Optimization of chemo-enzymatic epoxidation of cyclohexene mediated by lipases

Article abstract of DOI:10.1081/SCC-200066705

This work describes the lipase-mediated epoxidation of cyclohexene. Lipases were used to generate peroxyoctanoic acid directly from octanoic acid and hydrogen peroxide and applied in situ to obtain cyclohexene oxide. Various parameters, which could affect this reaction, were studied such as lipases from different sources, organic solvents, temperature and acyl donor concentrations. Highest conversions to cyclohexene epoxide were achieved using a two-phase system of toluene or xylene/water with ROL (Amano F-Ap15 free Rhizopus orizae lipase) (84 and 80%) or CALB (Novozymes 435-immobilized Candida antarctica lipase type B) (>9 and 84%) as biocatalysts. Using PSL (Amano PS-free Pseudomonas sp) the conversions were in the range of 12-53%, but an improvement was obtained by the use of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (20 to 41% in water/methyl dichloride). Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/SCC-200066705

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Optimization of
Chemo‐Enzymatic Epoxidation
of Cyclohexene Mediated by
Lipases
a
a
Marcelo Alves Moreira , Thiago Bergler Bitencourt
a
&
a
Maria da Graça Nascimento
Departamento de Química , Universidade Federal
de Santa Catarina , Florianópolis, SC, Brazil
Published online: 16 Aug 2006.
To cite this article: Marcelo Alves Moreira , Thiago Bergler Bitencourt & Maria
da Graça Nascimento (2005) Optimization of Chemo‐Enzymatic Epoxidation of
Cyclohexene Mediated by Lipases, Synthetic Communications: An International
Journal for Rapid Communication of Synthetic Organic Chemistry, 35:15, 2107-2114,
DOI: 10.1081/SCC-200066705
To link to this article: http://dx.doi.org/10.1081/SCC-200066705
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Synthetic Communications , 35: 2107–2114, 2005
Copyright # Taylor & Francis, Inc.
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1081/SCC-200066705
Optimization of Chemo-Enzymatic
Epoxidation of Cyclohexene Mediated
by Lipases
Marcelo Alves Moreira, Thiago Bergler Bitencourt, and
Maria da Gra c¸ a Nascimento
Departamento de Qu ´ı mica, Universidade Federal de Santa Catarina,
Florian o´ polis, SC, Brazil
Abstract: This work describes the lipase-mediated epoxidation of cyclohexene. Lipases
were used to generate peroxyoctanoic acid directly from octanoic acid and hydrogen
peroxide and applied in situ to obtain cyclohexene oxide. Various parameters, which
could affect this reaction, were studied such as lipases from different sources, organic
solvents, temperature and acyl donor concentrations. Highest conversions to cyclo-
hexene epoxide were achieved using a two-phase system of toluene or xylene/water
with ROL (Amano F-Ap15 free Rhizopus orizae lipase) (84 and 80%) or CALB
w
Novozymes 435 -immobilized Candida antarctica lipase type B) (.9 and 84%) as
(
biocatalysts. Using PSL (Amano PS-free Pseudomonas sp) the conversions were in
the range of 12–53%, but an improvement was obtained by the use of the ionic liquid
1-butyl-3-methylimidazolium tetrafluoroborate (20 to 41% in water/methyl dichloride).
Keywords: Two-phase system, epoxidation, lipases
INTRODUCTION
The use of lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) as biocatalysts
has been extensively explored in recent years and is still one of the most
useful tools in the resolution of chiral compounds to obtain enantiopure
[
1,2]
building blocks in organic synthesis.
Although these reactions are
usually carried out under “unnatural” conditions, it has appeared that the
high degree of selectivity and efficacy exhibited by lipases in their natural
Received in the USA February 18, 2005
Address correspondence to Maria da Gra c¸ a Nascimento, Departamento de Qu ´ı mica,
Universidade Federal de Santa Catarina, Florian o´ polis, SC 88040-970, Brazil. E-mail:
graca@qmc.ufsc.br
2
107

2108
M. A. Moreira, T. B. Bitencourt, and M. da Gra c¸ a Nascimento
[
3,4]
environment is also expressed in synthesis.
is the lipase-catalyzed perhydrolysis of carboxylic acids and esters and the use
An example of these reactions
[5,6]
of the resulting peroxyacids to form alkene oxides.
Chemo-enzymatic epoxidation is a very useful reaction in organic
synthesis. Skouridou et al. carried out the chemo enzymatic epoxidation of
a-pinene at room temperature using Candida antarctica lipase as the biocata-
[
7]
lyst (nearly 70% conversion). Epoxides are key raw materials for a wide
[
8]
variety of products and much effort has been devoted to the development
of new active and selective epoxidation catalysts for processes that
[9]
avoid the formation of large amounts of by-products. One of the most
used epoxidation processes is the stochiometric peracid route with meta-
[
10,11]
chloroperoxybenzoic acid (MCPBA),
is under threat owing to its hazardous nature.
but its commercial manufacture
Also, this epoxidation
[12]
method needs severe reaction conditions, for example, the use of a strong
mineral acid, which is considered responsible for side reactions such as the
[
13]
formation of diols and hydroxyesters.
Herein, the study of the experimental parameters, which affect the chemo-
enzymatic epoxidation of cyclohexene, is reported, such as the use of lipases
from different sources, temperature, organic solvents, and the effect of acyl
donor concentrations. In this reaction, octanoic acid was converted by
lipase-perhydrolysis to its respective peroxyoctanoic acid and then used in
the epoxidation of cyclohexene in an aqueous/organic solvent biphasic
system. For comparison, one reaction was also performed in a three-phase
system of water/methyl dichloride/ionic liquid (Scheme 1).
RESULTS AND DISCUSSION
In our initial attempts, lipases from various sources were investigated to
evaluate which of them could be used to produce cyclohexene oxide in
Scheme 1.

Chemo-Enzymatic Epoxidation
2109
highest yields. Nine lipases, CALB (immobilized C. antarctica lipase type B
immobilized in anionic resin, 10,000 PLU/g), ROL (free Rhizopus orizae
lipase, 150,000 u/g), PSL (free Pseudomonas sp lipase, 30,000 u/g), MJL
(
free Mucor javanicus lipase, 10,000 u/g), CRL (free Candida rugosa lipase,
0,000 u/g), RML (free Rhizomucor miehei lipase, 1,000 u/g), TML (free
Thermomices lanuginosus, 100,000 u/g), PPL (porcine pancreatic lipase,
35,000 u/g), and ANL (free Aspergillus niger lipase, 120,000 u/g) were
1
1
1
selected as the enzymes. The conversions were determined by H NMR.
Table 1 shows the variation of the conversion degrees (in %) using different
lipases.
These results demonstrated that the highest yields, 99%, were obtained
when CALB was used in a biphasic system of water/toluene. This is in
[
5,14]
agreement with some literature data.
This suggests that this lipase is
more resistant to the toxic media formed by the hydrogen peroxide and
organic solvent. However, good results were also achieved when ROL
and PSL were used in their native form, yielding 84% and 53% cyclo-
hexene oxide, respectively. When lipases from MJL, CRL, and RML were
employed, also in their native form, very low conversions to cyclohexene
oxide were obtained. These were 12, 2, and ,1%, respectively. TLL, PPL,
and ANL were not appropriate for this conversion and no product was
detected. Apparently, there is not a direct relation between the epoxide con-
version degree and the enzyme activity.
[15]
The shape and the properties of binding sites for each lipase,
and
the possible denaturation caused by hydrogen peroxide and the organic
solvent, must also be considered. It is well known that a denaturation
Table 1. Chemo-enzymatic epoxidation of
a
cyclohexene catalyzed by different lipases
Lipase
Conversion (%)
CALB
ROL
PSL
.99
84
53
12
MJL
CRL
RML
TML
PPL
2
,1
b
n.d.
b
n.d.
b
ANL
n.d.
a
The reactions were performed with
mmol of cyclohexene, 10 mmol of
5
octanoic acid, 1 mL of hydrogen peroxide
0%, 50 mg of each lipase, biphasic
3
system: water/toluene at 308C for 24 h.
b
n.d. ¼ not detected.

2110
M. A. Moreira, T. B. Bitencourt, and M. da Gra c¸ a Nascimento
can occur because of a change in the microenvironment that surrounds these
biocatalysts, which causes a change in their structural conformations and a
[
16,17]
loss of activity.
The influence of temperature was also evaluated for the epoxidation of
cyclohexene catalyzed by CALB for 24 h. In the range of 30–508C, the cor-
responding epoxide was obtained in quantitative yields (.99%). Above
[18]
5
08C, the thermostability of this enzyme was maintained;
however, the
experimental conditions were not adequate, mainly because of hydrogen-
peroxide decomposition and/or organic-solvent evaporation. Thus, a tempera-
ture of 308C was selected for the following assays.
The influence of the organic solvent on the chemo-enzymatic epoxidation
of cyclohexene was also studied. The three lipases chosen for these screenings
were CALB, ROL, and PSL.
In most cases, the conversion in epoxide was dependent on the polarity of
the organic solvent (expressed by log P values) and on the lipase source.
However, there was not a linear relation with the solvent polarity.
Table 2 shows that the best yields were obtained in the water/toluene
system regardless of the lipase source. This is in agreement with previous
[14]
results.
High yields of cyclohexene oxide were also achieved when
water/xylene was used, these being 53%, 80%, and 84% using PSL, ROL,
and CALB, respectively. When chloroform was the organic solvent in the
biphasic system, reasonable conversions in epoxide were also achieved.
These were 50% and 65%, respectively, using PSL and CALB, and 37%
Table 2. Chemo-enzymatic epoxidation of cyclohexene in different biphasic
a
systems
Lipase conversion (%)
Pseudomonas
sp
Rhizopus
orizae
Candida
antarctica
Organic solvent
log P
Ethyl ether
Methyl dichloride
Chloroform
Toluene
0.85
0.93
2.0
28
22
37
37
84
57
30
99
65
99
99
b
20/41
50
53
12
2.5
3.0
Methyl
tetrachloride
Xylene
Hexane
3.1
3.5
4.0
40
23
21
80
43
42
84
32
44
Heptane
a
The reactions were performed with 5 mmol of cyclohexene, 10 mmol of octanoic
acid, 1 mL of hydrogen peroxide 30%, 50 mg of each lipase, biphasic system: water/
toluene at 308C for 24 h.
b
4
In a three-phase system of water/methyl dichloride/[Bmim][BF ].

Chemo-Enzymatic Epoxidation
2111
using ROL. Using CALB in methyl tetrachloride, hexane, and heptane, the
conversions were 99%, 32%, and 44% respectively. When ROL was used in
these solvents, the chemo-enzymatic epoxidation yielded considerable con-
versions. These were 57%, 43%, and 42%, respectively.
Using PSL, lower conversions in cyclohexene oxide were obtained. These
were in the range of 12–53%. However, an improvement was obtained
by the use of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate
(
[C mim][BF ]) in a three-phase system of water/methyl dichloride/ionic
4 4
liquid, increasing conversion from 20 to 41%. This result is also in agreement
with those reported by Lau et al. for the transesterification of ethyl butanoate
[
19]
and ammoniolysis of ethyl octanoate.
The systems studied here involve a biocatalytic perhydrolysis followed by
a peroxyacid epoxidation. Thus, the solvent chosen must be suitable for the
two reaction conditions and it is expected that its effect should be significant.
In the first step the best organic solvents are those with a log P between 2.0 and
[
12]
4
.0, and in the second are those that can stabilize the transition state (1). See
Scheme 2.
The effect of octanoic acid (acyl donor) concentration on the process of
epoxidation of cyclohexene was also investigated using CALB, ROL, and
PSL (Figure 1).
As shown, the epoxide was obtained in quantitative yields using CALB
and no dependence on the amount of acyl donor was observed in the range
of 2.5–12.5 mmol. When ROL was the biocatalyst, the conversion in
epoxide was in the range of 50–99%. The conversion was quantitative
when 12.5 mmol of octanoic acid was the acyl donor. Reasonable conversions
were obtained using PSL. These were in the range of 37–52%.
These data show that the conversion in the corresponding epoxide is more
dependent on the nature of the lipase than the concentration of the acyl donor.
This study demonstrated that the lipase-mediated synthesis of cyclohexene
oxide under mild conditions depends on various parameters such as the
lipase source, organic solvent, temperature, and acyl-donor concentration.
These results showed that these systems for enzyme-mediated epoxidation
of alkenes in biphasic media are feasible. Further work is in progress in our
laboratory to investigate other factors that can affect lipase stability. Also,
more complex alkenes are currently under investigation.
Scheme 2.

2112
M. A. Moreira, T. B. Bitencourt, and M. da Gra c¸ a Nascimento
Figure 1. Degree of cyclohexene epoxidation as a function of octanoic acid concen-
tration catalyzed by CALB, ROL, and PSL at 308C for 24 h, H
biphasic system: water/toluene.
2 2
O (30%, 1 mL),
EXPERIMENTAL
General
w
Commercial lipases from C. antarctica (Novozym 435 ), R. miehei
Palatase M), and T. lanuginosus (Lipolase) were gently donated by
Novozymes Latin America Ltda. (Brazil). Lipases from porcine pancreas
Amano PP), C. rugosa (Amano AY), Pseudomonas sp (Amano PS), R.
(
(
orizae (Amano F-AP15), M. javanicus (Amano M), and A. niger (Amano
A) were donated by Amano Pharmaceutical Co. (Japan). Ethyl ether was
obtained from Din aˆ mica; methyl dichloride and chloroform from Nuclear;
toluene from Merck; methyl tetrachloride, hydrogen peroxide 30%, and
octanoic acid from Vetec; and xylene, heptane, and hexane from Grupo
Qu ´ı mica. Cyclohexene was obtained from Sigma/Aldrich Chemical
Co. The ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate
[
20]
(
[C mim][BF ]) was prepared as described by Dupont et al.
4 4
1
The H NMR spectra were recorded in CDCl (Aldrich) with TMS
Sigma) as the internal reference on a Bruker AC200F spectrometer. All
3
(
reagents and solvents were analytical grade.
Chemo-emzymatic Epoxidation
In a typical experiment, distillated water (5 mL), an organic solvent (6 mL),
hydrogen peroxide 30% (1 mL, 20 mmol), cyclohexene (5 mmol), lipases

Chemo-Enzymatic Epoxidation
2113
from different sources (50 mg), and octanoic acid (2.5–12.5 mmol) were used.
All reagents were added in a 125-mL, round-bottom flask, and this system was
kept in a controlled temperature reactor (20–508C) for 24 h. The yields of the
1
crude product were evaluated by H NMR by comparing the integral value of
the olefinic hydrogen signal centered at 5.5 ppm with the oxirane-ring
hydrogen signal centered at 3.1 ppm.
ACKNOWLEDGMENTS
The authors are grateful to Universidade Federal de Santa Catarina (UFSC),
Conselho Nacional de Pesquisa e Desenvolvimento (CNPQ) for financial
support and scholarships (M.A.M., T.B.B., and M.G.N.) and Amano Pharma-
ceutical Co. and Novozymes Inc. for the donation of lipases. J. Dupont (IQ-
UFRGS) is also acknowledged for his generous gift of ionic liquid.
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