Total production of (R)-3,5-bistrifluoromethylphenyl ethanol by asymmetric reduction of 3,5-bis(trifluoromethyl)-acetophenone in the submerged culture of Penicillium expansum isolate

Article abstract of DOI:10.1016/j.tetasy.2009.11.006

A total of 82 fungal isolates were screened for their ability to reduce 3,5-bis(trifluoromethyl)-acetophenone 1. Penicillium expansum was found to successfully reduce ketone 1 in the submerged culture. A second screening was performed on the active P. exp

Full text of DOI:10.1016/j.tetasy.2009.11.006

Tetrahedron: Asymmetry 20 (2009) 2759–2763
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Total production of (R)-3,5-bistrifluoromethylphenyl ethanol by asymmetric
reduction of 3,5-bis(trifluoromethyl)-acetophenone in the submerged culture
of Penicillium expansum isolate
b
a
c
Esabi B. Kurbanoglu a, , Kani Zilbeyaz , Mesut Taskin , Namudar I. Kurbanoglu
*
a Department of Biology, Science Faculty, Ataturk University, 25240 Erzurum, Turkey
b Department of Chemistry, Science Faculty, Ataturk University, 25240 Erzurum, Turkey
c Department of Chemistry, Hendek Faculty of Education, Sakarya University, 54300 Sakarya, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A total of 82 fungal isolates were screened for their ability to reduce 3,5-bis(trifluoromethyl)-acetophe-
none 1. Penicillium expansum was found to successfully reduce ketone 1 in the submerged culture. A sec-
ond screening was performed on the active P. expansum strains identified by the first screening. A number
of strains of P. expansum were found to produce (R)-3,5-bistrifluoromethylphenyl ethanol 2 with over 99%
enantiomeric excess (ee) from ketone 1. The most productive strain was identified as P. expansum EBK-9,
and this strain was selected for further experiments. The total production of 2 was carried out by P.
expansum EBK-9 in a laboratory-scale bioreactor employing optimized conditions as determined by our
experiments. P. expansum EBK-9 gave 2 with ee >99% and 76% yield. On a large scale, a total of 3.35 g/
L of 2 was produced from 1 after 56 h.
Received 22 October 2009
Accepted 11 November 2009
Available online 5 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
the other hand, the reduction by an alcohol dehydrogenase from
bacteria Rhodococcus erythropolis gave the corresponding (S)-alco-
Biocatalysts are used due to their high specific activities in the
pharmaceutical industry. A significant number of pharmaceutical
products are chiral and many have multiple stereogenic centers.
The efficient syntheses of these stereogenic centers require enan-
tioselective and/or regioselective catalysts. In recent years, en-
zymes have become the most efficient and most selective
catalysts available.1 It is well known that chiral alcohols are very
important precursors for a large number of pharmaceuticals and
(R)-3,5-bistrifluoromethylphenyl ethanol 2 is no exception. It is
an important intermediate in the synthesis of therapeutic agents
such as the NK-1 receptor antagonist used for the treatment of
emesis.2 A single approach for the synthesis of 2 is the asymmetric
reduction of 3,5-bis(trifluoromethyl)-acetophenone 1. There are
various catalytic methods available such as direct enantioselective
hydrogenation3 or borane reduction4 for the asymmetric reduction
of 1. However, they often require the utilization of hazardous
reagents. In light of this, the asymmetric reduction of 1 was
investigated using microbial resting cells and various enzymes.
Gelo-Pujic et al. showed that the reduction of 1-[3,5-bis(trifluoro-
methyl)phenyl]ethanone with resting cells of the bacteria Lactoba-
cillus kefir gave the corresponding (R)-alcohol with >99% ee.5 On
hol with 99% ee.6 The (S)-alcohol is currently under clinical evalu-
ation as an intermediate for antagonist. However, reactions
catalyzed by enzymes demand the use of cofactors such as NADH,
which are very expensive.
In recent years, there has been a growing interest in the isola-
tion of microorganisms from the environment for the synthesis
of organic materials. The screening of isolated microorganisms is
an important way to obtain new microorganisms and compounds,
or to improve the productivity of known compounds. In order to
improve the yield and the enantioselectivity of a product, many
synthetic chemists and biologists have adopted the methods of
screening microorganisms,7,8 which leads to constructing perfect
gene engineering strains.8 The addition of inhibitors to inhibit
the enzymes, which convert the substrate to the undesirable con-
figuration product9 and the addition of co-substrates to increase
the yield and control reaction stereoselectivity10 have also been
investigated and reported. In our laboratories, chiral alcohols have
been prepared with various microorganisms using a similar reduc-
tion system.11 However, there have been no reports regarding the
production of 2 by the bioreduction of 1 in the submerged culture.
Herein, we report the asymmetric reduction of 1 to the corre-
sponding chiral alcohol 2 in the submerged culture system with
screening of microorganisms. The effects of co-substrates and reac-
tion conditions on the yield and enantiomeric excess (ee) have also
been investigated in detail.
* Corresponding author. Tel.: +90 442 231 4342; fax: +90 442 236 0948.
E-mail address: ekurbanoglu@yahoo.com (E.B. Kurbanoglu).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.11.006

2760
E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 20 (2009) 2759–2763
production can be performed on a large scale so that it can be ap-
2. Results and discussion
plied in an industrial process using an engineered microorganism
and designed the reaction conditions.13,14 Therefore, it was decided
to determine the performance of the microorganism under opti-
mum reaction conditions. The first parameter that was investi-
gated was the effect of pH on the production of 2 from 1 in the
submerged culture. Table 2 shows the results for the effect for
the pH range from 3.0 to 8.0 on the product’s ee and conversion
of 1. It was observed that the reduction of 1 was greatly affected
by pH. The results indicate that ketone 1 could be reduced to alco-
hol 2 at pH 5.0–7.5. Above or below these values, no or weak
growth was observed. At low pH values (5.0–6.0), low ee’s were
obtained and at higher pH values (6.5–7.5) ee’s of >99% were ob-
tained. The highest conversion (54%) with >99% ee was obtained
at pH 6.5. At this pH the bioactivity of the EBK-9 strains was the
best for the reduction of ketone 1. All further studies were per-
formed at pH 6.5.
The second parameter that was investigated was the effect of
reaction temperature on the reduction of 1. Table 3 shows the re-
sults that were obtained. The reaction temperature was varied
from 22 to 30 °C and it was observed that maximum conversion
(62%) was achieved at 26 °C. Temperatures below and above
26 °C saw a rapid decline in conversion. Interestingly, the enantio-
purity of 2 remained at >99% at all the temperatures investigated.
The optimum temperature for the reduction of ketone 1 to alcohol
2 was set at 26 °C and hence, all the subsequent reduction experi-
ments were carried out at 26 °C.
The effect of agitation speed on the reaction rate was the third
parameter investigated and the results are given in Table 4. Agita-
tion speeds from 100 rpm to 300 rpm were chosen for the experi-
ments. The results obtained from the experiments showed that the
conversion of 1 was at a maximum at high agitation speeds (more
than 200 rpm). The enantiopurity, however, was recorded at >99%-
(R) for most agitation speeds but decreased significantly to 79% at
300 rpm. This decrease might be due to the effect of shear stress on
the fungus cells as well as on the enzyme structure. The best yield
of alcohol 2 with high ee was obtained at an agitation rate of
250 rpm. In light of this, an agitation speed of 250 rpm was used
for subsequent studies. We also added 1.5 mmol of 1 instead of
1 mmol of 1 to the reaction culture medium due to the 100%
conversion.
The effects of co-substrates and their concentrations on the con-
version and enantioselectivity for preparative scale were also
examined. In an attempt to improve the conversion of 1 to 2 by
P. expansum EBK-9, three different co-substrates, 2-propanol, glyc-
erol and ethanol, were assayed and compared in this study.
Employing the optimum conditions, the co-substrates at various
%/v concentrations and ketone 1 directly were added to the sub-
merged culture medium after the growth of the fungus for 48 h.
The results for these experiments are presented in Table 5. The
enantiopurity of the desired product was not affected by the addi-
A total of 82 fungal isolates isolated from various soils from
Erzurum, Turkey were tested for their ability to produce alcohol
2 from ketone 1. Among the isolates, the initial screening deter-
mined three isolates that produced 2 with both a highest conver-
sion (50–70%) and highest ee (88–99%). The three isolates were
identified as Penicillium expansum. The 14 strains of the P. expan-
sum determined by the first screening were then isolated from var-
ious samples and screened for their 2-producing activity. Substrate
tolerance of the biocatalyst is very important for its potential
industrial application. Higher substrate tolerance of the biocatalyst
can increase its production capability in industrial processes. In
light of this, different P. expansum strains with the same substrate
concentration (0.5 mmol substrate/100 mL culture) were tested for
the determination of the catalytic capacities of the strains. It has
been reported that reaction stereoselectivity and product yield
are related to the kind of reductases in microorganisms and the
molecular structure of the substrates.12 The reduction results of
compound 1 by 14 strains are listed in Table 1. It was found that
all strains produced optically active 2 from 1. Ten of the strains
examined (EBK-2, EBK-3, EBK-4, EBK-5, EBK-6, EBK-9, EBK-11,
EBK-12, EBK-13 and EBK-14) produced the desired product with
high enantiomeric purity (>99% ee). The conversion rates of 1 from
the 10 strains were compared and it was found that P. expansum
EBK-9 showed the highest activity (90% conversion and >99% ee)
in the second screening. The results showed that the conversion
and enantiomeric purity are strongly dependent on the different
strains of the same species. From these results, we suggest the care-
ful control of the strain used as biocatalyst for enantiomeric purity
and conversion in the asymmetric reduction. On the basis of these
results, P. expansum EBK-9 was selected as the best 2-producing
strain and was used for further experiments. Gelo-Pujic et al.5 re-
ported that the bacteria L. kefir is an effective biocatalyst for the
asymmetric bioreduction of 1 to its corresponding (R)-alcohol with
99% ee. They reported that their studies employed whole cells of L.
kefir, whereas our current study used a submerged culture of P.
expansum fungus. In order to improve the yield with high ee of 2,
the effects of fermentation conditions such as pH, temperature, reac-
tion time and agitation were studied with P. expansum EBK-9 in the
shake flasks. The aim was to achieve high conversion with high
enantiomeric purity. In order to minimize the error in the experi-
ments the substrate concentration (the concentration of 1 added
to the submerged culture of P. expansum EBK-9) was increased from
0.5 mmol 1 to 1.0 mmol 1 for all subsequent experiments.
One of the prime requirements of a microbial bioconversion is
the optimization of the reaction conditions. However, microbial
Table 1
Screening of P. expansum strains for the bioreduction of 1
Isolates
Conversions (%)
ee (%)-(R)-configuration
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
15
11
38
75
12
48
42
46
90
55
80
54
34
22
88
>99
>99
>99
>99
>99
79
85
99
80
Table 2
Effects of different pHs on the reduction of 1 by P. expansum EBK-9
pH
Conversions (%)
ee (%)-(R)-configuration
3.0
4.0
5.0
5.5
6.0
6.5
7.0
7.5
8
No growth
Weak growth
10
25
42
54
47
27
—
—
60
79
97
>99
>99
—
>99
>99
>99
>99
Weak growth
—
Reaction conditions: substrate 0.5 mmol, temperature 30 °C, reaction time 24 h, pH
Reaction conditions: substrate 1 mmol, temperature 30 °C, reaction time 24 h,
7.0, agitation 150 rpm.
agitation 150 rpm

E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 20 (2009) 2759–2763
2761
Table 3
Effects of temperature on the bioreduction of 1 by P. expansum EBK-9
Temperature (°C)
Conversion (%)
ee (%)-(R)-configuration
22
24
26
28
30
42
54
62
52
46
>99
>99
>99
>99
>99
Reaction conditions: substrate 1 mmol, pH 6.5, time 24 h, agitation 150 rpm.
Table 4
Effects of agitation speed on the bioreduction of 1 by P. expansum EBK-9
Agitation (rpm)
Conversion (%)
ee (%)-(R)-configuration
100
150
200
250
300
57
62
94
100
100
>99
>99
>99
>99
79
Reaction conditions: substrate 1 mmol, temperature 26 °C, pH 6.5, time 24 h.
Figure 1. Time course of the production on
a
preparative scale of (R)-3,5-
bistrifluoromethylphenyl ethanol by P. expansum EBK-9. Substrate concentration
is 20 mmol/L.
tion of any co-substrate. However, the conversion of 1 was found
to be strongly dependent on the structure and concentration of
the co-substrate. The conversion rate of 1 was highest when the
less bulky ethanol was used as a co-substrate compared to the
bulky glycerol. The conversion did not significantly increase when
using 2-propanol, however, when using ethanol the conversion in-
creased to 100%. It should be noted that the concentration of the
co-substrate also had an affect on the conversion. When ethanol
or 2-propanol was employed the conversion increased from con-
centration 0.2 to 0.6%/v and then rapidly decreased as the concen-
tration was increased above 0.6%/v to 0.8 and 1.0%/v. The reason
for such a low conversion must be related to the inhibition of the
enzyme involved in the bioreduction. Using ethanol at a concentra-
tion of 0.6%/v gave 100% conversion of ketone 1 to alcohol 2; this
was the highest conversion obtained for the various experiments
and thus ethanol at a concentration of 0.6%/v was selected as the
co-substrate and used for preparative scale production.
The preparative scale production is one of the most vital issues
that needs to be addressed in asymmetric reductions by biocata-
lysts. In light of this, we undertook a study of the preparative scale
productions of 2 which were carried out on a 1 L scale using a fer-
menter under the optimized conditions (pH 6.5, agitation 250 rpm,
temperature 26 °C substrate 20 mmol/L and ethanol as co-sub-
strate 6 mL/L). Figure 1 illustrates the product 2 concentration
and percentage ee over a 72 h incubation period. The product con-
centrations as reported were obtained after purification via column
chromatography. Initial experiments were carried out using
20 mmol/L of 1 and incubated for up to 72 h. After 72 h, complete
conversion was achieved and 15.6 mmol/L of 2 was produced.
However, the ee of the desired alcohol was only 54% ee. The unsat-
isfactory ee result obtained is due to the degradation of the micro-
organism cells as a result of the long incubation period. It is well
known that degradation products, which are often different en-
zymes, cause low ee’s. It was observed that the enantioselectivity
of P. expansum EBK-9 was dependent on the incubation time.
Although 100% conversion was achieved after 72 h, the enantiose-
lectivity was of greater importance and therefore, the optimum
incubation time for this bioprocess was selected as 56 h and was
thus used for further studies.
It is well known that in biocatalytic reactions, substrate concen-
tration is a very important parameter, since it either influences the
full utilization of enzyme activity or may result in substrate inhibi-
tion. The bioreduction was thus further studied by employing the
above-determined operational conditions and 17 mmol/L 1 instead
of 20 mmol/L 1. The results for the reaction are shown in Scheme 1.
The reaction was stopped after 56 h, to give a final concentration of
13 mmol/L of 2 in very high purity (>99% ee). The product, (R)-3,5-
bistrifluoromethylphenyl ethanol 2 was obtained in 76% yield
(3.35 g/L) from 4.35 g/L 1; ½a D20
¼ þ16:4 (c 0.85, CHCl3); >99% ee
ꢀ
as determined by HPLC on a OD-H chiral column; retention times
were 15.6 min for (ꢁ) (S) and 17.6 min for (+) (R) (Scheme 1). P.
expansum EBK-9 has been shown to be a potent biocatalyst for
the bioconversion of 3,5-bis(trifluoromethyl)-acetophenone to
the corresponding (R)-enantiomer with >99% ee, however, there
has been no report regarding the production of 2 by this organism
in a laboratory-scale bioreactor. This is thus the first report for the
enantioselective synthesis of 2 by this microorganism.
As aforementioned, Gelo-Pujic et al.5 also studied the enantiose-
lective bioreduction of 1 but they instead carried out the reactions
with whole cells of L. kefir and obtained the (R)-enantiomer with
99% ee. On the other hand, Pollard et al.6 produced the (S)-enantio-
Table 5
Effects of co-substrates and their concentrations on the bioreduction of 1 to 2 by P. expansum EBK-9
Co-substrate concn (%/v)
Glycerol
Conversion (%)
2-Propanol
Conversion (%)
Ethanol
Conversion (%)
ee (%)-(R)
ee (%)-(R)
ee (%)-(R)
0.2
0.4
0.6
0.8
1.0
68
67
67
68
68
>99
>99
>99
>99
>99
72
78
82
74
68
>99
>99
>99
>99
>99
88
96
100
97
>99
>99
>99
>99
>99
74
Reaction conditions: substrate 1.5 mmol, temperature 26 °C, pH 6.5, agitation 250 rpm, time 24 h.

2762
E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 20 (2009) 2759–2763
expansum EBK-9 gave 2 in 76% yield, complete conversion and
>99% ee. From the fermentative studies 13 mmol/L (3.35 g) of alco-
hol 2 was obtained from 17 mmol/L (4.35 g) of ketone 1. This is the
first report on the mass production of 2 with P. expansum as a
biocatalyst on a laboratory-scale bioreactor, which can be directly
applied for the industrialization of 2. Furthermore, this study
demonstrates the first example of the effective synthesis of
(R)-3,5-bistrifluoromethylphenyl ethanol by the fungus P. expansum.
14
16
18
4. Experimental
4.1. Materials
3,5-Bis(trifluoromethyl)-acetophenone 1 was bought from Al-
drich Organics (St. Louis, USA). The racemic 3,5-bistrifluoromethyl-
phenyl ethanol was produced via reductions of 1 catalyzed with
sodium borohydride. The other components of the culture media
and the chemical reagents were obtained from Merck (Darmstadt,
Germany) and Sigma (St. Louis, USA) in the highest purity
available.
14
16
18
OH
CH3
O
4.2. Isolation of microorganisms, identification and inoculation
F3C
F3C
Penicillium expansum
CH3
Microorganisms used in the screening were isolated from vari-
ous soil samples collected from around Erzurum, Turkey. The isola-
tion process was performed by serial dilution of the samples
according to standard techniques.15 Taxonomic identification of fil-
amentous fungi was identified in-house by using mature cultures
on a standard potato dextrose agar (PDA) in order to ensure a good
development of taxonomically relevant features, and following the
identification keys provided by Von Arx16 and Domsch et al.17
These cultures were maintained on PDA slants, incubated at
30 °C and stored at 4 °C. The conidia from 7-days-old cultures were
used for inoculation. The conidial suspension was prepared in
10 mL of sterilized distilled water by gently scratching conidia
with a sterile wire loop and then shaking vigorously to break the
clumps of conidia.
Glucose, H2O, ethanol 6 ml,
pH 6.5, 26 oC, 250 rpm, 56 h,
0.4 vvm
CF3
CF3
1
2
13 mmol (3.35 g)
ee >99%, yield 76%
17 mmol (4.35 g)
Conversion 100%
Scale 1 L
Scheme 1. (A) Chiral analysis of the product by HPLC. (B) Total production of the 2
in a laboratory-scale bioreactor. Yield (%) = 100 ꢂ 2/1.
mer with 99% ee using the enzyme obtained from R. erythropolis.
However, the reaction was catalyzed using cofactors, which are
very expensive. Herein, 3,5-bis(trifluoromethyl)-acetophenone 1
was reduced to the corresponding (R)-enantiomer with >99% ee
using a submerged culture of the P. expansum EBK-9. Active bio-
catalysts have been obtained by screening a broad variety of fungi
isolates. This fungus was firstly used for the production of a chiral
alcohol. This biotransformation was successfully carried out on a
preparative level as well. The results indicate that P. expansum fun-
gus is an excellent source of production of 2, which suggests that
the fungus has the potential to undergo genetic engineering in or-
der to improve its production level. Furthermore, the isolation of a
new microorganism from the environment has sparked the inter-
est of researchers because it is estimated that only very few exist-
ing microbial species are actually known. A new microorganism
can lead to the discovery of new enzymes able to catalyze various
types of reactions.
4.3. Growth medium and reduction reactions
The fermentation medium contained: glucose (30 g/L), yeast ex-
tract (3 g/L), KH2PO4 (2 g/L) and peptone (4 g/L). The initial pHs of
the culture media were adjusted to the appropriate level with 1 M
HCl and 1 M NaOH and the media sterilized at 121 °C for 15 min.
The experiments were carried out in 250 mL Erlenmeyer flasks
containing 100 mL of culture medium. Then, 1 mL of conidial sus-
pension was added to each flask. The flasks were incubated on a re-
ciprocal shaker at 150 rpm and 30 °C for 48 h. After the growth of
the fungus, 1 (0.5 mmol) was directly added to each culture. Then,
incubation of the flasks continued on a reciprocal shaker at
150 rpm, 25 °C for 24 h.
3. Conclusions
4.4. Preparative scale production
In the first set of experiments, submerged cultures of 82 fungus
isolates were screened for the bioreduction of 1 to optically active
2. Using a two-step screening strategy, P. expansum EBK-9 was
identified as an effective biocatalyst with high enantioselectivity
and a remarkable conversion value. In the second set of experi-
ments, the reaction parameters for the conversion of ketone 1 with
maximum ee by P. expansum EBK-9 were optimized. The optimum
reaction parameters (pH 6.5, temperature 26 °C, time 56 h, agita-
tion 250 rpm, co-substrate 6 mL/L) led to high conversion of 1
and with excellent ee (>99%) of 2. In the last set of experiments,
the production of gram amounts of alcohol 2 on a preparative scale
was investigated. The preparative scale bioreduction of 1 by P.
Preparative scale studies were conducted in a 2-L fermenter (Bio-
stat-M 880 072/3, Germany) with a working volume of 1 L. Ten mil-
liliters of the spore suspension was inoculated into the fermenter
containing 1 L of sterile culture medium. To prevent foam formation,
sterilized silicone oil (0.001%, w/v) was added twice, at the begin-
ning and after 24 h fermentation. After a 48 h incubation period, 1
(20 or 17 mmol/L) was directly added to the fermentation culture.
Agitation, pH, aeration (0.4 vol vol min) and temperature were auto-
matically controlled during the fermentation. The reaction time
(24–72 h) was optimized for the reduction of substrates 1 to product
2 in a submerged system. At regular intervals of 4 h during fermen-

E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 20 (2009) 2759–2763
2763
tation, the conversion and the ee were determined and the yields
References
were calculated (Fig. 1 and Scheme 1).
1. Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N. Acc. Chem. Res. 2007, 40,
1412–1419.
2. Hansen, K. B.; Chilenski, J. R.; Desmond, R.; Devine, P. N.; Grabowski, E. J. J.;
Heid, R.; Kubryk, M.; Mathre, D. J.; Varsolona, R. Tetrahedron: Asymmetry 2003,
14, 3581–3587.
4.5. Product purification and analytical procedures
After reduction, the mycelium was separated by filtration, the fil-
trate saturated with sodium chloride and then extracted with ethyl
acetate. The mycelia were separately extracted with ethyl acetate.
The ethyl acetate extracts were combined; dried with MgSO4 and
evaporated. For HPLC analysis, a small fraction of the product was
separated by preparative silica-gel TLC. The ee of the product was
determined by high performance liquid chromatography (HPLC)
with an OD-H column (Daicel) using n-hexane/i-PrOH (98/2) as elu-
ent, a flow rate of 0.5 mL minꢁ1 and the detection was performed at
220 nm. The crude product was purified by silica gel column chro-
matography. 1H and 13C NMR spectra were recorded on a Varian
400 spectrometer in CDCl3 and used as the identification tool for
purified 2. In addition the purity of the (R)-isomer of 2 produced
via the fermenter was also checked via HPLC analysis. The absolute
configuration of the compound was determined by comparing the
sign of its specific rotation with that in the literature.18 The conver-
sion for the flask cultures was determined by 1H NMR analysis with
diphenylmethane as internal standard; error ca. 5% of the stated
values. All experiments were replicated twice and averaged values
are presented in this study.
3. Brands, K. M. J.; Payack, J. F.; Rosen, J. D.; Nelson, T. D.; Candelario, A.; Huffman, M.
A.; Zhao, M. M.; Li, J.; Craig, B.; Song, Z. J.; Tschaen, D. M.; Hansen, K.; Devine, P. N.;
Pye, P. J.; Rossen, K.; Dormer, P. G.; Reamer, R. A.; Welch, C. J.; Mathre, D. J.; Tsou,
N. N.; McNamara, J. M.; Reider, P. J. J. Am. Chem. Soc 2003, 125, 2129–2135.
4. Noyori, R.; Ohkuma, T. Angew. Chem., Int. Ed. 2001, 40, 40–73.
5. Gelo-Pujic, M.; Le Guyader, F.; Schlama, T. Tetrahedron: Asymmetry 2006, 17,
2000–2005.
6. Pollard, D.; Truppo, M.; Pollard, J.; Chen, C.; Moore, J. Tetrahedron: Asymmetry
2006, 17, 554–559.
7. Kira, I.; Suzuki, T.; Onishi, N.; Watanabe, K.; Nakanishi, E. J. Mol. Catal. B: Enzym.
2009, 56, 283–287.
8. Borges, K. B.; Borges, W. D.; Duran-Patron, R.; Pupo, M. T.; Bonato, P. S.; Collado,
I. G. Tetrahedron: Asymmetry 2009, 20, 385–397.
9. Dahl, A. C.; Fjeldberg, M.; Madsen, J. O. Tetrahedron: Asymmetry 1999, 10, 551–559.
10. Yang, H.; Jonsson, A.; Wehtje, E. BBA–General Subjects 1997, 1336, 51–58.
11. (a) Kurbanoglu, E. B.; Zilbeyaz, K.; Kurbanoglu, N. I.; Taskin, M. Tetrahedron:
Asymmetry 2007, 18, 1529–1532; (b) Zilbeyaz, K.; Kurbanoglu, E. B. Bioresour.
Technol. 2008, 99, 1549–1552; (c) Kurbanoglu, E. B.; Taskin, M.; Zilbeyaz, K.;
Kurbanoglu, N. I. J. Chem. Technol. Biotechnol. 2009, 84, 1474–1479; (d)
Kurbanoglu, E. B.; Taskin, M.; Zilbeyaz, K.; Hasenekoglu, I. Chin. J. Catal. 2009,
30, 370–374; (e) Kurbanoglu, E. B.; Zilbeyaz, K.; Kurbanoglu, N. I.; Taskin, M.;
Kilic, H. Turk. J. Chem. 2008, 32, 685–692.
12. Ou, Z.; Wu, J.; Yang, L.; Cen, P. Korean J. Chem. Eng. 2008, 25, 124–128.
13. He, J. Y.; Sun, Z. H.; Ruan, W. Q.; Xu, Y. Process Biochem. 2006, 41, 244–249.
14. Singh, R. S.; Sooch, B. S.; Puri, M. Bioresour. Technol. 2007, 98, 2518–2525.
15. 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.
Acknowledgments
16. Von Arx, J. A.. Key to the Orders of Fungi, the Genera of Fungi Sporulating in Pure
Culture; Cramer: Hirschberg, Germany, 1981.
17. Domsch, K. H.; Gams, W.; Anderson, T. H. Key to the Genera, Compendium of Soil
Fungi; IHW, Eching: Germany, 1993.
18. Singh, R. P.; Twamley, B.; Fabry-Asztalos, L.; Matteson, D. S.; Shreeve, J. M. J.
Org. Chem. 2000, 65, 8123–8125.
This study was financially supported by the Scientific and Tech-
nological Research Council of Turkey (TUBITAK), Project No. TBAG-
107T670. The authors thank Professor Ismet Hasenekoglu for his
help with the identification of microorganism.