The influence of cosolvent concentration on enzymatic kinetic resolution of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives

Article abstract of DOI:10.3109/10242422.2015.1040005

A method to improve the enantioselectivity of lipase-catalyzed kinetic resolution (KR) of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives in water-acetone solution is presented. Two different approaches were compared: enzyme-catalyzed esterification and enzymatic hydrolysis of the target ester. A substantial influence of enzyme type, ethoxy group donor, and solvent on conversion and enantioselectivity of the enzymatic esterification was noted. While enzymatic esterification proceeds with poor enantioselectivity, the hydrolysis of target ester proceeds efficiently. Studies on the influence of cosolvent used for the enzymatic hydrolysis reaction showed that kinetic resolution can be performed in acetone and water buffer mixture predominantly containing organic solvent. Any change in organic solvent content resulted in a substantial decrease in enantioselectivity from almost E = 150 to less than 5.

Full text of DOI:10.3109/10242422.2015.1040005

Biocatalysis and Biotransformation, 2015; Early Online: 1–7
ORIGINAL ARTICLE
The influence of cosolvent concentration on enzymatic kinetic
resolution of trans-2-phenyl-cyclopropane-1-carboxylic acid
derivatives
CATALINA WIŚNIEWSKA, DOMINIK KOSZELEWSKI, MAŁGORZATA ZYSK
*
& RYSZARD OSTASZEWSKI
Institute of Organic Chemistry, Polish Academy of Sciences,Warsaw, Poland
Abstract
A method to improve the enantioselectivity of lipase-catalyzed kinetic resolution (KR) of trans-2-phenyl-cyclopropane-1-
carboxylic acid derivatives in water–acetone solution is presented. Two different approaches were compared: enzyme-
catalyzed esterification and enzymatic hydrolysis of the target ester. A substantial influence of enzyme type, ethoxy group
donor, and solvent on conversion and enantioselectivity of the enzymatic esterification was noted. While enzymatic
esterification proceeds with poor enantioselectivity, the hydrolysis of target ester proceeds efficiently. Studies on the
influence of cosolvent used for the enzymatic hydrolysis reaction showed that kinetic resolution can be performed in acetone
and water buffer mixture predominantly containing organic solvent. Any change in organic solvent content resulted in a
substantial decrease in enantioselectivity from almost Eϭ150 to less than 5.
Keywords: Kinetic resolution, hydrolases, lipases, enzymatic esterification, organic solvents, hydrolysis
Abbreviations: KR: kinetic resolution
Introduction
acid is used in the synthesis of (9R,10S)-dihy-
drosterculic acid, a common cyclopropyl fatty acid
of microorganisms and subtropical plants (Lou et al.
2004). Another important compound obtained from
chiral derivatives of trans-2-phenyl-cyclopropane-1-
carboxylic acid is tranylcypromine, an oral mono-
amine oxidase inhibitor type of antidepressant (Csuk
et al. 1996). In addition, the trans-2-phenylcyclo-
propylamine moiety is part of antibacterial active
gyrase inhibitors. The spiro 3,3’-cyclopropyl oxin-
dole core is a recurring structural motif present
in several agrochemically and pharmaceutically
active compounds, including herbicides, inhibitors,
and antagonists for treating cancer, mood disorders,
and human immunodeficiency virus infection. In
addition, spiro 3,3’-cyclopropyl oxindoles have
demonstrated synthetic utility as intermediates for
accessing relevant oxindole alkaloids (Ladd et al.
2013).
Enantiomerically pure trans-2-phenyl-cyclopropane-
1-carboxylic acid derivatives are an important family
of compounds that play a prominent role in organic
chemistry and are endowed with a wide spectrum of
biological properties (Youssef et al. 2012; Davi &
Lebel 2008). Reported biological activities include
enzyme inhibition, and antimicrobial, antibiotic,
antitumor, and antiviral activities (Rosenberg et al.
2011; van Oers et al. 2014).
They have a wide range of applications in the
synthesis of drugs and biologically active molecules
and are used as substrates in the synthesis of
b-amino acids (Chan et al. 2014; Silva et al 2013).
Interesting structural properties and significant
pharmacological activities are also crucial struc-
tural features of multiple natural products like
b-lactams and other pharmaceuticals (Miller et al.
2003). Trans-2-phenyl-cyclopropane-1-carboxylic
*
This work is dedicated to the memory of Jan Ramza (1958–2013).
Correspondence: Ryszard Ostaszewski, Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Kasprzaka 44/52 Poland. Tel. ϩ48 22
3432103. Faxϩ48 22 632 66 81. E-mail: ryszard.ostaszewski@icho.edu.pl
(Received 25 September 2014; revised 27 November 2014; accepted 08 April 2015)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2015 Informa UK, Ltd.
DOI: 10.3109/10242422.2015.1040005

2 C.Wiśniewska et al.
Since trans-2-phenyl-cyclopropane-1-carboxylic
acid derivatives are important substructures in many
synthetic and naturally occurring biologically active
compounds, great efforts have been made to develop
efficient stereoselective methods for the synthesis of
optically pure target acid (Youssef et al. 2012). A
widely used method for preparation of derivatives
of trans-2-phenyl-cyclopropane-1-carboxylic acid is
asymmetric cyclopropanation, reaction between ole-
fins and diazoacetates with different types of chiral
auxiliaries (Rosenberg et al. 2011). Several efficient
homogeneous catalysts have been developed for this
reaction, for example, (Salen)Ru(II) or (Salen)Ir(II)
catalysts (Miller et al. 2003; Kanchiku et al. 2007),
chloro(ŋ6-p-c ymene)(phosphoramidite)
ruthenium(II) (Huber et al. 2006), 2-aryl-5,
5-bisoxazoline-2-yl[1,3]dioxane (Knight & Belcher
2005), polymer-bound azabis(oxazoline) ligands
(Werner et al. 2006), 2,2-bipyridines, and pyridyl-
monooxazolines (pymox) (Bouet et al. 2007).Vitamin
B12 derivatives have also been found to be general
and efficient catalysts for asymmetric cyclopropana-
tion of alkenes with ethyl diazoacetate (Chen &
Zhang 2004; Ho et al. 2010). The enantiomerically
pure products were obtained with good yields but a
major inconvenience of this method is that substrates
containing diazo group are explosive, unstable, and
highly toxic, which limits its application.
Hydrolases (EC 3.1.x.x) catalyze the hydrolysis
of ester bonds (Schmid & Verger 1998) to release
free acids (Busto et al. 2010; Verma et al. 2012).
These enzymes are effective catalysts for various
inter-esterification and trans-esterification reactions
in the presence of low amounts of water and in
the presence of organic solvents (Bornscheuer &
Kazlauskas 2006; Schmid & Verger 1998; Drauz
et al. 2012). Their versatility means that lipases are
the enzyme of choice for potential applications in
the food and pharmaceutical industries (Sharma
et al. 2001; Andualema & Gessesse 2012; Liese
2006; Burton et al. 2002).
spectrometer. Chemical shifts are reported in parts
per million and coupling constants (J) given in Hz.
High-performance liquid chromatography (HPLC)
analyses were performed on a Chiracel OD-H
column (4.6 mm/250 mm, particle size: 5 μm from
Daicel Chemical Ind. Ltd.) equipped with a pre-
column (4 mm/• 10 mm, 5 l) using LC-6A Shimadzu
apparatus with UV SPD-6A detector and Chromatopac
C-R6A analyzer. All reactions were monitored by
thin-layer chromatography (TLC) on Merck silica
gel plates 60 F254. Column chromatography was
performed on Merck silica gel 60/230–400 mesh. All
chemicals were obtained from recognized suppliers and
the solvents were of analytical grade.The enantioselec-
tivity parameter E was calculated using equation:
Eϭln[1-c(1ϩeep)]/ln[1-c(1-eep)] (Yuryev et al. 2011).
Enzymes
The enzymes were used as obtained: wheat germ lipase
(Sigma-Aldrich), papain protease (Sigma-Aldrich),
Rhizopus oryzae lipase (Sigma-Aldrich), Candida
cylindracea lipase (Sigma-Aldrich), Amano PS lipase
(Sigma-Aldrich), C. antarctica lipase (Sigma-Aldrich),
hog pancreas lipase (Sigma-Aldrich), Mucor javanicus
lipase (Sigma-Aldrich), Rhizopus sp. protease (Sigma-
Aldrich), pig liver esterase (Sigma-Aldrich), Aspergillus
melleus acylase I (Sigma-Aldrich), C. lipolytica lipase
(Sigma-Aldrich), wild boar liver esterase acetone
powder (Institute of Organic Chemistry, PAS),
Alcalase CLEA (Sigma-Aldrich), Amano AK lipase
(Sigma-Aldrich), Novozym 435 (Novo Nordisk), and
lipase from Thermomyces lanuginosus (Lipolase L100,
Sigma-Aldrich).
Synthesis of ethyl trans-2-
phenylcyclopropanecarboxylate (2)
Trans-2-phenyl-cyclopropane-1-carboxylic acid (1) is
commercially available from Aldrich. Ethanol (50 mL,
1.35 mol), p-toluenesulfonic acid monohydrate
(50 mg, 0.25 mmol), and trans-2-phenyl-cyclopro-
pane-1-carboxylic acid (24.3 g, 0.15 mol) were heated
under reflux and the progress of reaction was con-
trolled by TLC. The mixture was then cooled, water
was added, and the ester formed was separated by
extraction with ethyl acetate (3ϫ50 mL).The organic
phase was washed with 10% aqueous sodium bicar-
bonate and dried with anhydrous MgSO4 and the sol-
vent was removed giving pure product as a colorless
liquid with 91% yield (25.9 g).1 H NMR (CDCl3, 200
MHz): d 7.09–7.31 (m, 5 H), 4.17 (q, Jϭ7.2 Hz, 2
H), 2.52 (m, 1 H), 1.91 (m, 1 H), 1.61 (m, 1 H), 1.31
(m, 1 H), 1.28 (t, Jϭ7.2 Hz, 3 H) ppm. 13C NMR
(CDCl3, 50 MHz): d 173.2, 140.0, 128.2, 126.3,
Enzymatic kinetic resolution (EKR) of esters of
the target acid has already been described (Csuk
et al. 1996). However, all of our efforts to repeat the
published data were unsuccessful.
In the present study, the scope and limitation of
hydrolase-catalyzed KR of trans-2-phenyl-cyclopro-
pane-1-carboxylic esters have been assessed.
Experimental procedure
General
Nuclear magnetic resonance (NMR) spectra were
recorded in CDCl3 with tetramethylsilane as internal
standard using a 200-MHz Varian Gemini 200

Influence of cosolvent concentration on enzymatic kinetic resolution
3
126.2, 60.6, 26.3, 24.1, 17.2, 14.1 ppm. 1H NMR and
13C NMR spectra were in accordance with spectra
presented in the literature (Shabtai et al. 1981).
(Cheng et al. 2011).The ee value could not be deter-
mined on chiral HPLC. Any derivatization to the
corresponding ethyl ester led to racemization.
General procedure for enzymatic esterification
Determination of enantiomeric excess of ester 2
A solution of rac-1 (0.5 mmol) in toluene (1 mL),
ethoxy group donor (3 equiv.), and 10 mg of enzyme
was placed in a 10-mL flask. The reaction was
conducted at 50°C.The progress of the reaction was
controlled by TLC and gas chromatography. The
enzymes were removed by filtration and the product
was isolated by silica gel column chromatography
(hexane/ethyl acetate) with increasing amounts of
ethyl acetate.
The ee of ester 2 was determined by HPLC using a
Chiralcel OD-H column, hexane/i-PrOH; 90/10;
flow rate:1 mL/min; and retention times: (-)-(R,R)-2:
7.2 min and (ϩ)-(S,S)-2: 8.5 min. HPLC tracers are
provided in Supplementary material
Results and discussion
The aim of our work was to develop an effective
EKR for readily available trans-2-phenyl-cyclopro-
pane-1-carboxylic acid (1).Two different approaches
can be applied: stereoselective enzyme-catalyzed
esterification of racemic carboxylic acid 1 or enzy-
matic hydrolysis of racemic ester 2. Only the second
one had previously been used with limited success.
EKR of racemic acids via esterification was recently
applied in our group for efficient synthesis of
enantiomerically pure 3-phenyl-4-pentenoic acid
esters (Brodzka et al. 2012). This method was
performed in organic solvents, and product purifica-
tion was very simple which makes it very attractive
from a practical point of view.
General procedure of enzymatic hydrolysis
A solution of rac-2 (0.5 mmol), 5 ml of mixture
(phosphate buffer 50 mM at pH of 7.4/acetone) and
10 mg of enzyme was placed in a 10-mL flask. The
reaction was conducted at 20°C.The progress of the
reaction was followed by TLC. After 2 hours, the
enzymes were removed by filtration and acetone was
removed under reduced pressure. The substrate
was extracted with ethyl acetate (3ϫ10 mL). The
combined extracts were washed with NaHCO3,
dried over MgSO4, and evaporated.
For both methods used, it was necessary to study
the influence of some important factors on the
reaction course, since EKR processes are strongly
determined by enzyme type, solvent, and ethoxy
group donor (Brodzka et al. 2013).
EKR via esterification of racemic acid 1 was
performed according to Scheme 1. Published meth-
odology was used in order to simplify all operations.
First, the influence of the enzyme on the reaction
course was studied using two different ethoxy
group donors: tetraethyl orthocarbonate and ethyl
orthoacetate, because in our previous studies they
gave satisfactory results (Brodzka et al.2012;Brodzka
et al. 2013). Table I shows the results of this study.
All experiments were conducted in toluene, at 50°C
Optimized conditions
To a solution of rac-2 (1.0 g, 5.25 mmol) in water/
acetone (15 mL/35 mL of acetone) at 20°C,
Novozyme 435 (100 mg) was added and the pH of
the reaction mixture was kept constant at 7.4 by the
addition of 0.1 M NaOH (7.93 mL corresponding
to a conversion of 49%). The reaction was termi-
nated by enzyme separation and the mixture was
extracted with ethyl acetate (3ϫ30 mL). The com-
bined extracts were washed with NaHCO3, dried
over MgSO4, and evaporated. Remaining ester 2 was
purified by column chromatography on silica gel
(hexane/ethyl acetate). 20[α]D ϩ 252 (c1.00, CHCl3),
92% enantiomeric excess (ee) [Lit. ϩ233(c1.00,
CHCl3), 82% ee for (S,S)-enantiomer] (Minuth
et al. 2009). The remaining aqueous reaction mix-
ture was acidified with cold concentrated hydrochlo-
ric acid to pH of 2 and extracted with ethyl acetate
(3ϫ30 mL).The combined extracts were dried over
MgSO4 and evaporated. The obtained product was
purified by crystallization from acetone to give
(R,R)-1 with 45% yield as colorless crystals; melting
point: 47–48°C (Lit. mp 49.5–50°C, (Elling et al.
1973); 20[α]D Ϫ398 (c 0.90, CHCl3) [Lit. Ϫ401.0
(c 0.88, CHCl3), Ͼ98% ee for (R,R)-enantiomer]
Ph
Ph
CO2H
CO2Et
(R,R)-1
enzyme,toluene
+
Ph
CO2H
ethoxy group donor
rac-1
(S,S)-2
Scheme 1. EKR by esterification.

4 C.Wiśniewska et al.
Table I. EKR of racemic trans-2-phenyl-cyclopropane-1-carboxylic acid.
Tetraethyl orthocarbonate
Ethyl orthoacetate
Yield (%) 2a ee (%) 2b
Entry
Enzyme
Yield (%) 2a
ee (%) 2b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Blank
Wheat germ lipase
Papain protease
0
16
22
55
22
43
18
23
22
20
23
38
88
49
22
24
63
nd
11
27
3
15
5
17
12
9
10
15
22
7
0
27
5
nd
2
rac
rac
3
Rhizopus oryzae lipase
Candida cylindracea lipase
Amano PS lipase
C. antarctica lipase
Hog pancreas lipase
Mucor javanicus lipase
Rhizopus sp. protease
Pig liver esterase
Aspergillus melleus acylase I
C. lipolytica lipase
Wild boar liver esterase
Alcalase CLEA
41
12
16
38
74
48
42
12
11
36
11
48
22
28
11
9
11
13
10
10
rac
21
rac
23
10
17
20
16
18
14
Amano AK lipase
Novozyme 435
aProduct was purified by column chromatography on silica gel (hexanes/ethyl acetate).
bValues of ee were determined by chiral HPLC (Chiracel OD-H column).
for 48 hours. The same reactions conducted
at 30°C yielded the corresponding ester with sub-
stantially lower yields and similar ee values. The
progress of the enzymatic reaction was measured by
gas chromatography. In the blank reaction without
catalyst, no product was found (Table I, entry 1).
For almost all enzymes applied to the reaction
(Table I, entries 2–17), the non-racemic product was
obtained; however, the ee values of ester 2 were low
and none of the enantioselectivities calculated for
results shown in the Table I exceeded 5.
In the next step, the influence of all the available
ethoxy group donors on the reaction course was
evaluated. Novozyme 435 was chosen as a model bio-
catalyst because the ee for both ethoxy donor groups
was the highest (Table I, entry 17). For most donors
applied, both stereoselectivity and conversion ratios
were relatively low (Table II, entries 1–8). Only for
ethyl orthobenzoate was product 2 was obtained in
40% yield and 51% ee which corresponds to an
enantioselectivity of Eϭ10 (Table II, entry 8).
The next set of experiments was carried out
with all 16 previously tested enzymes using ethyl
orthobenzoate as ethoxy group donor. Table III
shows the results for enzymes which exhibited rea-
sonable stereoselectivity. Once more, Novozyme 435
appeared to be suitable for further study (Table III,
entry 1).
It is clear that EKR of racemic acid 1 by esteri-
fication, independent of enzyme or alkoxy group
donors used, provides enantiomerically enriched
ester with low ee. As it was not possible to obtain
enantiomerically pure product by enzymatic esterifi-
cation, we turned our attention to EKR based on the
hydrolysis reaction (Scheme 2). The racemic
ethyl trans-2-phenylcyclopropanecarboxylate (2)
was synthesized chemically from trans-2-phenyl-
cyclopropane-1-carboxylic acid using a standard lit-
erature procedure (Charette et al. 2003).
Table II. The influence of ethoxy group donor on the conversion
and ee in KR catalyzed by Novozyme 435.
Entry
Ethoxy group donor
Yield (%) 2a
ee (%) 2b
Table III. EKR with ethyl orthobenzoate as an ethoxy group
donor.
1
2
3
4
5
6
7
8
Ethyl orthoacetate
Ethyl orthoformate
Tetraethyl orthocarbonate
1,2,-Diethoxyethane
Ethyl acetoacetate
Benzaldehyde acetal
Diethyl carbonate
Ethyl orthobenzoate
28
32
63
25
Ͻ1
16
17
18
14
10
nd
6
Entry
Enzyme
Yield (%) 2a
ee (%) 2b
1
2
3
4
Novozyme 435
38
55
62
71
52
15
10
20
Rhizopus arrhizus lipase
Wheat germ lipase
Rhizopus sp. protease
nd
51
Ͻ1
40
aProduct was purified by column chromatography on silica gel
(hexanes/ethyl acetate).
bValues of ee were determined by chiral HPLC (Chiracel OD-H
column).
aProduct was purified by column chromatography on silica gel
(hexanes/ethyl acetate).
bValues of ee were determined by chiral HPLC (Chiracel OD-H
column).

Influence of cosolvent concentration on enzymatic kinetic resolution
5
Table V. The influence of the concentration of acetone on
enantioselectivity for Novozyme 435.
Ph
Ph
CO2H
Yield (%) 1a
ee (%) 2b(%)
E
(R,R)-1
Entry
Acetone (%)
enzyme
+
Ph
CO2Et
rac-2
1
2
3
4
5
6
7
8
9
5
10
20
30
50
60
70
80
90
41
35
47
66
55
39
49
50
71
10
3
9
14
20
26
94
4
1.5
phosphate buffer
acetone
1.2
1.3
1.3
1.7
3
150
1.2
1.1
CO2Et
(S,S)-2
Scheme 2. Enzymatic hydrolysis of rac ethyl trans-2-
phenylcyclopropanecarboxylate (2).
4
To improve of the solubility of the starting
ethyl trans-2-phenylcyclopropanecarboxylate (2),
the enzymatic hydrolysis was conducted at ambient
temperature, in two solvent mixtures containing dif-
ferent percentages of acetone in phosphate buffer
(phosphate buffer pHϭ7.0; 1:4 and 3:4 v/v). The
spontaneous hydrolysis of substrate was found to be
very slow and formation of product was not observed
(Table IV, entry 1, Condition A). Hydrolysis was
found to be catalyzed by several different enzymes but
very low enantioselectivity was observed (Table IV,
Condition A). Since no efficient biocatalyst was
found, we turned our attention to enhancing the
selectivity of the screened hydrolases. It is already
known that enantioselectivity of enzymatic reactions
strongly depends on the type and amount of cosol-
vent (Bornscheuer 2002; Koszelewski et al. 2012;
Fryszkowska et al. 2005).Therefore, the next exper-
iments were carried out in a reaction mixture
containing 43% acetone as a cosolvent (Table IV,
Condition B). With Novozyme 435 the conversion
was lower, but non-racemic product was obtained.
Unfortunately, the ee values as well as productivity
were poor. Systematic studies were performed to
evaluate the influence of acetone content on enanti-
oselectivity. Novozyme 435 was chosen as the model
enzyme, and the amount of acetone used as a
cosolvent was systematically increased.
aProduct was purified by column chromatography on silica gel
(hexanes/ethyl acetate).
bValues of ee were determined by chiral HPLC (Chiracel OD-H
column).
In many cases, organic solvent can increase the
stereoselectivity of EKR (Schmid & Verger 1998;
Koeller & Wong 2001), but high amount of organic
cosolvent deactivate enzymes (Klibanov 2001; Reetz
2002). Table V shows the results obtained; with 5%
(v/v) acetone the enantioselectivity values were
low (Table V, entries 1). Gradually increasing the
concentration of acetone up to 60% resulted with a
little improvement in ee (Table V, entries 2–6), but a
substantial improvement in ee to 94% was observed
when the concentration of acetone was raised to
70%, what corresponds to an enantioselectivity of
Eϭ150 (Table V, entry 7). Further increasing the
acetone level to 80 and 90% caused deterioration of
enantioselectivity (Table V, entries 8–9).
Thus, we have found a significant effect of high
acetone cosolvent concentration on the reaction rate
and enantioselectivity. Finally, using optimized con-
ditions, the KR was performed on a preparative scale
and the remaining (S,S)-ester 2 was isolated with
48% yield and 92% ee. The corresponding (R,R)-
acid 1 was isolated with 45% yield and Ͼ95% ee
estimated from the optical rotation [α] 20 Ϫ398,
D
Table IV. EKR by hydrolysis of racemic ethyl 2-phenylcyclopropane carboxylate.
Condition Aa
Condition Bb
Yield (%) 1c ee (%) 2c
Entry
Enzyme
Yield (%) 1c
ee (%) 2d
1
2
3
4
5
6
Blank
Novozyme 435
Rhizopus sp. protease
Bacillus amyloliquefaciens lipase
Pseudomonas fluorescens lipase
Pig liver esterase
0
88
92
90
55
65
nd
11
rac
rac
rac
rac
0
78
44
22
36
72
Nd
40
Rac
9
5
6
aConditions: 0.5 mmol substrate, 1 mL of acetone, 4 mL of phosphate buffer at pH of 7.0, 10 mg of
enzyme, 3.5 h, 20°C.
bConditions: 0.5 mmol substrate, 1.5 mL of acetone, 2 mL of phosphate buffer at pH of 7.0, 10 mg of
enzyme, 24h, 20°C.
cProduct was purified by column chromatography on silica gel (hexanes/ethyl acetate).
dValues of ee were determined by chiral HPLC (Chiracel OD-H column).

6 C.Wiśniewska et al.
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the optically pure (R,R)-enantiomer (Cheng et al.
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Two different approaches to KR based on stereose-
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KR of racemic trans-2-phenylcyclopropanecar-
boxylic acid via esterification was catalyzed by
hydrolases, but the desired product was obtained
with a low ee. The second approach was based on
enzymatic hydrolysis of ethyl trans-2-phenylcyclo-
propanecarboxylate, and the influence of enzyme
and solvent on enantioselectivity was examined. It
was found that the concentration of acetone used as
a cosolvent has a major impact on ee. Systematic
studies on the concentration of acetone on the ste-
reoselectivity of hydrolysis confirmed this result.
Optimized conditions to obtain product with a high
ee were established.The results expand the scope and
limitation of biocatalytic processes to solvent systems
containing predominantly organic solvents. These
reaction conditions may be applied in many biocata-
lytic processes, thereby increasing productivity.
We believe that the method elaborated will be of
considerable use, due to its simplicity.
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synthesis. Weinheim: Wiley-VCH. pp. 1–1985.
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Acknowledgement
This research was partially financed by Polpharma
Starogard Gdański, by project “Biotransformations
for pharmaceutical and cosmetics industry” no.
POIG.01.03.01–00-158/09–01 and by the European
Union in the frame of the COST Action CM1303.
Huber D, Kumar PGA, Pregosin PS, MikhelI S, Mezzetti A. 2006.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
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Supplementary material available online
Supplementary material to be found online at http://
informahealthcare.com/doi/abs/10.3109/10242422.
2015.1040005