Production of (R)-1-phenylethanols through bioreduction of acetophenones by a new fungus isolate Trichothecium roseum

Article abstract of DOI:10.1002/chir.20775

A total of 120 fungal strains were isolated from soil samples and evaluated in the bioreduction of substituted acetophenones to the corresponding (R)-alcohols. 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.

Full text of DOI:10.1002/chir.20775

CHIRALITY 22:543–547 (2010)
Production of (R)-1-phenylethanols Through
Bioreduction of Acetophenones by a New Fungus Isolate
Trichothecium roseum
KANI ZILBEYAZ,1 MESUT TASKIN,2 ESABI B. KURBANOGLU,2 NAMUDAR I. KURBANOGLU,3 AND HAMDULLAH KILIC
1
*
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
ABSTRACT
A total of 120 fungal strains were isolated from soil samples and eval-
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
C
VV
preparation of (R)-1-phenylethanol is reported. Chirality 22:543–547, 2010.
2009
Wiley-Liss, Inc.
KEY WORDS: fungus; biocatalyst; enantioselective reduction; ketone; chiral alcohol
INTRODUCTION
with excellent ee under optimized conditions and allowed
gram scale preparation of (R)-1-phenylethanol (2a) from
acetophenone.
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.1–5 For example,
enantiopure (R)-1-phenylethanol (2a) is an important chi-
ral building block for pharmaceuticals, agrochemicals, and
natural products.6–10 For economic and environmental
reasons, biocatalysis has recently gained increasing impor-
tance for the preparation of enantiopure alcohols from
EXPERIMENTAL
Materials
Ram horns were obtained from a slaughterhouse in
Erzurum, Turkey. The other components of the culture
media and the chemical reagents were obtained from
prochiral ketones. For this purpose, ketoreductases have Merck and Sigma in the highest purity available. Produc-
been shown to be unique biocatalysts in the preparation tion of RHP was carried out using the method described
of enantiopure alcohols from prochiral ketones.11–19 by Kurbanoglu and Kurbanoglu.24
For example, Mandal et al. reported that whole cells of
Isolation of Microorganisms, Identification
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%.21 A 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.22 Recently, 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.23–29 In 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
C
and Inoculation
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.30 Filamentous 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
Additional Supporting Information may be found in the online version of
this article.
Contract grant sponsor: The Scientific and Technological Research Coun-
cil of Turkey (TUBITAK); Contract grant number: TBAG-107T670
*Correspondence to: Hamdullah Kilic, Faculty of Sciences, Department of
Chemistry, Ataturk University, Erzurum 25240, Turkey.
E-mail: hkilic@atauni.edu.tr
Received for publication 17 May 2009; Accepted 8 July 2009
DOI: 10.1002/chir.20775
Published online 9 September 2009 in Wiley InterScience
(www.interscience.wiley.com).
VV
2009 Wiley-Liss, Inc.

544
KANI ZILBEYAZ ET AL.
TABLE 1. Optimization of parameters for the bioreduction of acetophenone (1a) by Trichothecium roseuma
pH
Temperature
Incubation period
Agitation speed
Convn
(%)b
ee
Convn
(%)b
ee
Convn
ee
Convn
(%)b
ee
(%)c,d
8C
(%)c,d
h
(%)b
(%)c,d
rpm
(%)c,d
5.0
5.5
6.0
6.5
7.0e
30
50
60
50
0
65-(R)
80-(R)
95-(R)
87-(R)
26
28
30
32
34
68
75
80
74
30
90-(R)
95-(R)
95-(R)
87-(R)
60-(R)
24
48
72
96
80
92
100
100
90-(R)
95-(R)
77-(R)
77-(R)
100
150
200
250
300
20
80
100
100
100
70-(R)
95-(R)
99-(R)
60-(R)
40-(R)
aSubstrate 1 mM.
bConversion was determined by 1H 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.
eWeak growth.
at 258C, and stored at 48C. The conidia from 8-day-old cul- mycelia were also washed with ethyl acetate. Ethyl acetate
tures were used for inoculation. The conidial suspension extracts were combined and dried over Na2SO4. The con-
1
was prepared in 10 ml sterilized and distilled water by version was determined by H NMR analysis with diphe-
gently scratching conidia with a sterile wire loop and nylmethane as internal standard; error ca. 65% of
then it was shaken vigorously to break up the clumps of the stated values. After removal of the solvent, the crude
conidia.
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.33,34 The 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:35–40 76%
yield (1.85 g, 15.1 mmol); [a]D20 152.8 (c, 0.85, CHCl3);
>99% ee determined by HPLC on a OD chiral column;
retention times were 11.8 min for (1)-(R) and 13.0 min for
(2)-(S).
Medium, Culture Conditions and Screening for
Acetophenone Reduction
The fermentation medium per liter contained (g/l):
glucose 20, yeast extract 3, KH2PO4 1.5, 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.
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
Production of ( R)-2a by T. roseum EBK-18 in a
Laboratory-Scale Bioreactor
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.
Analytical Procedures
At the end of the incubation period, mycelium was sepa- chosen to monitor the progress of the bioreduction. The
rated by filtration, and the filtrate was saturated with so- complete conversion of 1a was observed after 72 and 96 h,
dium chloride and then extracted with ethyl acetate. The but the ee (77%) of 2a decreased. In contrast to these
Chirality DOI 10.1002/chir

PRODUCTION OF (R)-1-PHENYLETHANOLS
545
TABLE 2. Enantioselectivities for the microbial reduction of substituted acetophenones by Trichothecium roseum,
Aspergillus niger, and Alternaria alternate
ee (%)-Config.b,c
Trichothecium
roseuma
Aspergillus
nigerd
Alternaria
alternatae
Entry
Substrate
Product
H (2a)
Convn. (%)a
Yield (%)a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
H (1a)
o-F (1b)
100
. . .f
. . .f
. . .f
90
82
77
100
97
86
75
–
–
> 99-(R)
–
87-(R)
. . .g
99-(S)
96-(S)
. . .g
99-(S)
99-(S)
99-(S)
99-(S)
99-(S)
99-(S)
99-(S)
. . .g
o-F (2b)
o-Cl (1c)
o-Br (1d)
m-F (1e)
m-Cl (1f)
m-Br (1g)
m-NO2 (1h)
p-F (1i)
p-Cl (1k)
p-Br (1l)
p-NO2 (1m)
p-Me (1n)
p-MeO (1o)
p-Ph (1p)
o-Cl (2c)
o-Br (2d)
m-F (2e)
m-Cl (2f)
m-Br (2g)
m-NO2 (2h)
p-F (2i)
p-Cl (2k)
p-Br (2l)
p-NO2 (2m)
p-Me (2n)
p-MeO (2o)
p-Ph (2p)
–
–
–
66
58
56
79
75
63
55
78
74
–
> 99-(R)
> 99-(R)
> 99-(R)
> 99-(R)
> 99-(R)
> 99-(R)
> 99-(R)
> 99-(R)
> 99-(R)
–
57-(S)
66-(S)
. . .g
. . .g
. . .g
99-(R)
99-(R)
. . .g
95-(R)
90-(R)
65-(R)
29-(R)
49-(R)
20-(S)
. . .g
49-(R)
90-(S)
81
100
96
. . .f
. . .f
–
–
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.
results, the conversion increased up to 92% with 95% ee for position such as nitroacetophenones 1h and 1m furnished
48 h. Although the ee of 2a remained steady after 48 h, quantitative conversions. The reduction of the meta- or
the conversion rate was obviously different. The best con- para-substituted acetophenone was more favorable as com-
dition (48 h) obtained was used for further optimization of pared to ortho-substituted acetophenone. There was no
conversion and ee. The highest values for both ee (>99%) reaction for any ortho-substituted acetophenones. Presum-
and conversion (100%) were obtained at 200 rpm and ably, steric repulsion between the catalytically active site
thus this agitation speed was determined as optimum for and the ortho-substituents hinders the transfer of the
fermentation.
hydrogen atom.
Under the optimum conditions (pH 6.0, temperature
After successful determination of the reaction parame-
308C, time 48 h, and agitation 200 rpm) asymmetric biore- ters, we decided to conduct the transformation of 1a
ductions of the other derivatives of 1a by T. roseum EBK- to (R)-2a on a gram scale to demonstrate industrial
18 were investigated in the shake flask scale. The results feasibility.
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
Scheme 1. A gram scale production of (R)-1-phenylethanol (2a)
Chirality DOI 10.1002/chir

546
KANI ZILBEYAZ ET AL.
hol dehydrogenase (LSADH). Tetrahedron: Asymmetry 2005;16:
Preparative scale production of 2a was performed on a
2539–2549.
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
gel column.
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-
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