Improving the enantioselective bioreduction of aromatic ketones mediated by Aspergillus terreus and Rhizopus oryzae: the role of glycerol as a co-solvent

Article abstract of DOI:10.1016/j.tetasy.2009.05.033

The improvement of the enzymatic performance of Aspergillus terreus and Rhizopus oryzae in enantioselective bioreductions by using glycerol as a co-solvent has been studied. In the most of the bioreductions, glycerol has demonstrated its potential for improved conversions (up to >99%) and enantioselectivities (up to >99%) when compared to reactions in aqueous or other aqueous-organic media (THF, diethyl ether, toluene, DMSO and acetonitrile). Moreover, high isolated yields of the desired chiral alcohols have been obtained on a preparative scale showing the great potential of this green solvent in biocatalysis.

Full text of DOI:10.1016/j.tetasy.2009.05.033

Tetrahedron: Asymmetry 20 (2009) 1521–1525
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Improving the enantioselective bioreduction of aromatic ketones mediated
by Aspergillus terreus and Rhizopus oryzae: the role of glycerol as a co-solvent
*
Leandro H. Andrade , Leandro Piovan, Mônica D. Pasquini
University of São Paulo, Institute of Chemistry, Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo—SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The improvement of the enzymatic performance of Aspergillus terreus and Rhizopus oryzae in enantiose-
lective bioreductions by using glycerol as a co-solvent has been studied. In the most of the bioreductions,
glycerol has demonstrated its potential for improved conversions (up to >99%) and enantioselectivities
Received 6 May 2009
Accepted 29 May 2009
Available online 24 June 2009
(up to >99%) when compared to reactions in aqueous or other aqueous–organic media (THF, diethyl ether,
toluene, DMSO and acetonitrile). Moreover, high isolated yields of the desired chiral alcohols have been
obtained on a preparative scale showing the great potential of this green solvent in biocatalysis.
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Biocatalytic asymmetric reactions have been widely used in or-
CCT 4964. Herein, we report an improvement in the enantioselec-
tive bioreduction of haloacetophenones using glycerol as co-
solvent.
1
–3
ganic synthesis.
As the enzymatic reactions are usually carried
out in water, the use of organic compounds as solvent or co-solvent
has proven to be useful for expanding the applicability of biocata-
lytic processes to poorly water-soluble organic compounds. There-
fore, the presence of a hydrophilic solvent can improve the
solubility of organic compounds and facilitates the enzyme–sub-
2
2
. Results and discussion
.1. Screening of solvents for bioreduction mediated
by A. terreus and R. oryzae
4
strate interaction. Several solvent systems have been applied in
Despite all the relevant properties of glycerol discussed above,
we also decided to evaluate the influence of some hydrophobic sol-
vents (toluene, diethyl ether and tetrahydrofuran) and hydrophilic
solvents (dimethyl sulfoxide and acetonitrile) in the bioreduction
of haloacetophenones by cells of A. terreus and R. oryzae. The
hydrophobic solvents gave biphasic systems that were used for
comparison with the bioreductions in glycerol. For this study, we
5
–11
reactions mediated by purified enzymes or microorganisms.
In some cases, organic co-solvents have improved the yield and
enantioselectivity of enzymatic transformations.12 Nowadays, the
choice of an appropriate solvent for enantioselective reactions
has to take into account environment concerns, and green solvents,
such as supercritical CO
2
and ionic liquids have been studied with
1
3–15
this purpose.
Another green solvent that has received atten-
0
selected 2 -chloroacetophenone 1 as a model substrate and the
tion is glycerol, especially because it is a by-product of biodiesel
production, which can be obtained from renewable sources. Glyc-
erol is an environmentally friendly, non-toxic, biodegradable sol-
reactions were performed in different concentrations of organic
solvent (Table 1).
As can be seen from the results presented in Table 1, cells of A. ter-
reus and R. oryzae promoted the reduction of the ketone 1 into the
vent. It has already been applied in biocatalytic reactions, and
demonstrated its potential for biocatalytic reactions.1
6,17
More-
(
S)-alcohol with good results. For example, the bioreduction cata-
lyzed by cells of A. terreus in phosphate buffer solution (PBS) led to
S)-1a alcohol with low concentration (24%) and enantiomeric ex-
over, the water–glycerol combination leads to a homogenous sys-
tem and avoids mass transfer limitations, which is common in
biphasic systems. Furthermore, glycerol solubilizes several organic
compounds, but is immiscible with hydrophobic solvents (e.g.,
ethyl acetate, diethyl ether). This feature allows easy product
recovery by simple extraction with a hydrophobic solvent. Due to
these properties, we decided to study the effect of the glycerol as
a co-solvent in the bioreduction of haloacetophenones mediated
by whole cells of Aspergillus terreus SSP 1498 and Rhyzopus oryzae
(
cess (65%; Table 1, entry 1). However, by using PBS–glycerol (9:1),
the values of concentration and ee of the (S)-1a alcohol increased
to 49% and 92%, respectively (Table 1, entry 2). Similarly, PBS–glyc-
erol (4:1) gave better results (44% concentration, 99% ee) than the
reaction using PBS as solvent (Table 1, entry 3). When PBS–DMSO
(
9:1) was used with cells of A. terreus, moderate concentration and
excellent enantioselectivity were observed for (S)-1a alcohol (51%
and >99%, respectively; Table 1, entry 4). However, by using PBS–
DMSO(4:1) a significant decrease in theconcentration(10%) was ob-
served with maintenance of the enantioselectivity (Table 1, entry 5).
*
Corresponding author. Tel.: +55 1130912287; fax: +55 1138155579.
E-mail address: leandroh@iq.usp.br (L.H. Andrade).
0
957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.05.033

1
522
L. H. Andrade et al. / Tetrahedron: Asymmetry 20 (2009) 1521–1525
Table 1
0
a
Influence of co-solvents in the bioreduction of the 2 -chloroacetophenone
O
OH
micro-organism
solvent
*
Cl
Cl
1
1a
PBS: Phosphate buffer solution [Na
2
HPO
4
/KH
2
PO
4
buffer (pH 7)].
a
0
Reaction conditions: 3.0 g of fungal cells, 20
lL of 2 -chloroacetophenone, 50 mL of solvent, 48 h, 32 °C, 160 rpm.
b
c
Concentration of product was determined by chiral GC analysis.
Enantiomeric excess was determined by chiral GC analysis; (ꢀ) not determined due to low conversion.
The bioreduction of ketone 1 with cells of R. oryzae in PBS
explained the stabilizing-action of glycerol in terms of energy by a
showed good concentration and ee of the product (88% and 94%,
respectively; Table 1, entry 1). However, the use of PBS–glycerol
model called preferential exclusion that was formulated based on
the preferential hydration of proteins. According to this model, in a
protein–water–glycerol system, the glycerol is preferentially ex-
cluded from the immediate proximity of the protein (unfavourable
interaction), and the protein will tend to be preferentially hy-
(
4:1) increased the ee to 97% and the concentration was slightly
decreased to 50% (Table 1, entry 3). By using PBS–DMSO (9:1 and
:1) excellent enantioselectivity (>99%) was observed but the
product concentration was low (27% and 19%, respectively; Table
, entries 4 and 5). No significant bioreduction was observed
4
1
9
drated. Thus, it will stabilize the protein native structure and pre-
vent its denaturation; consequently the enzymatic activity can be
1
2
0
employing A. terreus or R. oryzae in acetonitrile, toluene, diethyl
ether and THF (Table 1, entries 6–13). The reason for such a low
conversion can be related to the strong inhibition of the alcohol
dehydrogenase involved in the bioreduction. Moreover, the bipha-
sic system obtained by hydrophobic solvents (toluene, diethyl
ether and THF) can make the enzyme–substrate interaction diffi-
cult, and consequently the bioreduction reaction fails.
maintained. As we know that glycerol can penetrate the cell wall
2
1
and the cytoplasmic membrane, the same protein-stabilizing ac-
tion can be occurring with alcohol dehydrogenases from A. terreus
and R. oryzae.
Based on the remarkable enhancement of both conversion and
enantioselectivity by the use of glycerol as co-solvent, we decided
to apply it in the bioreduction of different haloacetophenones.
However, we initially investigated the ideal ratio of PBS and glyc-
erol in order to evaluate its influence on the conversion and enanti-
oselectivity of the bioreduction.
As can be seen in Table 1, the bioreduction with A. terreus and R.
oryzae using glycerol or DMSO as co-solvent afforded (S)-1a alcohol
insimilar enantioselectivities (entries 2–5). However, itwas observed
that the use of glycerol as a co-solvent gave higher conversions than
the reaction with DMSO. By using glycerol, the concentration range
of the product was 44–72% (Table 1, entries 2 and 3), and 10–50% by
using DMSO. Based on theseresults weselected glycerol as the co-sol-
venttobeappliedinthe following studies. Moreover, thechoiceofthe
co-solvent can be reinforced by the growing availability of glycerol in
the market, since it is a by-product from biodiesel production.
In general, the use of the glycerol improved the enantioselectiv-
ity and conversion in the bioreductions, with the desired chiral
alcohols being obtained with higher concentrations, and mainly
excellent enantiomeric excesses in comparison to the bioreduc-
tions in a phosphate buffer solution (Table 1, entries 2 and 3).
We can associate the successful application of glycerol as a co-
solvent in the bioreductions mediated by A. terreus and R. oryzae to
different factors: (i) It was created as a homogeneous system by
the glycerol–water combination that avoids the mass transfer limi-
tation, thus increasing the enzyme–substrate interaction; (ii) glyc-
2.2. Study of the ratio of glycerol to aqueous media for
bioreduction reactions
To optimize the ratio of glycerol to aqueous media for the biore-
0
duction, we selected 2 -chloroacetophenone 1 as the substrate mod-
el and a phosphate buffer solution as the aqueous media (Table 2).
As we can see from the results summarized in Table 2, the concen-
tration of glycerol between 5% (PBS–glycerol 9.5:0.5) to 20% (PBS–gly
cerol4:1)gave goodvaluesofconversion and ee (Table 2, entries2–4).
It is also noteworthy that the enantiopreference was not affected by
the different concentrations of glycerol. In all cases, the formation of
alcohol (S)-1a was observed. Above 30% of glycerol, a gradual and sig-
nificant decrease in the conversion was observed, especially for the
reactions using 100% of glycerol in which no conversion was
observed (Table 2, entries 5–8). Based on these results, PBS–glycerol
(9:1) and PBS–glycerol (4:1) were selected as the ideal solvent
systems for the bioreduction of different haloacetophenones 1–9.
1
8
erol has a protein-stabilizing action. Timasheff and Gekko

L. H. Andrade et al. / Tetrahedron: Asymmetry 20 (2009) 1521–1525
1523
Table 2
0
a
Study of the ratio of glycerol to aqueous media in the bioreduction of 2 -chloroacetophenone
O
OH
micro-organism
solvent
*
Cl
Cl
1
1a
Entry
Solvent—PBS–glycerol (glycerol,%)
A. terreus SSP 1498
R. oryzae CCT 4964
eec (%)
94 (S)
cb (%)
eec (%)
cb (%)
1
2
3
4
5
6
7
8
0
5
24
49
49
44
14
10
7
65 (S)
92 (S)
95 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
—
88
75
72
50
13
5
95 (S)
96 (S)
97 (S)
>99 (S)
—
10
20
30
40
50
100
4
<1
—
—
<1
a
0
Reaction conditions: 3.0 g of fungal cells, 20
l
L of 2 -chloroacetophenone, 50 mL of solvent [Na
2
HPO
4
/KH
2
PO
4
buffer (pH 7):glycerol], 48 h, 32 °C, 160 rpm.
b
c
Concentration of product was determined by chiral GC analysis.
Enantiomeric excess was determined by chiral GC analysis; (ꢀ) not determined due to low conversion.
2.3. Bioreduction of chloro, fluoro and bromoacetophenones 1–9
2.3.1. Bioreduction of bromoacetophenones
The important influence of glycerol as co-solvent was observed
in the reduction of the bromoacetophenones 3, 6 and 9. In compar-
ison to the PBS system, in all cases, the use of PBS–glycerol (9:1)
improved the conversion of ketones 3, 6 and 9 into the correspond-
Having in hand the appropriate reaction conditions, they were
applied to the bioreduction of chloro-, fluoro- and bromoacetoph-
enones 1–9 (Table 3).
Table 3
a
Bioreduction of haloacetophenones 1–9 in phosphate buffer solution (PBS) and PBS–glycerol system
O
OH
micro-organism
solvent
X
X
1
-9
1a-9a
PBS: Phosphate buffer solution [Na
2
HPO
4
/KH
2
PO
4
buffer (pH 7)].
a
Reaction conditions: 3.0 g of fungal cells, 20
lL of haloacetophenone, 50 mL of solvent [PBS–Glycerol], 48 h, 32 °C, 160 rpm.
b
c
Concentration of product was determined by chiral GC analysis.
Enantiomeric excess was determined by chiral GC analysis.

1
524
L. H. Andrade et al. / Tetrahedron: Asymmetry 20 (2009) 1521–1525
0
ing alcohols. The conversion of 2 -bromoacetophenone 3 into the
S)-3a alcohol was improved from 27% in PBS to 48%, in reactions
catalyzed by A. terreus (Table 3, entry 3), and from 21% in PBS to
9% in reactions catalyzed by R. oryzae (Table 3, entry 12). For both
5. In all cases, low ee was observed (37%). A slight decrease in
the concentration of alcohol (S)-5a (96–88%) was observed when
PBS–glycerol (4:1) was used (Table 3, entry 14).
(
0
2
In the reduction of 4 -fluoroacetophenone 8 by A. terreus, PBS
cases, excellent enantiomeric excesses (up to >99%) were observed.
was the best solvent leading to (R)-8a with moderate concentra-
tion and ee (58% and 61%, respectively). In this case, the use of
PBS–glycerol (9:1) system improved the concentration to 66%,
but the ee was reduced to 40% (Table 3, entry 8). When PBS–glyc-
erol (4:1) was used both conversion and ee were decreased to 48%
and 27%, respectively (Table 3, entry 8). The reductions of 8 by R.
oryzae occurred with very low concentration of the (S)-8a alcohol
(up to 6%) and moderate ee (60%) (Table 3, entry 17).
In general, the use of PBS–glycerol, especially in a 9:1 ratio was
shown to be an excellent system for the reduction of haloacetoph-
enones. Glycerol has demonstrated its potential for the improve-
ment of the enzymatic performance of A. terreus and R. oryzae in
terms of conversion and enantioselectivity.
In the reaction catalyzed by A. terreus, the use of PBS–glycerol
0
(
9:1) improved the conversion of 3 -bromoacetophenone 6 from
6
9% in PBS to 85% (Table 3, entry 6). In this case, no alteration
was observed in the enantioselectivity and the alcohol (S)-6a was
obtained with 86% ee. In the reaction catalyzed by R. oryzae with
PBS–glycerol (9:1), the concentration of alcohol (S)-6a was im-
proved from 12% in PBS to 19% and with 70% ee (Table 3, entry 15).
0
In the bioreduction of 4 -bromoacetophenone 9 by A. terreus in
PBS–glycerol (9:1) the ee of alcohol (R)-9a was improved from 18%
in PBS to 28%. In all cases high concentrations of (R)-9a (up to 98%)
were observed (Table 3, entry 18). When the reaction was cata-
lyzed by R. oryzae, the concentration of the product was improved
from 30% in PBS to 40% PBS–glycerol 10%. For this case, no remark-
able change in the ee was observed, and alcohol (S)-9a was ob-
tained with 98% ee (Table 3, entry 18).
In order to evaluate the application of the bioreduction in a
PBS–glycerol system on a higher scale, we decided to carry out dif-
0
ferent assays for the reduction of 2 -chloroacetophenone 1: (i) A set
of 10 Erlenmeyer flasks (150 mL) each one containing solvent
(
PBS–glycerol, 9:1) or (PBS–glycerol, 4:1), ketone 1 and cells of A.
2
.3.2. Bioreduction of chloroacetophenones
The enantioselectivity of the bioreduction of 2 -chloroacetophe-
terreus. (ii) One Erlenmeyer flask (2000 mL) with solvent (PBS–
glycerol, 9:1 or PBS–glycerol, 4:1), ketone 1 and fungi cells of A.
terreus.
0
none 1 using cells of A. terreus was strongly influenced by the addi-
tion of glycerol. When the reaction was carried out in only PBS, a
low concentration of alcohol (S)-1a (24%) and the ee (65%) were
observed. However, the reaction in PBS–glycerol (4:1) improved
the concentration of (S)-1a to 44% and the enantiomeric excess
to >99% (Table 3, entry 1). For the reactions catalyzed by cells of
R. oryzae, an improvement in the ee of the alcohol (S)-1a from
Both reaction systems carried out with PBS–glycerol (4:1) led to
(S)-1a with excellent enantioselectivity (>99% ee) and high concen-
tration (90%). The product was obtained in 67% isolated yield from
the assay carried out in a set of 10 Erlenmeyer flasks, and 72% from
the assay with one flask. Both reaction systems in PBS–glycerol
(
9:1) showed high enantioselectivity (95% ee) and >99% concentra-
9
4% in PBS to 97% in PBS–glycerol (4:1) was observed. Moreover,
tion of the (S)-1a alcohol. The isolated yield from the assay using a
set of 10 Erlenmeyer flasks was 71%, while for the assay with one
flask, the yield was 80%. These similar results (high yield and
enantioselectivity) showed us that the chiral alcohol production,
a decrease in the concentration to 50% by using PBS–glycerol sys-
tem (4:1) was also observed (Table 3, entry 10).
0
The bioreduction of 3 -chloroacetophenone 4 by A. terreus was
not positively affected by glycerol. In this case, the best result
was observed in the PBS system where the concentration of the
(S)-1a, can be easily carried out in a simple one reaction system.
(
S)-4a alcohol was 72% with >99% ee (Table 3, entry 4). On the
3
. Conclusion
other hand, in the reactions catalyzed by R. oryzae, the addition
of glycerol improved the concentration of (S)-4a from 23% in PBS
to 34% in PBS–glycerol (9:1). The ee of the (S)-4a alcohol was also
improved from 38% to 54% by the use of glycerol as co-solvent (Ta-
ble 3, entry 13).
In conclusion, we have shown that glycerol, a by-product from
biodiesel production, is an excellent option as a co-solvent to im-
prove the bioreduction of halo-acetophenones, in terms of concen-
tration and enantioselectivity of the products. For both fungi, A.
terreus and R. oryzae, bioreduction provided the desired chiral alco-
hols in high enantiomeric excess (up to >99%) and conversion (up
to >99%). Moreover, these data gave us support for further studies
using new alcohol dehydrogenases from A. terreus and R. oryzae.
The use of PBS–glycerol (9:1) was shown to be the best system
0
for reducing 4 -chloroacetophenone 7. When the ketone 7 was re-
duced by A. terreus an improvement in the ee of (R)-7a from 46% in
PBS to 53% in PBS–glycerol (9:1) with high concentration of the
product (87%; Table 3, entry 7) was observed. When R. oryzae
was used as a biocatalyst, the reduction of 7 in PBS led to (S)-7a
alcohol in 20% concentration and 90% ee. By using PBS–glycerol
4
4
. Experimental
(
9:1), the concentration and ee of (S)-7a were improved to 30%
and 93%, respectively (Table 3, entry 16).
.1. General methods
0
0
0
2
.3.3. Bioreduction of fluoroacetophenones
The bioreduction of 2 by A. terreus using all solvent systems
2 -Bromoacetophenone, 3 -bromoacetophenone, 4 -bromoace-
0
0
0
tophenone, 2 -chloroacetophenone, 3 -chloroacetophenone, 4 -
chloroacetophenone, 2 -fluoroacetophenone, 3 -fluoroacetophe-
none and 4 -fluoroacetophenone were purchased from Aldrich
0
0
gave alcohol (S)-2a with excellent concentration (>99%) and ee
up to 94%) (Table 3, entry 2). In the reactions catalyzed by R. ory-
0
Ò
(
zae, the addition of glycerol decreased the concentration of (S)-2a
from 90% in PBS to 56% in PBS–glycerol (4:1). However, excellent
enantiomeric excesses (>99%) were observed for the three solvent
and used as supplied. Microorganisms were manipulated in a Vec-
co laminar flow cabinet, and all incubation experiments were car-
ried out using sterile materials. A Technal TE-421 orbital shaker
was employed in the bioreductions. The compound purification
was performed by chromatographic column over silica gel (230–
400 mesh) eluted with mixtures of n-hexane and ethyl acetate as
eluent. Column effluents were monitored by TLC on pre-coated Sil-
ica Gel 60 F254 layers (aluminium-backed: Merck) with mixtures of
n-hexane and ethyl acetate. Products from the enzyme-catalyzed
systems evaluated (Table 3, entry 11).
0
3
-Fluoroacetophenone 5 was best reduced by cells of A. terreus
into (S)-5a in PBS–glycerol (9:1), in which the ee was improved to
0% while the bioreduction carried out in PBS led to (S)-5a with
9
low ee (14%; Table 3, entry 5). Moreover, the ee was not affected
by glycerol when cells of R. oryzae were used in the reduction of

L. H. Andrade et al. / Tetrahedron: Asymmetry 20 (2009) 1521–1525
1525
reactions were analyzed by a Shimadzu model GC-17A gas chro-
matograph equipped with a flame ionization detector (FID). A chi-
ral capillary column (Chirasil-Dex CB B-cyclodextrin—25 m ꢁ
umn (Chirasil-Dex CB B-cyclodextrin—25 m ꢁ 0.25 mm) for
determination of the conversion and enantiomeric excesses. The
products of the biocatalyzed reactions were compared to a racemic
mixture. The preparation of the racemic alcohols 1a–9a was carried
out by reduction of the corresponding acetophenones 1–9 with so-
dium borohydride in methanol.
0
.25 mm) was used for determination of the conversion and enan-
tiomeric excesses. Optical rotation values were determined with a
Jasco DIP-378 polarimeter using a 1 dm cuvette and the reported
values refer to the Na-D line.
2
2
2
Racemic compounds: GC conditions (carrier gas H ; 100 kPa;
injector 220 °C; detector 220 °C): (RS)-1-(2-bromophenyl)ethanol
(140 °C, 5 °C/min up to 180 °C; (R)-enantiomer 5.11 min; (S)-enan-
tiomer 5.88 min); (RS)-1-(3-bromophenyl)ethanol (130 °C, 3 °C/min
up to 160 °C; (R)-enantiomer 7.89 min; (S)-enantiomer 8.27 min);
(RS)-1-(4-bromophenyl)ethanol (110 °C, 3 °C/min up to 150 °C;
(R)-enantiomer 5.33 min; (S)-enantiomer 5.65 min); (RS)-1-(2-
chlorophenyl)ethanol (125 °C, 5 °C/min up to 150 °C (3 min); (R)-
enantiomer 5.72 min; (S)-enantiomer 6.44 min); (RS)-1-(3-chloro-
phenyl)ethanol (130 °C, 3 °C/min up to 160 °C; (R)-enantiomer
6.03 min; (S)-enantiomer 6.37 min); (RS)-1-(4-chlorophenyl)etha-
nol (130 °C, 5 °C/min up to 160 °C); (R)-enantiomer 5.25 min; (S)-
enantiomer 5.54 min); (RS)-1-(2-fluorophenyl)ethanol (113 °C,
3 °C/min up to 145 °C; (R)-enantiomer 6.03 min; (S)-enantiomer
4
4
.2. General procedure for the bioreduction reactions
.2.1. Growth conditions for the fungi cultures
The fungi were grown in Erlenmeyer flasks (2000 mL) contain-
ing 1000 mL of culture medium (malt extract—20 g/L) at 32 °C
4 days) in an orbital shaker (160 rpm). After this stage, the cells
were filtered off and used for bioreduction assays.
(
4
.2.2. Small scale reactions
Cells of fungi (3 g) were resuspended in 50 mL of the appropri-
ate solvent system (Tables 1 and 2) in an Erlenmeyer flask
(
(
150 mL) followed by the addition of the desired haloacetophenone
20 L; Table 3). The reaction mixture was stirred in an orbital sha-
l
6.35 min); (RS)-1-(3-fluorophenyl)ethanol (110 °C, (isotherm -
ker (32 °C; 160 rpm) for 48 h. The bioreductions were analyzed by
GC (Section 4.3).
9 min); (R)-enantiomer 5.99 min; (S)-enantiomer 6.67 min); (RS)-
1-(4-fluorophenyl)ethanol (110 °C, 3 °C/min up to 180 °C; (R)-enan-
tiomer 11.92 min; (S)-enantiomer 13.47 min).
4
.2.3. Preparative scale reactions
Assays 1 and 2: In a set of 10 different Erlenmeyer flasks
References
(
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0
were added cells of A. terreus SSP 1498 (3 g) and 2 -chloroaceto-
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1
at 32 °C, 160 rpm for 48 h. After this time the content of the flasks
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4
over MgSO and filtered. The solvent was removed in vacuum,
and the residue was purified by column chromatography on silica
gel using a mixture of n-hexane and ethyl acetate (9:1) as eluent to
afford (S)-1-(2-chlorophenyl)ethanol. Isolated yield for bioreduc-
4
.
For some reviews, see: (a) Ghanem, A. Tetrahedron 2007, 63, 1721–
754; (b) Nakamura, K.; Yamanaka, R.; Matsuda, T. Curr. Org. Chem.
1
2006, 10, 1217–1246; (c) Carrera, G.; Riva, S. Angew. Chem., Int. Ed.
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2
D
2
tion in PBS–glycerol (4:1) system: 67%; >99% ee; ½
aꢂ
¼ ꢀ59:8 (c
1
7
5
.00, CHCl
3
).
Isolated yield for bioreduction in PBS–glycerol (9:1) system:
2
D
2
1%; 95% ee; ½
aꢂ
¼ ꢀ59:6 (c 1.00, CHCl
3
).
5
00.
Assays 3 and 4: In one Erlenmeyer flask (2000 mL) containing
00 mL of PBS–glycerol (9:1 or 4:1) were added cells of A. terreus
SSP 1498 (30 g) followed by 2 -chloroacetophenone (200
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0
l
L). The
8
9
.
.
Halling, P. J. Curr. Opin. Chem. Biol. 2000, 4, 74–80.
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reaction was carried out in an orbital shaker at 32 °C, 160 rpm
for 48 h. After this time, the content of the flask was filtered and
washed with ethyl acetate (200 mL). The aqueous phase was ex-
tracted with ethyl acetate (2 ꢁ 200 mL). The organic phases were
10. Gainer, J. L.; Gervais, T. R.; Carta, G. Biotechnol. Prog. 2003, 19, 389–
95.
3
1
1
1. Castro, G. R.; Knubovets, T. Crit. Rev. Biotechnol. 2003, 23, 195–231.
2. For recent examples see: (a) Banerjee, U. C.; Kansal, H. Biosour. Technol.
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Schaffenberger, M.; Gross, J.; Glieder, A.; de Wildeman, S. ChemSusChem
4
combined and dried over MgSO and filtered. The solvent was re-
moved in vacuum and the residue was purified by column chroma-
tography on silica gel using a mixture of n-hexane and ethyl
acetate (9:1) as eluent to afford (S)-1-(2-chlorophenyl)ethanol.
Isolated yield for bioreduction in PBS–glycerol (4:1) sys-
2008, 1, 431–436; (d) Banerjee, U. C.; Fatima, Y.; Kansal, H.; Soni, P. Process.
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2
D
2
13. Van Rantwijk, F.; Sheldon, R. A. Chem. Rev. 2007, 107, 2757–2785.
tem = 72%; >99% ee; ½
a
ꢂ
¼ ꢀ60:0 (c 1.00, CHCl
3
).
1
1
4. Thomas, N. R.; Hobbs, H. R. Chem. Rev. 2007, 107, 2786–2820.
5. Basso, A.; Cantone, S.; Hanefeld, U. Green Chem. 2007, 9, 954–971.
Isolated yield for bioreduction in PBS–glycerol (9:1) sys-
2
2
tem = 80%; 95% ee; ½
a
ꢂ
¼ ꢀ59:5 (c 1.00, CHCl
3
).
16. Wolfson, A.; Dlugy, C.; Shotland, Y. Environ. Chem. Lett. 2007, 5, 67–71.
D
1
1
7. Wolfson, A.; Dlugy, C.; Tavor, D.; Blumenfeld, J.; Shotland, Y. Tetrahedron:
Asymmetry 2006, 17, 2043–2045.
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N.; Gekko, K. Biochemistry 1981, 20, 4677–4686.
4
.3. Determination of the enzymatic activity of the
microorganisms
1
2
9. Gangadhara, K. P. R.; Praskash, V. Protein J. 2008, 27, 440–449.
0. (a) Yan, Y.-B.; Feng, S. Proteins 2008, 71, 844–854; (b) Fields, P. A.; Wahlstrand,
B. D.; Somero, G. N. Eur. J. Biochem. 2001, 268, 4497–4505.
The reaction progress was monitored after 48 h by collecting
0 mL samples. These samples were extracted by stirring with ethyl
1
21. Hubalék, Z. Cryobiology 2006, 46, 205–209.
22. Andrade, L. H.; Porto, A. L. M.; Comasseto, J. V.; Rodrigues, D. F.; Pellizari, V. H. J.
acetate (2 mL) followed by centrifugation (6000 rpm, 5 min). The or-
ganic phase was analyzed by GC (1 L) using a chiral capillary col-
Mol. Catal. B: Enzym. 2005, 33, 73–79.
l