Enantioselective reduction of substituted acetophenones by Aspergillus niger

Article abstract of DOI:10.1016/j.tetasy.2007.05.017

Fourteen strains of Aspergillus niger were isolated from soil samples. These isolates were evaluated for the reduction of acetophenone to 1-phenylethanol. Among the tested isolates, whole cells of the A. niger EBK-9 isolate were found to be an effective biocatalyst for the enantioselective reduction of acetophenone. Under optimized conditions substituted acetophenones were converted to the corresponding optically active alcohol in up to 99% ee.

Full text of DOI:10.1016/j.tetasy.2007.05.017

Tetrahedron: Asymmetry 18 (2007) 1159–1162
Enantioselective reduction of substituted acetophenones
by Aspergillus niger
Eshabi B. Kurbanoglu,a,* Kani Zilbeyaz,b Namudar I. Kurbanogluc and Hamdullah Kilicb,*
aDepartment of Biology, Ataturk University, 25240 Erzurum, Turkey
bDepartment of Chemistry, Ataturk University, 25240 Erzurum, Turkey
cHendek Faculty of Education, Department of Chemistry, Sakarya University, Sakarya, Turkey
Received 5 February 2007; revised 8 May 2007; accepted 15 May 2007
Abstract—Fourteen strains of Aspergillus niger were isolated from soil samples. These isolates were evaluated for the reduction of
acetophenone to 1-phenylethanol. Among the tested isolates, whole cells of the A. niger EBK-9 isolate were found to be an effective
biocatalyst for the enantioselective reduction of acetophenone. Under optimized conditions substituted acetophenones were converted
to the corresponding optically active alcohol in up to 99% ee.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
A. niger strains isolated from soil samples as enantioselec-
tive reducing agents for substituted acetophenones.
Chiral alcohols are useful starting materials for the synthe-
sis of various biologically active compounds. The need for
optically active drugs has increased in pharmaceutical and
agrochemical fields in recent years and thus, the demand
for chiral alcohols has increased.1 Among the current
methodologies to obtain chiral alcohols are the biocatalytic
reduction2 of the corresponding ketones, and kinetic reso-
lution3 with lipases of the racemic alcohols via esterifica-
tion or hydrolysis of the appropriate esters. The use of
microbial whole cells as biocatalysts is particularly advan-
tageous for carrying out the desired reduction, since they
contain multiple dehydrogenases that are able to accept a
broad spectrum of unnatural substrates, as well as all the
necessary co-factors and metabolic pathways for their gen-
eration. Furthermore, all the enzymes and co-factors are
well protected within their natural cellular environment.4,5
In this way screening for new microorganisms strains from
different natural environments can be used in the search for
efficient enzymatic systems, which can promote the
biotransformation with no need for external additives.
Recently, we reported that ram horn hydrolysate (RHH)
can be utilized as a source of peptone for microbial growth
media.6 Herein, we report a study on the potential of
2. Results and discussion
Fourteen different isolates of A. niger isolated from soil
samples were evaluated for reduction of acetophenone 1a
to 1-phenylethanol 2a. The microbial reduction was per-
formed by suspending the wet cells in a 50 ml fresh med-
ium, 1a was then added and the mixture was incubated
for 48 h in an orbital shaker at 28 °C. The reaction progress
1
was monitored by 400 MHz H NMR spectroscopy and a
chiral HPLC column was used for the determination of the
enantiomeric excess of 2a. As can be observed in Table 1,
the strains under study produced (R)-1-phenylethanol7 2a
ranging from 41% to 75% ee. The best results for the bio-
reduction were obtained when whole cells of A. niger
EBK-9 were used. The reaction occurred with moderate
conversion and produced 2a in 75% ee (Table 1, entry 9).
Reaction conditions, such as reaction time, the amount of
wet biocatalyst, and pH were investigated using A. niger
EBK-9 under the standard conditions. The best ee (80%)
and yield (44%) were observed before 48 h, but an exten-
sion of the reaction time led to a decrease in ee, but an in-
crease in the conversion (entry 15). The optimum pH for
the enantioselective reduction was found to be 7.0, which
yielded 2a with 82% ee (entry 16). Under the optimum
*
Corresponding authors. Tel.: +90 442 231 4426; fax: +90 442 236 0948
(H.K.); e-mail: hkilic@atauni.edu.tr
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.017

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E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 1159–1162
Table 1. Screening for Aspergillus niger isolates in the bioreduction of
acetophenone 1aa
pends on the steric and electronic effects of the bromine
or chlorine atom at the ortho- and meta-position on the
aromatic ring. The A. niger EBK-9 reduced para haloge-
nated acetophenones 1f and 1g to the anti-Prelog (R)-p-
bromo- and p-chloro-arylethanols9 2f and 2g with complete
conversion and excellent enantiomeric excess (99%). Elec-
tron donating substituents methyl, methoxy and phenyl
groups at the para position on the aromatic ring led to a
slight decrease in the conversion and enantiomeric excess.
Thus, 1h–k were converted to the corresponding (R)-alco-
hols10 2h–k with 95, 90, 60% ee, respectively. Since several
acetophenones afforded excellent enantioselectivities for
the reduction on a small scale, we decided to conduct the
transformation of 1f to (R)-2f on a large scale to demon-
strate the viability of the present system as industrially fea-
sible. The bioreduction in a 2 L Erlenmeyer flask with
10 mmol of 1-(4-chloro-phenyl)ethanone 1f produced (R)-
2f in 70% yield and 99% enantiomeric excess (Scheme 1).
In this way, (R)-2f7 was isolated in gram amount. The data
concerning the observed stereoselectivities for the sub-
strates clarified that A. niger EBK-9 produced (R)-alcohols
2f–k from para-substituted acetophenone derivatives 1f–k
and (S)-alcohols 2b–e from ortho- and meta-substituted
acetophenone derivatives 1b–e. This observation can be ex-
plained in terms of steric hindrance of the substituents. The
bioreduction of sterically unhindered aryl alkyl ketones 1a
and 1f–k was very fast the anti-Prelog-(R) enantiomer pre-
dominated, but acetophenones 1b–e with substituent prox-
imate to the carbonyl group were converted to the Prelog
product (S)-enantiomer.11
O
OH
CH3
isolates of A. niger
CH3
1a
2a
Entry
Isolates of
A. niger
Conversionb
(%)
Yieldc (%)
ee of (R)-2a
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15d
16e
17f
EBK-1
EBK-2
EBK-3
EBK-4
EBK-5
EBK-6
EBK-7
EBK-8
EBK-9
EBK-10
EBK-11
EBK-12
EBK-13
EBK-14
EBK-9
EBK-9
EBK-9
30
28
35
36
35
36
38
41
46
41
18
36
34
39
53
57
63
23
15
23
27
26
24
30
34
39
32
14
28
28
52
44
47
54
64
41
46
67
67
57
65
70
75
64
48
46
48
65
80
82
87
a Reaction conditions: biocatalyst 5 g (wet weight); acetophenone: 1 mmol;
temperature: 28 °C; time: 24 h; pH: 6.8.
b Conversion was determined by 1H NMR analysis with diphenylmethane
as an internal standard; error ca. 5% of the stated values.
c Isolated yields.
d Biocatalyst: 5 g (wet weight); time: 48 h; temperature: 28 °C; pH: 6.8.
e Biocatalyst: 5 g (wet weight); time: 48 h; temperature: 28 °C; pH: 7.0.
f Biocatalyst: 7.5 g (wet weight); time: 48 h; pH: 7.0.
3. Conclusions
In conclusion, 14 strains of Aspergillus niger were isolated
from soil samples. The para- and ortho-substituted aceto-
phenones are reduced by whole cells of the A. niger
EBK-9 to the corresponding chiral alcohols with high
enantiomeric excess (up to 99%). The observed Prelog
and anti-Prelog enantioselectivity depends on the substitu-
ents position in the benzene ring. The present bioreduction
protocol would be applicable to the production of some
enantiomerically pure alcohols.
conditions (pH 7.0, 48 h), the effect of a wet biocatalyst
amount on the conversion and enantioselectivity of ace-
tophenone 1a reduction was investigated. The best ee (87%)
and moderate conversion (63%) were obtained when 7.5 g
biocatalyst was applied (entry 17). A biocatalyst amount
higher than 7.5 g led to complete conversion of 1a to 2a,
but with very low ee (27%). The experiments were conducted
with A. niger EBK-9, which gave the best results in terms of
conversion and enantiomeric excess.
After successfully optimizing the reaction conditions, we
turned our attention to the biocatalytic reduction of substi-
tuted acetophenones 1b–1k. Acetophenone derivatives with
different substituents (chlorine, bromine, methyl, methoxy
and phenyl groups) on the benzene ring were selected to
assess the efficiency and stereoselectivity of the ketone func-
tionality bioreduction by the dehydrogenase present in
the enzymatic system of A. niger EBK-9. As can be seen
in Table 2, an enantioselectivity of more than 96% ee was
obtained for the reduction of o-chloro and o-bromo aceto-
phenones 1b and 1c, which are transformed into (S)-alco-
hols8 2b and 2c with high enantioselectivity and in low
conversion for 48 h. However, the bioreduction of
m-chloro and m-bromo derivatives 1d and 1e led to the
formation of alcohols 2d and 2e with moderate ee in low
conversion for 48 h. The low conversions observed for
substrates 1b–1e show that the rate of the reduction de-
4. Experimental
Ram horns were obtained from the slaughterhouse of Erzu-
rum, Turkey. The other components of the culture media
and the chemical reagents were obtained from Acros Chem-
ical and from Sigma in the highest purity available. 1H and
13C NMR spectra were recorded on a Varian 400 spectro-
meter in CDCl3. Enantiomeric excesses were determined
by HPLC analysis using a chiral column (Chiralcel OD)
with eluent n-hexane–i-PrOH, 90:10, flow rate of 0.6 ml/
min, detection performed at 254 nm. Optical rotation was
measured with a Bellingham + Stanley, ADP220, 589 nm
spectropolarimeter. A polarimetric Chiralyser detector
was also employed to assess the configuration of the enan-
tiomer formed. The racemic 2a–k were obtained by reacting
the corresponding 1a–k with NaBH4 in methanol at rt.

E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 1159–1162
1161
Table 2. Enantioselective reduction of substituted acetophenones 1b–1ka
Substrate Product
Cl OH
eeb,c (%)
Conversiond (%)
16
Yielde (%)
Cl
O
>99 (S)
12
CH3
CH3
1b
2b
Br
O
Br OH
CH3
96 (S)
57 (S)
11
25
7
CH3
1c
2c
O
OH
Cl
Cl
Br
19
16
87
89
47
28
52
CH3
1d
CH3
2d
O
O
OH
Br
66 (S)
21
CH3
1e
CH3
2e
OH
>99 (R)
>99 (R)
95 (R)
90 (R)
65 (R)
100
100
60
CH3
1f
CH3
2f
Cl
Cl
O
O
OH
CH3
1g
CH3
2g
Br
Br
OH
CH3
1h
CH3
2h
Me
MeO
Ph
Me
MeO
Ph
O
OH
33
CH3
1i
CH3
2i
O
OH
CH3
67
CH3
1k
2k
a Reaction conditions: biocatalyst 7.5 g (wet weight); substrate: 1 mmol; temperature: 28 °C; time: 48 h; pH: 7.0.
b Determined by HPLC using Chiralcel OD column.
c Absolute configurations were assigned by comparison of the sign of optical rotations relative to the literature values.
d Conversion was determined by 1H NMR analysis with diphenylmethane as an internal standard; error ca. 5% of the stated values.
e Isolated yields.
in-house by using mature cultures on standard potato dex-
trose agar (PDA) in order to ensure a good development of
taxonomically relevant features, and following the identifi-
cation keys provided by von Arx13 and Domsch et al.14
These cultures were maintained on PDA slants, incubated
at 30 °C and stored at 4 °C. The conidia from 5 days old
cultures 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 for breaking the clumps of conidia.
O
OH
CH3
EBK-9, D-glucose
pH 7.0, 28 oC, 48 h
CH3
Cl
Cl
2f, 70% yield, ee >99%
(1.5 g, 10 mmol)
1f
scale: 1 L
Scheme 1. Bioreduction of 1f to 2f.
4.1. Isolation of microorganisms, identification and
inoculation
4.2. Mycelium production from A. niger strains and culture
conditions
All the microorganisms used in this study were isolated
from soil samples collected from around Erzurum, Turkey.
The isolation process was performed by serial dilution of
the samples according to standard techniques.12 Taxo-
nomic identification of filamentous fungi were identified
The per liter fermentation medium contained 15 g glucose,
3 g yeast extract and 30 ml RHH. Ram horn hydrolysate
was prepared as described previously.6 The initial pH of

1162
E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 1159–1162
the culture medium was adjusted to 7.0 with 1 M HCl and
1 M NaOH. All the cultures were grown in 250 ml flasks
containing 100 ml of medium. One ml of conidial suspen-
sion was added to each flask. Flasks were incubated on a
reciprocal shaker at 200 rpm, 28 °C for 48 h.
meric excess of the alcohol was then determined by chiral
HPLC analysis.
Acknowledgements
The authors are indebted to the Ataturk University and
The State Planning Organization of Turkey (D.P.T.) for
the financial support of this work. The authors thank Pro-
fessor Ismet Hasanekoglu for his help in identification of
fungus.
4.3. General procedure for bioreduction of ketones 1a–k
After 48 h of fermentation, mycelia were separated from
the culture broth by filtration and cell pellets were washed
twice with sterile 0.1 M KH2PO4 (pH 7.0). Approximately
7.5 g wet mycelia were put in a 250 ml conical flask con-
taining 50 ml of the fresh culture medium and incubated
at 28 °C for 24 h under static conditions. After the growth
of the fungus, the substrate (1 mmol) was added directly to
medium and then the incubation continued on a reciprocal
shaker at 200 rpm, 28 °C for 48 h. At the end of the incu-
bation period, the mycelium was separated by filtration,
and the filtrate was saturated with sodium chloride and
then extracted with diethyl ether. The mycelia were washed
with diethyl ether. Diethyl ether extracts were combined
and dried over Na2SO4. After removal of the solvent under
reduced pressure (25 °C, 100 Torr) the crude product was
purified by silica gel column chromatography and identi-
fied by NMR analysis. The absolute configuration was
determined by the sign of the specific rotation and compar-
ison with the literature. The enantiomeric excess of the
alcohol was then determined by chiral HPLC analysis.
References
1. Kataoka, M.; Kita, K.; Ada, M.; Yasohara, Y.; Hasegawa,
J.; Shimizu, S. Appl. Microbiol. Biotechnol. 2003, 62, 437–445.
2. (a) Wong, C. H.; Drueckhammer, D. G.; Sweers, H. M.
J. Am. Chem. Soc. 1985, 107, 4028–4031; (b) Yang, W.; Xu,
J.-H.; Xie, Y.; Xu, Y.; Zhao, G.; Lin, G.-Q. Tetrahedron:
Asymmetry 2006, 17, 1769–1774; (c) Bruni, R.; Fantin, G.;
Maietti, S.; Medici, A.; Pedrini, P.; Sacchetti, G. Tetrahedron:
Asymmetry 2006, 17, 1769–1774.
3. Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic
Organic Chemistry; Pergamon, 1994.
4. Mandal, D.; Ahmad, A.; Khan, M. I.; Kumar, R. J. Mol.
Catal. B: Enzym. 2004, 27, 61–63.
5. Faber, K. Biocatalytic Application. In Biotransformations in
Organic Chemistry; Springer, 2000; pp 192–193.
6. (a) Kurbanoglu, E. B.; Kurbanoglu, N. I. J. Ind. Microbiol.
Biotechnol. 2004, l31, 289–294; (b) Kurbanoglu, E. B.;
Kurbanoglu, N. I. Process Biochem. 2003, 38, 1421–1424;
(c) Kurbanoglu, E. B. Energ. Convers. Manage. 2003, 44,
2125–2135; (d) Kurbanoglu, E. B.; Kurbanoglu, N. I. J.
Biosci. Bioeng. 2002, 94, 202–206.
7. Inoue, K.; Makino, Y.; Itoh, N. Tetrahedron: Asymmetry
2005, 16, 2539–2549.
8. Andrade, L. H.; Comasseto, J. V.; Rodrigues, D. F.; Pellizari,
V. H.; Porto, A. L. M. J. Mol. Catal. B: Enzym. 2005, 33, 73–
79.
4.4. Preparative scale bioreduction of 1f
After 48 h of fermentation, mycelia were separated from
the culture broth by filtration and cell pellets were washed
twice with sterile 0.1 M KH2PO4 (pH 7.0). Approximately
75 g wet mycelia were put in a 2 L conical flask containing
500 ml of the fresh culture medium and incubated at 28 °C
for 24 h under static conditions. After the growth of the
fungus, 1-(4-chlorophenyl)ethanone 1f (1.54 g, 10 mmol)
was added directly to the medium and then the incubation
continued on a reciprocal shaker at 200 rpm, 28 °C for
48 h. At the end of the incubation period, the mycelium
was separated by filtration, the filtrate saturated with
sodium chloride and then extracted with diethyl ether.
The mycelia were washed with diethyl ether. The diethyl
ether extracts were combined and dried over Na2SO4. After
removal of the solvent under reduced pressure (25 °C,
100 Torr) (R)-1-(4-chlorophenyl)ethanol 2f was purified
by silica gel column chromatography and identified by
NMR analysis (1.1 g, 7 mmol, 70% yield). The absolute
configuration was determined by the sign of the specific
rotation and comparison with the literature. The enantio-
9. Stampfer, W.; Kosjek, B.; Faber, K.; Kroutil, W. J. Org.
Chem. 2004, 68, 402–406.
10. Comasseto, J. V.; Andrade, L. H.; Omori, A. T.; Assis, L. F.;
Porto, A. L. M. J. Mol. Catal. B: Enzym. 2004, 29, 55–61.
11. Prelog, V. Pure Appl. Chem. 1964, 9, 119–130.
12. Nakayama, K. Sources of Industrial Microorganisms. In
Biotechnology, Microbial Fundamentals; Rehm, H.-J., Reed,
G., Eds.; Verlag Chemie: Weinheim, 1981; Vol. 1, pp 355–
410.
13. Von Arx, J. A. Key to the Orders of Fungi, The Genera of
Fungi Sporulating in Pure Culture; Cramer: Hirschberg,
Germany, 1981.
14. Domsch, K. H.; Gams, W.; Anderson, T. H. Key to the
Genera, Compendium of Soil Fungi; IHW, Eching: Germany,
1993.