mild
conditions with high enantiomeric excess.[18,19] Whole cell microorganisms or pure
enzymes are usually used as catalysts in asymmetric bioreduction. There are advantages
for the use of whole cell biocatalysts compared to isolated enzymes. For instance, using
whole cell biocatalyst does not require purification of the enzyme, and thus the cost is
reduced. In addition, expensive cofactors such as NADPH should be used in asymmet-
ric reduction reactions using pure enzymes. Furthermore, unlike with purified enzymes,
asymmetric reductions performed with whole cells do not require expensive co-factors
since these are reproduced in the whole cell.[20] These advantages are some of the fac-
tors that encourage the use of whole cells as biocatalysts compared to the pure enzymes.
In the literature, many different biocatalysts were successfully demonstrated to be used
for the bioreduction reactions.[4,10,11,21] These examples showed that different biocata-
lysts can act as different bioreduction agents with different yields and enantiomeric
excesses. Thus, finding a new biocatalyst with bioreduction potential is very important.
One of the most common examples of biocatalysts used in these asymmetric reduction

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SYNTHETIC COMMUNICATIONSV
3
Table 1. Screening for LAB strain in the bioreduction of acetophenone 1.
Entry
Bacteria
Lactobacillus kefiri P2
Lactobacillus fermentum P1
Lactobacillus fermentum H3
Conversiona (%)
eeb,c
%
1
2
3
95
2
25
66 (R)
18 (R)
4 (R)
Reaction condition: substrate 1 mmol, temperature: 25oC, time 64 h, 150 rpm.
aThe conversions were determined by HPLC. bDetermined by HPLC using Chiralcel OD-H column. cAbsolute configur-
ation were assigned by comparison of the sign of optical rotations relative to the values in literature
reactions are lactic acid bacteria (LAB) that are food originated bacteria which have
GRAS (generally considered as safe) status.[22–24] In these studies, three species of LAB
that are Lactobacillus kefiri P2, Lactobacillus fermentum P1, and Lactobacillus fermentum
H3 were demonstrated to have bioreduction capacity in order to obtain enantio-
merically pure secondary alcohols.[25,26] As LAB is a safe microbial group and have
different strains, it is important to find novel LAB strains as a biocatalysts.
Herein, we reported the asymmetric bioreduction of acetophenones by LAB strains
including Lactobacillus fermentum P1, Lactobacillus kefiri P2 and Lactobacillus fermen-
tum H3 isolated from traditional dairy products.[27,28] A series of aromatic alcohols is
prepared within the system. Moreover, to obtain maximum enantiomeric excess (ee) of
product and conversion of substrate, bioreduction reaction conditions such as tempera-
ture, pH, agitation speed and incubation period have been investigated comprehensively.
This study provides an easy and practical method for the synthesis of aromatic
chiral alcohols.
Results and discussion
Acetophenones are very useful aromatic ketones for enantioselective bioreduction reac-
tions, as they are important precursors in the production of different pharmaceutically
significant molecules.[29] The strategy of screening of biocatalysts has been widely used
to find desired biocatalysts for asymmetric bioreduction of ketones to corresponding
chiral alcohols. Substrate acetophenone 1 was selected as model substrate for screening
LAB strains and optimization conditions in the enantioselective bioreduction reaction
(Table 1). As can be seen in Table 1, these strains produced (R)ꢀ1-phenylethanol (1a)
with a range of 4–66% ee. When evaluated in terms of conversion of substrate and ee
of the products, it was found that Lactobacillus kefiri P2 had the best reduction activity.
In this strain, the product was obtained with moderate ee (66%) and high conversion
(95%) (Table 1, Entry 1). Lactobacillus kefiri P2 was thus selected for further studies as
a biocatalyst.
Bioreduction optimization conditions are important in the enantiomerically pure pro-
duction of an aromatic chiral secondary alcohols since optimization of key conditions
such as incubation period, pH, agitation speed and temperature were shown to affect

4
Y. BAYDAŞ ET AL.
Table 2. Optimization of asymmetric reduction conditions using Lactobacillus kefiri P2 bacteria strain
in the bioreduction of acetophenone 1.
pH
Temperature
Incubation period
Agitation Speed
Conv.
(%)a
ee
Conv.
(%)a
ee
ee
Conv. ee
(%)b,c
oC
(%)b,c
h
Conv. (%)a
(%)b,c
rpm
(%)a
(%)b,c
4.5
5
5.5
6
6.5
7
99
96
98
99
97
99
98
80
99(R)
97(R)
98(R)
97(R)
97(R)
96(R)
96(R)
95(R)
25
28
30
32
34
99
94
93
88
83
99(R)
82(R)
96(R)
96(R)
80(R)
24
48
64
72
96
67
96
99
92
84
96 (R)
97 (R)
99 (R)
96 (R)
92 (R)
100
150
200
250
95
99
92
94
97 (R)
99 (R)
97 (R)
97 (R)
7.5
8
Substrate 1 mmol. The conversions were determined by chiral HPLC. bDetermined by HPLC using a Daicel Chiralcel OD-
a
c
H column. Determination of absolute configuration was carried out by comparison of the sign of optical rotation rela-
tive to the values in the literature.
the ee of the product and conversion of the substrate.[11] So, these parameters were
investigated to increase the conversion of substrate and ee of the product using
Lactobacillus kefiri P2 (Table 2).
The pH can change the molecular structure of the enzyme and the charge density on
the cell surface. This may cause a change in the rate of substrate entry and exit into the
cell, and thus may result in a change in the activity and selectivity of the enzyme.[29,30]
Therefore, the pH of the reaction medium plays an important role on the activity of the
enzyme. In order to determine the optimum pH of the catalytic asymmetric bioreduc-
tion of model substrate acetophenone to (R)ꢀ1-phenylethanol (1a), various reactions
were carried out in different pH ranging from 4.5 to 8.0 in the reaction conditions of
1 mmol substrate, at 25 ꢁC, 150 rpm and 64 h. Reactions at different pHs show that the
conversion and ee are not significantly affected by the pH change, but when the pH
was higher than 7.5, the conversion was reduced. The highest ee and conversion were
obtained when the pH was 4.5, so the optimum pH was determined to be 4.5. Since
temperature plays an important role in the thermodynamic balance of the reactions, it
significantly affects the selectivity and activity of the enzyme.[31] The effect of reaction
temperature on selectivity and conversion was investigated as another parameter in the
reaction conditions of pH 4.5, 64 h and 150 rpm and the results are shown in Table 2.
It can be seen that when the temperature was increased from 25 ꢁC to 34 ꢁC, both con-
version and ee decreased gradually. This indicates that the elevated temperatures lead to
partial inactivation of the enzyme, which might significantly have impaired the selectiv-
ity and activity of the enzyme. Therefore, the reaction temperature of 25 ꢁC was deter-
mined as the optimum temperature for subsequent reactions. As a third optimization
parameter, incubation period of the enantioselective reduction of acetophenone 1 cata-
lyzed by Lactobacillus kefiri P2 whole cell biocatalyst was investigated in the reaction
conditions of pH 4.5, 25 ꢁC and 150 rpm. Table 2 shows that both conversion and ee
increased up to 64 hours. When the reaction time was more than 64 hours, both the
conversion and the ee values decreased. This might be probably originated from the fact
that the enzyme selectivity decreased as the incubation period increased. Taking into
account the incubation period that resulted in the best conversion and ee, subsequent
bioreductions were performed at 64 h and optimized incubation period was determined
as 64 h. The agitation speed effect, the last parameter of optimization, on selectivity and

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5
conversion was investigated in the reaction conditions of pH 4.5, 25 ꢁC and 64 h, and
the results are shown in Table 2. It can be seen from these results that agitation speed
had a minor effect on both conversion and ee. Despite these minor changes in the ee
and conversion, 150 rpm was selected as the optimum agitation speed, because the best
results were obtained at this agitation speed. After parameters such as temperature,
reaction time, pH and agitation speed (pH: 4.5; time: 64 hours; temperature: 25 ꢁC and
agitation speed: 150 rpm) have been optimized, we applied the optimization conditions
to investigate the potential of Lactobacillus kefiri P2 to reduce different substituted
(bromine, chlorine, methoxy, nitro and methyl groups) acetophenones (2–16) (Table 3).
Under optimized conditions, acetophenone (1) was reduced to (R)-1a by 99%
conversion and ee (Table 3, entry 1). In the literature, ADH enzyme isolated from
Lactobacillus kefiri was used as biocatalysts for bioreduction of acetophenone and differ-
ent prochiral ketones to corresponding chiral alcohols with high ee and conversion.[22]
Asymmetric bioreduction of ethyl 4-chloro acetoacetate to ethyl (S)ꢀ4-chloro-3-hydrox-
ybutanoate using Lactobacillus kefiri as a whole cell biocatalyst was carried out in high
ee and yield by Amidjojo et al.[23] Bioreduction of (2,5)-hexanedione to enantiopure
(5 R)-hydroxyhexane-2-one (ee >99%) with whole cell immobilized Lactobacillus kefiri
was achieved by Tan et al.[24] Although these studies show that Lactobacillus kefiri is an
important biocatalyst, extensive studies, especially the use of Lactobacillus kefiri as a
whole cell biocatalyst and investigation of the reducing potential in the wide variety of
substrates, are not known in the literature. The bioreduction of ortho-chloro and ortho-
bromo acetophenones 2 and 3 were reduced to 2a and 3a with 46 and 40% ee, respect-
ively with a low conversion (Table 3, entry 2, 3). The substrate where nitro, another
electron-withdrawing group, in the ortho position, was reduced to 5a with a 50% con-
version and 82% ee (Table 3, entry 5). However, the substrate of the methoxy, which is
the electron donating group, in the ortho position, was reduced to 4a with 97% ee and
100% conversion (Table 3, entry 4). The lowest ee and conversion from ketones with
halogen in the ortho position was obtained in substrate 3 (Table 3, entry 3). The low
conversions and ee for substrates 2 and 3 show that the rate of the reduction depends
on the electronic and steric effects of the ortho-position on the aromatic ring and these
findings were consistent with steric (atomic size Br > Cl) influence of the groups. In the
ortho position, the best result was obtained in the substrate 4, which contains the elec-
tron-donating group, methoxy (Table 3, entry 4). The bioreduction of meta-chloro,
meta-bromo and meta-nitro substrates 6, 7 and 9 led to the formations of alcohols 6a,
7a and 9a with high ee and conversions (Table 3, entry 6, 7 and 9). When these meta
halogenated substrates were compared to ortho, it can be seen that there was a signifi-
cant increase in both conversion and ee. This can be explained by the fact that these
bulky groups move away from the reaction center. When meta-methoxy substrate 8 was
compared with ortho-methoxy substrate 4, it can be seen that there was a slight decrease
in ee and conversion. This situation can be explained by the removal of the electron
donating methoxy group from the reaction center. The bioreduction of meta-methyl
substrate 10 was converted to 10a with 98% ee and moderate conversion (62%) (Table
3, entry 10). When para-chloroacetophenone (11), para-bromoacetophenone (12), and
para-nitroacetophenone (14) were treated with Lactobacillus kefiri P2 under the opti-
mized bioreduction conditions employed, the chiral alcohols 11a, 12a and 14a were

6
Y. BAYDAŞ ET AL.
Table 3. Enantioselective reduction of acetophenones catalyzed by whole cells of Lactobacillus
kefiri P2.
Deney
1
Substrat
Product
ee (%)a,b
Conv. (%)c
Yield (%)d
99 (R)
99
94
2
3
4
5
6
7
8
9
46 (R)
40(S)
53
33
47
30
95
78
80
69
84
89
97 (R)
50 (R)
100 (S)
94 (R)
90 (S)
94(S)
100
82
86
72
88
91
(continued)

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SYNTHETIC COMMUNICATIONSV
7
Table 3. Continued.
Deney
10
Substrat
Product
ee (%)a,b
Conv. (%)c
62
Yield (%)d
58
98 (R)
11
12
13
14
98 (R)
94 (S)
74 (R)
88 (R)
96
97
83
96
93
94
79
92
15
16
100 (S)
94 (R)
100
93
95
89
Reaction conditions: substrate 1 mmol, pH 4.5, 25oC, 64 h, 150 rpm.
aDetermined by HPLC using a chiral column. bDetermination of absolute configuration was carried out by comparison
of the sign of optical rotation relative to the values in the literature. cThe conversions were determined by chiral
HPLC. dIsolated yield.
obtained with high conversion and good enantioselectivity as 98%, 94% and 88%
respectively (Table 3, entry 11, 12, 14). The substrate 13 was reduced to the correspond-
ing (R)-alcohol 13a with a good ee (74% ee) and conversion (83%) (Table 3, entry 13).
Other derivatives with electron-donating substituents para-methylacetophenone (15)
was reduced to the (S)-alcohol 15a in the highest ee (>99%) and conversion (>99%)
(Table 3, entry 15). The para-phenylacetophenone (16) was reduced to (R)-alcohol 16a

8
Y. BAYDAŞ ET AL.
in the optimized condition with 94% ee and 93% conversion (Table 3, entry 16).
Clearly, the activity of Lactobacillus kefiri P2 for the bioreduction of acetophenones was
different and substantially influenced by electronic and steric factors of the substituents.
The configurations of the alcohols obtained in this study varied according to the pos-
ition and structure of the substituent groups in the aromatic ring. The probable reason
for this can be explained by the fact that different configurations might occur due to
the interaction of substrates with the active center of the enzyme depending on the
structure and position of the substituents in the aromatic ring. Based on these data, it
can be concluded that this new biocatalyst might work as substrate selective. When the
reactions were carried out in the absence of the catalyst in the medium, practically no
transformation occurred, which indicated that the cause of the transformation in the
reactions was the biocatalyst. From the perspective of the environment and economy,
this biosynthesis method is of great application prospects, making it an ideal green and
sustainable synthesis route.
Conclusion
In conclusion, we have produced chiral (R) or (S) aromatic alcohols with excellent ee
(40–99%) and good conversion (33–99%) by reduction reactions, revealing Lactobacillus
kefiri P2 as a promising biocatalyst with enantioselectivity for the synthesis of organic
molecules of industrial interest. In addition, we also obtained that the presence of sub-
stituents (electron-donating or electron-withdrawing) in the benzene ring can have elec-
tronic and steric effects on bioreduction of substrates and ee of product. The
biocatalysts exhibited high enantioselectivity in catalyzing various aromatic ketones,
thereby highlighting its potential applications for the industrial production of chiral
alcohols. The results obtained from this study showed that Lactobacillus kefiri P2 is a
precious biocatalyst for the synthesis of chiral secondary alcohols, which can be of inter-
est for the pharmaceutical industry as a biocatalyst. In addition, studies testing this bio-
catalyst in the asymmetric bioreduction of different substrates are underway in
our laboratory.
Experimental section
General
MRS medium, solvents and substrates were purchased from Sigma–Aldrich (Milwaukee,
WI, USA). Purification of obtained chiral secondary alcohols was carried out using col-
umn chromatography with a mixture of hexane: ethyl acetate (90:10, v/v) solvent. The
progress of the reactions was determined by analysis of the reaction mixture by TLC
using the mobile phase of hexane: ethyl acetate (90:10, v/v). Racemic alcohol 1a-16a
was obtained by reducing the corresponding substrate (1–16) with NaBH4 in the
MeOH at room temperature. The products were characterized using Bruker NMR spec-
trometer (Bruker Ltd., Germany). The conversion of the substrates and the enantio-
meric excesses of the products were determined using the Agilent 1260 HPLC system
with UV and chiral detector. Optical rotations of the products were determined using
Bellingham þ Stanley (ADP 220, 589 nm) spectropolarimeter. The crude product was

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SYNTHETIC COMMUNICATIONSV
9
used as the sample to determine the conversion of substrate, by comparing the alcohol
and ketone peak.
Culture condition and bacterial strain
Lactobacillus kefiri and Lactobacillus fermentum strains used in this study was previously
isolated from kefir[27] and koumiss[28] respectively and these strains were grown in
MRS broth as described previously[27]
.
General asymmetric bioreduction process
Lactobacillus kefiri P2 was added from its glycerol stock by inoculation to 10 mL MRS
mixture (MgSO4.7H2O 11.5% (w/v), K2HPO4 2 g/L, pepton [Oxoid] 10 g/L, yeast
extract 2% glucose, [Difco] 5 g/L, C2H3NaO2.3H2O 5 g/L, salt solution [MgSO4.7H2O
11.5% (w/v), triamonium citrate 2 g/L], Tween 80 1 mL/L) followed by 48 h growth at
37 ꢁC. From this mixture, overnight grown bacterial cells were inoculated to 100 mL
MRS mixture at 10% concentration. Then, pH of medium was adjusted to 4.5 with 1 M
HCI. This mixture was shaken for 2 hours, and then 1 mmol of substrate (1–16) was
directly added to this mixture and incubated on an orbital shaker at 150 rpm with an
incubation temperature of 25 ꢁC for 64 h incubation period. After the catalytic reaction
was complete, the supernatant was separated. The aqueous phase was saturated with
NaCI and extracted with dichloromethane. After the organic phases were combined,
they were dried over anhydrous Na2SO4 and filtered to remove the salt. The liquid was
removed in a vacuum to remove dichloromethane and the crude product was character-
ized by NMR analysis. After the crude product was purified using column chromatog-
raphy, ee was determined using chiral HPLC. The absolute configurations of the
alcohols were determined by comparing the literature values of the specific rotation.
1
Copies of H, 13C-NMR spectra, spectroscopic datas, racemic and chiral HPLC chro-
matograms of 1a–16a related to this article can be found at Supplementary data.
Acknowledgements
We are thankful to Central Research Laboratory of Bayburt University for HPLC analysis.
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