Preparation of Enantiomerically Pure (S)-(?)-1-(1′-naphthyl)-ethanol by the Fungus Alternaria alternata

Article abstract of DOI:10.1002/chir.22629

(S)-(?)-1-(1′-napthyl)-ethanol (S-NE) is an important intermediate for the preparation of mevinic acid analogs, which is used for the treatment of hyperlipidemia. The objectives of the study were to isolate a microorganism that could effectively reduce 1-acetonaphthone (1-ACN) to S-NE, to determine the influence that the physicochemical parameters would have on the reduction by the isolated microorganism, and to attempt large-scale studies with the microorganism. Over the years fungi have been considered a promising biocatalyst and it has been presumed that many fungal species have not been isolated and therefore the current study focused on possible isolation of these microorganisms. A total of 72 fungal isolates were screened for their ability to reduce 1-ACN to its corresponding alcohol. The isolate, EBK-62, identified as Alternaria alternata, was found to be the most successful at reducing the ketone to the corresponding alcohol in the submerged culture. The reaction conditions were systematically optimized for the reducing agent A. alternata EBK-62, which showed high stereospecificity and good conversion for the reduction. The preparative scale study was carried out in a 2?L bioreactor and a total of 4.9?g of S-NE in optically pure form (>99% enantiomeric excess) was produced in 48?h. Chirality 28:669–673, 2016.

Full text of DOI:10.1002/chir.22629

CHIRALITY 28:669–673 (2016)
Preparation of Enantiomerically Pure (S)-(À)-1-(1′-naphthyl)-ethanol
by the Fungus Alternaria alternata
1
2
KANI ZILBEYAZ, ESABI BASARAN KURBANOGLU AND HAMDULLAH KILIC3
*
1Department of Chemistry, Agri Ibrahim Cecen University, Agri, Turkey
2Department of Biology, Ataturk University, Erzurum, Turkey
3Department of Chemistry, Ataturk University, Erzurum, Turkey
ABSTRACT
(S)-(À)-1-(1′-napthyl)-ethanol (S-NE) is an important intermediate for the
preparation of mevinic acid analogs, which is used for the treatment of hyperlipidemia. The
objectives of the study were to isolate a microorganism that could effectively reduce
1-acetonaphthone (1-ACN) to S-NE, to determine the influence that the physicochemical
parameters would have on the reduction by the isolated microorganism, and to attempt large-
scale studies with the microorganism. Over the years fungi have been considered a promising
biocatalyst and it has been presumed that many fungal species have not been isolated and
therefore the current study focused on possible isolation of these microorganisms. A total of
72 fungal isolates were screened for their ability to reduce 1-ACN to its corresponding alcohol.
The isolate, EBK-62, identified as Alternaria alternata, was found to be the most successful at
reducing the ketone to the corresponding alcohol in the submerged culture. The reaction
conditions were systematically optimized for the reducing agent A. alternata EBK-62, which
showed high stereospecificity and good conversion for the reduction. The preparative scale
study was carried out in a 2 L bioreactor and a total of 4.9 g of S-NE in optically pure form
(>99% enantiomeric excess) was produced in 48 h. Chirality 28:669–673, 2016. © 2016 Wiley
Periodicals, Inc.
KEY WORDS: asymmetric reduction; biotransformations; enantioselective; ketone; submerged
culture
Chirality has become a grave concern in various industries
including aroma/fragrance, agrochemical, fine chemical, and
pharmaceutical. The growing interest within the pharmaceu-
tical industry is fueled by regulatory agencies, who are
demanding the use of single enantiomers in the production
of drug formulations due to the vast differences in many
enantiomer pairs, which may have different pharmacological
activities and different pharmacokinetic and pharmacody-
namic effects.1,2 Over the past 20 years there has been an
accelerated and continuous growth of single enantiomer drug
sales. In light of this, it is not surprising that new
enantioselective synthetic methodologies have been growing
at a rapid rate.3 One methodology that has been very success-
ful and has become a common one for preparation of chiral
compounds is biotransformation. An extensive collection of
biocatalysts have been reported to assist biotransforma-
tions.4–6 Biocatalysts have many advantages compared to
chemical catalysts. Chemical catalysts produce toxic waste
and a large range of by-products, whereas biocatalysts are
biodegradable, and provide a clean and environment-friendly
way to carry out chemical reactions under mild reaction
conditions and great selectivity for the substrate.7,8 Biocataly-
sis can be achieved by employing isolated enzymes or whole
cells, with both having advantages and disadvantages.9
Isolated enzymes catalyze specific reactions, eliminating
by-product formation or product breakdown, and product
purification is often much easier.9 However, enzymes are
very expensive and very unstable.9 In addition, enzymes
catalyze a single reaction, whereas a plethora of industrial
products are afforded after a series of biochemical reactions
using whole cells. Whole cells are sustainable, are much
more stable and efficient, and more readily and
cost-effectively prepared.10 Hence, whole cells are preferred
as biocatalysts in biotransformation processes. Fungi have
been one of the most studied whole-cell systems for biotrans-
formations.11 However, it is believed that very few fungal
species are known and have been isolated from the ecosys-
tem and therefore can be considered as a promising source
of new biocatalysts.11
Optically active alcohols are among the vast amount of
chiral compounds that have become important building
blocks in the pharmaceutical industry. A large number of
optically active alcohols have been produced by means of
biocatalytic processes.12–16 Optically active phenylethanol
and its derivatives have become key building blocks for the
preparation of large complex molecules. This is attributable
to the alcohol functional group, which can be easily trans-
formed to other desired functional groups. (S)-(À)-1-(1′-
napthyl) ethanol (S-NE) is an intermediate for the synthesis
of mevinic acid analogs used for the treatment of hyperlipid-
emia, which acts as potent inhibitors of 3-hydroxy-3-
methylglutaryl co-enzyme A reductase (HMGR).17 Several
methods for the preparation of optically active S-NE have
been reported in the literature, including the asymmetric
reduction of 1-acetonaphthone (1-ACN) catalyzed by
Rhodotorula glutinis, Baker’s yeast, Geotrichum candidum,
Candida viswanathii, Rhizopus arrhizus, Merulius tremellosus
*Correspondence to: Kani Zilbeyaz, Department of Chemistry, Faculty of
Science, Agri Ibrahim Cecen University, 4100, Agri, Turkey. E-mail:
kbeyaz25@yahoo.com
Received for publication 7 June 2016; Accepted 14 July 2016
DOI: 10.1002/chir.22629
Published online 1 September 2016 in Wiley Online Library
(wileyonlinelibrary.com).
© 2016 Wiley Periodicals, Inc.

670
ZILBEYAZ ET AL.
ono991, and Daucus carota.18–24 However, in light of the
belief that not many fungal species are known, we set off on
a search to find new fungal species for the biotransformation
of 1-ACN. There are several advantages of mold utilization in
a bioprocess. The principal advantage is the easy separation
of the mold from the reaction mixture.
In the current communication we report the asymmetric
reduction of 1-ACN to the corresponding chiral alcohol
S-NE in the submerged culture system by the fungus isolate
Alternaria alternata. The effects of the reaction conditions
such as pH, temperature, and agitation speed on the yield,
conversion, and enantiomeric excess (ee) were also investi-
gated in detail and reported.
silica-gel thin-layer chromatography (TLC) for analysis by high-
performance liquid chromatography (HPLC).
Optimization of the various parameters involved systematically
changing the conditions for the reaction as outlined in the Results and
Discussion section.
The scale-up for the production of S-NE was carried out in a 2 L
bioreactor (Biostat-M 880072/3, Germany) with a working volume of
1 L under the optimized conditions obtained through process optimiza-
tion. The medium as described before was sterilized at 121 °C for
15 min and inoculated with 10 mL of the spore suspension selected in
the screening process. In order to prevent foam formation, sterilized
silicone oil (0.001%, w/v) was added to the reaction at two different times,
once at onset and once after 24 h of fermentation. After a 48-h incubation
period, 1-ACN (40 mmol/L for the initial reaction and 35 mmol/L for
subsequent reactions) was added directly to the fermentation culture.
Agitation, pH, and temperature were set to the optimum values and were
automatically controlled during the fermentation. The aeration was set to
0.4 vol/vol/min. The reaction was carried out for 60 h to determine the
optimum time for the reduction of substrate 1-ACN to product S-NE in a
submerged system. Samples were withdrawn periodically at 4-h intervals,
purified, and analyzed by HPLC. The conversion of the substrate and the
ee of the product were determined and the yields calculated. After 60 h
the product was extracted as described before and purified by silica gel
column chromatography. S-NE was identified by its 1H and 13C NMR
spectra. In addition, the purity of S-NE produced via the fermenter was
checked by HPLC. The absolute configuration and specific rotation of
the product was determined and compared with that of an authentic
sample as well as the literature value, which were all in agreement.
MATERIALS AND METHODS
Chemicals
The components of the culture media and the chemical reagents were
bought from Sigma-Aldrich (St. Louis, MO) in the highest purity
available. 1-ACN and S-NE was bought from Fluka (Steinheim,
Germany). Racemic 1-(1′-naphthyl) ethanol was prepared by NaBH4
1
reduction of 1-ACN and its H and 13C NMR spectra were in agreement
with those reported in the literature.21,25
Analysis
All analysis was performed on a Thermo Spectra Analysis HPLC
System equipped with a UV detector using a chiral OD-H column
(4.6 mm, 250 mm, 5 μm, Daicel, France). 1-ACN, S-NE, and racemic
1-(1′-naphthyl) ethanol were analyzed using n-hexane-i-PrOH (90:10) as
the eluent, a flow rate of 0.6 ml/min, and the detection performed at a
wavelength of 220 nm. The retention times of S-(À)- and R-(+)-1-(1′-
naphthyl) ethanol were determined as 15.7 min and 23.3 min, respec-
tively. Their ee was determined directly from their respective areas under
the curve. 1H and 13C NMR spectra were recorded on a Varian (Palo Alto,
CA) 400 MHz spectrometer in CDCl3. A polarimetric Chiralyser detector
was used to assess the sign of configuration of the enantiomer formed.
RESULTS AND DISCUSSION
Analytical Scale Bioreduction and Screening of
Microorganism
The screening of the fungal microorganisms for the
production of S-NE from 1-ACN found that isolate EBK-62,
isolated from soil, was the most productive isolate, with
considerably high activity and enantioselectivity. The isolate
was identified as Alternaria alternata. Scheme 1 outlines the
reaction conditions for selection of the best active biocatalyst.
A good biocatalyst should be highly active and stable for
continuous use. In our previous study, we investigated the
biocatalytic activity and stability of A. alternata EBK-4 and
we showed that A. alternata possessed these required
qualities. (S)-1-phenylethanol was obtained up to gram scale
Isolation, Identification, and Inoculation of Microorganism
The microorganisms used in the screening process were isolated from
various soil, plant, and fruit samples that were collected from Erzurum,
Turkey. Standard techniques were used for the isolation process, which
involved serial dilution of the samples.26 Cultures were prepared and
identified as previously reported.27–29
with this microorganism from acetophenone using
a
Culture Media and Conditions
bioreactor.29 In light of this, it was not surprising that of the
large number of isolates screened A. alternata EBK-62 gave
the best ee and conversion (>99 ee and 50% conversion; data
for the screening experiments are not shown in this article).
All the other isolates screened were able to reduce the
substrate but gave low ee’s (8–86%). Screening a diverse
collection of microorganisms is a great way to obtain the
desired ee and conversion for a substrate.11,30,31 However,
the literature has described the qualities of a good biocatalyst
and this could aid researchers in the production of superior
intermediates and products.32 Cheetham reported that a good
active biocatalyst can be developed from an isolated
The culture medium contained (g/L): glucose 20, yeast extract 3, and
peptone 4. The initial pH of the culture medium was adjusted to 6.0 with
1 N HCl and 1 N NaOH and autoclaved at 121 °C for 15 min.
Microbial Reduction of 1-Acetonaphthone
Screening of the microorganisms and optimization of the reaction
parameters were done on an analytical scale, which was followed by the
investigation of the reduction on a preparative scale. All experiments
were done in duplicate and averaged values are presented in this study.
All isolated microorganisms were screened for their asymmetric reduc-
tion capability of 1-ACN to S-NE. The reactions for the screening process
and the optimization of the reaction parameters were carried out in 250 ml
Erlenmeyer flasks containing 100 mL of culture medium. One mL of
spore suspension was added to each flask. For the screening process
the flasks were incubated on a reciprocal shaker at standard conditions
of 100 rpm and 30 °C for 48 h. After sufficient growth of the fungi in
the flasks, ketone 1-ACN was directly added to each culture. The flasks
were then incubated for 24 h on a reciprocal shaker at 100 rpm and
25 °C. The cells were then separated by filtration, the supernatant
saturated with sodium chloride, and then extracted with ethyl acetate.
The organic phase was dried over Na2SO4 and the solvent removed under
reduced pressure. A small amount of sample was purified by preparative
Scheme 1. Screening of fungal isolates for the asymmetric reduction of
1-ACN to S- or R-NE in flask cultures containing 100 ml medium.
Chirality DOI 10.1002/chir

PREPARATION OF ENANTIOMERICALLY PURE (S)-(À)-1-(1′-NAPHTHYL)-ETHANOL BY THE FUNGUS ALTERNARIA ALTERNATA
671
microorganism with the needed enzyme activities.32 This
may be achieved by using methods such as fermentation
optimization, gene cloning, or mutagenesis.32 The current
biocatalysts known are still limited and therefore it is
necessary and of vital importance to discover new biocatalysts
with novel and improved activities. Not many publications on
the biocatalytic activity of A. alternata have been reported and
we believe that it is a good biocatalyst, since it has good
reaction activity and high selectivity.
were obtained at lower speeds and we thus compromised
conversion for maximum ee. Optimum agitation speed for
the reduction was set at 150 rpm.
The optimum conditions for the asymmetric bioreduction
of 1-ACN to S-NE were pH .5, temperature 30 °C, and
agitation speed 150 rpm.
Preparative Bioreduction of 1-ACN TO S-NE
The final objective of the study was to attempt large-scale
production of S-NE using the determined optimum reaction
conditions. The bioreduction of 1-ACN for the production of
S-NE by A. alternata EBK-62 in the bioreactor is outlined in
Figure 1. The conversion of the substrate to product
increased steadily with time. After 60 h a 100% conversion
was observed and gave an 85% product yield. The ee
remained consistent (ee >99%) up to 48 h; however, thereaf-
ter it decreased drastically. By the end of the 60 h reaction
time an ee of 58% was observed. Enantiomeric excess cannot
be compromised for conversion, and thus 48 h was selected
as the optimal reaction time for the bioreduction. The
concentration of S-NE after 48 h was 27.3 mmol/L, which
corresponds to a 68% product yield. It has been shown that
a change in substrate concentration has an effect on the
conversion and outcome of the product yield, higher
concentrations decrease the conversion, which has been
Optimization of Physicochemical Parameters
To address the second objective of the study the reduction
of 1-ACN was carried out with A. alternata EBK-62 at various
pH values, temperatures, and agitation speeds to determine
the optimum reaction conditions for the reduction. The first
parameter investigated was the pH. The reactions were
carried out for 24 h using 2 mmol/100 ml 1-ACN in shake
flasks. pH values ranging from 4.5 to 7.5 were chosen.
pH .0, 6.5, and 7.0 were found to be optimum for
enantioselectivity (ee >99%) and pH .5 was optimum for
conversion (38%). The results are shown in Table 1. pH .5
was thus used for all further optimization studies.
The second parameter studied was the reaction tempera-
ture. Various temperatures ranging from 24–36 °C were
investigated. The best results for cellular growth, conversion,
and enantioselectivity were observed at 30 °C. From Table 1
it can be seen that the conversion of the substrate increased
steadily from 24–30 °C, with a maximum conversion of 48%
at 30 °C and then decreased once again from 32–36 °C.
Lower and higher temperatures showed a drastic reduction
in conversion of the substrate. The ee of the product formed
with cells grown at higher temperatures of 32, 34, and 36 °C
was 96, 72, and 40%, respectively, while at lower temperatures
24, 26, 28, and 30 °C the ee of the product was >99%. Taking
into account the temperature that gave the best conversion
and best ee, subsequent reactions were carried out at 30 °C.
The effect of agitation speed on the reduction was the last
parameter investigated. It was observed that agitation exerted
a strong influence on both the ee and conversion. A progres-
sive increase in conversion was observed with an increase of
the agitation rate (from 100–250 rpm) with a maximum
conversion of 86% obtained at a speed of 250 rpm. However,
at this speed a low ee of 60% was obtained. This decrease
can be attributed to the effect of shear stress on the A.
alternata cells at high agitation speeds, which alters the cell
internal structure and in turn lowers their activity. Good ee’s
Fig. 1. Time course of the production in gram scale of (S)-(À)-1-
(1′-napthyl)-ethanol by Alternaria alternata EBK-62 (ee (%) and mmol) by means
of a bioreactor over a period of 60 h (substrate concentration: 40 mmol/L).
TABLE 1. Optimization of reaction conditions for the asymmetric reduction of 1-ACN by Alternaria alternataa
pH
Temperature
Conv. (%)b
Agitation speed
Conv. (%)b
d
d
d
pH
Conv. (%)b
ee (%)c,
oC
ee (%)c,
rpm
ee (%)c,
4.5
5.0
5.5
6.0
6.5
7.0
7.5
12
18
22
25
38
36
25
55
70
86
>99
>99
>99
96
24
26
28
30
32
34
36
20
32
36
48
35
24
18
>99
>99
>99
>99
96
72
40
100
150
200
250
48
68
77
86
>99
>99
92
60
aSubstrate: 2 mmol/100 mL, time: 24 h.
bConversion was determined by 1H NMR analysis with diphenylmethane as an internal standard; error ~ 5% of the stated values.
cDetermined by HPLC using a Chiralcel OD-H column.
dAbsolute configurations were assigned by comparison of the sign of optical rotations relative to the values of the authentic samples and those in the literature.
Chirality DOI 10.1002/chir

672
ZILBEYAZ ET AL.
ascribed to cell toxicity and substrate/product inhibition.19 In
light of this, the substrate concentration was slightly varied
and investigated. Higher concentrations (45 and
50 mmol/L) decreased the product concentration. The best
results were obtained with a substrate concentration of
35 mmol/L (Scheme 2). Complete conversion and an ee of
>99% was observed. The product concentration after the
48 h reduction was 28.6 mmol/L, indicating a product yield
increase from 68% to 82%. These results show that A. alternate
can be used on a large scale in the synthesis of important
building blocks.
Biocatalytic multigram studies on the total production of
S-NE have been explored by several groups and reported in
many articles. Roy et al. reported the multigram synthesis of
S-NE (0.93 g, ee >99%, 84% conversion) from 2 g 1-ACN with
resting cells of Geotrichum candidum in phosphate buffer.19
Similarly, Kamble et al. accomplished the synthesis of S-NE
from 2 g 1-ACN with 97% conversion and >99% ee with resting
cells of Candida viswanathii.20 We also previously reported
the reduction of 3.4 g 1-ACN to S-NE (2.7 g, 78% product
yield, ee >99%) by Rhodotorula glutinis. Many more have
been reported but most of these processes either gave
relatively lower conversion, product yield, or low substrate
tolerance.
of 28.6 mmol (4.9 g) S-NE from 35 mmol (5.9 g) 1-ACN with
an 81% product yield. This is the first report on the asymmet-
ric reduction of ketone 1-ACN with A. alternata as a biocata-
lyst using a submerged culture system. In addition, optically
pure S-NE was obtained with excellent selectivity.
CONCLUSION
In summary, this study presented the fungus A. alternata
as a biocatalyst for the production of optically active (S)-(À)-1-
(1′-napthyl)-ethanol. The fungus shows promise as a highly
attractive candidate for the biocatalytic preparation of other
chiral aryl alcohols. It was demonstrated that the asymmetric
reduction of 1-ACN can be performed at high substrate
concentration by submerged culture of A. alternata EBK-62
fungus. Vital parameters such as pH, temperature, incubation
time, and agitation speed were optimized for maximum prod-
uct yield with excellent enantioselectivity. Enantiomerically
pure (S)-(À)-1-(1′-napthyl)-ethanol was produced in gram
scale with excellent ee (up to >99%) and good yield (up to
82%). A biocatalyst with high selectivity and stereospecificity
is vital and indispensable for the development of a biocatalytic
process, a green process that offers higher yields and purer
products.
The present study offers advantages of higher substrate
tolerance (5.9 g/L), use of the fungus isolate without using
an expensive cofactor such as NADPH, complete conversion,
and >99% ee. The various reaction parameters and conditions
for the bioreduction were optimized and the reaction was
successfully carried out on a preparative scale. As illustrated
in Scheme 2, the fermentative studies allowed the production
ACKNOWLEDGMENTS
This study was financially supported by Ataturk University.
The authors thank Professor Ismet Hasenekoglu for help in
the identification of the fungus.
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Chirality DOI 10.1002/chir