Production of (R)-1-phenylethanols Through
Bioreduction of Acetophenones by a New Fungus Isolate
KANI ZILBEYAZ,1MESUT TASKIN,2ESABI B. KURBANOGLU,2NAMUDAR I. KURBANOGLU,3AND HAMDULLAH KILIC1*
1Faculty of Sciences, Department of Chemistry, Ataturk University, Erzurum 25240, Turkey
2Faculty of Sciences, Department of Biology, Ataturk University, Erzurum 25240, Turkey
3Hendek Faculty of Education, Department of Chemistry, Sakarya University, Sakarya, Turkey
uated in the bioreduction of substituted acetophenones to the corresponding (R)-alco-
hols. Among these strains, isolate Trichothecium roseum EBK-18 was highly effective in
the production of (R)-alcohols with excellent enantioselectivity (ee > 99%). Gram scale
preparation of (R)-1-phenylethanol is reported. Chirality 22:543–547, 2010.
A total of 120 fungal strains were isolated from soil samples and eval-
KEY WORDS: fungus; biocatalyst; enantioselective reduction; ketone; chiral alcohol
Enantiomerically pure secondary chiral alcohols are im-
portant building blocks for the synthesis of bioactive com-
pounds such as pharmaceuticals, pesticides, pheromones,
flavors, fragrances, and natural products.1–5For example,
enantiopure (R)-1-phenylethanol (2a) is an important chi-
ral building block for pharmaceuticals, agrochemicals, and
natural products.6–10For economic and environmental
reasons, biocatalysis has recently gained increasing impor-
tance for the preparation of enantiopure alcohols from
prochiral ketones. For this purpose, ketoreductases have
been shown to be unique biocatalysts in the preparation
of enantiopure alcohols from prochiral
For example, Mandal et al. reported that whole cells of
Trichothecium sp. are an effective biocatalyst for the enan-
tioselective bioreduction of acetophenone and its analo-
gous compounds to their corresponding (R)-alcohols, e.g.
(R)-2a (93.5 ee), (R)-2k (98.5 ee), and (R)-2n (90.5 ee).20
Ou et al. reported the production of 2a by chemoenzy-
matic route and the ee obtained was 97%.21A pure enzy-
matic method has also been applied for the production of
2a with high ee; however, the reactions catalyzed by
isolated enzymes require cofactors, which are often too
expensive.22Recently, we found that ram horn peptone
(RHP) could be utilized as a source of peptone for micro-
bial growth media, as a supplement in fermentation
medium for the asymmetric reduction of substituted aceto-
phenones to the corresponding chiral alcohols.23–29In this
work, we screened the submerged culture of Trichothe-
cium roseum strain for the biocatalytic reduction of substi-
tuted acetophenone series to the corresponding chiral
alcohols with R-configuration using RHP in fermentation
medium. We found that 10 of the assayed 120 isolates of
Trichothecium roseum enabled the formation of R enan-
tiomer through the reduction of the substituted acetophe-
nones. One isolate, namely EBK-18, yielded (R)-alcohols
with excellent ee under optimized conditions and allowed
gram scale preparation of (R)-1-phenylethanol (2a) from
Ram horns were obtained from a slaughterhouse in
Erzurum, Turkey. The other components of the culture
media and the chemical reagents were obtained from
Merck and Sigma in the highest purity available. Produc-
tion of RHP was carried out using the method described
by Kurbanoglu and Kurbanoglu.24
Isolation of Microorganisms, Identification
The microorganisms used in this study were isolated
from soil samples collected from the region around Erzu-
rum, Turkey. The isolation process was performed by
serial dilution of the samples according to standard techni-
ques.30Filamentous fungi were taxonomically identified
in-house using mature cultures on standard potato
dextrose agar (PDA) to ensure good development of taxo-
nomically relevant features, and following the identifica-
tion keys provided by Von Arx and Domsch et al.31,32
These cultures were maintained on PDA slants, incubated
*Correspondence to: Hamdullah Kilic, Faculty of Sciences, Department of
Chemistry, Ataturk University, Erzurum 25240, Turkey.
Received for publication 17 May 2009; Accepted 8 July 2009
Published online 9 September 2009 in Wiley InterScience
Additional Supporting Information may be found in the online version of
Contract grant sponsor: The Scientific and Technological Research Coun-
cil of Turkey (TUBITAK); Contract grant number: TBAG-107T670
CHIRALITY 22:543–547 (2010)
C 2009 Wiley-Liss, Inc.
at 258C, and stored at 48C. The conidia from 8-day-old cul-
tures were used for inoculation. The conidial suspension
was prepared in 10 ml sterilized and distilled water by
gently scratching conidia with a sterile wire loop and
then it was shaken vigorously to break up the clumps of
Medium, Culture Conditions and Screening for
The fermentation medium per liter contained (g/l):
glucose 20, yeast extract 3, KH2PO41.5, and RHP 4. The
initial pH of the culture medium was adjusted to 7.0 with
1 M HCl and 1 M NaOH and sterilized at 1218C for
15 min. All the cultures were grown in 250 ml flasks con-
taining 100 ml of medium. Then 1 ml of conidial suspen-
sions was added to each flask. The flasks were incubated
on a reciprocal shaker at 150 rpm and 258C for 72 h. After
the growth of the fungal strains, acetophenone (1a)
(1 mmol) was added directly to each medium and then the
incubation continued on a reciprocal shaker at 150 rpm
and 258C for 24 h. A total of 120 fungal strains were
screened to produce (R)-2a from 1a with RHP as nitrogen
and mineral sources. Among them, ten fungal strains
reduced 1a to (R)-2a. The most productive strain (EBK-
18) was identified as Trichothecium roseum. This strain
was selected for further research.
Production of ( R)-2a by T. roseum EBK-18 in a
All production experiments under optimum fermenta-
tion conditions were performed in a 2 l fermenter (Biostat-
M 880072/3, Germany) with a working volume of 1 liter.
Ten milliliters of the spore suspension were inoculated
into the fermenter containing 1 l of sterile medium. Agita-
tion, pH, aeration (vol/vol/min), and temperature were
automatically controlled during fermentation. At regular
intervals (6 h) during fermentation, the conversion, yield,
and ee were determined.
At the end of the incubation period, mycelium was sepa-
rated by filtration, and the filtrate was saturated with so-
dium chloride and then extracted with ethyl acetate. The
Chirality DOI 10.1002/chir
mycelia were also washed with ethyl acetate. Ethyl acetate
extracts were combined and dried over Na2SO4. The con-
version was determined by1H NMR analysis with diphe-
nylmethane as internal standard; error ca. 65% of
the stated values. After removal of the solvent, the crude
products were purified by short silica gel column chroma-
tography and identified by NMR analysis. The absolute
configuration was determined by the sign of the specific
rotation and comparison with the literature.33,34The ee of
the alcohols was then determined by HPLC analysis using
Chiralcel OD and OB columns. The purity of (R)-1-phenyl-
ethanol (2a) produced via a fermenter was checked by
HPLC analysis. The specific rotation was measured with a
polarimeter at 589.3 nm. (R)-1-phenylethanol 2a:35–4076%
yield (1.85 g, 15.1 mmol); [a]D
>99% ee determined by HPLC on a OD chiral column;
retention times were 11.8 min for (1)-(R) and 13.0 min for
20152.8 (c, 0.85, CHCl3);
RESULTS AND DISCUSSION
To establish the optimal reaction conditions for the
asymmetric reduction with Trichothecium roseum, pH, tem-
perature, incubation period, and agitation speed were
investigated in the reduction of acetophenone (1a). The
results of these optimizations are given in Table 1. Differ-
ent pH ranges (5.0, 5.5, 6.0, 6.5, and 7.0) were chosen
to monitor the progress of the bioreduction. The highest
conversion (60%) and ee (95%) were achieved when the
medium pH was controlled at 6.0. Under suitable culture
conditions, the effects of different culture temperatures
were examined by carrying out the fermentation processes
within different temperature ranges (26–348C). The high-
est conversion (80%) was obtained at 308C with 95% ee.
Temperatures over 308C, both ee and conversion dropped
substantially. For example, the lowest ee (60%) and
conversion (30%) were obtained at 348C. These results
suggest that an increase in temperature had a negative
effect on the ee and conversion. Therefore, we continued
the research with 308C Different incubation times were
chosen to monitor the progress of the bioreduction. The
complete conversion of 1a was observed after 72 and 96 h,
but the ee (77%) of 2a decreased. In contrast to these
TABLE 1. Optimization of parameters for the bioreduction of acetophenone (1a) by Trichothecium roseuma
pHTemperatureIncubation periodAgitation speed
aSubstrate 1 mM.
bConversion was determined by1H NMR analysis with diphenylmethane as an internal standard; error ca. 65% of the stated values.
cDetermined by HPLC using Chiralcel OD column.
dAbsolute configurations were assigned by comparison of the sign of optical rotations relative to the values in the literature.
KANI ZILBEYAZ ET AL.
results, the conversion increased up to 92% with 95% ee for
48 h. Although the ee of 2a remained steady after 48 h,
the conversion rate was obviously different. The best con-
dition (48 h) obtained was used for further optimization of
conversion and ee. The highest values for both ee (>99%)
and conversion (100%) were obtained at 200 rpm and
thus this agitation speed was determined as optimum for
Under the optimum conditions (pH 6.0, temperature
308C, time 48 h, and agitation 200 rpm) asymmetric biore-
ductions of the other derivatives of 1a by T. roseum EBK-
18 were investigated in the shake flask scale. The results
are shown in Table 2. All resulting alcohols had an R-con-
figuration with >99% ee. In the first set of experiments, we
studied the influence of the ketone structure. The promi-
nent trend displayed in Table 2 is that the conversion of
the substrates decreases with the degree of electron
donating-withdrawing groups at the aromatic ring. Clearly,
the electron-deficient substrates show higher reactivity.
When para-substituted acetophenones were reduced, elec-
tron-donating groups provided no conversions (Table 2,
entries 13–14) except for para-methyl derivative 1n (entry
13), while electron-withdrawing substituents afforded con-
versions in the range of 77–100%. Moreover, ketones with
a strong electron-withdrawing group at the para or meta
position such as nitroacetophenones 1h and 1m furnished
quantitative conversions. The reduction of the meta- or
para-substituted acetophenone was more favorable as com-
pared to ortho-substituted acetophenone. There was no
reaction for any ortho-substituted acetophenones. Presum-
ably, steric repulsion between the catalytically active site
and the ortho-substituents hinders the transfer of the
After successful determination of the reaction parame-
ters, we decided to conduct the transformation of 1a
to (R)-2a on a gram scale to demonstrate industrial
TABLE 2. Enantioselectivities for the microbial reduction of substituted acetophenones by Trichothecium roseum,
Aspergillus niger, and Alternaria alternate
Entry SubstrateProductConvn. (%)a
aFrom this work with Trichothecium roseum. Isolated yields after column chromatography on silica gel.
bDetermined by HPLC using Chiralcel OD and OB columns.
cAbsolute configurations were assigned by comparison of the sign of optical rotations relative to the values in the literature.
dSee Ref. 25.
eSee Ref. 26.
fNo conversion was observed.
gData not available.
Scheme 1. A gram scale production of (R)-1-phenylethanol (2a)
PRODUCTION OF (R)-1-PHENYLETHANOLS
Chirality DOI 10.1002/chir
Preparative scale production of 2a was performed on a
1l scale in a 2 l fermenter (Scheme 1). Bioreduction of 1a
(3.0 g, 25 mmol) after 62 h resulted in complete conver-
sion, but the ee of the desired product was rather low
(60%). It was noted that the enantioselectivity of T. roseum
EBK-18 depended on the incubation time used for cultiva-
tion and substrate concentration. Therefore, 1a (2.4 g, 20
mmol) was directly added to the fermentation medium.
Complete conversion of 1a was achieved after 56 h of
incubation, and then the mixture was extracted with
EtOAc (3 X 25 ml) and dried over Na2SO4. After evapora-
tion of the solvent the product 2a was purified on a silica
Recently, we reported the bioreduction of acetophe-
nones by Aspergillus niger and Alternaria alternata.25–27
In comparison, Trichothecium roseum is sensitive to the
position and electronic effect of the substituent. Thus, the
derivatives 1b-d and 1o-p did not afford the correspond-
ing alcohols 2 (Table 2, entries 2–4, and 13–14); however,
in contrast to this observation, Aspergillus niger and Alter-
naria alternata are not substrate structure-dependent
reducers. While Trichothecium roseum exhibits R selectiv-
ity in all cases, Aspergillus niger and Alternaria alternata
do not show any preference in enantioselectivity. Thus,
depending on the substrates, they produce either (R)- or
In the present study, acetophenone (1a) and its deriva-
tives were reduced to the corresponding (R)-enantiomer
with >99% ee using submerged culture of T. roseum EBK-
18. We have demonstrated a novel microbial system to
obtain enantiopure sec-alcohols that possess several advan-
tages: conversion and enantioselectivity are controlled by
the substituent position and electronic effect, and the
process can be scaled up. This is a convenient system
that exhibits excellent enantioselectivity and can be
applied for the clean synthesis of valuable enantiopure
1. Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Sturmer R,
Zelinski T. Industrial methods for the production of optically active
intermediates. Angew Chemie Int Ed 2004;43:788–824.
2. Gladiali S, Alberico E. Asymmetric transfer hydrogenation: chiral
ligands and applications. Chem Soc Rev 2006;35:226–236.
3. Ikariya T, Blacker AJ. Asymmetric transfer hydrogenation of ketones
with bifunctional transition metal-based molecular. Acc Chem Res
4. Ikariya T, Murata K, Noyori R. Bifunctional transition metal-based mo-
lecular catalysts for asymmetric syntheses. Org Biomol Chem
5. Wu XF, Xiao JL. Aqueous-phase asymmetric transfer hydrogenation of
ketones—a greener approach to chiral alcohols. Chem Commun
6. Bui V, Hansen TV, Stenstrom Y, Ribbons DW, Hudlicky T. Toluene
dioxygenase-mediated oxidation of aromatic substrates with remote chi-
ral centers. J Chem Soc Perkin Trans 1 2000;11:1669–1672.
7. Bui VP, Hansen TV, Stenstrom Y, Hudlicky T, Ribbons DW. A study
of substrate specificity of toluene dioxygenase in processing aromatic
compounds containing benzylic and/or remote chiral centers. N J
8. Inoue K, Makino Y, Itoh N. Production of (R)-chiral alcohols by a
hydrogen-transfer bioreduction with NADH-dependent Leifsonia alco-
hol dehydrogenase (LSADH). Tetrahedron: Asymmetry 2005;16:
9. Persson BA, Larsson ALE, Le Ray M, Backvall JE. Ruthenium- and
enzyme-catalyzed dynamic kinetic resolution of secondary alcohols. J
Am Chem Soc 1999;121:1645–1650.
10. Yamada M, Ichikawa T, Yamano T, Kikumoto F, Nishikimi Y, Tamura
N, Kitazaki T. Optically active cyclohexene derivative as a new anti-
sepsis agent: an efficient synthesis of ethyl (6R)-6-[N-(2-chloro-4-fluo-
rophenyl)-sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242). Chem
Pharm Bull 2006;54:58–62.
11. Seelbach K, Riebel B, Hummel W, Kula MR, Tishkov VI, Egorov AM,
Wandrey C, Kragl U. A novel, efficient regenerating method of
NADPH using a new formate dehydrogenase. Tetrahedron Lett
12. Yuan R, Watanabe S, Kuwabata S, Yoneyama H. Asymmetric electro-
reduction of ketone and aldehyde derivatives to the corresponding
alcohols using alcohol dehydrogenase as an electrocatalyst. J Org
13. Yun H, Yang YH, Cho BK, Hwang BY, Kim BG. Simultaneous synthe-
sis of enantiomerically pure (R)-1-phenylethanol and (R)-alpha-methyl-
benzylamine from racemic alpha-methylbenzylamine using omega-
transaminase/alcohol dehydrogenase/glucose dehydrogenase cou-
pling reaction. Biotechnol Lett 2003;25:809–814.
14. Ferloni C, Heinemann M, Hummel W, Daussmann T, Buchs J. Opti-
mization of enzymatic gas-phase reactions by increasing the long-term
stability of the catalyst. Biotechnol Prog 2004;20:975–978.
15. De Temino DM, Hartmeier W, Ansorge-Schumacher MB. Entrapment
of the alcohol dehydrogenase from Lactobacillus kefir in polyvinyl
alcohol for the synthesis of chiral hydrophobic alcohols in organic sol-
vents. Enzyme Microb Technol 2005;36:3–9.
16. Edegger K, Gruber CC, Faber K, Hafner A, Kroutil W. Optimization
of reaction parameters and cultivation conditions for biocatalytic
hydrogen transfer employing overexpressed ADH-‘A’ from Rhodococ-
cus ruber DSM 44541 in Escherichia coli. Engeerg Life Sci 2006;6:149–
17. Trivedi AH, Spiess AC, Daussmann T, Buchs J. Study on mesophilic
and thermophilic alcohol dehydrogenases in gas-phase reaction. Bio-
technol Prog 2006;22:454–458.
18. Voss CV, Gruber CC, Kroutil W. Deracemization of secondary alco-
hols through a concurrent tandem biocatalytic oxidation and reduc-
tion. Angew Chemie Int Ed 2008;47:741–745.
19. Borges KB, Borges WD, Duran-Patron R, Pupo MT, Bonato PS, Col-
lado IG. Stereoselective biotransformations using fungi as biocata-
lysts. Tetrahedron: Asymmetry 2009;20:385–397.
20. Mandal D, Ahmad A, Khan MI, Kumar R. Enantioselective bioreduc-
tion of acetophenone and its analogous by the fungus Trichothecium
sp. J Mol Catal B-Enzym 2004;27:61–63.
21. Ou L, Xu Y, Ludwig D, Pan J, Xu JH. Chemoenzymatic deracemization
of chiral secondary alcohols: process optimization for production of
22. Theil F. Lipase-supported synthesis of biologically-active compounds.
Chem Rev 1995;95:2203–2227.
23. Kurbanoglu EB, Kurbanoglu NI. Production of citric acid from ram
horn hydrolysate by Aspergillus niger. Process Biochem 2003;38:
24. Kurbanoglu EB, Kurbanoglu NI. Ram horn peptone as a source of cit-
ric acid production by Aspergillus niger, with a process. J Ind Micro-
biol Biotechnol 2004;31:289–294.
25. Kurbanoglu EB, Zilbeyaz K, Kurbanoglu NI, Kilic H. Enantioselective
reduction of substituted acetophenones by Aspergillus niger. Tetrahe-
dron: Asymmetry 2007;18:1159–1162.
26. Kurbanoglu EB, Zilbeyaz K, Kurbanoglu NI, Kilic H. Asymmetric
reduction of acetophenone analogues by Alternaria alternata using
ram horn peptone. Tetrahedron: Asymmetry 2007;18:2332–2335.
27. Kurbanoglu EB, Zilbeyaz K, Kurbanoglu NI, Taskin M. Highly enan-
tioselective reduction of acetophenone by locally isolated Alternaria
alternata using ram horn peptone. Tetrahedron: Asymmetry 2007;
KANI ZILBEYAZ ET AL.
Chirality DOI 10.1002/chir
28. Kurbanoglu EB, Zilbeyaz K, Kurbanoglu NI, Taskin M, Kilic H.
Production of (S)-(-)-1-(10-Napthyl) ethanol by Rhodotorula glutinis
isolate using ram horn peptone. Turkish J Chem 2008;32:685–
29. Zilbeyaz K, Kurbanoglu EB. Production of (R)-1-(4-Bromo-phenyl)-
ethanol by locally isolated Aspergillus niger using ram horn peptone.
Bioresour Technol 2008;99:1549–1552.
30. Nakayama K.Rehm H J,Reed G, editors.
microorganisms, Vol. 1. Weinheim: Verlag Chemie; 1981. 355–410.
31. Von Arx JA. Key to the orders of fungi, the genera of fungi sporulat-
ing in pure culture, Hirschberg, 11. Germany: Cramer; 1981.
32. Domsch KH, Gams W, Anderson TH. Key to the genera, compen-
dium of soil fungi, Eching, Germany: IHW; 1993.
33. Matsumura Y, Ogura K, Kouchi Y, Iwasaki F, Onomura O. New effi-
cient organic activators for highly enantioselective reduction of aro-
matic ketones by trichlorosilane. Org Lett 2006;8:3789–3792.
34. Nakamura K, Matsuda T. Asymmetric reduction of ketones by the ac-
etone powder of Geotrichum candidum. J Org Chem 1998;63:8957–
Sources of industrial
35. Rao SI, Duffel MW. Benzylic alcohols as stereospecific substrates and
inhibitors for aryl sulfotransferase. Chirality 1991;3:104–111.
36. Suginaka K, Hayashi Y, Yamamoto Y. Highly selective resolution of
secondary alcohols and acetoacetates with lipases and diketene in or-
ganic media. Tetrahedron: Asymmetry 1996;7:1153–1158.
37. Reetz MT, Kuhling KM, Hinrichs H, Deege A. Circular dichroism as a
detection method in the screening of enantioselective catalysts. Chir-
38. Le Barbu K, Zehnacker A, Lahmani F, Mons M, Piuzzi F, Dimicoli I.
Spectroscopic studies of enantiomeric discrimination in jet-cooled chi-
ral complexes. Chirality 2001;13:715–721.
39. Fabian WMF, Stampfer W, Mazur M, Uray G. Modeling the chro-
matographic enantioseparation of aryl- and hetarylcarbinols on
ULMO, a brush-type chiral stationary phase, by 3D-QSAR techniques.
40. Inoue K, Makino Y, Itoh N. Purification and characterization of a
novel alcohol dehydrogenase from Leifsonia sp strain S749: a promis-
ing biocatalyst for an asymmetric hydrogen transfer bioreduction.
Appl Environ Microbiol 2005;71:3633–3641.
PRODUCTION OF (R)-1-PHENYLETHANOLS
Chirality DOI 10.1002/chir