Enantioselective Bioreduction of Acetophenone into (R)- and (S)-1-Phenylethanols

Article abstract of DOI:10.1134/S1070363217130229

The possibility of enantioselective bioreduction of acetophenone into (R)- and (S)-1-phenylethanol used in the synthesis of antifeedant, antidiabetic, antidepressant, antirabic, antiparasitic, antimicrobial, and antifungal agents is investigated. The infl

Full text of DOI:10.1134/S1070363217130229

ISSN 1070-3632, Russian Journal of General Chemistry, 2017, Vol. 87, No. 13, pp. 3259–3262. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © A.R. Chanysheva, E.N. Vorobyova, V.V. Zorin, 2017, published in Ekologicheskaya Khimiya, 2017, Vol. 26, No. 6, pp. 296–300.
Enantioselective Bioreduction of Acetophenone
into (R)- and (S)-1-Phenylethanols
A. R. Chanysheva*, E. N. Vorobyova, and V. V. Zorin
Ufa State Petroleum Technological University, ul. Kosmonavtov 1, Ufa, Bashkortostan, 450062 Russia
*e-mail: aliyach@mail.ru
Received October 20, 2017
Abstract―The possibility of enantioselective bioreduction of acetophenone into (R)- and (S)-1-phenylethanol
used in the synthesis of antifeedant, antidiabetic, antidepressant, antirabic, antiparasitic, antimicrobial, and
antifungal agents is investigated. The influence of various exogenous reducing agents (isopropanol, ethanol,
and glucose) on the yields of the target products and the enantioselectivity of the bioreduction of acetophenone
is studied.
Keywords: acetophenone, bioreduction, stereoinversion, enantioselective biocatalysis, (R)-(+)-1-phenylethanol,
(S)-(-)-1-phenylethanol, environmentally safe technologies
DOI: 10.1134/S1070363217130229
S- and R-1-phenylethanol enantiomers have found
application in organic synthesis. (S)-(–)-1-Phenyl-
ethanol is used in the synthesis of antidepressant,
antidiabetic, and antiarabic drugs, as well as anti-
feedants and antiparasitic, antimicrobial, and anti-
fungal agents [1]. (S)-(–)-1-Phenylethanol derivatives
are used in the synthesis of liquid crystals and optically
active polymers for separation of racemic mixtures of
organic compounds. R-(+)-1-Phenylethanol is a chiral
intermediate in the synthesis of pharmaceutical and
agrochemical products. R-1-(+)-Phenylethanol is also
used in ophthalmology and also as an inhibitor of
cholesterol adsorption in the digestive tract [2].
accessible biocatalyst on the basis of D. carotа culture
and found conditions which allow (S)-(–)-1-phenyl-
ethanol to be prepared with a yield of 95% and an
enantioselectivity of 97% ee.
Aimed at optimizing the synthesis and searching for
other environmentally friendly biocatalysts, in the
present work we have explored the use of a Petro-
selinum crispum cell culture in the synthesis of (S)-(–)-
1-phenylethanol (Scheme 1).
As known, P. Crispum has a high reducing power
[26] and allows enantioselective reduction of prochiral
ketones to enantiomerically pure secondary alcohols in
high yields.
A lot of synthetic approaches to optically pure S-
enantiomers of secondary alcohols are known. To this
end, enantioselective metal complex catalysts and
bacterial cells are used [3‒24]. We recently proposed
[25] an environmentally friendly synthesis of (S)-(–)-1-
phenylethanol with the use of a convenient and
It was found that the reduction of acetophenone
with P. crispum cells in water at room temperature for
144 h gives (S)-(–)-1-phenylethanol with a yield of
59% and an optical purity of 48% ее.
Scheme 1.
P. crispum
+
O
OH
OH
(R)-(+)-1-Phenylethanol
Acetophone
(S)-(−)-1-Phenylethanol
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CHANYSHEVA et al.
Table 1. Yields and optical purities of (S)-(–)-1-phenylethanol at different concentrations of ethanol in acetophenone
bioreductionа
Time, h
Ethanol
concentration,
%
24
48
72
144
optical
optical
optical
optical
yield, %
yield, %
yield, %
yield, %
purity, % ee
purity, % ee
purity, % ee
purity, % ee
0
1
2
3
4
5
17
8
91
69
86
81
87
85
37
8
97
87
97
97
99
98
26
10
34
32
94
34
96
88
94
98
99
99
59
12
38
32
96
34
48
88
82
90
99
98
7
28
26
97
24
6
4
4
а T = 20‒25°С, solvent water, substrate concentration 1.4 g/L.
As the intrinsic potential of the cells is not too high,
to increase the conversion of the substrate and the
yield and optical purity of the target (S)-(–)-1-phenyl-
ethanol, we made use of exogenous reducers (ethanol,
isopropanol, and glucose) to regenerate the oxidized
biocatalyst coenzymes, specifically, to convert NAD+
into NADH.
another enzyme with the opposite stereochemical
preference [27].
Increasing the concentration of isopropanol to 2‒
5% (48 h) does not increase, in general, the reaction
yield and does not increase essentially the reaction
enantioselectivity. Increasing the reaction time in the
presence of isopropanol to 144 h always (Table 2)
leads to stereoinversion resulting in the preferential
formation of (R)-(+)-1-phenylethanol.
It was found that after the addition of ethanol (4%)
to the reaction mixture the yield of (S)-(–)-1-phenyl-
ethanol reaches 97% already within 48 h and the
enantiomeric excess of the product reaches 99% ее,
which is much better than in the absence of the
exogenous reducer (Table 1).
The bioreduction of acetophenone in the presence
of glucose as the exogenous reducer was also studied.
The reaction was performed in water at room tem-
perature for 144 h. An equimolar amount of glucose
was added to the reaction mixture. The yield of (S)-(–)-
1-phenylethanol in this reaction was 48%, and the enetio-
selectivity appreciably increased (91% ее) (Table 3).
With isopropanol (1%) as the exogenous reducer,
the yield of (S)-(–)-1-phenylethanol after 48-h reaction
is 69% (68% ее). If the bioreaction time is prolonged
to 72 h, the yield of (S)-(–)-1-phenylethanol decreases
to 32% (57% ее), and, therewith, the concentration of
the starting ketone increases. If the biotransformation
time under the same conditions is further prolonged
(144 h), the reaction results in preferential formation of
(R)-(+)-1-phenylethanol with a total yields of 98%
(29% ее) (Table 2), which suggests the stereoinversion
of (S)-(–)-1-phenylethanol to R-1-(+)-phenylethanol.
The reaction progress was followed by chromato-
graphy on the enantioselective column AstecChiraldexb-
PM (30 m × 0.25 mm×0.12 µm), from which (R)-(+)-
1-phenylethanol is eluted earlier than (S)-(–)-1-
phenylethanol; the reference was the synthesized
authentic racemic phenylethanol.
The structure of (S)-(–)-1-phenylethanol was
1
confirmed by H and С13 NMR spectroscopy and gas
As known, deracemization of racemic alcohols can
occur via stereoinversion of one enantiomer to the
other. It is suggested that stereoinversion involves redox
transformations. One of the enantiomers of the racemic
mixture of alcohols is oxidized to the corresponding
ketone under the action of dehydrogenase, whereas its
mirror analog (optical antipode) remains unoxidized.
At the next stage, the resulting ketone is reduced by
chromatography‒mass spectrometry. The configura-
tions of (R)-(+)-1- and (S)-(–)-1-phenylethanols was
assessed by polarimetry.
Comparison of the yields and optical densities of
the (S)-(–)-1-phenylethanol samples synthesized with
D. carota and P.crispum biocatalysts shows that the
synthesis with the latter in the presence of 4% of
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 13 2017

ENANTIOSELECTIVE BIOREDUCTION OF ACETOPHENONE
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Table 2. Yields and optical purities of (R)- и (S)-1-phenylethanols at different concentrations of isopropanol in acetophenone
bioreductionа
Time, h
Ethanol
concentration,
%
24
48
72
144
optical
optical
optical
optical
yield, %
yield, %
yield, %
yield, %
purity, % ee
purity, % ee
purity, % ee
purity, % ee
0
1
2
3
4
5
91 S
37
69
24
44
34
8
97 S
26
32
33
56
54
33
96 S
59
98
79
87
88
59
48 S
17
10
9
89 S
68 S
57 S
29 R
94 S
47 S
19 R
27 R
90 S
37 S
14 R
23 R
8
89 S
73 S
19 R
13 R
7
92 S
73 S
18 R
50 R
2
а
T = 20‒25°С, solvent water, substrate concentration 1.4 g/L.
Table 3. Yields and optical purities of (S)-(–)-1-phenylethanol at different durations of acetophenone bioreduction in the
presence of glucoseа
Time, h
Ethanol
concentration,
%
24
48
72
144
optical
optical
optical
optical
yield, %
17
yield, %
37
yield, %
26
yield, %
59
purity, % ee
purity, % ee
purity, % ee
purity, % ee
Without
glucose
91
97
96
48
With glucose
45
91
50
91
50
92
48
91
а
T = 20‒25°С, solvent water, substrate concentration 1.4 g/L.
ethanol ensures a higher yield (97%) and a higher
optical purity (99% ее) of the product. Therewith, if
with D. carota the best result was obtained in the
presence of isopropanol (2%) as the exogenous
reducer, with P. crispum the best result was obtained
in the presence of ethanol (4%). The use of P. crispum
allows the transformation time to be decreased to 48 h
against 144 h with D. carota.
ion source temperature 200°C; chromatographic separa-
tion was done on an HP-1MS capillary column
(30 m×0.25 mm×0.25 µm) in the programmed
temperature mode (initial 100°C, final 280°C, ramp
rate 20°C/min), carrier gas helium, flow rate 1.1 mL/min.
Polarimetry was performed on an Optical Activity
АА-55 polarimeter with a halogen lamp (standard
wavelength 589 nm), chloroform solutions were used
for measurements.
EXPERIMENTAL
Gas chromatographic analysis was performed on a
Khromatek Crystal 5000.2 system with a flame
ionization detector, using an Astec Chiraldexb-PM
enantioselective column (30 m×0.25 mm×0.12 µm).
1
The Н and 13С NMR spectra were recorded on
Bruker АМ-300 and AMX-300 spectrometers at 300
(1Н) and 75 MHz (13С) against internal TMS (1Н, 13С).
The 13С NMR spectra were recorded with complete
proton decoupling in the JMOD mode.
Synthesis of (R)- and (S)-1-phenylethanols. Aceto-
phenone (0.1 g) was added to a suspension of P.
crispum biocatalyst (15 g) in 70 mL of water. The
reaction mixture stirred in an orbital shaker (150 rpm)
for 15 min at room temperature. After the required
The mass spectra were measured on a Shimadzu
GCMS-QP2010S instrument (electron ionization at
70 eV, mass range 33‒500), injector temperature 280°C,
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CHANYSHEVA et al.
11. Patel, R., McNamee, C.G., Banerjee, A., Howell, J.M.,
Robison, R.S., and Szarka, L.J., Enzym. Microbial.
Technol., 1992, vol. 14, p. 731.
12. Medson C., Smallridge A.J., Trewhella M.A., Tetra-
hedron: Asymmetry, 1997, vol. 8, p. 1049.
13. Hamada, H., Miura, T., Kumobayashi, H., Matsuda, T.,
Harada, T., and Nakamura, K., Biotechnol. Lett., 2001,
vol. 23, p. 1603.
14. Ishihara, K., Yamaguchi, H., Hamada, H., Nakajima, N.,
and Nakamura, K., J. Mol. Catal. B, 2000, vol. 10,
p. 429.
conversion had been reached, the suspension was
filtered, and biomass was washed with three portion of
water. The filtrates were extracted with diethyl ether
(3×125 mL). The ether extracts were dried over
Na2SO4, and the solvent was removed on a rotary
1
evaporator. H NMR spectrum, δ, ppm: 1.42–1.49 d
(3H, CH3), 2.6 s (1H,ОН), 4.2–4.4 q (1Н, СН), 7.2-
7.35 m (5Н, СНАr). 13C NMR spectrum, δ, ppm: 20.28
(C, CH3), 70.35 (C,CH), 125.27 (2C, СНАr), 127.35
(C, СНАr), 128.12 (2C, СНАr), 145.8 (C, СНАr). Mass
spectrum, m/z (Irel., %): 105.1 (100), 77.1 (93.8), 51.1
(36.7), 120.1 (32.9), 79.1 (26.4), 107.1 (25.3), 43.0
(21.7), 78.1 (14.0), 50.1 (13.7), 122.2(9.7).
15. Matsuda, T., Harada, T., Nakajima, Y., Itoh, T., and
Nakamura, K., J. Org. Chem., 2000, vol. 65, p. 157.
16. Zarevucka, M., Wimmer, Z., Saman, D., Demnerova, K.,
and Mackova, M., Tetrahedron: Asymmetry, 2004,
vol. 15, p. 1325.
Racemic (R),(S)-1-phenylethanol was synthesized
as described in [28].
17. Kazici, H., Bayraktar, E., Mehmetoglu, Ü., Green
Process. Synth., 2006, vol. 5, no. 2. p. 131.
ACKNOWLEDGMENTS
18. Shakirov, A.N., Del’mukhametov, R.R., Petukhova, N.I.,
Shakhmaev, R.N., and Zorin, V.V., Ekol. Khim., 2012,
vol. 21, no. 3, p. 187.
19. Shakirov, A.N., Petukhova, N.I., Del’mukhametov, R.R.,
Koreneva, S.V., and Zorin, V.V., Bashkir. Khim. Zh.,
2012, vol. 19, no. 2, p. 71.
The work was performed under the State contract
with the Ministry of Education and Science of the
Russian Federation (contact no. 4.6451.2017/8.9).
REFERENCES
20. Zorin, V.V., Petukhova, N.I., and Shakhmaev, R.N.,
Ross. Khim. Zh., 2011, vol. 55, no. 1, p. 77.
1. Semenov, A.A., Ocherk khimii prirodnykh soedinenii
(Essay on the Chemistry of Natural Compounds),
Novosibirsk: Nauka, 2000.
21. Vasilova, L.Ya., Shakirov, A.N., Petukhova, N.I.,
Shakhmaev, R.N., and Zorin, V.V., Bashkir. Khim. Zh.,
2010, vol. 17, no. 5, p. 32.
2. Suan, C.L. and Sarmidi, M.R., J. Mol. Catal. B: Enzym.,
22. Kalimullina, L.Ia., Petukhova, N.I., and Zorin, V.V.,
2004, vol. 28, p. 111.
Bashkir. Khim. Zh., 2008, vol. 15, no. 1, p. 8–10.
3. Nakamura, K., Matsuda, T., Shimizu, M., and Fujisawa, T.,
23. Zorin, V.V., Petukhova, N.I., an Shakhmaev, R.N.,
Russ. J. Gen. Chem., 2012, vol. 82, no. 5, p. 1013. doi
10.1134/S1070363212050362
24. Petukhova, N.I., Kon’shina, I.I., Spivak, A.Yu.,
Odinokov, V.N., and Zorin, V.V., Appl. Biochem.
Microbiol., 2017, vol. 53, no. 2. p. 187. doi 10.1134/
S0003683817020144
25. Chanysheva, A.R., Yunusova, G.V., Vorobyova, T.E.,
and Zorin, V.V., Russ. J. Gen. Chem., 2016, vol. 86,
no. 13. p. 3021. doi 10.1134/S107036321613017X
26. Patent US 2004/0082043 A1.
27. Biocatalysis in the Pharmaceutical and Biotechnology
Industries, Patel, R.N., Ed., Boca Raton: CRC, 2006.
28. Agronomov, A.E., Laboratornye raboty v organi-
cheskom praktikume (Laboratory Manual in Organic
Chemistry), Moscow: Khimiya, 1974.
Tetrahedron, 1998, vol. 54, p. 8393.
4. Matsuda, T., Harada, T., Nakajima, Y., Itoh, T., and
Nakamura, K., J. Org. Chem., 2000, vol. 65, p. 157.
5. Lappin, G.J., Stride, J.D., and Tampion, J., Phyto-
chemistry, 1987, vol. 26, p. 995–997.
6. Hamada, H., Nakamura, N., Ito, S., Kawabe, S., and
Funamoto, T., Phytochemistry, 1988, vol. 27, p. 3807.
7. Yadav, J. S., Reddy, P.T., Nanda, S., and Rao, A.B.,
Tetrahedron: Assymetry, 2001, vol. 12, p. 63.
8. Suga, T., Hirata, T., and Lee, Y.S., Chem. Lett., 1982,
p. 1595.
9. Ishihara, K., Hamada, H., Hirata, T., and Nakajima, N.,
J. Mol. Catal. B: Enzym., 2003, 23, p. 145.
10. Pawlowicz, P. and Siewinski, A., Phytochemistry, 1987,
vol. 26, p. 1001.
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