Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

Article abstract of DOI:10.1002/cbdv.201700121

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1-phenylethanol as biocatalysts. Of these strains, C.?zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4-nitroacetophenone (1m) by C.?zeylanoides P1 gave (S)-1-(4-nitrophenyl)ethanol (2m) with 89% yield and >?99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C.?zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.

Full text of DOI:10.1002/cbdv.201700121

Article Type: Full Paper
Highly Enantioselective Production of Chiral Secondary Alcohols with
Candida Zeylanoides as a New Whole Cell Biocatalyst
Engin Şahin1*, Enes Dertli1
1Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt, Turkey
*Corresponding author: Engin Şahin, Department of Food Engineering, Faculty of
Engineering, Bayburt University, Bayburt, 69000, Turkey. Tel: +90 (0) 458 2111153, Fax: +90
(0) 458 2111128, Email: esahin@bayburt.edu.tr
The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols
results from the outstanding characteristics of biocatalysts in reaction, economic, ecological
issues. Herein fifteen yeast strains belonging to three food originated yeast species Candida
zeylanoides, Pichia fermentans and Saccharomyces uvarum were tested for their capability for
asymmetric reduction of acetophenone to 1-phenylethanol as biocatalysts. Of these strains,
Candida zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized
conditions, substituted acetophenones were converted to corresponding optically active
secondary alcohols in up to 99% enantiomeric excess and at high yield. The preparative scale
asymmetric bioreduction of 4-nitro acetophenone 1m by Candida zeylanoides P1 gave (S)-1-(4-
nitrophenyl) ethanol 2m 89% yield, and >99% enantiomeric excess. Compound 2m has been
obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate
that Candida zeylanoides P1is a promising biocatalyst for the synthesis of chiral alcohols in
industry.
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Keywords: Bioreduction, Asymmetric reduction, Whole yeast cells, Candida zeylanoides,
Biotransformations
Introduction
Production of single enantiomers of different chiral compounds is one of the main targets of
different industries including pharmaceutical industry as majority of the drugs in the market are
optically pure compounds at least bearing one chiral center [1-5]. Chiral secondary alcohols are
good examples of chiral compounds that can be used in the production of different drugs. For
instance, isoproterenol[6] which is used for the treatment of bradycardia, prozac[7] which is used in
the treatment of depression and neobeodine[8] which display antihistaminic and anticholinergic
action are synthesized from chiral secondary alcohols (Figure 1). These enantioselective products
can be produced with different methodologies although biotransformation is one of the most
preferred methodologies among them that have many advantages compared to chemical catalysts
[9-11]. For instance, biocatalysts are providing clean and eco-friendly way to perform chemical
reactions under mild conditions and high selectivity for the substrate whereas chemical catalysts
produce toxic waste and a large amount of waste product [12,13]. Biotransformation generally
relies on the use of isolated enzymes or whole cell microorganism as both methods have some
advantages and drawbacks. Isolated enzymes are extremely selective and recyclable catalysts, but
are usually very expensive and not readily accessible due to the limited availability of
cosubstrates and cofactors of the biocatalyst [14]. On the other hand, whole cells are small
factories, especially advantageous since they are equipped with all the metabolic ways for the
regenerations of necessary cofactors, well protected within their natural cellular environment and
several different microorganisms were successfully shown to be used for these biotransformation
reactions [15-18]. These examples showed that different microorganisms belonging to bacteria,
yeast or fungi can act as different biotransformation agents with different final yields and ee
values and finding new microbial species with biotransformation potential is crucial. So three
different yeast species Candida zeylanoides, Pichia fermentans and Saccharomyces uvarum,
were isolated from fermented pastırma, orange juice and sourdough, respectively as potential
whole cell biotransformation agents. These yeast species were further tested for the asymmetric
reduction of acetophenone and its derivatives in order to investigate the percent conversion and
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enantiomeric excess. A Candida zeylanoides strain showed good characteristics among the tested
strains and reaction conditions including pH, incubation temperature, time, agitation level was
optimised to achieve higher conversion and ee values. Following the optimisation, this strain was
used as a new biocatalyst for production of a chiral secondary alcohol 2m which has not been
obtained enantio pure in the gram scale previously. Importantly, this compound can be easily and
inexpensively converted to (S)-1-(4-nitrophenyl) ethanamine in future works which was
previously reported to be producible in an expensive way19. Our findings clearly showed the
usage of whole cells as a cheap and effective methodology to produce different secondary
alcohols including (S)-1-(4-nitrophenyl) ethanol.
Results and Discussion
Fifteen different strains belonging to three different yeast species that were isolated from
fermented pastırma, orange juice and sourdough namely, Candida zeylanoides, Pichia fermentans
and Saccharomyces uvarum were tested for the bioreduction of acetophenone 1a to 1-
phenylethanol 2a (Table 1). The bioreduction ability of these strains were determined by addition
of 5 ml wet yeast cells to the 50 ml culture environment in which 1 mmol substrate 1a was added
and cells with substrate were incubated at 30oC for 24 h under 100 rpm agitation conditions. The
1
yield and the conversion rate of the reaction 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 (41-74% ee). The good
results for the bioreduction of acetophenone was obtained when Candida zeylanoides P1 was
used as a whole cell biocatalyst. The conversion rate to (S)-1-phenylethanol by Candida
zeylanoides P1 was at moderate conversion (67%) and the ee 74%. Candida zeylanoides P1 was
thus selected for further studies, which includes reduction of acetophenone derivatives.
Firstly, we investigate the effects of reaction conditions on the product’s enantioselectivity and
conversion. Reaction conditions are important in the successful production of a secondary chiral
alcohols [20]. Thus, reaction conditions such as pH, temperature, incubation period and agitation
speed were optimized using Candida zeylanoides P1 as a biocatalyst. The results of these
optimizations can be seen in Table 1. Different pH ranges (4.5-8.0) were chosen to monitor the
progress of the bioreduction reaction in the reaction conditions of 30oC, 100 rpm and 24 h. The
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highest conversion (81%) and enantiomeric excess (89%) were obtained when the medium pH
was controlled at 6.5. This effect of pH on conversion and enantioselectivity can be due to the
fact that the three-dimensional structures of the enzymes can vary depending on the pH which
affects both enantioselectivity and conversion as previously discussed [15]. The effects of several
temperatures were investigated by carrying out the fermentation process within different
temperature ranges (25-34oC) in the reaction condition of 100 rpm, 24h and pH 6.5. The highest
conversion (81%) and ee (89%) were obtained at 30oC. When the temperature rises above 30
degrees, both the conversion and the ee decreased significantly. For instance, the lowest
conversion (40%) and ee (60%) were obtained at 34oC. These results show that the increase in
temperature above 30 oC has negative effects on conversion and enantioselectivity which might
be related with optimum temperature range of the corresponding enzyme as the optimum
o
temperature conditions for yeast species is around 30 C. Therefore, the temperature of the
reduction reaction was chosen to be 30°C. Several incubation times were selected to follow the
progress of the bioreduction reaction in the reaction conditions of pH 6.5 and 30oC. The highest
conversions (94%) and (97%) were obtained after 72 and 96 hours, respectively, but the
enantioselectivity (81% and 77%) decreased during these periods. The increase in the incubation
period positively affected the conversion, but negatively affected the enantioselectivity. This
decrease in the enantioselectivity might be due to the degradation of the yeast cells as a result of
the long incubation period [21]. The period of incubation was chosen to be 48 hours because the
best enantioselectivity (90%) was obtained during this time. Different agitation speeds (100-250
rpm) were selected to monitor the progress of the bioreduction reaction in the reaction conditions
of pH 6.5, 30oC and 48 h. The highest values for both ee (>99%) and conversion (>99%) were
achieved at 150 rpm and thus this agitation speed was chosen as optimum for fermentation.
Complete conversion of 1a was obtained at 200 and 250 rpm, but enantioselectivity dropped
sharply at these agitation speeds. This decrease might be owing to the effect of stress on the yeast
cells as well as on the enzyme structure [15]
.
Under the optimized conditions (pH 6.5, temperature 30oC, time 48 h and agitation speed 150
rpm) asymmetric reduction of the derivatives of acetophenone by Candida zeylanoides P1 were
investigated. The stereoselectivity of the reaction was investigated for the acetophenones bearing
electron-donating groups (Me, OMe and phenyl) and electron-withdrawing groups (CI, Br, NO2)
at the ortho-, metha- and para-position (Table 3). The bioreduction of ortho-, and metha-
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substituted chloro, bromo, methoxy and nitro acetophenones, as well as the para-nitro
acetophenones were transformed to the corresponding alcohols with excellent enantioselectivity
(Table 3, entry 2-7, 12). However, depending on the steric and electronic effects of these groups,
the conversions are slightly reduced especially in the electron-donating methoxy group (Table 3,
entry 4, 7). The reduction of 4-nitroacetophenone 1m occurred with complete conversion,
yielding the corresponding (S)-secondary alcohol with excellent ee (>99%) (Table 3, entry 12).
Electron-donating substituents methyl and phenyl groups at the para position on the aromatic
ring let to a slight decrease in enantiomeric excess and conversion (Table 3, entry 11, 13). The
reduction of 4-methoxyacetophenone 1j occurred with good enantiomeric excess (96%) and
lowest conversion (65%) (Table 3, entry 10). The Candida zeylanoides P1 reduced para
halogenated acetophenones 1i with good enantiomeric excess (97%) and moderate conversion.
Similar selectivity was obtained in the reduction of ortho- and para-nitro isomers highlighting the
importance of electronic effects for the conversion and selectivity of bioreduction, with a
negligible steric effects of the substituent. The enantioselectivity of the acetophenone derivatives
were dependent on the position of substitutes at benzene ring [22]. Since several acetophenone
derivatives demonstrated perfect enantioselectivities for the asymmetric reduction on small scale,
we decided to conduct the asymmetric reduction of 1m to (S)-2m on a large scale to prove the
applicable of the current system as industrially feasible. The asymmetric bioreduction in a 1 L
Erlenmeyer with 12 mmol of 4-nitoacetophenone 1m was converted to the (S)-1-(4-nitophenyl)
ethanol 2m with 89% yield and >99% enantiomeric excess (Figure 2). This result demonstrates
and confirms that the system is feasible for industrial purpose. In this way, (S)-2m was obtained
in gram scale. So in this study Candida zeylanoides P1 has been shown to be effective as a whole
cell biocatalyst for the reduction of acetophenone and its derivatives.
Conclusions
In this study, different acetophenones 1a-n were reduced corresponding chiral secondary alcohols
2a-n with high enantiosectivity using Candida zeylanoides P1 as yeast species. We have showed
a new yeast strain to obtain enantiopure chiral secondary alcohols. The present study has different
benefits, because enantioselectivity 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
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gram scale chiral secondary alcohols. This system shows perfect enantioselectivity and can be
carried out for the synthesis of valued enantiopure secondary alcohols. By using Candida
zeylanoides P1 yeast whole cell as an alternative to other classic enantioselective catalysts that
frequently required the use of costly ligands or metal complexes, an easy and productive process
has been developed for the preparation of chiral secondary alcohols in excellent enantiomeric
excess. This new biocatalyst has high selectivity for the asymmetric reduction reaction in a
biocatalytic process that offers higher yields and optically pure products. Candida zeylanoides P1
can be considered to be an outstanding catalyst for asymmetric synthesis of various important
chiral alcohols industrially.
Experimental Section
General
The chemical reagents were obtained from Sigma-Aldrich in the highest purity available. 1H and
13C NMR spectra were recorded on Bruker 400 spectrometer in CDCl3. Enantiomeric excesses
were determined by chiral HPLC analysis using chiral columns OD-H, OD and AS. Enantiomeric
excess was determined directly from the areas under the curve. The racemic 2a-n were obtained
from corresponding 1a-n with NaBH4 in methanol at room temperature used as a standard for the
determination 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 by preparative TLC.
Microorganisms, identification and culture conditions
Candida zeylanoides strains were previously isolated from pastırma a fermented Turkish meat
product[23] and Pichia fermentans and Saccharomyces uvarum strains were isolated from orange
juice and sourdough, respectively. The genotypic identification of the latter two species was
conducted by PCR analysis. Briefly, each isolate was grown in YPD medium (Merck) and 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 NL1 (5′-
GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′).
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The PCR mix was prepared with1 μL of DNA template, 10 μL 5× PCR buffer, 0.4 μL dNTPs, 1
μL of 20 mM primers NL1 and NL4, 0.25 μL 5U Taq polymerase (Promega) and up to 50 μL of
sterile H2O and PCR conditions were conducted as described previously[24] and after BLAST
search of the obtained sequence both species were identified with 97–100% similarity criterion.
General Bioreduction Reactions
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 growth at 30°C. From this
cultures, exponentially grown yeast cell was inoculated to 50 ml YPD medium at 10%
concentration and pH was adjusted 6.5 and shaken 2 hours than 1 mmol substrate was directly
added to the medium and incubated on a shaker (150 rpm) at 30oC for 48h. At the end of the
incubation period, the cells were separated by centrifugation at 6000 × g for 5 min at 4°C and the
supernatant saturated with NaCl, then extracted with diethyl ether. Diethyl ether extracts were
combined and dried over Na2SO4. After removal of the solvent under reduced pressure the crude
product identified by NMR analysis. The absolute configuration was determined by sign of
specific rotation and comparison with the literature. The enantiomeric excess of the seconder
alcohol was determined by chiral HPLC analysis.
Production of gram scale of 2m
The same transformation was performed on the preparative scale in 2000 mL flask. Yeast strain
was propagated from stock by incubation to 100 mL YPD medium followed by 2 d growth at 30
oC, then grown yeast cell was inoculated to 1000 mL sterilized fresh YPD medium in 2 L flask
and following 12 mmol 1m was directly added to the reaction medium and incubated for 48 h.
Then the mixture was extracted with EtOAc and dried over Na2SO4. After evaporation of the
solvent the product 2m was purified on silica gel column (Figure 2) and obtained as an optically
pure secondary alcohol (1.78 g, 10.68 mmol, 89% yield)
Spectral data of secondary alcohols
(S)-1-phenylethanol (2a)[25]
Colourless oil, Yield 89%, 1H NMR (400 MHz, CDCl3) δ= 7.38-7.33 (m, 4H), 7.31-7.26 (m, 1H),
4.84 (q, J = 6.45 Hz, 1H), 2.62 (bs, 1H), 1.48 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ= 146.0, 128.4, 127.3, 125.5, 70.2, 25.2; [α]D25 = -42.0 (c 1.1, CHCI3), >99% ee; retention time:
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9.3 min (minor), 11.0 min (major), Chiralcel OD-H, n-hexane/i-PrOH, 95:5, flow rate of 1.0
mL/min, 210 nm.
(R)-1-(2-chlorophenyl) ethanol (2b)[26,27]
Colourless oil, Yield 91%, 1H NMR (400 MHz, CDCl3) δ= 7.58-7.56 (m, 1H), 7.32-7.17 (m, 3H),
5.27 (q, J = 6.4 Hz, 1H), 2.25 (bs, 1H), 1.47 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=
25
143.0, 131.6, 129.4, 128.4, 127.2, 126.4, 66.9, 23.5; [α]D = +66.1(c 1.0, CHCI3), 97% ee;
retention time: 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.
(R)-1-(2-bromophenyl) ethanol (2c)[27,28]
Colourless oil, Yield 90%, 1H NMR (400 MHz, CDCl3) δ= 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 = 6.4 Hz, 1H), 2.79 (bs, 1H), 1.44 (d, J = 6.4
13
Hz, 3H); C NMR (100 MHz, CDCl3) δ= 144.7, 132.6, 128.7, 127.8, 126.7, 121.6, 69.1, 23.6;
25
[α]D = +56.6 (c 1.0, CHCI3), >99% ee; retention time: 13.6 min (major), Chiralcel OD-H, n-
hexane/i-PrOH, 98:2, flow rate of 1.0 mL/min, 220 nm.
(S)-1-(2-methoxyphenyl) ethanol (2d)[27]
Colourless oil, Yield 93%, 1H NMR (400 MHz, CDCl3) δ= 7.36-7.43 (m, 1H), 7.28-7.23 (m, 1H),
6.97 (t, J = 7.5 Hz, 1H), 6.88 (d, J = 8.20 Hz, 1H), 5.13-5.07 (m, 1H), 3.86 (s, 1H), 2.78 (d, J =
4.3 Hz, 1H), 1.50 (d, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ= 156.5, 133.4, 128.3,
126.3, 120.8, 110.4, 66.4, 55.2, 22.9; [α]D25 = -24.7 (c 1.1, CHCI3), >99% ee; retention time: 17.0
min (major), Chiralcel OD-H, n-hexane/i-PrOH, 98:2, flow rate of 1.0 mL/min, 220 nm.
(S)-1-(2-nitrophenyl) ethanol (2e)[29]
1
Colourless oil, Yield 87%, H NMR (400 MHz, CDCl3) δ= 7.84 (dd, J = 8.2, 1.1 Hz, 1H), 7.79
(dd, J = 7.9, 1.1 Hz, 1H), 7.63-7.59 (m, 1H), 7.40-7.36 (m, 1H), 5.35 (q, J = 6.4 Hz, 1H), 2.85
13
(bs, 1H), 1.51 (d, J = 6.4 Hz, 3H); C NMR (100 MHz, CDCl3) δ= 147.7, 141.0, 133.6, 128.0,
127.5, 124.2, 65.4, 24.2; [α]D25 = -29.8 (c 0.4, CHCI3), 99% ee; retention time: 19.1 min (minor),
20.3 min (major), Chiralcel AS, n-hexane/i-PrOH, 97:3, flow rate of 0.8 mL/min, 220 nm.
(R)-1-(3-chlorophenyl) ethanol (2f)[25]
1
Colourless oil, Yield 93%, H NMR (400 MHz, CDCl3) δ= 7.34-7.19 (m, 4H), 4.82 (q, J = 6.4
13
Hz, 1H), 2.43 (bs, OH), 1.45 (d, J = 6.5 Hz, 3H); C NMR (100 MHz, CDCl3) δ= 147.8, 134.3,
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129.8, 127.5, 125.6, 123.5, 69.7, 25.2; [α]D25 = +38.20 (c 0.60, CHCI3), >99% ee; retention time:
22.0 min (major), Chiralcel OD-H, n-hexane/i-PrOH, 99:1, flow rate of 1.0 mL/min, 210 nm.
(R)-1-(3-methoxyphenyl) ethanol (2g)[27]
Colourless oil, Yield 94%, 1H NMR (400 MHz, CDCl3) δ= 7.27-7.23 (m, 1H), 6.93-6.91 (m, 2H),
6.81-6.78 (m, 1H), 4.83 (q, J = 6.4 Hz, 1H), 3.79 (s, 3H), 2.41 (bs, OH), 1.46 (d, J = 6.5 Hz, 3H);
25
13C NMR (100 MHz, CDCl3) δ= 159.7, 147.6, 129.5, 117.7, 112.8, 110.9, 70.2, 55.2, 25.1; [α]D
= +58.8 (c 0.9, CHCI3), 99% ee; retention time: 12.1 min (minor), 20.1 (major), Chiralcel OD-H,
n-hexane/i-PrOH, 95:5, flow rate of 0.8 mL/min, 210 nm.
(R)-1-(4-chlorophenyl) ethanol (2h)[25,26]
1
Colourless oil, Yield 95%, H NMR (400 MHz, CDCl3) δ= 7.29-7.22 (m, 4H), 4.79 (q, J = 6.5
13
Hz, 1H), 2.77 (bs, 1H), 1.41 (d, J = 6.5 Hz, 3H); C NMR (100 MHz, CDCl3) δ= 144.2, 132.9,
128.5, 126.8, 69.6, 25.2; [α]D25 = +39.8 (c 1.0, CHCI3), 88% ee; retention time: 13.3 min (minor),
16.1 (major), Chiralcel OD-H, n-hexane/i-PrOH, 98:2, flow rate of 1 mL/min, 220 nm.
(R)-1-(4-bromophenyl) ethanol (2i)[30]
Colourless oil, Yield 85%, 1H NMR (400 MHz, CDCl3) δ= 7.43-7.40 (m, 2H), 7.18-7.15 (m, 2H),
4.75 (q, J = 6.5 Hz, 1H), 2.93 (bs, 1H), 1.39 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=
144.7, 131.5, 127.2, 121.0, 69.6, 25.2; [α]D25 = +38.9 (c 1.0, CHCI3), 97% ee; retention time: 8.7
min (major), 9.6 (minor), Chiralcel OD-H, n-hexane/i-PrOH, 95:5, flow rate of 1 mL/min, 220
nm.
(R)-1-(4-methoxyphenyl) ethanol (2j)[30]
Colourless oil, Yield 88%, 1H NMR (400 MHz, CDCl3) δ= 7.30-7.27 (m, 2H), 6.89-6.85 (m, 2H),
13
4.83 (q, J = 6.4 Hz, 1H), 3.79 (s, 3H), 2.11 (bs, 1H), 1.46 (d, J = 6.5 Hz, 3H); C NMR (100
MHz, CDCl3) δ= 158.9, 138.0, 126.7, 113.8, 69.9, 55.3, 25.0; [α]D25 = +58.6 (c 1.0, CHCI3), 96%
ee; retention time: 10.8 min (major), 12.0 (minor), Chiralcel OD-H, n-hexane/i-PrOH, 95:5, flow
rate of 0.8 mL/min, 220 nm.
(R)-1-(p-tolyl) ethanol (2k)[31]
Colourless oil, Yield 95%, 1H NMR (400 MHz, CDCl3) δ= 7.27 (d, J = 8.0 Hz, 1H), 7.17 (d, J =
13
7.9 Hz, 1H), 4.84 (q, J = 6.5 Hz, 1H), 2.42 (bs, 3H), 2.37 (s, 3H), 1.48 (d, J = 6.5 Hz, 3H); C
25
NMR (100 MHz, CDCl3) δ= 142.9, 137.0, 129.1, 125.4, 70.1, 25.1, 21.1; [α]D = +54.4 (c 1.0,
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CHCI3), 91% ee; retention time: 21.1 min (minor), 22.9 (major), Chiralcel OD, n-hexane/i-PrOH,
90:10, flow rate of 0.5 mL/min, 220 nm.
(S)-1-(4-nitrophenyl) ethanol (2m)[29]
Colourless oil, Yield 91%, 1H NMR (400 MHz, CDCl3) δ= 8.10-8.14 (m, 2H), 7.51-7.48 (m, 2H),
5.00-4.94 (m, 1H), 2.75 (d, J = 3.4 Hz, 1H), 1.47 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz,
25
CDCl3) δ= 153.3, 147.0, 126.1, 123.7, 69.4, 25.4; MS: m/z 168 (M+1)+; [α]D = -25.4 (c 0.5,
CHCI3), >99% ee; retention time: 13.2 min (minor), 15.4 (major), Chiralcel AS, n-hexane/i-
PrOH, 95:5, flow rate of 0.8 mL/min, 220 nm.
(S)-1-(p-biphenyl) ethanol (2n)[32]
1
White solid, mp 92-94oC, yield 92%, H NMR (400 MHz, CDCl3) δ= 7.63-7.60 (m, 4H), 7.49-
7.45 (m, 4H), 7.40-7.36 (m, 1H), 4.95 (qd, J = 6.4, 2.9 Hz, 1H), 2.24 (d, J = 2.8 Hz, 1H), 1.56 (d,
J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ= 144.9, 140.9, 140.4, 128.8, 127.3, 127.3, 127.1,
125.9, 70.1, 25.2; [α]D25 = -44.3 (c 0.55, CH2CI2), 85% ee; retention time: 16.8 min (major), 18.2
(minor), Chiralcel OD, n-hexane/i-PrOH, 90:10, flow rate of 0.6 mL/min, 220 nm.
Acknowledgements
Financial support from Bayburt University (BAP-2014/1-15) is gratefully acknowledged.
Supplementary data
1
13
Copies of H, C NMR spectra and chiral HPLC profiles of 2a-n related to this article can be
found at supplementary data
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Tables and Figures
Table 1. Screening of yeast strains for the bioreduction of acetophenone
Table 2. Optimization of parameters for the bioreduction of acetophenone (1a) with Candida
zeylanoides P1
Table 3. Enantioselective reduction of acetophenone derivatives 1a-n by Candida zeylanoides P1
Figure 1. Some examples of pharmaceutical compounds
Figure 2. A gram scale production of (S)-1-(4-nitrophenyl) ethanol (2m)
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Table 1.
Microbial straina
Conversionb (%)
M.B.b,c
--
%
eed % (S)
--
Saccharomyces uvarum S1
Saccharomyces uvarum S2
Saccharomyces uvarum S3
Saccharomyces uvarum S4
Saccharomyces uvarum S5
Pichia fermentans O1
No growth
54
51
43
39
89
94
97
89
--
41
46
57
59
Weak growth
--
Pichia fermentans O1
Pichia fermentans O1
Pichia fermentans O1
Pichia fermentans O1
59
37
29
51
67
56
54
50
41
94
90
91
88
93
95
97
89
90
49
64
53
61
74
58
49
54
Candida zeylanoides P1
Candida zeylanoides P2
Candida zeylanoides P3
Candida zeylanoides P4
Candida zeylanoides P5
48
Reaction condition: acetophenone 1 mmol, temperature: 30 oC, time 24 h, 100 rpm
a Comparison of the best micobial strain.
b
1
The conversions and mass balances (MB) were determined by H NMR analysis with diphenyl methan as an
internal standard.
c MB refers to the sum of the yields of the characterized adduct 2a, and the unreacted acetophenone 1a.
d Determined by HPLC using Chiralcel OD-H column.
Table 2.
pH
Temperature
oC Conv.
ee
(%)b (%)c,d
Incubation period
Agitation speed
Conv.
ee
h
Conv.
(%)b
81
ee
rpm Conv.
ee
(%)b (%)c,d
(%)c,d
(%)b
88
(%)c,d
90 (S)
>99 (S)
55 (S)
35 (S)
4.5
5
5.5
6
6.5
7
54
67
71
70
81
65
60
0
65 (S) 25
67
70
81
77
40
71 (S) 24
72 (S) 48
89 (S) 72
81 (S) 96
60 (S)
89 (S) 100
90 (S) 150
81 (S) 200
77 (S) 250
74 (S) 28
79 (S) 30
82 (S) 32
89 (S) 34
84 (S)
88
94
97
99
99
99
7.5
81 (S)
8e
-
a Substrate 1mmol.
b The conversion was determined by 1H NMR analysis with diphenylmethane as an internal standard.
c Determined by HPLC using Chiralcel OD-H column.
d
Absolute configuration were assigned by comparison of the sign of optical rotations relative to the values in
literature
e weak growth
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Table 3.
Entry Substrate
Product
ee (%)b,c
Conversion (%)d M.B. (%)d
>99 (S)
>99
94
87
91
90
93
1
2
3
4
5
97 (R)
>99 (R)
>99 (S)
99 (S)
77
75
70
97
>99 (R)
99 (R)
88 (R)
87
77
94
89
93
94
6
7
8
97 (R)
96 (R)
91 (R)
87
65
70
92
88
94
9
10
11
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>99 (S)
85 (S)
>99
77
95
94
12
13
a Bireduction conditions: Substrate: 1 mmol, temperature: 30 oC, time: 48 h, pH: 6.5, 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 literatüre values.
d
1
The conversions and mass balances (MB) were determined by H NMR analysis with diphenylmethane as an
internal standard.
Figure 1.
Figure 2.
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