Debaryomyces hansenii as a new biocatalyst in the asymmetric reduction of substituted acetophenones

Article abstract of DOI:10.1080/10242422.2017.1348500

Chiral secondary alcohols are convenient mediator for the synthesis of biologically active compounds and natural products. In this study fifteen yeast strains belonging to three food originated yeast species Debaryomyces hansenii, Saccharomyces cerevisiae and Hanseniaspora guilliermondii were tested for their capability for the asymmetric reduction of acetophenone to 1-phenylethanol as biocatalyst microorganisms. Of these strains, Debaryomyces hansenii P1 strain showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to the corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high conversion rates. This is the first report on the enantioselective reduction of acetophenone by D. hansenii P1 from past?rma, a fermented Turkish meat product. The preparative scale asymmetric bio reduction of 3-methoxy acetophenone 1g by D. hansenii P1 gave (R)-1-(3-methoxyphenyl) ethanol 2g 82% yield, and >99% enantiomeric excess. Compound 2g can be used for the synthesis of (+)-NPS-R-568 [3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenly) ethyl] propan-1-amine] which have a great potential for the treatment of primary and secondary hyper-parathyroidism. In addition, D. hansenii P1 successfully reduced acetophenone derivatives. This study showed that this yeast can be used industrially to produce enantiomerically pure chiral secondary alcohols, which can be easily converted to different functional groups.

Full text of DOI:10.1080/10242422.2017.1348500

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Debaryomyces hansenii as a new biocatalyst
in the asymmetric reduction of substituted
acetophenones
Engin Şahin
To cite this article: Engin Şahin (2017): Debaryomyces hansenii as a new biocatalyst in the
asymmetric reduction of substituted acetophenones, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2017.1348500
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1348500
View supplementary material
Published online: 11 Jul 2017.
Submit your article to this journal
Article views: 9
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ibab20
Download by: [Australian National University]
Date: 15 July 2017, At: 06:49

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
https://doi.org/10.1080/10242422.2017.1348500
RESEARCH ARTICLE
Debaryomyces hansenii as a new biocatalyst in the asymmetric reduction of
substituted acetophenones
Engin S¸ ahin
Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt, Turkey
ABSTRACT
ARTICLE HISTORY
Chiral secondary alcohols are convenient mediator for the synthesis of biologically active com-
pounds and natural products. In this study fifteen yeast strains belonging to three food origi-
nated yeast species Debaryomyces hansenii, Saccharomyces cerevisiae and Hanseniaspora
guilliermondii were tested for their capability for the asymmetric reduction of acetophenone to
Received 4 January 2017
Revised 8 March 2017
Accepted 22 June 2017
1-phenylethanol as biocatalyst microorganisms. Of these strains, Debaryomyces hansenii P1 strain
KEYWORDS
showed an effective asymmetric reduction ability. Under optimized conditions, substituted aceto-
phenones were converted to the corresponding optically active secondary alcohols in up to 99%
enantiomeric excess and at high conversion rates. This is the first report on the enantioselective
reduction of acetophenone by D. hansenii P1 from pastırma, a fermented Turkish meat product.
The preparative scale asymmetric bio reduction of 3-methoxy acetophenone 1g by D. hansenii
P1 gave (R)-1-(3-methoxyphenyl) ethanol 2g 82% yield, and >99% enantiomeric excess.
Compound 2g can be used for the synthesis of (þ)-NPS-R-568 [3-(2-chlorophenyl)-N-[(1R)-1-
Bio reduction: asymmetric
reduction; whole yeast cells;
Debaryomyces hansenii; bio
transformations
(
3-methoxyphenly) ethyl] propan-1-amine] which have a great potential for the treatment of
primary and secondary hyper-parathyroidism. In addition, D. hansenii P1 successfully reduced
acetophenone derivatives. This study showed that this yeast can be used industrially to produce
enantiomerically pure chiral secondary alcohols, which can be easily converted to different func-
tional groups.
Introduction
(Legnani and Morandi 2016), (þ)-NPS-R-568 (Cohen
and Silverberg 2002; Joy et al. 2006; Nagano 2006)
and (S)-orphenandrine (Wujkowska et al. 2016) (Figure
Biocatalysts has earned major importance in the asym-
metric synthesis of optically pure compounds owing
to its high environmental convenience, high enantiose-
lectivity and very slight reaction conditions (Yamada
and Shimizu 1988; Wichmann and Vasic-Racki 2005;
Patel 2008; Huang et al. 2010; Strohmeier et al. 2011).
Biocatalysts have many superiorities compared to
chemical catalysts. Chemical catalysts generate toxic
waste while biocatalysts provide a clean and environ-
ment friendly way to perform chemical reaction if bio-
catalysts have specific safety properties such as GRAS
status. The exploration of new biocatalysts to produce
optically pure compounds in mild conditions with high
selectivity is an issue of great interest. One of the
most popular enantioselective synthesis is the asym-
metric reduction of prochiral ketones to secondary
alcohols. Optically pure secondary alcohols are useful
as important intermediates in the synthesis of pharma-
ceutical and agricultural chemicals at low production
cost in industry. For example, (R)-phenylephrine
1
) are pharmacologically active compounds based
upon optically pure secondary alcohols. Optically pure
-phenylethanol is a chiral building block used for the
1
synthesis of pharmaceuticals such as inhibitors of chol-
esterol intestinal absorption (Kurbanoglu et al. 2007).
Besides, (S)-1-(2-cholorophenyl)ethanol has been used
as a key intermediate for the synthesis of anti-asthma
compound L-cloprenaline (Kurbanoglu et al. 2009).
Hence, preparation of optically pure secondary alco-
hols has received more and more attention. The sig-
nificance of these compounds have resulted in the
stunning need for new, efficient, and enantioselective
ways for making them (Comasseto et al. 2004; Patel
et al. 2004; Murzin et al. 2005). Optically pure phenyl
ethanol and its derivatives can be converted without
racemization into other functional groups (Pollard and
Woodley 2006). Fungus or yeast are often used as
reducing agents because of the fact that they are
cheap and easily available. For this purpose, either
CONTACT Engin S¸ ahin
esahin@bayburt.edu.tr
Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt, 69000, Turkey
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
E. S¸ AHIN
Figure 1. Some examples of pharmaceutical compounds.
pure enzyme or whole cell microorganism is used as a
biocatalyst. Actually, there are many benefits of using
whole cell microorganism as biocatalysts instead of
pure enzymes. Pure enzymes are very costly and
hardly accessible; there is a limited availability of
enzymes and they need co-substrates like cofactors.
The use of whole cell is especially favorable to achieve
the desired reduction since they do not need addition
of cofactors for their renewal and enantioselective
reaction can be carried out with inexpensive equip-
ment (Patel et al. 2004; Murzin et al. 2005; Pollard and
Woodley 2006). But, some unconventional biocatalysts
can also be used for these purposes (Zilbeyaz et al.
OD and AS chiral columns. Enantiomeric excess was
determined directly from the areas under the curve
(Supplementary file). The racemic 2a–n were obtained
from corresponding 1a–n with NaBH in methanol at
4
room temperature used as a standard for the deter-
mination of the (R) or (S)-enantiomers. Reactions were
monitored by thin layer chromatography (TLC) using
aluminum-backed Merck Silica-Gel 60 F254 plates. For
analysis purpose a small fraction of the product was
prepared using preparative TLC.
Microorganisms identification and culture
conditions
2
010) which should also be in consideration during
finding new biocatalysts as microorganisms.
All yeast species used in this study were previously
isolated from different food products. Debaryomyces
hansenii strains were previously isolated from pastırma,
a fermented Turkish meat product (Ozturk 2015), and
Saccharomyces cerevisiae and Hanseniaspora guillier-
mondii strains were isolated from sourdough and
orange juice, respectively, in this study. The genotypic
identification of the yeast strains was conducted by
PCR analysis. Briefly, each isolate was grown in YPD
medium (Merck) and the DNA of the yeast samples
were isolated by phenol-chloroform methodology.
For the identification, the D1/D2 domain of 26S
rDNA region was PCR amplified using the primers
Herein, we report the potential of fifteen yeast
strains belonging to three yeast species Debaryomyces
hansenii, Saccharomyces cerevisiae and Hanseniaspora
guilliermondii, isolated from fermented meat product,
sourdough and orange juice, respectively as whole cell
biocatalysts to explore the bioreduction of prochiral
ketones, with the aim to screen the biocatalyst sub-
strate specificities and the enantioselectivities depend
on molecular structure of the prochiral ketone to be
reduced. Importantly, as food isolates, these strains
have great potential to be used safely for the indus-
trial purposes which increase their value as biocatalyst.
The objective of this study was to find successful
yeast strain in terms of percent conversion and
enantiomeric excess, for the asymmetric reduction of
prochiral ketones. Besides, we demonstrate the use of
Debaryomyces hansenii P1 (D. hansenii P1) as a new
biocatalyst for production of a chiral secondary alcohol
on a preparative scale.
0
0
NL1 (5 -GCATATCAATAAGCGGAGGAAAAG-3 ) and NL4
0
0
(5 -GGTCCGTGTTTCAAGACGG-3 ). The PCR mix was pre-
pared with 1ll of DNA template, 10ll of 5x PCR buffer,
0.4 ll dNTPs, 1ll of 20 mM primers NL1 and NL4,
0.25 ll 5U Taq polymerase (Promega) and up to 50 ll
of sterile H O and PCR conditions were conducted as
2
described previously (Yilmaz et al. 2016) and later
BLAST search of the obtained sequence of both spe-
cies were identified with 97–100% similarity criterion.
Materials and methods
The chemical reagents were obtained from Sigma-
1
13
General bioreduction reactions
Aldrich in the highest purity available. H and C NMR
spectra were recorded on Bruker 400 spectrometer in
CDCl3 (Supplementary file). Enantiomeric excesses
were determined by chiral HPLC analysis using OD-H,
Yeast strain was propagated from their glycerol stocks
by inoculation to 10 ml YPD medium (1% yeast extract,
1% peptone from casein, 2% Glucose) followed by 2 d

BIOCATALYSIS AND BIOTRANSFORMATION
3
Figure 2. A gram scale production of (R)-1-(3-methoxyphenyl) ethanol (2g).
ꢀ
growth at 30 C. From this culture, exponentially
grown yeast cell was inoculated to 50 ml YPD medium
at 10% concentration and pH was adjusted to 5.5. This
was shaken for 2 hours, then 1 mmol substrate was dir-
J ¼ 6.4 Hz, 1H), 2.25 (bs, OH: exchanges with D O), 1.47
2
1
3
(d, J ¼ 6.4 Hz, 3H); C NMR (100 MHz, CDCl ) d ¼ 143.0,
3
131.6, 129.4, 128.4, 127.2, 126.4, 66.9, 23.5;
2
5
[a] ¼ þ66.1 (c 1.0, CHCl ) 97% ee; retention time:
D
3
ectly added to the medium and incubated on a shaker
15.1 min (major), 16.9 min (minor), Chiralcel OD-H, n-
hexane/i-PrOH, 98:2, flow rate of 1.0 mL/min, 220 nm.
1-(2-Bromophenyl) ethanol (2c) (Zhang et al. 2011;
ꢀ
(
150 rpm) at 30 C for 48 h. At the end of the incuba-
tion period, the cells were separated by centrifugation
ꢀ
at 6000 ꢁ g for 5 min at 4 C and the supernatant was
Li et al. 2014)
1
saturated with NaCl , then extracted with diethyl
Colorless oil, Yield 90%, H NMR (400 MHz, CDCl
3
)
2
ether. Diethyl ether extracts were combined and was
dried over Na SO . After removal of the solvent under
d ¼ 7.56–7.54 (m, 1H), 7.50–7.48 (m, 1H), 7.33–7.29 (m,
1H), 7.12–7.08 (m, 1H), 5.39 (q, J ¼ 6.4 Hz, 1H), 2.79 (bs,
2
4
1
3
reduced pressure the crude product was identified by
NMR analysis. The absolute configuration was deter-
mined by sign of specific rotation and comparison
with the literature. The enantiomeric excess of the sec-
ondary alcohol was determined by chiral HPLC
analysis.
OH: exchanges with D
2
O), 1.44 (d, J ¼ 6.4 Hz, 3H);
C
NMR (100 MHz, CDCl
) d ¼ 144.7, 132.6, 128.7, 127.8,
3
25
D
126.7, 121.6, 69.1, 23.6; [a]
¼ þ56.6 (c 1.0, CHCl
);
3
99% ee; retention time: 11.7 min (minor), 13.3 min
(major), Chiralcel OD-H, n-hexane/i-PrOH, 98:2, flow
rate of 1.0 mL/min, 220 nm.
1
-(2-Methoxyphenyl) ethanol (2d) (Zhang et al.
011)
Colorless oil, Yield 93%, H NMR (400 MHz, CDCl )
2
Production of gram scale of 2 g
1
3
Yeast strain was propagated from stock by incubation
to 100 mL YPD medium followed by 2 d growth at
d ¼ 7.36–7.43 (m, 1H), 7.28–7.23 (m, 1H), 6.97 (t,
J ¼ 7.5 Hz, 1H), 6.88 (d, J ¼ 8.20 Hz, 1H), 5.13–5.07 (m,
ꢀ
3
1
1
0 C, then grown yeast cell was inoculated to
000 mL YPD medium in 2L fermenter and following
5 mmol 1g was directly added to the reaction
1H), 3.86 (s, 1H), 2.78 (bs, OH: exchanges with D O),
2
1
3
1.50 (d, J ¼ 7.1 Hz, 3H); C NMR (100 MHz, CDCl3)
d ¼ 156.5, 133.4, 128.3, 126.3, 120.8, 110.4, 66.4, 55.2,
2
5
medium and incubated for 48 h. Then the mixture was
extracted with diethyl ether and was dried over
Na SO . After evaporation of the solvent, the product
22.9; [a] ¼ ꢂ11.2 (c 1.1, CHCl ) 45% ee; retention
D
3
time: 16.9 min (major), 18.0 min (minor), Chiralcel OD-
H, n-hexane/i-PrOH, 98:2, flow rate of 1.0 mL/min,
220 nm.
2
4
2
g was purified on silica gel column (Figure 2) and
obtained as a optically pure secondary alcohol (1.87 g,
2.3 mmol, 82% yield).
-Phenylethanol (2a) (Wei and Du 2014; Perna
et al. 2016)
1-(2-Nitrophenyl) ethanol (2e) (Vieira et al. 2010)
1
1
Colorless oil, Yield 87%, H NMR (400 MHz, CDCl )
3
1
d ¼ 7.84 (dd, J ¼ 8.2, 1.1 Hz, 1H), 7.79 (dd, J ¼ 7.9,
1.1 Hz, 1H), 7.63–7.59 (m, 1H), 7.40–7.36 (m, 1H), 5.35
1
Colorless oil, Yield 89%, H NMR (400 MHz, CDCl )
(q, J ¼ 6.4 Hz, 1H), 2.85 (bs, OH: exchanges with D O),
3
2
1
3
d ¼ 7.38–7.33 (m, 4H), 7.31–7.26 (m, 1H), 4.84 (q,
1.51 (d, J ¼ 6.4 Hz, 3H); C NMR (100 MHz, CDCl )
3
J ¼ 6.45 Hz, 1H), 2.62 (bs, OH: exchanges with D O),
d ¼ 147.7, 141.0, 133.6, 128.0, 127.5, 124.2, 65.4, 24.2;
2
1
3
25
D
1
.48 (d, J ¼ 6.5 Hz, 3H); C NMR (100 MHz, CDCl )
[a] ¼ ꢂ29.8 (c 0.4, CHCl ); 99% ee; retention time:
3
3
2
5
d ¼ 146.0, 128.4, 127.3, 125.5, 70.2, 25.2; [a] ¼ ꢂ45.3
19.1 min (minor), 20.3 min (major), Chiralcel AS, n-hex-
ane/i-PrOH, 97:3, flow rate of 0.8 mL/min, 220 nm.
D
(
c 1.1, CHCl ) 95% enantiomeric excess (ee); retention
3
time: 7.5 min (minor), 8.7 min (major), Chiralcel OD-H,
n-hexane/i-PrOH, 95:5, flow rate of 1.0 mL/min, 210 nm.
1-(3-Chlorophenyl) ethanol (2f) (Wei and Du 2014)
1
Colorless oil, Yield 93%, H NMR (400 MHz, CDCl
)
3
1
-(2-Chlorophenyl) ethanol (2b) (Zhang et al. 2011;
d ¼ 7.34–7.19 (m, 4H), 4.82 (q, J ¼ 6.4 Hz, 1H), 2.43 (bs,
13
Liu et al. 2015)
OH: exchanges with D O), 1.45 (d, J ¼ 6.5 Hz, 3H);
C
2
1
Colorless oil, Yield 91%, H NMR (400 MHz, CDCl )
d ¼ 7.58–7.56 (m, 1H), 7.32–7.17 (m, 3H), 5.27 (q,
NMR (100 MHz, CDCl ) d ¼ 147.8, 134.3, 129.8, 127.5,
3
3
25
D
125.6, 123.5, 69.7, 25.2; [a] ¼ ꢂ35.6 (c 0.60, CHCl );
3

4
E. S¸ AHIN
9
2% ee; retention time: 22.1 min (major), 23.6 min
21.1 min (minor), 22.9 (major), Chiralcel OD, n-hexane/
i-PrOH, 90:10, flow rate of 0.5 mL/min, 220 nm.
(
minor), Chiralcel OD-H, n-hexane/i-PrOH, 99:1, flow
rate of 1.0 mL/min, 210 nm.
-(3-Methoxyphenyl) ethanol (2g) (Zhang et al.
011)
Colorless oil, Yield 94%, H NMR (400 MHz, CDCl )
1-(4-Nitrophenyl) ethanol (2m) (Vieira et al. 2010)
1
1
Colorless oil, Yield 91%, H NMR (400 MHz, CDCl
3
)
2
d ¼ 8.10–8.14 (m, 2H), 7.51–7.48 (m, 2H), 5.00–4.94 (m,
1H), 2.75 (bs, OH: exchanges with D O), 1.47 (d,
J ¼ 6.5 Hz, 3H); C NMR (100 MHz, CDCl ) d ¼ 153.3,
47.0, 126.1, 123.7, 69.4, 25.4; [a] ¼ ꢂ25.4 (c 0.5,
1
3
2
1
3
d ¼ 7.27–7.23 (m, 1H), 6.93-6.91 (m, 2H), 6.81-6.78 (m,
H), 4.83 (q, J ¼ 6.4 Hz, 1H), 3.79 (s, 3H), 2.41 (bs, OH:
3
2
5
1
1
D
1
3
CHCl ) 99% ee; retention time: 13.3 min (minor), 15.4
exchanges with D O), 1.46 (d, J ¼ 6.5 Hz, 3H); C NMR
3
2
(
major), Chiralcel AS, n-hexane/i-PrOH, 95:5, flow rate
of 0.8 mL/min, 220 nm.
-(p-Biphenyl) ethanol (2n) (Zilbeyaz et al. 2012)
(
1
100 MHz, CDCl ) d ¼ 159.7, 147.6, 129.5, 117.7, 112.8,
3
2
5
10.9, 70.2, 55.2, 25.1; [a] ¼ þ58.8 (c 0.9, CHCl ); 99%
D
3
1
ee; retention time: 12.6 min (minor), 20.4 (major),
ꢀ
1
White solid, m.p. 92–94 C, yield 92%, H NMR
Chiralcel OD-H, n-hexane/i-PrOH, 95:5, flow rate of
(
4
2
400 MHz, CDCl ) d ¼ 7.63–7.60 (m, 4H), 7.49–7.45 (m,
3
0
.8 mL/min, 210 nm.
-(4-Chlorophenyl) ethanol (2h) (Wei and Du 2014;
Liu et al. 2015)
H), 7.40–7.36 (m, 1H), 4.95 (qd, J ¼ 6.4, 2.9 Hz, 1H),
1
.24 (bs, OH: exchanges with D O), 1.56 (d, J ¼ 6.5 Hz,
2
1
3
1
3H); C NMR (100 MHz, CDCl ) d ¼ 144.9, 140.9, 140.4,
3
Colorless oil, Yield 95%, H NMR (400 MHz, CDCl )
3
1
28.8, 127.3, 127.3, 127.1, 125.9, 70.1, 25.2;
d ¼ 7.29–7.22 (m, 4H), 4.79 (q, J ¼ 6.5 Hz, 1H), 2.77 (bs,
2
5
1
3
[a] ¼ ꢂ46.9 (c 0.55, CH Cl ); 90% ee; retention time:
D
2
2
OH: exchanges with D O), 1.41 (d, J ¼ 6.5 Hz, 3H);
C
2
1
6.5 min (major), 17.9 (minor), Chiralcel OD, n-hexane/
NMR (100 MHz, CDCl ) d ¼ 144.2, 132.9, 128.5, 126.8,
3
2
5
i-PrOH, 90:10, flow rate of 0.6 mL/min, 220 nm.
6
9.6, 25.2; [a] ¼ ꢂ34.6 (c 1.0, CHCl ); 74% ee; reten-
D
3
tion time: 13.3 min (major), 14.9 (minor), Chiralcel OD-
H, n-hexane/i-PrOH, 98:2, flow rate of 1 mL/min,
Results and discussion
2
20 nm.
Fifteen different strains belonging to three different
yeast species that were isolated from fermented
pastırma, sourdough and orange juice namely D. hanse-
nii, S. cerevisiae and H. guilliermondii were tested for the
bioreduction of acetophenone 1a to 1-phenylethanol 2a
1-(4-Bromophenyl) ethanol (2i) (Singh and Chopra
2
016)
1
Colorless oil, Yield 85%, H NMR (400 MHz, CDCl )
3
d ¼ 7.43–7.40 (m, 2H), 7.18–7.15 (m, 2H), 4.75 (q,
J ¼ 6.5 Hz, 1H), 2.93 (bs, OH: exchanges with D O), 1.39
2
(Table 1). The bioreduction ability of these strains were
1
3
(
1
d, J ¼ 6.5 Hz, 3H); C NMR (100 MHz, CDCl ) d ¼ 144.7,
3
determined by the addition of 5 ml wet yeast cells to
the 50 ml culture environment in which 1 mmol sub-
strate of 1a was added and cells with substrates were
2
5
31.5, 127.2, 121.0, 69.6, 25.2; [a] ¼ þ32.8 (c 1.0,
D
CHCl ); 82% ee; retention time: 8.7 min (major), 9.6
3
(
minor), Chiralcel OD-H, n-hexane/i-PrOH, 95:5, flow
rate of 1 mL/min, 220 nm.
-(4-Methoxyphenyl) ethanol (2j) (Singh and
Chopra 2016)
ꢀ
incubated at 30 C for 24 h under 100 rpm agitation con-
ditions. The yield and the conversion rate of the reaction
1
1
were determined by H-NMR using internal standard.
The enantiomeric specificity of the final product was
determined by chiral HPLC. As can be seen in Table 1,
the strains produced (S)-1-phenylethanol 2a (39–71%
ee). Good results for the bio reduction of acetophenone
was obtained when D. hansenii P1 was used as a whole
cell biocatalyst for further studies. The conversion rate
to (S)-1-phenylethanol by D. hansenii P1 was a moderate
conversion (60%) and the ee was 71%. D. hansenii P1
was thus selected for further studies which includes the
reduction of acetophenone derivatives.
Firstly, we investigated the effects of reaction condi-
tions on the product’s enantioselectivity and conver-
sion. Reaction conditions are important in the
successful production of a secondary chiral alcohols
(Pereira 1988). Hence, reaction conditions such as pH,
reaction time and agitation speed were optimized
using D. hansenii P1 as a biocatalyst. The results of
1
Colorless oil, Yield 88%, H NMR (400 MHz, CDCl )
3
d ¼ 7.30–7.27 (m, 2H), 6.89–6.85 (m, 2H), 4.83 (q,
J ¼ 6.4 Hz, 1H), 3.79 (s, 3H), 2.11 (bs, OH: exchanges
1
3
with D O), 1.46 (d, J ¼ 6.5 Hz, 3H);
C
NMR
2
(
5
100 MHz, CDCl ) d ¼ 158.9, 138.0, 126.7, 113.8, 69.9,
3
5
2
5.3, 25.0; [a] ¼ þ3.7 (c 1.0, CHCl ); 6% ee; reten-
D
3
tion time: 14.9 min (major), 16.6 (minor), Chiralcel
OD-H, n-hexane/i-PrOH, 95:5, flow rate of 0.8 mL/min,
2
20 nm.
-(p-Tolyl) ethanol (2k) (Wakeham et al. 2015)
1
1
Colorless oil, Yield 95%, H NMR (400 MHz, CDCl )
3
d ¼ 7.27 (d, J ¼ 8.0 Hz, 1H), 7.17 (d, J ¼ 7.9 Hz, 1H), 4.84
(
2
q, J ¼ 6.5 Hz, 1H), 2.42 (bs, OH: exchanges with D O),
2
1
3
.37 (s, 3H), 1.48 (d, J ¼ 6.5 Hz, 3H); C NMR (100 MHz,
CDCl ) d ¼ 142.9, 137.0, 129.1, 125.4, 70.1, 25.1, 21.1;
3
2
5
[
a] ¼ þ53.8 (c 1.0, CHCl ) 90% ee; retention time:
D
3

BIOCATALYSIS AND BIOTRANSFORMATION
5
Table 1. Screening of yeast strains for the bio reduction of acetophenone.
a
b
b,c
d
Microorganism
Conversion (%)
M.B.
%
ee % (S)
Saccharomyces cerevisiae SD1
Saccharomyces cerevisiae SD2
Saccharomyces cerevisiae SD3
Saccharomyces cerevisiae SD4
Saccharomyces cerevisiae SD5
Debaryomyces hansenii P1
Debaryomyces hansenii P2
Debaryomyces hansenii P3
Debaryomyces hansenii P4
Debaryomyces hansenii P5
Hanseniaspora guilliermondii OJ1
Hanseniaspora guilliermondii OJ2
Hanseniaspora guilliermondii OJ3
Hanseniaspora guilliermondii OJ4
Hanseniaspora guilliermondii OJ5
Weak growth
35
47
38
17
60
47
39
46
54
51
28
57
67
54
41
46
57
71
58
48
51
48
41
55
39
77
69
88
87
91
83
89
83
89
91
80
No growth
61
88
47
ꢀ
Reaction conditions: biocatalyst 5 ml, substrate: 1mmol, temperature: 30 C, time: 24 h, pH: 6.5, 100 rpm
a
Comparison of the best microorganism.
b
1
The conversions and mass balances (MB) were determined by H NMR analysis with diphenyl methane as an internal
standard.
MB refers to the sum of the yields of the characterized adduct 2a and the unreacted acetophenone 1a.
c
d
Determined by HPLC using Chiralcel OD-H column.
Table 2. Optimization of parameters for the bioreduction of acetophenone 1a with D. hansenii P1.
pH
Temperature
Incubation period
Agitation speed
b
c,d
ꢀ
b
c,d
b
c,d
b
c,d
Conv. (%)
ee (%)
C
Conv. (%)
ee (%)
h
Conv. (%)
ee (%)
rpm
Conv. (%)
ee (%)
4
5
5
6
6
7
7
8
a
.5
.5
.5
38
50
78
55
60
40
38
0
92 (S)
91 (S)
95 (S)
90 (S)
92 (S)
88 (S)
84 (S)
25
28
30
32
34
65
68
80
65
45
83 (S)
90 (S)
95 (S)
87 (S)
60 (S)
24
48
72
96
60
90
87
97
71 (S)
100
150
200
250
60
97
98
99
71 (S)
95 (S)
67 (S)
60 (S)
95 (S)
83 (S)
65 (S)
.5
e
Substrate: 1 mmol.
b
c
1
The conversion was determined by H NMR analysis with diphenyl methane as an internal standard.
Determined by HPLC using Chiralcel OD-H column.
d
e
Absolute configuration were assigned by comparison of the sign of optical rotations relative to the values in literature.
Weak growth.
optimizations are shown in Table 2. Several pH’s
enantioselectivity. Thus, we continued the study with
30 C. Various incubations times (24–96 h) were
ꢀ
(
4.5–8) were selected to monitor the progress of the
asymmetric reduction reaction in the reaction condi-
tions of 100 rpm, 30 C and 24h. The highest conver-
selected to observed the progress of the reduction
ꢀ
reaction in the reaction conditions of pH ¼ 5.5,
ꢀ
sion (78%) and ee (95%) were obtained when the pH
was controlled at 5.5. The impacts of different culture
temperatures were investigated by using fermentation
100 rpm and 30 C. The highest conversion (97%) was
observed at 96 h, but the ee (65%) was decreased. As
incubations time increased, the conversions of bio
reduction increased but ee decreased substantially.
Hence, the best time chosen was 48 hours because the
best ee was obtained in this process. Finally, the rate
of agitation speeds was investigated for the best con-
version and ee in the reaction conditions of pH ¼ 5.5,
ꢀ
processes at different temperatures (25–34 C) in the
reaction conditions of pH ¼ 5.5, 100 rpm and 24h. The
highest conversion (80%) and ee (95%) were obtained
ꢀ
ꢀ
at 30 C. When the temperature increased over 30 C,
not only ee but also conversion dropped drastically.
For instance, low ee (60%) and conversion (45%) were
ꢀ
30 C and 48h. As seen in Table 1, when the agitation
ꢀ
seen at 34 C. This suggest that high temperature had
speed is increased, conversion of the reaction
increased, but the enantioselectivity reduced
a negative action on conversion of reduction and

6
E. S¸ AHIN
Table 3. Enantioselective reduction of acetophenone derivatives 1a–n by D hansenii P1.
b,c
d
d
Entry
1
Substrate
Product
ee (%)
Conversion (%)
97
M.B. (%)
94
95 (S)
2
97 (R)
90
87
3
4
5
6
7
8
99 (R)
45 (S)
99 (S)
92 (S)
99 (R)
74 (S)
87
73
93
93
85
95
91
88
94
90
89
93
9
82 (R)
6 (R)
91
77
72
92
94
90
1
0
1
1
90 (R)
(
continued)

BIOCATALYSIS AND BIOTRANSFORMATION
7
Table 3. Continued
Entry
b,c
d
d
Substrate
Product
ee (%)
Conversion (%)
M.B. (%)
1
2
99 (S)
>99
92
1
3
90 (S)
80
93
a
ꢀ
Bio reduction conditions: Substrate: 1 mmol, temperature: 30 C, time: 48 h, pH: 5.5, 150 rpm.
b
Determined by HPLC using Chiralcel OD-H,OD and AS columns.
c
Configurations were assigned by comparison with the sign of specific rotation relative to the literature values.
d
1
The conversions and mass balances (MB) were determined by H NMR analysis with diphenylmethane as an internal standard.
drastically. The highest ee (95%) and conversion (97%)
rate was obtained at 150 rpm and therefore this agita-
tion speed was selected for optimum conditions.
conversion and enantiomeric excess (Table 3, entry
11). 1-(p-biphenyl) ethanol 2n was obtained with mod-
erate conversion rate and enantiomeric excess (Table
3, entry 13). However, the lowest enantioselectivity
(6%) and low conversion rate (77%) were obtained in
the bio reduction of p-methoxy acetophenone 1j
(Table 3, entry 10). The lowest decrease in conversions
was observed for substrates 1b and 1c, which sug-
gests that the rate of the reduction depends on the
steric and electronic effects of bromine and chlorine
atom at the ortho-position on the phenyl ring.
Enantioselectivity decreases as chlorine and bromine
groups move away from the reaction center. The
decrease in bromine was less because bromine was
larger than the chlorine in terms of volume. This
shows that the steric effect of chlorine and bromine is
important in enantioselectivity. Clearly, the electron-
deficient substrate showed higher selectivity, while
electron-rich substrate showed moderate selectivity
except for m-methoxy derivative 1g (Table 3, entry 7).
The methoxy group provides more electrons in the
ortho and para positions while it cannot provide more
electrons in the meta position. Therefore, the methoxy
group in the meta position has significantly increased
enantioselectivity. It can be said that electronic effect
of the methoxy group is greater than steric effect.
Similar selectivity in the reductions of the ortho- and
para-nitro derivatives was observed which highlighted
the importance of electronic effects for the selectivity
of bio reduction with a negligible steric effects on the
substituent for these samples. The enantioselectivity of
the acetophenone derivatives were dependent on the
position of substitutes at benzene ring (Prelog 1964).
Since several acetophenone derivatives demonstrated
perfect enantioselectivities for the asymmetric reduc-
tion on small scale, we decided to conduct the asym-
metric reduction of 1g to (R)-2g on a large scale to
prove the applicability of the current system as
Under the optimum conditions (pH 5.5, temperature
ꢀ
3
0 C, time 48h and agitation speed 150 rpm) asym-
metric reduction of acetophenone derivatives 1a–n
with D. hansenii P1 were investigated, since the corre-
sponding chiral secondary alcohols 2a–n are important
starting materials in the synthesis of pharmacological
and bioactive compounds. The bio reduction products
demonstrated in Table 3. The stereoselectivity of
the reaction was initially studied for acetophenones
bearing electron-donating groups (Me and OMe) and
electron-withdrawing groups (CI, Br and NO ) at the
2
ortho-, meta- and para-position on aromatic benzene
ring. As can be seen Table 3, the enantioselectivity of
more than 99% was obtained from reduction of o-nitro
and o-bromo acetophenones 1c and 1e, which are
converted into corresponding alcohols 2c and 2e with
highest ee (99%) and high conversions 93 and 87%
respectively (Table 3, entry 3,5). o-chloro acetophe-
none 1b was reduced into the (R)-alcohol 2b with
high ee (97%) and conversion (90%) (Table 3, entry 2).
However, o-methoxy acetophenone 1d was reduced
into the (S)-alcohol 2d with low ee (45%), and conver-
sion (73%) (Table 3, entry 4). The bioreduction of m-
chloro 1f led to the observe of alcohol 2f with high ee
(
92%) and conversion (93%) (Table 3, entry 6). m-
methoxy acetophenone 1g was reduced in excellent
ee (99%) and moderate conversion (75%) (Table 3,
entry 7). Reduction of 4-chloro acetophenone 1h and
4
-bromo acetophenone 1i occurred with high conver-
sions (95%, 91%), and ee (74%, 82%) respectively
Table 3, entry 8, 9). However, 4-nitroacetophenone
m was reduced in the (S)-alcohol 2m with excellent
(
1
conversion (99%) and ee (99%) (Table 3, entry 12).
Electron-donating group methyl at the para position
on the aromatic ring led to a slight decrease in the

8
E. S¸ AHIN
industrially feasible. The asymmetric bioreduction of 3-
methoxyacetophenone 1g (15 mmol) was carried out
in a 1 L Erlenmeyer and (R)-1-(3-methoxyphenyl) etha-
nol 2g was obtained in 82% yield and 99% enantio-
meric excess, which is a good result for a preparative
scale (Figure 2). In this way, (R)-2g which can be used
for treatment of hyper-parathyroidism was obtained in
gram scale. Transformations using isolated enzyme as
biocatalysts for reduction of prochiral ketones can pro-
duce excellent yields and enantioselectivity. But, these
bioreduction reactions require very costly cofactors
such as NADPH. These cofactors can be regenerated
by using whole cell as biocatalysts throughout the bio-
reduction reaction (Bradshaw et al. 1992; Kataoka et al.
Funding
Financial support from Bayburt University [BAP-2015/1-02] is
gratefully acknowledged.
References
Bradshaw CW, Hummel W, Wong CH. 1992. Lactobacillus kefir
alcohol dehydrogenase: a useful catalyst for synthesis.
J Org Chem 57:1532–1536.
Chen Q, Hu Y, Zhao W, Zhu C, Zhu B. 2010. Cloning,
Expression and Characterization of a Novel (S)-Specific
Alcohol Dehydrogenase from Lactobacillus kefir. Appl
Biochem Biotechnol 160:19–29.
Cohen A, Silverberg SJ. 2002. Calcimimetics: therapeutic poten-
tial in hyperparathyroidism. Curr Opin Pharmacol 2:734–739.
Comasseto JV, Andrade LH, Omori AT, Assis LF, Porto ALM.
1
997; Chen et al. 2010). In this study, similar to the bio
2004. Deracemization of aryl ethanols and reduction of
reduction method, the substrate was directly added to
the reaction medium without using cofactors and buf-
fers. D. hansenii P1 was used effectively as a whole cell
biocatalyst for reduction of acetophenone and its
derivatives.
acetophenones by whole fungal cells of Aspergillus terreus
CCT 4083, A. terreus CCT 3320 and Rhizopus oryzae CCT
4964. J Mol Catal B Enzym 29:55–61.
Huang Y, Liu N, Wu XM, Chen Y. 2010. Dehydrogenases/
reductases for the synthesis of chiral pharmaceutical inter-
mediates. Curr Org Chem 14:1447–1460.
Joy MS, Kshirsagar AV, Franceschini N. 2006. Calcimimetics
and the treatment of primary and secondary hyperpara-
thyroidism. Ann Pharmacother 38:1871–1880.
Kataoka M, Rohani LP, Yamamoto K, Wada M, Kawabata H,
Kita K. 1997. Enzymatic production of ethyl (R)-4-chloro-3-
hydroxybutanoate: asymmetric reduction of ethyl 4-
chloro-3-oxobutanoate by an Escherichia coli transformant
expressing the aldehyde reductase gene from yeast. Appl
Microbiol Biotechnol 48:699–703.
Kurbanoglu EB, Taskin M, Zilbeyaz K, Hasenekoglu I. 2009.
Efficient Synthesis of (S)-1-(2-chlorophenyl) ethanol in the
Submerged Culture of Alternaria alternata Isolate. Chin J
Cat 30:370–374.
Kurbanoglu EB, Zilbeyaz K, Kurbanoglu NI, Kilic H. 2007.
Asymmetric reduction of acetophenone analogues by
Alternaria alternata using ram horn peptone. Tetrahedron:
Asymmetry 18:2332–2335.
Conclusion
In this study, different acetophenones 1a–m were
reduced to the corresponding chiral secondary alco-
hols 2a–m with high enantioselectivity using D. hanse-
nii P1 yeast strain. We have showed a new yeast to
obtain enantiopure chiral secondary alcohols. The pre-
sent study has many benefits because enantioselectiv-
ity and conversion of the bioreduction are controlled
by the substituent position and electronic effect on
phenyl ring, and this yeast can be used to obtain
gram scale chiral secondary alcohols. This system is
suitable and shows perfect enantioselectivity which
can be used to carried out synthesis of valued enantio-
pure secondary alcohols. An easy and productive pro-
cess has been developed for the preparation of chiral
secondary alcohols in excellent enantiomeric excess by
using D. hansenii P1 yeast whole cell as an alternative
to other classic enantioselective catalysts that fre-
quently required the use of costly ligands or metal
complex. D. hansenii P1 can be beneficial for the pro-
duction of important building blocks in pharmaceutical
synthesis.
Legnani L, Morandi B. 2016. Direct catalytic synthesis of
unprotected 2-amino-1-phenylethanols from alkenes by
using iron(II) phthalocyanine. Angew Chem Int Ed Engl
55:2248–2251.
Li Y, Yu S, Wu X, Xiao J, Shen W, Dong Z, Gao J. 2014. Iron
catalyzed asymmetric hydrogenation of ketones. J Am
Chem Soc 136:4031–4039.
Liu WP, Yuan ML, Yang XH, Li K, Xie JH, Zhou QL. 2015.
Efficient asymmetric transfer hydrogenation of ketones in
ethanol with chiral iridium complexes of spiro PAP ligands
as catalysts. Chem Commun 51:6123–6125.
Murzin DY, Maki-Arvela P, Toukoniitty E, Salmi T. 2005.
Asymmetric heterogeneous catalysis: science and engin-
eering. Catal Rev 47:175–256.
Acknowledgements
Financial support from Bayburt University (BAP-2015/1-02) is
gratefully acknowledged.
Nagano N. 2006. Pharmacological and clinical properties of
calcimimetics: calcium receptor activators that afford an
innovative approach to controlling hyperparathyroidism.
Pharmacol Ther 109:339–365.
Ozturk I. 2015. Presence, changes and technological proper-
ties of yeast species during processing of pastirma, a
Turkish dry-cured meat product. Food Control 50:76–84.
Disclosure statement
No potential conflict of interest was reported by the author.

BIOCATALYSIS AND BIOTRANSFORMATION
9
Patel RN, Goswami A, Chu L, Donovan MJ, Nanduri VBJ,
Goldberg S, Johnston R, Siva PJ, Nielsen B, Fan J. 2004.
Enantioselective microbial reduction of substituted aceto-
phenones. Tetrahedron: Asymmetry 15:1247–1258.
Patel RN. 2008. Synthesis of chiral pharmaceutical intermedi-
ates by biocatalysis. Coord Chem Rev 252:659–701.
Wei S, Du H. 2014. A Highly Enantioselective hydrogenation
of silyl enol ethers catalyzed by chiral frustrated Lewis
Pairs. J Am Chem Soc 136:12261–12264.
Wichmann R, Vasic-Racki D. 2005. Cofactor regeneration
at the lab scale. Adv Biochem Eng Biotechnol 92:
225–260.
Pereira RS. 1988. The use of Baker’s yeast in the generation
of asymmetric centers to produce chiral drugs and other
compounds. Crit Rev Biotechnol 18:25–64.
Perna PM, Ricci MA, Scilimati A, Mena MC, Pisano I, Palmieri
L, Agrimi G. 2016. Cheap and environmentally sustainable
stereoselective arylketones reduction by Lactobacillus reu-
teri whole cells. J Mol Cat B Enzym 124:29–37.
ꢀ
ꢀ
Wujkowska Z, Jarzynski S, Pieczonka AM, Lesniak S,
Rachwalski M. 2016. Highly enantioselective addition of
arylzinc reagents to aldehydes promoted by chiral aziri-
dine alcohols. Tetrahedron: Asymmetry 27:1238–1244.
Yamada H, Shimizu S. 1988. Microbial and enzymatic proc-
esses for the production of biologically and chemically
useful compounds [New Synthetic Methods (69)]. Angew
Chem Int Ed Engl 27:622–642.
Pollard DJ, Woodley JM. 2006. Biocatalysis for pharmaceutical
intermediates: the future is now. Trends Biotechnol
Yilmaz FA, Bozkurt F, Cicek PK, Dertli E, Durak MZ, Yilmaz
MT. 2016. A novel antifungal surface-coating application
to limit postharvest decay on coated apples: molecular,
thermal and morphological properties of electrospun
zein–nanofiber mats loaded with curcumin. Innov Food
Sci Emerg Technol 37:74–83.
Zhang Z, Jain P, Antilla JC. 2011. Asymmetric reduction of
ketones by phosphoric acid derived catalysts. Angew
Chem Int Ed Engl 50:10961–10964.
25:66–73.
Prelog V. 1964. Specification of the stereoselectivity of some
oxido-reductases by diamond lattice sections. Pure Appl
Chem 9:119–130.
Singh A, Chopra HK. 2016. New benzimidazolium-based
chiral ionic liquids: synthesis and application in enantiose-
lective sodium borohydride reductions in water.
Tetrahedron: Asymmetry 27:448–453.
Strohmeier GA, Pichler H, May O, Gruber-Khadjawi M. 2011.
Application of designed enzymes in organic synthesis.
Chem Rev 111:4141–4164.
Zilbeyaz K, Kilic H, Sisecioglu M, Ozdemir H, G u€ ng o€ r AA.
2
012. Preparation of enantiomerically pure p-substituted
phenylethyl hydroperoxides by kinetic resolution and their
use as enantioselective oxidants in the asymmetric
Weitz–Scheffer epoxidation of E-chalcone. Tetrahedron:
Asymmetry 23:594–601.
Zilbeyaz K, Taskin M, Kurbanoglu EB, Kurbanoglu NI, Kilic H.
2010. Production of (R)-1-phenylethanols through bio
reduction of acetophenones by a new fungus isolate
Trichothecium roseum. Chirality 22:543–547.
Vieira GAB, Araujo DMF, Lemos TLG, de Mattos MC, de
Oliveira MCF, Melo VMM, de Gonzalo G, Gotor-Fernandez
V, Gotor V. 2010. Candida tropicalis CE017: a new Brazilian
enzymatic source for the bioreduction of aromatic prochi-
ral ketones. J Braz Chem Soc 21:1509–1516.
Wakeham RJ, Morris JA, Williams MJ. 2015. Alternative hydro-
gen source for asymmetric transfer hydrogenation in the
reduction of ketones. Chemcatchem 7:4039–4041.