Potential application of native lipases in the resolution of (RS)-phenylethylamine

Article abstract of DOI:10.1590/S0103-50532010000600005

The performance of two native lipases (lipase from Aspergillus niger and Rhizoupus oligosporus) in the resolution of (RS)-phenylethylamine (1), varying the temperature, acyl donor type (ethyl acetate, vinyl acetate, iso-propenyl acetate and acetic anhydride) and organic medium, was studied. The effect of the nature of the anion using native A. niger lipase in n-heptane with a series of imidazolium-based ILs [BMIm][X], where X = BF₄, PF₆, SCN and Cl, was also evaluated. Using the lipase from A. niger, the R-2b amide was obtained with conversions from 6 to > 99percent and E-values from 2 to > 200, with n-heptane or n-hexane. This lipase showed better E-values in a two-phase system using n-heptane and [BMIm][PF₆] or [BMIm][BF₄] 9:1 (v/v), obtaining values of 9 and 7, respectively, when vinyl acetate was used as the acyl donor, compared to the use of pure n-heptane (E = 2). The series for the anions in terms of decreasing performance was as follows: PF ₆->BF₄-> SCN-> Cl^-.

Full text of DOI:10.1590/S0103-50532010000600005

J. Braz. Chem. Soc., Vol. 21, No. 6, 973-977, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Potential Application of Native Lipases in the Resolution of (RS)-Phenylethylamine
a
b
,a
Cristiane Pilissão, Patrícia de O. Carvalho and Maria da G. Nascimento*
a
Departamento de Química, Universidade Federal de Santa Catarina,
8
8040-900 Florianópolis-SC, Brazil
b
Curso de Farmácia, Universidade São Francisco, 12916-900 Bragança Paulista-SP, Brazil
O desempenho de duas lipases nativas (lipase de Aspergillus niger e Rhizopus oligosporus), e a
influência da temperatura, doador acila (acetato de etila, acetato de vinila, acetato de iso-propenila
e anidrido acético) e do meio orgânico foi avaliado na resolução da (RS)-feniletilamina (1). O efeito
de anions em uma série de líquidos iônicos (LIs) baseados em derivados do imidazol [BMIm][X],
onde X = BF , PF , SCN e Cl em n-heptano e acetato de vinila, também foi verificado com a lipase
4
6
nativa de A. niger. Com esta lipase, a amida R-2b foi obtida com conversões de 6 até > 99% e
valores de E de 2 até > 200, em n-heptano ou n-hexano. Os maiores valores de E obtidos, usando
sistema bifásico formado por n-heptano e [BMIm][ PF ] ou [BMIm][ BF ] 9:1 (v/v), foram de 9
6
4
e 7, respectivamente, quando comparado com o solvente puro (E = 2). A influência dos ânions
–
6
–
4
–
–
segue a seguinte ordem: PF >BF > SCN > Cl .
The performance of two native lipases (lipase from Aspergillus niger and Rhizoupus
oligosporus) in the resolution of (RS)-phenylethylamine (1), varying the temperature, acyl donor
type (ethyl acetate, vinyl acetate, iso-propenyl acetate and acetic anhydride) and organic medium,
was studied. The effect of the nature of the anion using native A. niger lipase in n-heptane with a
series of imidazolium-based ILs [BMIm][X], where X = BF , PF , SCN and Cl, was also evaluated.
4
6
Using the lipase from A. niger, the R-2b amide was obtained with conversions from 6 to > 99%
and E-values from 2 to > 200, with n-heptane or n-hexane. This lipase showed better E-values
in a two-phase system using n-heptane and [BMIm][PF ] or [BMIm][BF ] 9:1 (v/v), obtaining
6
4
values of 9 and 7, respectively, when vinyl acetate was used as the acyl donor, compared to the
use of pure n-heptane (E = 2). The series for the anions in terms of decreasing performance was
–
6
–
–
–
as follows: PF >BF > SCN > Cl .
4
Keywords: native lipases, amine, resolution, ionic liquids
Introduction
species belong to the genera Rhizopus, Rhizomucor, Mucor,
Geotrichum, Aspergillus and Penicillinium.
4
Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3)
are the most widely applied enzymes in regio-selective
and enantioselective biotransformations, since they are
inexpensive, stable and easy to recycle. Lipases can be
found in many animal and plant tissues. They can also be
produced by fermentation processes using several microbial
species, namely fungi and bacteria. From the economic
and industrial standpoints, microorganisms are preferable
to animals and plants as enzyme sources. Filamentous
fungi are preferred sources of lipases since they tend to
produce extracellular enzymes, facilitating extraction from
the fermentation growth medium. The most productive
Lipases possess wide substrate specificity and catalyze
many reversible reactions in aqueous and non-aqueous
media, such as esterification, transesterification, amidation,
1,5-10
hydrolysis, epoxidation and dipeptide synthesis.
1-3
Amides are a very important class of organic compounds
with a wide range of applications. Some amide derivatives
exhibit biological properties such as anthelmintic,
11,12
antihistamine, antifungal and antibacterial properties.
Numerous studies involving enzymatic aminolysis
7
,11-13
have been reported recently.
The catalytic activity
and selectivity of lipases can be improved by changing the
1
1,14
solvent, the acyl donor or both.
The lipase-catalyzed
resolution of racemic amines has been successfully carried
1
5
*e-mail: graca@qmc.ufsc.br
out in low polarity organic solvents.

9
74
Potential Application of Native Lipases in the Resolution of (RS)-Phenylethylamine
J. Braz. Chem. Soc.
7
Wen et al. reported the aminolytic activity of an
gained wide popularity not only in conventional synthetic
chemistry, but also in biotransformation processes.
2
1
immobilized extracellular lipase (LIP2) from Yarrowia
lipolytica using (±)-a-phenylethylamine with an acetic
ester in a medium containing a co-solvent. When the
resolution of the (±)-a-phenylethylamine was carried out in
a single organic solvent (viz. hexane), the enantioselectivity
of the lipase was extremely low, but with the addition of
Considering the extraordinary microbial diversity and
importance of the fungi as enzyme producers, this paper
reports the performance of two native lipases (Aspergillus
niger and Rhizopus oligosporus) in the resolution of
(RS)-phenylethylamine (1). Other parameters included the
influence of temperature, acyl donors (ethyl acetate, vinyl
acetate, iso-propenyl acetate and acetic anhydride) and the
organic medium. The effect of anions on the resolution of
(RS)-1 using A. niger in pure n-hexane or n-heptane or in
mixtures with ionic liquids from a series of imidazolium-
based ILs [BMIm][X], where X = BF , PF , SCN and Cl
3
% (v/v) DMSO to the hexane, the enantiomeric excess
of product (ee ) markedly increased from 0.35 (in pure
p
hexane) to 0.96 and the enantiomeric ratio (E) improved
from 2.5 (in pure hexane) to 190. The immobilized lipase
could be reused for at least five consecutive batches at
7
high E (@ 190).
4
6
Recently, biocatalysis in non-aqueous media has been
extensively used for the resolution of alcohols and amines,
but the use of solvents has certain disadvantages, such as
being volatile and toxic to the environment, particularly
was also evaluated (Scheme 1).
Experimental
13
when used on a large scale. The use of ionic liquids (ILs)
has emerged contemporarily in organic synthesis, and in
some cases can be highly efficient in biocatalysis. These
are salts that have an organic cation and an inorganic
anion and are often fluid at room temperature with a
Materials and methods
-
1
Lipases from A. niger AC-54 (19.4 U mL ) and R.
-
1
oligosporus (14.9U mL ) were isolated and purified,
and the activity determined as previously described
o
23
4,20
melting point below 100 C. Ionic liquids can be used in
by Carvalho et al. and Carvalho and co-workers
an enzymatic system in three different ways: as co-solvent
in the aqueous phase, as a pure solvent and as a two-phase
system together with other solvents. These solvents possess
several interesting properties, such as ease of preparation,
reuse, high thermal stability and low vapor pressure. They
also have widely regulated assets with regard to polarity,
hydrophobicity and solvent miscibility behavior, by means
(RS)-phenylethylamine was purchased from Sigma-
Aldrich. The reagents n-hexane, n-heptane, ethyl acetate,
vinyl acetate, acetic anhydride and iso-propenyl acetate
were obtained fromVetec (Brazil) and Sigma-Aldrich in the
highest purity available. The ionic liquids 1-butyl-3-methyl
imidazolium thiocyanide [BMIm][SCN] (≥ 98%), 1-butyl-
3-methyl imidazolium chloride [BMIm][Cl] (≥ 98%),
1-butyl-3-methyl imidazolium hexafluorphosphate
1
4,15
of appropriate modification of the cation and anion.
In addition, the use of ILs in biocatalysis can enhance
enzyme stability, substrate and/or product selectivity, and
[BMIm][PF ](≥ 98%) and 1-butyl-3-methyl imidazolium
6
tetrafluoroborate [BMIm][BF ] (≥ 97%) were purchased
4
1
3,14-17
can suppress side reactions.
Although enzymatic
from Sigma-Aldrich or Fluka.
transformations in ionic liquids have only been considered
since the beginning of the 21 century, a wide number
The amount of water in the free enzymes was evaluated
by Karl-Fisher titration and they contained 10-12%, which
is the amount necessary to preserve the catalytic activity.
All other solvents were of analytical grade and were dried
by storing over activated 3 Å molecular sieves before use.
st
24
of applications have already been tested in this field,
such as esterification, transcyanidation, oxidoreduction,
16,18-22
hydrogenation, amidation and acylation,
and ILs have
Scheme 1. Resolution of (RS)-phenylethylamine (1) with acyl donors mediated by native lipases.

Vol. 21, No. 6, 2010
Pilissão et al.
975
General procedure for the lipase-catalyzed resolution of
solvents, and in mixtures of the organic solvents with ionic
liquids. Also, the influence of the temperature and acyl
donors was evaluated in subsequent experiments.
(RS)-1 with different acyl donors
A solution of (RS)-phenylethylamine (1) (2 mmol;
0.26 mL) and ethyl acetate, vinyl acetate, acetic anhydride
Effect of reaction temperature
or iso-propenyl acetate (8 mmol) was added to the lipases
(
50 mg) in n-heptane or hexane (25 mL) or in mixtures with
Temperature has a significant influence on the activity,
selectivity and stability of a biocatalyst and also on the
equilibrium of a reaction. Thus this effect was investigated
in the resolution of (RS)-1 at three different temperatures
ranging from 25 to 45 C. The results showed that both the
conversions and the enantiomeric ratios were dependent on
this parameter (Figure 1).
o
ionic liquids (expressed by v/v) at 25-45 C. In each case,
the mixture was shaken on an orbital shaker (100 rpm).
The reaction progress and enantiomeric excess values
were measured using a gas chromatograph (GC) equipped
with a chiral column (CP-chirasil-Dex CB, packed with
b-cyclodextrin, 25m × 0.25mm × 0.25mm,Varian). For the
o
analysis, H was used as the carrier gas, the detector and
2
injector were set at 230 °C, and the column was raised from
o
o
-1
6
0 to 200 C at 3 C min . The retention times of S-amide 2a
and R-amide 2b were 24.6 and 25.5 min, respectively, and
these data were also compared with enantiopure standard
compounds using chiral gas chromatography.
The enantiomeric ratio (E-value) was calculated from
the enantiomeric excess of the product (ee ), and the
p
conversion degree (c) according to the method described
2
5
by Chen et al.
Results and Discussion
Figure 1. Effect of temperature (°C) on the conversion degree (%) in the
resolution of (RS)-phenylethylamine (1) with ethyl acetate catalyzed by
native lipase from A. niger (50 mg) in n-heptane (25 mL) at () 25 °C,
() 35 °C, (p) 45 °C.
Screening of native lipases
In the first stage of this study, two native lipases
obtained from A. niger and R. oligosporus were screened
for the resolution of (RS)-phenylethylamine (1) with ethyl
The results showed that the conversion degrees
increased (from 7 to 34%) as the temperature increased
from 25 to 45 C, after 96 h of reaction. The conversion
o
o
acetate in pure heptane or hexane at 35 C.
degrees were similar in the temperature range from
o
When the lipase from R. oligosporus was employed
in its native form in heptane or hexane, no product was
detected until 144 h of reaction time.
However, better results were obtained when the lipase
from A. niger in its native form was employed using
n-hexane or n-heptane as the organic solvent, obtaining
conversion degrees of 1 and 30%, respectively, with an
enantiomeric excess of the product of 27% and > 99%,
resulting in E-values of 2.4 and > 200 after 96 h of reaction.
Recently, when CAL-B was used as the biocatalyst,
35-45 C. These results are in agreement with those reported
4
in the literature. Carvalho and co-workers and Carvalho et
2
3
al. reported that the lipases isolated from Brazilian soil
samples, such as those from A. niger, Geotrichum candidum
and Penicillium solitum, were stable and maintained 100%
o
of activity in the temperature range from 35-45 C.
o
At temperatures of 25, 35 and 45 C, the E-values
remained constant (> 200) after 96 h of reaction.
o
Considering the above results, a temperature of 35 C was
selected for use in the study of the influence of the acyl
similar conversion, ee and E-values were obtained for the
donor and of ionic liquids in the resolution of (RS)-1.
p
resolution of (RS)-1, giving values for these parameters of
18
2
3-45%; > 99% and > 200, respectively. These results
Influence of the acyl donor
showed that the lipase from A. niger presented good
potential for use in this type of reaction. This native lipase
was previously used in organic media in the resolution of
For the resolution of (RS)-phenylethylamine (1)
catalyzed by the lipase from A. niger in n-heptane, four
different acyl donors (ethyl acetate, iso-propenyl acetate,
vinyl acetate and acetic anhydride) were screened, and the
results are presented in Table 1.
4
,20,26
Ibuprofen.
Based on these results, A. niger lipase was selected as
the catalyst for the evaluation of the effect of the organic

9
76
Potential Application of Native Lipases in the Resolution of (RS)-Phenylethylamine
J. Braz. Chem. Soc.
Table1.Effectoftheacyldonorintheresolutionof(RS)-phenylethylamine(1)
catalyzed by native lipase from A. niger
9
:1 (v/v) where X = Cl, SCN, PF and BF were selected,
6
4
in order to evaluate the influence of the IL and the anions
in the enzymatic resolution of (RS)-1 with vinyl acetate.
The four ILs employed for the current study [BMIm]
a
b
Entry
Acyl donor
iso-propenyl acetate
acetic anhydride
vinyl acetate
time / h c / (%) eep / (%) E-value
1
2
3
4
5
6
0.30
0.30
0.30
48
74
> 99
58
6
1.2
1
[
PF ], [BMIm][BF ], [BMIm][SCN] and [BMIm][Cl] have
1
6
4
distinctly different properties in terms of hydrophobicity,
polarity, anion nucleophilicity, hydrogen-bond basicity and
viscosity. Since these properties influence the conformation
of the lipases and consequently their reactivity, these
22
2
ethyl acetate
6
> 99
> 99
> 99
> 200
> 200
> 200
ethyl acetate
72
27
ethyl acetate
96
30
7,28
properties are of primary concern.
Reaction conditions: (RS)-phenylethylamine (1) (2 mmol); acyl donor
8 mmol); A. niger (50 mg); n-heptane (25 mL), at 35 ºC. Enantiomeric
The results obtained were compared with pure
a
(
n-heptane after 30 min of reaction (Figure 2). For the
b
excess of amide 2b; Enantiomeric ratio.
–
–
weakly chaotropic anions Cl and SCN , the conversion
The conversion degrees were 74%, > 99% and 58%
degrees were 67 and 25%, and the ee values 3 and 13%,
p
when iso-propenyl acetate, acetic anhydride and vinyl
resulting in E-values of 1 and 1.5, respectively.
acetate were used, respectively. However, the ee values and
p
E-values were 6, 1 and 22% and 1.2, 1 and 2, respectively,
for 30 min of reaction.
When ethyl acetate was used low conversion degrees
were obtained, but the enantiomeric excess of product and
E-values were much better. The conversion degrees were
from 6-30%, resulting in ee values > 99% and E-values >
p
2
00 after 96 h of reaction.
In the resolution of (RS)-1, the native lipase from A.
niger gave a better result than that obtained in the resolution
2
0
of (RS)-Ibuprofen (E 2.4-6.4). Thus, the choice of a
particular substrate structure is also of interest and may
influence the E-values of the biocatalytic process.
Figure 2. Effect of the anions of the ILs on the resolution of
As observed and discussed, the structure of the acyl
donor is crucial. Vinyl acetate, iso-propenyl acetate and
acetic anhydride proved to be good acyl donors, forming the
product with good conversion values, but the E-values were
low. Using ethyl acetate, the conversions were moderate
(
RS)-phenylethylamine (1) (2 mmol) with vinyl acetate (8 mmol) using
native lipase from A. niger (50 mg), n-heptane:[BMIm][X] (9:1 v/v) at
3
5 °C, 30min.
Better results were obtained when the strongly
–
–
but gave high ee and E-values.
chaotropic anions, BF4 and PF6 were investigated. The
p
These results may improve by using ionic liquids
conversion degrees were 27 and 49%, and the ee values
p
(
ILs) in mixtures with n-heptane, as will be discussed in
62 and 73%, resulting in E-values of 7 and 9, respectively.
It should also be noted that the E-value increased from 2
in pure n-heptane to 9 using the n-heptane:[BMIm][PF6]
mixture. Thus, these data show the dependence of the
E-values on the presence of the anions in the ILs. In the
reaction studied, the ion effectiveness in enhancing the
the following section, and vinyl acetate was selected for
this study.
The stereopreference was always towards the
(
R)-enantiomer and these results were verified by
comparison with enantiopure samples of S- and R-amides
a and 2b using chiral gas chromatography.
–
–
–
2
enzyme selectivity followed the series: PF > BF > SCN >
6 4
–
Cl in [BMIm][X]/n-heptane mixtures, with vinyl acetate
as the acyl donor. The decreasing order of the anion effect
was the same as that obtained in the reaction catalyzed by
CAL-B in the resolution of (RS)-phenylethylamine, using
Effect of ionic liquid
Ionic liquids are increasingly used as reaction media
in organic chemistry since they offer a wide range of
advantages over classical organic solvents. Basically, the
interest in using ionic liquids in biocatalysis is the desire
to replace volatile organic solvents by non-volatile ionic
1
8
ethyl acetate in chloroform:[BMIm][X] mixtures.
In the presence of iso-octane:[BMIm][PF ] mixtures in
6
various proportions, using the native lipase from A. niger
in the resolution of (RS)-Ibuprofen, the E-value increased
2
7
20
liquids. Initially mixtures of n-heptane:[BMIm][X]
from 2.1 to 4.6.

Vol. 21, No. 6, 2010
Pilissão et al.
977
These studies showed that, in general, enzymes can
maintain their activity and stability in certain ionic media.
However, not all ILs are suitable for maintaining the activity
of an enzyme in a particularly biocatalytic reaction. Thus,
there is no simple answer to whether an enzyme is active in
a certain IL because the enzyme activity is also dependent
on the enzyme-medium-substrate relationship.
5. Wang, Y.; Li, Q.; Zhang, Z.; Ma, J.; Feng, Y.; J. Mol. Catal. B:
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. Carvalho, P. O.; Campos, P. R. B.; Noffs, M. D.; Fregolente, P.
Conclusions
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1
0. Liria, C. W.; Romagna, C. D.; Rodovalho, N. N.; Marana, S.
Inthispaper, theresolutionof(RS)-phenylethylamine(1)
was studied using two native lipases (Aspergillus niger and
Rhizoupus oligosporus) in pure n-hexane or n-heptane
and in mixtures with ionic liquids from a series of
imidazolium-based ILs [BMIm][X], where X = BF , PF ,
R.; Miranda, M. T. M.; J. Braz. Chem. Soc. 2008, 19, 1574.
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4
6
SCN and Cl. The influence of temperature and the acyl
donors was also considered. With respect to conversion,
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14. González-Sabín, J.; Gotor, V.; Rebolledo, F.; J. Org. Chem.
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ee and E-values, better results were obtained using the
p
lipase from A. niger in n-heptane and ethyl acetate as
the acyl donor at 35 C (E > 200). Using n-heptane:ILs
15. Kim, K. W.; Song, B.; Choi, M.Y.; Kim, M. J.; Org. Lett. 2001,
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o
mixtures with vinyl acetate, the E-values were not very high
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(
E = 1-9) but were better than that obtained with pure
n-heptane (E = 2). The series for the anions was as follows:
–
–
4
–
–
PF >BF > SCN > Cl .
18. Pilissão, C.; Carvalho, O. P.; Nascimento, M. G.; Process
Biochem. 2009, 44, 1352.
6
The results presented in this paper show that the
appropriate choice of reaction medium and the nature of the
acyl donor appear to be the key to obtaining products with a
high enantiomeric ratio and good conversion values, and the
search for new biocatalysts with particular characteristics
and possible biocatalytic applications is of great interest.
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Acknowledgments
2
2. Torres-Gavelán, A.; Escalante, J.; Regla, I.; López-Munguía,
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This study was supported by the Universidade Federal
de Santa Catarina (UFSC-Brazil), the Universidade
de São Francisco (USF), the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq-Brazil)
and FAPESP (Process 02/06807-9), all of which provided
financial support and/or scholarships (M.G.N and C.P).
23. Carvalho, P. O.; Calafatti, S. A.; Marassi, M.; Silva, D. M.;
Contesini, F. J.; Bizaco, R.; Quim. Nova 2005, 28, 614.
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