Asymmetric reduction of acetophenone analogues by Alternaria alternata using ram horn peptone

Article abstract of DOI:10.1016/j.tetasy.2007.09.009

Alternaria alternata EBK-4 fungus isolated from a plant sample was evaluated for the asymmetric reduction of acetophenone analogues. In a previous study, this isolate was used for the reduction of acetophenone to 1-phenylethanol in excellent enantiomeric excess. The substituted acetophenones were converted to the corresponding optically active alcohol by A. alternata EBK-4 under optimized conditions in up to >99% enantiomeric excess (ee). This is the first report on the enantiomeric reduction of acetophenone analogues by A. alternata using ram horn peptone from waste material.

Full text of DOI:10.1016/j.tetasy.2007.09.009

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 18 (2007) 2332–2335
Asymmetric reduction of acetophenone analogues by
Alternaria alternata using ram horn peptone
Esabi B. Kurbanoglu,a,* Kani Zilbeyaz,b Namudar I. Kurbanogluc and Hamdullah Kilicb,*
aDepartment of Biology, Faculty of Arts and Sciences, Ataturk University, 25240 Erzurum, Turkey
bDepartment of Chemistry, Faculty of Arts and Sciences, Ataturk University, 25240 Erzurum, Turkey
cHendek Faculty of Education, Department of Chemistry, Sakarya University, 54300 Sakarya, Turkey
Received 9 August 2007; accepted 7 September 2007
Available online 10 October 2007
Abstract—Alternaria alternata EBK-4 fungus isolated from a plant sample was evaluated for the asymmetric reduction of acetophenone
analogues. In a previous study, this isolate was used for the reduction of acetophenone to 1-phenylethanol in excellent enantiomeric
excess. The substituted acetophenones were converted to the corresponding optically active alcohol by A. alternata EBK-4 under opti-
mized conditions in up to >99% enantiomeric excess (ee). This is the first report on the enantiomeric reduction of acetophenone ana-
logues by A. alternata using ram horn peptone from waste material.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
compounds and combines inexpensive raw materials with
environmentally friendly processes.7 The fermentation
medium can represent almost 30% of the cost for a micro-
bial fermentation, with micronutrients representing the
most significant cost of production.
Chiral alcohols are important as bioactive compounds and
as starting materials for the synthesis of various biologi-
cally active compounds. The need for optically active drugs
has increased in pharmaceutical and agrochemical fields in
the recent years. Therefore, the asymmetric reduction of
ketones to their corresponding alcohols is useful in organic
synthesis.1,2 Optically active phenylethanol and its deriva-
tives are useful building blocks for the synthesis of complex
molecules, because the alcohol functionality can be easily
transformed without racemization into other useful func-
tional groups.3 There has been much interest in the use
of fungus or yeast for the enantioselective reduction of aro-
matic ketones. They are used as a reducing agent because
they are easily available and cheap. There are many advan-
tages to using microorganisms as biocatalysts instead of
purified enzymes. Microorganisms are generally much less
expensive, and in some cases, enzymes are more stable
within the cell, thus extending the life of the biocatalyst.
In addition, the use of microbial cells is particularly advan-
tageous for carrying out the desired reduction since they do
not require the addition of cofactors for their regenera-
tion.4–6 On the other hand, biotechnology opens up future
prospects in the chemical field for the synthesis of complex
By-products can supply unique micronutrients to replace
expensive peptone and yeast extract. The consistency of
ingredients used in commercial medium formulations and
significant increase in product yield or cost reduction are
critical for industrial fermentation utilization of any by-
product.8
Recently, we reported that ram horn peptone (RHP) can be
utilized as a source of peptone for microbial growth media
in chiral alcohol synthesis.9f Herein, we report a study on
the potential of Alternaria alternata EBK-4 as an asymmet-
ric reducing agent for acetophenone analogues by using the
RHP in fermentation medium.
2. Results and discussion
A series of acetophenone derivatives were targeted for the
biotransformation using A. alternata isolate in RHP med-
ium, and ortho-, meta- and para-substituted fluoro, chloro,
bromo, methyl methoxy and phenyl acetophenones 1b–l
were reduced to the corresponding (R)- or (S)-alcohols.
*
Corresponding authors. Tel.: +90 442 231 4342; fax: +90 442 236 0948
(E.B.K.); e-mail: ekurbanoglu@yahoo.com
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.09.009

E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 2332–2335
2333
In a previous study, A. alternata fungus isolated from a
plant sample was evaluated for reduction of acetophenone
1a to 1-phenylethanol 2a.9 The asymmetric reductions of
1a derivatives with the same fungus were performed under
previously determined optimum fermentation conditions.
The reaction progress was monitored by 400 MHz 1H
NMR spectroscopy and a chiral HPLC column was used
for the determination of the enantiomeric excess of 1a
derivatives. As can be seen from Table 1, the fungus was
found to catalyze the asymmetric reduction of 1a deriva-
tives, such as 1b–l, to give the corresponding chiral alco-
hols. The experiments were carried out on a millimole
scale in 100 mL culture medium and the corresponding chi-
ral alcohols according to substrate conversion rate were
isolated in 17–78% yields. Reduction of o-, m-fluoro, o-,
m-chloro and o-, m-bromo acetophenones provided excel-
lent enantioselectivity (>99%). In contrast, reduction of
p-chloro, p-bromo, p-nitro, methoxy and phenyl acetophe-
nones was observed in low yields (17–68) and enantioselec-
tivity (29–90%). The results obtained from 1b–g
demonstrated the applicability of this process and the
fungus in the preparation of 2b–g. Acetophenone deriva-
tives with different substituents, such as chlorine, bromine,
methyl, methoxy and phenyl groups on the benzene ring
were selected to assess the efficiency and stereoselectivity
of the ketone functionality bioreduction by the dehydroge-
nase present in the enzymatic system of A. alternata EBK-
4. As can be seen in Table 1, an enantioselectivity of more
than 99% ee was obtained for the reduction of o-fluoro,
o-chloro, o-bromo, m-fluoro, m-chloro and m-bromo ace-
tophenones 1b–g, which are transformed into the (S)-alco-
hols10 2b–g with high enantioselectivity in complete
conversion. However, the bioreduction of para halogenated
acetophenones p-chloro and p-bromo derivatives 1h and 1i
led to the formation of alcohols 2h and 2i with moderate ee
in low conversion. The low conversions or ees observed for
the substrate 1h–l testify that the rate of the reduction
depends on the steric and the electronic effects of the bro-
mine or chlorine atom at the ortho-, meta- or para-position
on the aromatic ring. Electron donating or withdrawing
substituents nitro, methoxy and phenyl groups at the
para-position on the aromatic ring led to a dramatical de-
crease in the conversion and enantiomeric excess. Thus, 1h,
1i and 1k were converted to the corresponding (R)-alco-
hols11 2h, 2i and 2k with 29, 49 and 49% ee, respectively.
It was found that the electronic effect of the substituents
on the aromatic ring has a defined role in the enantioselec-
tivity and the configuration of the reaction products. The
data concerning the observed stereoselectivities for the sub-
strates clarified that A. alternata EBK-4 produced (S)-alco-
hols 2b–g from o- and m-substituted acetophenone
derivatives 1b–g and (R)-alcohols 2h,i,k from para-substi-
tuted acetophenone derivatives 1h,i,k. This observation
can be explained in terms of steric hindrance of the substit-
uents. The bioreduction of sterically unhindered aryl alkyl
ketones 1b–g and 1j,l was very fast; the Prelog-(S) enantio-
mer predominated, but the acetophenones 1h,i,k with a
substituent proximate to the carbonyl group were con-
verted to the anti-Prelog product (R)-enantiomer.12 These
results suggest that the reduction of o-, m-fluoro, chloro
and bromo acetophenones by A. alternata by using RHP
will be very useful for the practical preparation of (S)-alco-
hols. The A. alternata fungus catalyzed the reactions in an
eco-friendly environment when compared to chemical reac-
tions. Moreover, the RHP from waste material as an inex-
pensive substrate for microbial growth was used. Meat
industry wastes are an important environmental contami-
nation source. The importance of this research must be
high because of the formation of little waste, use of accept-
able solvents, transformation of waste materials into valu-
able products and the highly asymmetric synthesis of the
many desired products.
3. Conclusions
In conclusion, A. alternata EBK-4 isolated from plant sam-
ple is the first report regarding the use of a biocatalyst for
the asymmetric reduction of acetophenone derivatives. The
ortho- and meta-substituted acetophenones are reduced in
submerged culture of the A. alternata EBK-4 to the corres-
ponding chiral alcohols with high enantiomeric excess
(>99%). The observed Prelog and anti-Prelog enantioselec-
tivity depends on the substituent’s position in the benzene
ring. In previous study, (S)-1-phenylethanol was success-
fully produced on a gramme scale with the present pro-
cess.9 This bioreduction protocol is applicable to the
production of some enantiomerically pure alcohols.
4. Experimental
4.1. Materials
Ram horns were obtained from the Slaughterhouse of
Erzurum, Turkey. The other components of the culture
media and the chemical reagents were obtained from
Merck and Sigma in the highest purity available. The pro-
duction of ram horn peptone was carried out with the
method of Kurbanoglu and Kurbanoglu.9
The microorganism used in this study was isolated from a
plant sample. The isolation process was performed by the
serial dilution of the samples according to standard tech-
niques. Taxonomic identification of filamentous fungi was
identified in-house by using mature cultures on standard
potato dextrose agar (PDA) in order to ensure a good
development of taxonomically relevant features. The fun-
gus used was maintained on PDA slants, incubated at
25 °C and stored at 4 °C. The conidia from 10 days old cul-
tures were used for inoculation. The conidial suspension
was prepared in sterilized 10 mL distilled water by gently
scratching conidia with a sterile wire loop and then it was
shaken vigorously to break the clumps of conidia.9f
4.2. Culture conditions and reduction of acetophenone
analogues
The culture conditions were prepared according to opti-
mum fermentation parameters found for A. alternata in a
previous study.9 The per liter fermentation medium con-
tained (g/L): glucose 20, yeast extract 3 and RHP 4. The
initial pH of the culture medium was adjusted to 6.5 with
1 M HCl and 1 M NaOH and sterilized at 121 °C for

2334
E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 2332–2335
Table 1. Asymmetric reduction of acetophenone analogues 1b–l by A. alternata EBK-4a
Substrates
Products
OH
eeb,c (%)
Conversionsd (%)
Yields (%)
86e
O
(S)-> 99e
100e
CH3
CH3
1a
2a
F
O
F
OH
(S)- >99
(S)- >99
(S)- >99
(S)- >99
100
100
100
100
78
76
74
72
CH3
CH3
1b
2b
Cl
Br
O
O
Cl
Br
OH
CH3
1c
CH3
2c
OH
CH3
1d
CH3
2d
OH
O
F
F
CH3
2e
OH
CH3
1e
O
Cl
Br
Cl
Br
(S)- >99
(S)- >99
100
100
73
74
CH3
1f
CH3
2f
O
O
OH
CH3
1g
CH3
2g
OH
(R)-29
(R)-49
92
85
68
61
CH3
1h
CH3
2h
Cl
Br
Cl
Br
OH
O
CH3
1i
CH3
2i
O
OH
CH3
CH3
(S)-20
(R)-49
(S)-90
70
36
25
52
27
17
1j
2j
O2N
MeO
Ph
O2N
MeO
Ph
O
OH
CH3
1k
CH3
2k
O
OH
CH3
2l
CH3
1l
a Bioreduction conditions: substrate: 1 mmol; temperature: 28 °C; time: 24 h; pH: 6.5, 150 rpm.
b Determined by HPLC using Chiralcel OD and OB columns.
c Absolute configurations were assigned by comparison of the sign of specific rotations relative to the literature values.
d Conversion was determined by 1H NMR analysis with diphenylmethane as internal standard; error ca 5% of the stated values.
e From Ref. 9f.

E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 2332–2335
2335
15 min. All the cultures were grown in 250 mL flasks con-
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