Esterification of (RS)-Ibuprofen by native and commercial lipases in a two-phase system containing ionic liquids

Article abstract of DOI:10.1016/j.tetasy.2006.07.020

Four commercially available lipases and two native lipases from Aspergillus niger AC-54 and Aspergillus terreus AC-430 were used for the resolution of (RS)-Ibuprofen in systems containing the ionic liquids [BMIM][PF₆] and [BMIM][BF₄]. The lipases showed higher conversion in a two-phase system using [BMIM][PF₆] and isooctane compared to that in pure isooctane. Although the best enzyme was a commercially available lipase from Candida rugosa (E = 8.5), another native lipase, produced in our laboratory, from A. niger gave better enantioselectivity (E = 4.6) than the other lipases tested (E = 1.9-3.3.). After thorough optimization of several reaction conditions (type and ratios of isooctane/ionic liquid, amount of enzyme, and reaction time), the E-value of A. niger lipase (15% w/v) could be duplicated (E = 9.2) in a solvent system composed of [BMIM][PF₆] and isooctane (1:1) after 96 h of reaction.

Full text of DOI:10.1016/j.tetasy.2006.07.020

Tetrahedron: Asymmetry 17 (2006) 2069–2073
Esterification of (RS)-Ibuprofen by native and commercial lipases
in a two-phase system containing ionic liquids
Fabiano Jares Contesini and Patr ´ı cia de Oliveira Carvalho*
Curso de Farm a´ cia, Universidade S a˜ o Francisco, Av. S a˜ o Francisco de Assis, 218, 12916-900 Bragan c¸ a Paulista, SP, Brazil
Received 5 June 2006; accepted 17 July 2006
Available online 10 August 2006
Abstract—Four commercially available lipases and two native lipases from Aspergillus niger AC-54 and Aspergillus terreus AC-430 were
used for the resolution of (RS)-Ibuprofen in systems containing the ionic liquids [BMIM][PF ] and [BMIM][BF ]. The lipases showed
6 4
higher conversion in a two-phase system using [BMIM][PF ] and isooctane compared to that in pure isooctane. Although the best
6
enzyme was a commercially available lipase from Candida rugosa (E = 8.5), another native lipase, produced in our laboratory, from
A. niger gave better enantioselectivity (E = 4.6) than the other lipases tested (E = 1.9–3.3). After thorough optimization of several
reaction conditions (type and ratios of isooctane/ionic liquid, amount of enzyme, and reaction time), the E-value of A. niger lipase
(
15% w/v) could be duplicated (E = 9.2) in a solvent system composed of [BMIM][PF ] and isooctane (1:1) after 96 h of reaction.
6
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
Ibuprofen is marked as racemic drug, but its activity
belongs to the (S)-(+)-enantiomer. It has been reported
that (S)-(+)-Ibuprofen is 160-fold more active than its anti-
pode in the synthesis of prostaglandin ‘in vitro’. Based
upon this, many studies has been made attempting to
resolve (RS)-Ibuprofen by different reactions, such as
Lipases are very used in several industrial applications
because of their ability in catalyzing different reactions such
as hydrolysis, esterification, and transesterification. These
enzymes are capable of recognizing the enantiomers of a
racemate resulting in an enantiopure compound. The use
of lipases in an organic solvent makes synthetic reactions
possible, that do not occur in the natural media of these
enzymes. However, these solvents can have disadvantages,
1
0
1
2
11
12
13
esterification, transesterification, and hydrolysis. Lip-
ases from Candida rugosa (cylindracea), Candida antarctica
(Novozyme 435) and Rhizomucor miehei have been used
1
4,15
to resolve the enantiomers of Ibuprofen,
napr-
3
16,17
18,19
such as being volatile and toxic for the environment, par-
oxen,
and ketoprofen
in organic solvents. A few
2
0
ticularly when they are used on a large scale. An alternative
for these organic solvents is the use of ionic liquids (ILs).
These are salts that have one or both of the large ions, an
organic cation and an inorganic anion, often fluid at room
temperature. These solvents possess several interesting
properties, such as ease of preparation, reuse, high thermal
stability, and low vapor pressure and the possibility to
manipulate at will its structure using different cations or an-
ions.4 The most commonly used ILs are 1-butyl-3-methyl
imidazolium hexafluorophosphate ([BMIM][PF6]) and
authors such as Hongwei et al. have exploited lipase
enantioselectivity in the resolution of (RS)-Ibuprofen in
solvent systems containing ILs. Herein, we report the
performance of native and commercial lipases in the enan-
tioselective esterification of Ibuprofen with 1-propanol
in systems containing the ILs [BMIM][PF6] and
[BMIM][BF ], with the results compared to those in pure
4
isooctane. Selected enzymes were optimized with respect
to the type of IL, ratios between isooctane and
,5
[BMIM][PF ], amount of enzymes, and reaction time.
6
tetrafluoroborate ([BMIM][BF ]). Different enzymes, such
4
as hydrolases have been shown to keep their activity and
6
,7
enantioselectivity in some ILs. Moreover, in some cases
2. Results and discussion
8
,9
they are higher than in some common organic liquids.
2.1. Enzyme screening
*
Corresponding author. Tel.: +11 4034 8298; fax: +11 4034 1825; e-mail:
patcarvalho@saofrancisco.edu.br
To study the influence of the ionic liquids (ILs) in the
resolution of (RS)-Ibuprofen catalyzed by lipases, we
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.07.020

2070
F. J. Contesini, P. de Oliveira Carvalho / Tetrahedron: Asymmetry 17 (2006) 2069–2073
designed an experiment wherein [BMIM][PF6] and
BMIM][BF ] were added as an additive (co-solvent) to
significantly (improved ꢀ50% compared to that in isooc-
[
tane) in a solvent system containing [BMIM][PF ]. This
4
6
the solvent isooctane. In the first stage, this experiment
was carried through using equal parts of IL (250 lL) and
isooctane (250 lL). This results in a system of two-phases,
since isooctane is immiscible with the ILs employed. Enzy-
matic reactions in pure isooctane systems were also carried
out in order to compare the efficiency of ILs for use as reac-
tion media. Figure 1 shows the esterification activity
expressed as a conversion degree (%) exhibited by both
lipases after 48 h of reaction.
was not observed when the mixture [BMIM][BF ] + isooc-
4
tane was used. In some cases such as RM and CR lipases,
this IL was harmful to the enzyme activity (reduction
ꢀ40% compared to that in pure isooctane). The two liquids
employed for the current study, [BMIM][PF ] and
6
[BMIM][BF ], are distinctly different in their properties,
4
such as hydrophobicity, polarity, anion nucleophilicity,
3
hydrogen-bond basicity, and viscosity. The [BMIM][BF ]
4
is highly hydrophilic in nature, whereas [BMIM][PF ] is
6
highly hydrophobic due to the different anions associated
with the common organic cation. These properties are of
primary concern in enzyme-catalyzed reactions, since they
50
45
40
35
30
25
20
15
10
5
0
% (isooctane)
%
%
(isoctane+[BMIM][PF6])
(isoctane+[BMIM][BF4])
are capable of influencing the conformation of the lipases
3,21
and consequently their reactivity.
The conversion
degree in the resolution of (RS)-Ibuprofen reduced in
the presence of [BMIM][BF ] probably due to stripping
4
6
of the essential water of the lipases. However, this area
is very new and much effort is needed in order to under-
stand the effect of different ionic liquids in biotransforma-
tion reactions.
As shown in Table 1, the conversion degree (c), the enan-
tiomeric excess (ee), and enantioselectivity (E-value) of
the different lipases in the esterification of the (RS)-Ibupro-
AN
AT
CA
CR
RM
TL
fen in absence or presence of [BMIM][PF ] were rather
6
heterogeneous.
Figure 1. Conversion degree (%) of the resolution of (RS)-Ibuprofen
obtained in the screening of several lipases in pure isooctane, iso-
octane + [BMIM][PF ] (1:1) and isooctane + [BMIM][BF ] (1:1). Lipase
6 4
abbreviations: RM: Rhizomucor miehei (Lipozyme RM IM); TL: Ther-
momyces lanuginose (Lipozyme TL IM); CA: Candida antarctica type B
The best conversion degree (48%), enantiomeric excess of
remaining acid (60%), and the E-value (8.5) were attained
with the C. rugosa lipase in a two-phase system containing
(Novozyme 435), CR: Candida rugosa, AN: Aspergillus niger AC-54, and
isooctane + [BMIM][PF ] (1:1) compared with a system in
6
AT: A. terreus AC-430.
pure isooctane. The conversion degree in the presence of an
ionic liquid was not significantly improved (42% to 48%),
but the E-value was doubled (4.1 to 8.5). The same behav-
ior was observed using a lipase native of A. niger. The E-
value was significantly increased (2.1 to 4.6) in the presence
The most efficient lipases in the esterification of (RS)-Ibu-
profen were from C. rugosa (CR), C. antarctica (CA),
and R. miehei (RM). The highest conversion degree (48%,
1
1
3
5%, and 25%, respectively) was obtained when two-phase
of [BMIM][PF ]. As described in our previous works, this
6
system containing [BMIM][PF ] + isooctane (1:1) was
lipase showed a stereopreference toward the (R)-(ꢁ)-enan-
tiomer and a moderate E-value has been achieved, which is
considerably higher than those results previously reported
6
used. The conversion degree increased 20–25% compared
to using pure isooctane as solvent media. The activity of
the native lipase from Aspergillus niger (AN) increased
1
8,22
using this lipase.
Table 1. Kinetic resolution of (RS)-ibuprofen catalyzed by native and commercial lipases in reaction media in pure isooctane or iso-
octane + [BMIM][PF
6
] (1:1) after 48 h of reaction
Lipases
Solvent
c (%)
ee (%)
E
Stereo preference
a
b
a
a
a
b
a
a
a
a
Aspergillus niger AC-54
Candida antarctica
Candida rugosa
Isooctane
Isooctane + [BMIM][PF
Isooctane
Isooctane + [BMIM][PF
Isooctane
Isooctane + [BMIM][PF
Isooctane
Isooctane + [BMIM][PF
Isooctane
Isooctane + [BMIM][PF
8
12
29
35
42
48
20
25
12
10
3
8
2.1
4.6
3.1
3.3
4.1
8.5
2.6
2.7
1.7
1.9
R
R
R
R
S
6
6
6
6
6
]
]
]
]
]
18
24
35
60
10
13
3
S
Rhizomucor miehei
Thermomyces lanuginosa
R
R
R
R
4
c = Conversion is given as the percentage of initial racemic ibuprofen esterified after the reaction time, ee = enantiomeric excess of the (S)-Ibuprofen
active, E = enantiomeric ratio.
Reaction conditions: (RS)-Ibuprofen (4 mM), 1-propanol (12 mM), lipase (5.0% w/v), pure solvent (250 lL), or mixture of organic solvent/ionic liquids
(1:1), 35 ꢁC, 300 rpm, 48 h.
The E-value means for each lipase with different superscript letters are significantly different p 60.05.

F. J. Contesini, P. de Oliveira Carvalho / Tetrahedron: Asymmetry 17 (2006) 2069–2073
2071
R. miehei and C. antarctica lipases displayed a good con-
version, but poor enantioselectivity, since they catalyzed
the esterification of both the (R)- and (S)-enantiomers of
Ibuprofen. These results indicated that commercial
C. rugosa and native A. niger lipases exhibited the highest
esterification activity and enantioselectivity in the presence
Ionic liquids have been studied as pure solvents27 and in a
two-phase system with another solvent such as organic sol-
2
4
28
vent and with supercritical carbon dioxide. Ganske and
2
9
Bornscheuer, reported that C. antarctica lipase (CALB-B
Chirazyme L2) do not show activity in the synthesis of
sugar esters in pure ionic liquids. However, it was possible
using a biphasic mixture containing 60% ionic liquid
([BMIM][PF ] or [BMIM][BF ]) and 40% of t-butanol as
of [BMIM][PF ] and were therefore chosen as the model
6
enzymes for continuity of this study.
6
4
solvent for the esterification reaction.
2
.2. Influence of ratios between isooctane and [BMIM][PF6]
2.3. Influence of time reaction
The amount of [BMIM][PF ] was progressively increased
6
from 0% to 100% v/v in the reaction mixture and the con-
version and enantiomeric excess were monitored after 48 h
Table 3 shows the conversion degree, the enantiomeric
excess of remaining acid, and the E-values as a function
of time reaction. The reactions were catalyzed by A. niger
and C. rugosa lipases in a solvent system composed of
(
Table 2).
5
0% and 30% of [BMIM][PF ], respectively.
6
6
Table 2. Influence of ratios between isooctane/[BMIM][PF ] (%) in the
kinetic resolution of (RS)-Ibuprofen catalyzed by Aspergillus niger and
Candida rugosa lipases
For A. niger lipase, in the range of 24–96 h, the conversions
were 6% and 15%, with enantiomeric excesses of 3% and
12%, resulting in E-values of 2.4 and 6.4. For lipase C. rug-
osa, after 24 h, more than 40% Ibuprofen propyl ester is
synthesized in a solvent system composed of 70% of iso-
Isooctane/
6
BMIM][PF ] (%)
Aspergillus niger lipase Candida rugosa lipase
[
c (%) ee (%)
E
c (%) ee (%)
E
a
a
b
b
a
a
100/0
70/30
50/50
30/70
0/100
8
13
12
18
9
3
7
8
13
5
2.1
3.2
4.6
4.1
3.1
42
45
48
38
38
35
56
60
36
34
4.1
9.1
8.5
5.3
4.7
octane and 30% of [BMIM][PF ], resulting in the E-value
b,c
d
6
of 9.3 with no further increase after prolonged reaction
times.
b,d
c
Reaction conditions: (RS)-Ibuprofen (4 mM), 1-propanol (12 mM), lipase
5.0% w/v), isooctane + [BMIM][PF ] (500 lL), 35 ꢁC, 48 h.
Table 3. Influence of reaction time in the kinetic resolution of (RS)-
Ibuprofen catalyzed by Aspergillus niger and Candida rugosa lipases
(
6
E-value means for each lipase with different superscript letters are signi-
ficantly different p 60.05.
Reaction time (h)
Aspergillus niger
lipase
Candida rugosa
lipase
c (%) ee (%)
E
c (%) ee (%)
E
The results indicate that the resolution of Ibuprofen occurs
more easily in the biphasic system, with isooctane and
a
b
b
b
b
1
2
48
72
96
2
4
6
6
12
14
15
0
3
8
10
12
ND 36
33
51
56
53
47
5.1
9.3
9.1
8.9
8.9
a
b
b
c
2.4
4.6
4.9
6.4
42
45
44
40
[
BMIM][PF ] than in pure ionic liquid or pure isooctane.
6
The best E-values were achieved by C. rugosa lipase at
0% [BMIM][PF ] (E = 9.1) and 50% [BMIM][PF ]
3
6
6
(
E = 8.5). For lipase from A. niger, a good result was found
ND: not detected.
using 50% of the [BMIM][PF ]. Similar results were found
Reaction conditions: (RS)-Ibuprofen (4 mM), 1-propanol (12 mM),
6
using 70% of the [BMIM][PF ]. This indicates that the
lipase (5.0% w/v), isooctane + [BMIM][PF ] (500 lL), 35 ꢁC.
6
6
presence of both isooctane and [BMIM][PF ] in the reac-
E-value means for each lipase with different superscript letters are signi-
ficantly different p 60.05.
6
tion mixtures, on the one hand causes the solubility of
the drug to increase, while on the other hand the esterifica-
tion causes the reaction to proceed with higher enantio-
selectivity compared to reactions in conventional
solvents. This IL, despite being polar due its ionic nature,
is hydrophobic, contrary to what is observed in the case
2.4. Effect of enzyme loading
The influence of the amount of lipases on the resolution of
(RS)-Ibuprofen in biphasic system is shown in Figures 2
and 3. The amounts used were 5%, 15%, 30%, and 50%
(w/v) and the products were determined after 96 and 24 h
in reactions catalyzed by A. niger and C. rugosa lipases,
respectively.
6
of most of the common polar organic solvents. This can
justify the highest enantioselectivity compared with pure
isooctane, since the enzyme needs a small amount of water
to maintain its structure.
It has previously been reported that the enantioselectivity
of lipases in different reactions can be increased by the
Using 5% and 15% (w/v) of lipase from A. niger the conver-
sion was 15% and 19%, with enantiomeric excesses of 12%
and 17%, resulting in E-value of 6.4 and 9.2, respectively.
Using 30% and 50% (w/v), better conversions (32% and
40%) and ee values (28% and 31%) were obtained. How-
ever, the E-values were 5.2 and 3.7 indicating the low selec-
tivity of A. niger lipase under these experimental
conditions. On the other hand, Ibuprofen can be obtained
2
3,24
use of ionic liquids,
while in other cases the enantio-
selectivity was similar in ionic liquid and in organic
2
5,26
20
solvents.
Hongwei et al. recently described a kinetic
resolution of Ibuprofen catalyzed by C. rugosa lipase
conducted in seven pure ionic liquids. Only in the case of
[
BMIM][PF ] was the E-value (E = 24.1) almost twice that
6
(
E = 13.0) in isooctane.

2072
F. J. Contesini, P. de Oliveira Carvalho / Tetrahedron: Asymmetry 17 (2006) 2069–2073
5
4
3
2
1
0
0
0
0
0
0
80
zymes Latin America Ltda (Arauc a´ ria, PR, Brazil). C.
rugosa lipase was purchased from Sigma Chemicals (St.
Louis, MO, USA) and native lipases from A. niger AC-
70
60
50
40
30
20
10
0
conversion degree (%)
enantiomeric excess (%)
ꢁ1
ꢁ1
54 (lipase activity 0.56 lmol min mg for the hydrolysis
of olive oil at pH 7.0 and 40 ꢁC) and Aspergillus terreus
ꢁ
1
ꢁ1
AC-430 (lipase activity 0.47 lmol min mg
for the
hydrolysis of olive oil at pH 6.0 and 40 ꢁC) were obtained
3
0
in our laboratory and stored from the culture collection
of the Laboratory of the University S a˜ o Francisco (Brazil).
Ionic liquids, 1-butyl-3-methyl imidazolium tetrafluoro-
borate [BMIM][BF ], 1-butyl-3-methyl imidazolium hexa-
4
fluorophosphate [BMIM][PF6], isooctane, 1-propanol,
and (S)-Ibuprofen were purchased from Sigma–Aldrich
Chemical Co. (St. Louis, MO, USA). All other chemical
reagents were obtained from Merck (Darmstadt, Germany)
or Sigma–Aldrich Chemical Co. in the highest purity
available.
0
10
20
30
40
50
native lipase Aspergillus niger (% (w/v))
Figure 2. Effect of amount of Aspergillus niger lipase (% w/v) in the kinetic
resolution of (RS)-Ibuprofen in solvent system composed of 50% of
isooctane and 50% of [BMIM][PF
6
] after 96 h.
4.2. General procedure for enzymatic reactions
5
4
3
2
1
0
0
0
0
0
0
80
In a typical experimental procedure, 4 mM (RS)-Ibuprofen
and 12 mM 1-propanol were placed into screw-capped vials
and dissolved in 250 lL of the isooctane. Five percent
(w/v) enzyme, ten percent (w/v) activated molecular
70
60
50
40
30
20
10
0
˚
sieves (4 A), and 250 lL of the ionic liquids [BMIM][PF ]
6
(1-butyl-3-methyl imidazolium hexafluorophosphate) or
[
BMIM][BF ] (1-butyl-3-methyl imidazolium tetrafluoro-
4
borate) were added. The reaction mixtures were shaken
in a New Brunswick with orbital magnetic stirring at
conversion degree (%)
enantiomeric excess (%)
300 rpm at 35 ꢁC. At regular time intervals, 500 lL of the
isooctane was added and the biphasic mixture strongly sha-
ken for 3 min to extract all substrates and products into the
isooctane phase. Then, 50 lL aliquots of the resultant solu-
tion were withdrawn in the isooctane phase and the
amount of ester (conversion degree) formed during the
reaction and the enantiomeric excess of the (S)-enantiomer
determined by GC and by HPLC, respectively, are as
described below.
0
10
20
30
40
50
commercial lipase Candida rugosa (% (w/v))
Figure 3. Effect of the amount of Candida rugosa lipase (% w/v) in the
kinetic resolution of (RS)-Ibuprofen in solvent system composed of 70%
6
of isooctane and 30% of [BMIM][PF ] after 24 h.
4.3. Chromatography analysis
with good ee (69%) and E-value (12) quenching the reac-
tion near 50% conversion after 24 h reaction using 50%
Gas chromatography was performed using a CHROM-
PACK CP 9001 gas chromatography equipped with flame
ionization detector (FID) and a CP-Sil 5 CB column
(
w/v) of C. rugosa lipase (Fig. 3).
(10 m · 0.25 mm · 0.12 lm). Injector temperature was
3
. Conclusion
300 ꢁC and the detector was 350 ꢁC; an oven temperature
was maintained at 150 ꢁC. The carrier gas was hydrogen
with a flow rate of 12 mL/min. An external standard meth-
od was employed to quantify the formed ester and the
remaining acid. The enantiomers of the unreacted substrate
were separated by HPLC using a chiral column (Chiralcel
OD, Daicel Chemical Industries, Ltd., Japan). The mobile
phase was a mixture of n-hexane/isopropanol/trifluoroace-
tic acid (100:1:0.1 v/v/v) at a flow rate of 1.0 mL/min and
detection was by UV at 254 nm.
Although the E-values throughout this study were not high
enough for industrial application, the significant increase in
the enantioselectivity in the PF anion containing ionic
liquids is promising in a biocatalysts reaction especially
when we consider the fact that ionic liquids are considered
as green solvents and can be reused.
6
4
. Experimental
4.4. Enantioselectivity-value measurements
4
.1. Enzymes and chemical
The value of enantioselectivity (E-value) was calculated
Immobilized lipases from R. miehei (Lipozyme RM IM),
Thermomyces lanuginosa (Lipozyme TL IM), and C. ant-
arctica type B (Novozyme 435) were donated by Novo-
from the enantiomeric excess of the substrate (ee ) and
s
the conversion degree (c) according to the method
3
1
described by Chen et al. (Eq. 1):

F. J. Contesini, P. de Oliveira Carvalho / Tetrahedron: Asymmetry 17 (2006) 2069–2073
2073
ln½ð1 ꢁ cÞð1 ꢁ ee Þꢂ
9. Ulbert, O.; B e´ lafi-Bak o´ , K.; Tonova, K.; Gubicza, L.
Biocatal. Biotransform. 2005, 23, 177–183.
s
E ¼
ð1Þ
ln½ð1 ꢁ cÞð1 þ ee Þꢂ
s
1
0. Adams, S. S.; Bresloff, P.; Mason, G. C. J. Pharm. Pharma-
col. 1976, 28, 156–157.
Using the equations discussed above, the expected enantio-
meric excess of substrate (ee ) and product can be calcu-
11. Carvalho, P. O.; Calafatti, A. S.; Marassi, M.; Silva, D. M.;
s
Contesini, F. J.; Bizaco, R. Quim. Nova 2005, 28, 614–621.
lated for a chosen point of conversion and the E-value
can be determined as a convenient constant value for the
selectivity of an enzymatic resolution.
1
1
2. Henke, E.; Schuster, S.; Yang, H.; Bornscheuer, U. T.
Monatsh. Chem. 2000, 131, 633–638.
3. Kumar, I.; Manju, K.; Jolly, R. Tetrahedron: Asymmetry
2
001, 12, 1431–1434.
4
.5. Statistical analysis
1
1
4. Ikeda, Y.; Kurokawa, Y. J. Biosci. Bioeng. 2002, 93, 98–100.
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17. Sakaki, K.; Giorno, L.; Drioli, E. J. Membr. Sci. 2001, 184,
27–38.
One-way analysis of variance (ANOVA) and Tukey’s stu-
dentized range test were used to determine the differences
in mean E-values based on data collected from three repli-
cations of each measurement. Significance was established
at p 6 0.05.
1
1
2
2
8. D’ Antona, N.; Lombardi, P.; Nicolosi, G.; Salvo, G. Process
Biochem. 2002, 38, 373–377.
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The authors gratefully acknowledge financial support from
FAPESP (Processo 02/06807-9) and CNPq/PIBIC.
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Hammock, B. D.; Morisseau, C. J. Mol. Catal. B: Enzym.
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