Enzymatic hydrolytic resolution of racemic ibuprofen ethyl ester using an ionic liquid as cosolvent

Article abstract of DOI:10.3390/molecules21070905

The aim of this study was to develop an ionic liquid (IL) system for the enzymatic resolution of racemic ibuprofen ethyl ester to produce (S)-ibuprofen. Nineteen ILs were selected for use in buffer systems to investigate the effects of ILs as cosolvents for the production of (S)-ibuprofen using thermostable esterase (EST10) from Thermotoga maritima. Analysis of the catalytic efficiency and conformation of EST10 showed that [OmPy][BF₄] was the best medium for the EST10-catalyzed production of (S)-ibuprofen. The maximum degree of conversion degree (47.4%), enantiomeric excess of (S)-ibuprofen (96.6%) and enantiomeric ratio of EST10 (177.0) were achieved with an EST10 concentration of 15 mg/mL, racemic ibuprofen ethyl ester concentration of 150 mM, at 75°C, with a reaction time of 10 h. The reaction time needed to achieve the highest yield of (S)-ibuprofen was decreased from 24 h to 10 h. These results are relevant to the proposed application of ILs as solvents for the EST10-catalyzed production of (S)-ibuprofen.

Full text of DOI:10.3390/molecules21070905

molecules
Article
Enzymatic Hydrolytic Resolution of Racemic
Ibuprofen Ethyl Ester Using an Ionic Liquid
as Cosolvent
Tao Wei 1,2, Kunpeng Yang 1,2, Bing Bai 1, Jie Zang 1, Xuan Yu 1 and Duobin Mao 1,2,
*
1
School of Food and Biological Engineering, Zhengzhou University of Light Industry, 5 Dongfeng Rd.,
Zhengzhou 450002, China; weit8008@zzuli.edu.cn (T.W.); 15238358950@163.com (K.Y.);
baibing82@163.com (B.B.); pds0188@126.com (J.Z.); 15038262577@126.com (X.Y.)
Collaborative Innovation Center of Food Production and Safety, 136 Science Avenue,
Zhengzhou 450002, China
2
*
Correspondence: duobinmao@126.com; Tel./Fax: +86-371-8660-9631
Academic Editor: Steven Bull
Received: 12 June 2016; Accepted: 7 July 2016; Published: 13 July 2016
Abstract: The aim of this study was to develop an ionic liquid (IL) system for the enzymatic resolution
of racemic ibuprofen ethyl ester to produce (S)-ibuprofen. Nineteen ILs were selected for use in
buffer systems to investigate the effects of ILs as cosolvents for the production of (S)-ibuprofen using
thermostable esterase (EST10) from Thermotoga maritima. Analysis of the catalytic efficiency and
conformation of EST10 showed that [OmPy][BF4] was the best medium for the EST10-catalyzed
production of (S)-ibuprofen. The maximum degree of conversion degree (47.4%), enantiomeric
excess of (S)-ibuprofen (96.6%) and enantiomeric ratio of EST10 (177.0) were achieved with an EST10
concentration of 15 mg/mL, racemic ibuprofen ethyl ester concentration of 150 mM, at 75 ˝C , with
a reaction time of 10 h. The reaction time needed to achieve the highest yield of (S)-ibuprofen was
decreased from 24 h to 10 h. These results are relevant to the proposed application of ILs as solvents
for the EST10-catalyzed production of (S)-ibuprofen.
Keywords: cosolvent; enzymatic; ionic liquid; racemic ibuprofen; (S)-ibuprofen
1. Introduction
Ibuprofen (2-(4-isobutylphenyl)propionic acid) is an important non-steroidal anti-inflammatory
drug that is widely used for the treatment of headaches, rheumatoid arthritis, and muscular
strains [1–3]. Commercially available ibuprofen is usually a racemic mixture, as the racemate is
easily obtained through chemical synthesis. However, (S)-ibuprofen is about 160 times more active
than (R)-ibuprofen. Moreover, the presence of (R)-ibuprofen can cause serious side effects, such as
gastrointestinal tract irritation, and disruption of normal lipid metabolism and membrane function [4,5].
Therefore, much effort has been therefore devoted to the production of pure (S)-ibuprofen. Among the
approaches for obtaining a single enantiomer of ibuprofen, hydrolase-catalyzed enzymatic kinetic
resolution has attracted considerable interest because of its enantioselectivity, regioselectivity, and
substrate specificity under mild conditions [6,7].
Ionic liquids (ILs) are salts made up of ions that remain liquid at room temperature or
˝
temperatures below 100 C. ILs have unique properties that make them useful as green solvents
for many biocatalytic reactions [ ]. They are non-volatile, non-flammable, and have excellent
chemical and thermal stability, which can provide enzymes with excellent levels of activity, stability,
and stereoselectivity [10 11]. For enzyme catalysis, the activity and stability of enzymes are closely
8,9
,
related to the physicalchemical properties of ILs (e.g., polarity, viscosity, hydrogen bond basicity,
hydrophobicity and anion concentration) [12]. The use of enzymes, especially lipases, in ionic liquids,
Molecules 2016, 21, 905; doi:10.3390/molecules21070905
www.mdpi.com/journal/molecules

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has demonstrated many advantages, such as high conversion rates, good enantioselectivity, better
recoverability and recyclability. For example, the stability of lipases such as CaLB [13 16] and
17] are catalytically active in imidazolium and pyridinium ILs
14]. [OmPy][BF4] provide high enantioselectivity to hesperidinase
–
Pseudomonas cepacia l´ipase (PcL)´[15
,
´
containing BF4 , PF6 and Tf2N
[
when used as co-solvent in the enzymatic conversion of rutin to isoquercitrin. The reaction time
decreased 0.33-fold, and the rutin conversion and isoquercitrin yield increased 1.67-fold and 2.33-fold,
respectively, in [OmPy][BF4]-containing buffer systems. Besides acting as a reaction media, ILs can also
be used to pre-treat enzyme. There are many literature data on enhanced the activity and stability of
enzyme by coating it with ILs which had been used for transesterification, esterification and hydrolysis
reaction with higher enantioselecticity and yields [12,18].
Several thermophilic esterases have been reported to have the ability to resolve mixtures of chiral
esters. So far only one thermophilic esterase APE147 from archaeon Aeropyrum pernix K1 with a strict
stereoselectivity toward (S)-ibuprofen was identified and characterized. The enantioselectivity of
EST10 was higher than that of APE147, which showed eep of 99% and E value of 38.1 at 57% conversion
in 96 h [19]. In an earlier study, the thermostable esterase (EST10) from Thermotoga maritima was
identified and characterized [20], and EST10 showed high thermostability and gave a high hydrolytic
resolution of racemic ketoprofen ethyl ester compared with other esterases. However, EST10 exhibited
low enantioselectivity toward (R,S)-racemic ibuprofen ethyl ester (enantioselectivity, E-value < 10)
(Scheme 1) in both aqueous and organic solvents [20]. The objective of this study was to develop an IL
system for high-yield enzymatic production (EST-catalyzed) of (S)-ibuprofen racemic ibuprofen ethyl
ester. The effects of different ILs and reaction parameters on the enantioselectivity toward racemic
ibuprofen ethyl ester were also investigated.
Scheme 1. EST10-catalyzed enantioselective hydrolysis of racemic eaters.
2. Results and Discussion
2.1. Effects of ILs
The effects of 19 different ILs were evaluated for the production of (S)-ibuprofen from the
EST-catalyzed hydrolysis of racemic ibuprofen ethyl ester. Table 1 shows that the structure of the IL
had a large impact on the degree of conversion (X = 2.3%–46.5%), enantiomeric excess of the product
(eep = 1.4%–96.2%), and enantiomeric ratio (E = 1.0–134.7) of EST10. The catalytic efficiencies of the
different anions were in the following order: [BF4]´ > [Tf2N]´ > [PF6]´ > [NO3]´ > [CH3COO]´
> [TfO]´ > [CH3SO3]´ > [Cl]´. The highest enantioselectivity for the hydrolysis of ibuprofen ethyl
ester was achieved using [OmPy][BF4] in the buffer system, giving X 46.5%, eep of 96.2%, and E of
´
´
´
134.7. The (S)-ibuprofen yields for ILs containing [BF4] , [Tf2N] , and [PF6] anions were higher than
those observed for the ILs containing [NO3]´, [CH3COO]´, [TfO]´, [Cl]´, and [CH3SO3]´ anions.
ILs containing structurally different anions could potentially interact with protein molecule in different
ways, including hydrogen bonding, van der Waals, ionic and dipolar interactions. The hydrogen bond
basicity and nucleophilicity of anions have been found to affect the enzyme activity and stability in
IL-based solvent media [12,21]. Anions having lower hydrogen bond basicity and nucleophilicity are
found to be enzyme-compatible, because low hydrogen bond basicity and nucleophilicity minimize
their interference with the internal hydrogen bonds and interaction with the positively charged
sites in the enzyme and hence reduce the tendency to change the conformation of the enzyme’s
structure [12
,21–23]. This difference in (S)-ibuprofen yields could be attributed to the fact that ILs
containing [NO3]´, [CH3COO]´, [TfO]´, [Cl]´, and [CH3SO3]´ anions have higher hydrogen bond

Molecules 2016, 21, 905
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´
´
´
basicity and nucleophilicity than those containing [BF4] , [Tf2N] , and [PF6] anions. This difference
in hydrogen bond basicity and nucleophilicity could result in a change in the conformation of the
enzyme, which could lead to a loss of activity [24,25]. Therefore, the EST-catalyzed synthesis of
(S)-ibuprofen would occur much more effectively in enzymatic media containing ILs with weakly
coordinating anions such as [BF4]´, [Tf2N]´, and [PF6]´. This conclusion is in agreement with the
results of several previous studies using ILs in the reaction media [26,27]. A comparison of the
´
´
enantioselectivity achieved with ILs containing the same anions (i.e., [BF4] and [PF6] and different
cations (i.e., [Emim]+, [Bmim]+, [Hmim]+, [Omim]+ and [Dmim]+) revealed that the (S)-ibuprofen
yield increased as the length of the alkyl chain attached to the imidazolium ring of the IL increased
(Table 1). This increase in yield with increasing chain length could be attributed to the cations
becoming increasingly hydrophobic with increasing chain length, which would lead to a reduction in
the water-stripping capacity of the ILs and the occurrence of unwanted protein–ion interactions [28].
Our results show that [OmPy][BF4] is the most suitable of all of the ILs tested in the current study as
a reaction medium for the EST-catalyzed production of (S)-ibuprofen.
Table 1. Effect of ionic liquid on the enantioselective hydrolysis of (R,S)-racemic ibuprofen ethyl ester
by EST10.
Ionic Liquids
X (%)
eep (%)
E
Control
[Emim][PF6]
[Bmim][PF6]
[Hmim][PF6]
[Omim][PF6]
[Dmim][PF6]
[Emim][BF4]
[Bmim][BF4]
[Omim][BF4]
[OmPy][BF4]
[Bmim][Tf2N]
[Emim][TfO]
[Bmim][TfO]
[Bmim][CH3SO]
[Omim][CH3SO]
[Bmim][Cl]
19.5
25.3
39.2
42.1
43.2
44.2
34.5
40.6
44.9
46.5
44.6
10.2
8.3
45.2
75.4
85.6
91.2
93.5
94.8
76.9
87.6
92.3
96.2
95.2
35.0
19.5
1.4
2.95
9.1
22.4
43.3
63.5
84.9
11.3
27.8
56.9
134.7
94.5
2.2
1.5
1.0
1.1
1.2
4.3
5.8
6.8
4.5
9.5
[Emim][Cl]
2.3
0.6
1.0
[Omim][Cl]
3.5
3.8
1.1
[Bmim][NO3]
[Bmim][CH3COO]
34.1
32.1
82.2
66.9
15.5
6.8
Values given are the average of three replicates.
2.2. Conformational Studies of EST
Conformational changes in the EST enzyme could cause the enantioselectivity differences
observed with the different ILs. Circular dichroism (CD) has been widely used to gather information
about the structural features of proteins. CD spectroscopy was conducted to determine the effects
of the different ILs on the secondary structure of the EST enzyme. After treatment with selected
ILs containing different anions (e.g., [PF6]´ and [BF4]´) and cations (e.g., [Emim]+, [Bmim]+,
[Hmim]+, [Omim]+, and [Dmim]+), there were large changes in the secondary structural elements
of the EST enzyme, including a decrease in the
α
-helical content and an increase in the
β-sheet
content (Table 2). This result indicated that the observed decrease in the
α
-helical content of
the enzyme would most likely have an impact on the active site of EST, which would lead to
an increase in its enantioselectivity. Similar results have been reported for lipases from Candida rugosa,
Burkholderia cepacia, Chromobacterium viscosum, and Thermomyces lanuginose in non-aqueous media, and
these confirm that higher catalytic activity can be related to a decrease in the α-helical content of the

Molecules 2016, 21, 905
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enzyme [27,29,30]. Further studies are required to develop a deeper understanding of the relationship
between the enhanced enantioselectivity of EST and changes to its structure.
Table 2. The secondary structure elements of EST in ILs by CD spectroscopy.
Ionic Liquids
α-Helix (%)
β-Sheet (%)
β-Turn (%)
Random Coil (%)
Control
[Emim][PF6]
[Bmim][PF6]
[Hmim][PF6]
[Omim][PF6]
[Dmim][PF6]
[Emim][BF4]
[Bmim][BF4]
[Omim][BF4]
[OmPy][BF4]
[Bmim][Tf2N]
[Emim][TfO]
[Bmim][TfO]
[Bmim][CH3SO3]
[Omim][CH3SO3]
[Bmim][Cl]
21.5 ˘ 0.3
21.3 ˘ 0.2
20.5 ˘ 0.2
19.2 ˘ 0.5
18.2 ˘ 0.2
17.9 ˘ 0.3
20.5 ˘ 0.3
18.8 ˘ 0.1
16.4 ˘ 0.1
15.6 ˘ 0.2
16.5 ˘ 0.3
22.3 ˘ 0.6
24.9 ˘ 0.4
25.3 ˘ 0.2
26.8 ˘ 0.4
25.3 ˘ 0.2
25.7 ˘ 0.1
26.3 ˘ 0.2
26.5 ˘ 0.3
26.3 ˘ 0.4
26.5 ˘ 0.5
26.0 ˘ 0.4
27.8 ˘ 0.3
28.9 ˘ 0.4
30.7 ˘ 0.3
31.3 ˘ 0.2
28.2 ˘ 0.5
30.1 ˘ 0.3
31.2 ˘ 0.5
32.3 ˘ 0.3
33.0 ˘ 0.2
28.2 ˘ 0.2
26.3 ˘ 0.3
25.9 ˘ 0.4
26.0 ˘ 0.2
27.2 ˘ 0.3
28.3 ˘ 0.3
28.5 ˘ 0.4
28.3 ˘ 0.3
28.1 ˘ 0.3
35.2 ˘ 0.2
36.3 ˘ 0.2
34.7 ˘ 0.1
33.7 ˘ 0.2
33.1 ˘ 0.3
32.5 ˘ 0.4
34.9 ˘ 0.1
33.7 ˘ 0.2
32.2 ˘ 0.3
32.0 ˘ 0.3
32.3 ˘ 0.2
37.8 ˘ 0.1
39.3 ˘ 0.2
40.3 ˘ 0.3
40.1 ˘ 0.2
39.0 ˘ 0.5
38.3 ˘ 0.4
38.2 ˘ 0.5
39.3 ˘ 0.1
39.1 ˘ 0.2
16.8 ˘ 0.2
16.4 ˘ 0.1
17.0 ˘ 0.3
17.8 ˘ 0.1
18.0 ˘ 0.2
18.3 ˘ 0.1
16.4 ˘ 0.4
17.4 ˘ 0.1
20.2 ˘ 0.4
20.1 ˘ 0.4
18.2 ˘ 0.3
11.7 ˘ 0.3
9.5 ˘ 0.1
8.5 ˘ 0.2
7.1 ˘ 0.3
8.5 ˘ 0.2
7.7 ˘ 0.5
7.0 ˘ 0.3
5.9 ˘ 0.5
6.5 ˘ 0.1
[Emim][Cl]
[Omim][Cl]
[Bmim][NO3]
[Bmim][CH3COO]
Values given are the average of three replicates.
2.3. Effects of IL Concentration
The effects of [OmPy][BF4] concentration on the X, eep and E of the enzyme in the EST-catalyzed
production of (S)-ibuprofen were investigated (Figure 1). The IL concentration in the medium (0%–60%)
greatly affected the catalytic efficiency of EST10. The highest X (47.0%) and eep (96.4%) were achieved
at an IL concentration of 30% ([OmPy][BF4]/buffer, v/v). The X and eep decreased sharply as the IL
concentration was increased from 30% to 60%. The highest E of EST10 (150.3) was also achieved in
the [OmPy][BF4]–buffer (30:70, v/v) system. A higher concentration of IL not only increases the ionic
strength in the enzymatic reaction medium, which might inactivate the enzyme, but also increases
the viscosity of the reaction mixture, which limits diffusion of reaction substrates to the enzyme
active site [31].
Figure 1. Effect of IL concentrations on the conversion degree, enantiomeric excess of product and
enantioselectivity of EST10 using [OmPy][BF4] as a co-solvent in the presence of enzyme EST10
(15 mg/mL) at 75 ˝C for 10 h. The concentration of racemic ibuprofen ethyl ester was 2.5 mg/mL.
The values are means of three independent experiments.

Molecules 2016, 21, 905
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2.4. Effects of Time
The effects of reaction time (0–16 h) on the conversion degree, enantiomeric excess of product and
enantioselectivity of the enzyme in the EST-catalyzed production of (S)-ibuprofen are shown in Figure 2.
After an enzymatic reaction time of 10 h, no significant improvement in the production of (S)-ibuprofen
was observed. The X, eep and E of EST10 were 46.8%, 96.8%, and 168.2, respectively. The reaction
time of industrial procedure of (S)-ibuprofen (<10 h), such as enantioselective crystallization and
chemical asymmetric synthesis, is shorter than that of the enzymatic resolution. However, almost all
industrial methods involves a complicated synthesis that requires harsh separation techniques [32
Enzymatic hydrolysis of racemic ibuprofen ethyl ester usually requires a long reaction time (over
24 h) to achieve the maximum yield in both aqueous and organic solvents [31 34 35]. However, the
,33].
,
,
maximum conversion degree, enantiomeric excess of product and enantioselectivity of enzyme were
achieved after only 10 h in the [OmPy][BF4]–buffer (30:70, v/v) system.
Figure 2. Effect of reaction time (from 0 to 16 h) on the conversion degree, enantiomeric excess of
product and enantioselectivity of EST10 using [OmPy][BF4] as a co-solvent in the presence of enzyme
EST10 (15 mg/mL) at 75 ˝C. The concentration of racemic ibuprofen ethyl ester was 2.5 mg/mL.
The values are means of three independent experiments.
2.5. Effects of Temperature
˝
The effects of temperature (45–95 C) on X, eep, and E of the enzyme in the EST-catalyzed
production of (S)-ibuprofen were investigated (Figure 3).
Figure 3. Effect of reaction temperature (from 45 ˝C to 95 ˝C) on the conversion degree, enantiomeric
excess of product and enantioselectivity of EST10 using [OmPy][BF4] as a co-solvent in the presence
of enzyme EST10 (15 mg/mL). The concentration of racemic ibuprofen ethyl ester was 2.5 mg/mL.
The values are means of three independent experiments.

Molecules 2016, 21, 905
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When the reaction was conducted in the [OmPy][BF4]–buffer (30:70, v/v) system, the X and
eep increased sharply˝when the temperature was increased from 45 to 75˝C. The optimum reaction
temperature was 75 C (Figure 3). When the reaction was conducted in the [OmPy][BF4]–buffer
(30:70, v/v) system, the conversion degree and enantiomeric excess of product increased sharply with
˝
˝
increasing temperature from 45 to 75 C, with the optimum reaction temperature being 75 C (Figure 3).
˝
This is slightly higher than that used in solvent-free systems (70 C) [20].
2.6. Effects of pH
The effect of pH on the enzymatic hydrolysis of racemic ibuprofen ethyl ester was examined at
75 C from pH 3.0 to 8.0 (Figure 4). When the pH value was adjusted to 5.5 in the [OmPy][BF4]–buffer
˝
(30:70, v/v) system, the enzymatic hydrolysis of racemic ibuprofen ethyl ester reached the highest
yield of (S)-ibuprofen. The results of X, eep and E were 47.4%, 96.8%, and 177, respectively. The optimal
pH value of the buffer solution in the 30% (v/v) [OmPy][BF4]–containing system was 5.5. These results
demonstrated that the IL co-solvents did not change the optimal pH values of EST, which was similar
to that of EST in an aqueous buffer medium (5.5).
Figure 4. Effect of pH (from 3 to 8) on the degree of conversion, enantiomeric excess of product
and enantioselectivity of EST10 using [OmPy][BF4] as a co-solvent in the presence of enzyme EST10
(15 mg/mL) at 75 ˝C. The concentration of racemic ibuprofen ethyl ester was 2.5 mg/mL. The values
are means of three independent experiments.
2.7. Effects of Substrate Concentration
The effects of substrate concentration (25–400 mM) on the enzymatic hydrolysis of racemic
ibuprofen ethyl ester were investigated (Figure 5).
Figure 5. Effect of ibuprofen ethyl ester concentration on the conversion degree, enantiomeric excess of
product and enantioselectivity of EST10 using [OmPy][BF4] as a co-solvent in the presence of enzyme
EST10 (15 mg/mL) at 75 ˝C for 10 h. The values are means of three independent experiments.

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In the [OmPy][BF4]–buffer (30:70, v/v) system, the highest enantioselectivity for the hydrolysis
of ibuprofen ethyl ester (X = 46.9%, eep = 96.8%, and E = 153.8) was achieved with 150 mM of
racemic ibuprofen ethyl ester. Any further increase or decrease in the substrate concentration resulted
in a decrease in the (S)-ibuprofen yield. This indicates that increasing the substrate concentration
(from 25 to 150 mM) facilitates contact between the enzyme and the substrate in the [OmPy][BF4]–buffer
(30:70, v/v) system.
2.8. Effects of Enzyme Concentration
The effects of enzyme concentration (0.2–5 U/mL) on X, eep, and E in the EST-catalyzed production
of (S)-ibuprofen were investigated. The c and eep increased considerably with increasing enzyme
concentration (from 0.2 to 3.0 U/mL, Figure 6). The highest X (46.8%) and eep of (S)-ibuprofen (96.9%)
were obtained at an enzyme concentration of 3 U/mL. However, the presence of excess catalyst
(>3 U/mL) could result in enzyme agglomeration and diffusion problems, which could decrease
the reaction efficiency [36]. An EST loading of 3 U/mL provided good catalytic efficiency in the
[OmPy][BF4]–buffer (30:70, v/v) system.
Figure 6. Effect of enzyme concentration on the conversion degree, enantiomeric excess of product and
enantioselectivity of EST10 using [OmPy][BF4] as a co-solvent at 75 ˝C for 10 h. The concentration of
racemic ibuprofen ethyl ester was 2.5 mg/mL. The values are means of three independent experiments.
3. Experimental Section
3.1. Chemicals
Nineteen ILs, including [Bmim][CH3SO3], [Omim][CH3SO3], [Bmim][Cl], [Emim][Cl], [Emim][Cl],
[Emim][TfO], [Bmim][TfO], [Bmim][BF4], [Emim][BF4], [Omim][BF4], [OmPy][BF4], [Bmim][PF6],
[Emim][PF6], [Hmim][PF6], [Omim][PF6], [Dmim][PF6], [Bmim][Tf2N], [Bmim][CH3COO], and
[Bmim][NO3], with purities
ě
99.9%, were purchased from Shanghai Cheng Jie Chemical Co. Ltd
(Shanghai, China). (R,S)-Ibuprofen ethyl ester, (S)-ibuprofen and (R)-ibuprofen were purchased from
J & K Scientific Ltd. (Beijing, China). All other chemicals were of the highest grade available and
obtained from Sangon (Shanghai, China). The EST10 enzyme was purified according to a previously
reported procedure [20].
3.2. Enzyme Assay
The EST10 enzyme was used with 19 different ILs containing six different cations and eight
different anions to investigate their effects on the enantioselectivity and enzymatic production of
(S)-ibuprofen. The standard reaction was carried out in a 10-mL round-bottom flask containing
0.3 mL of IL, 10 mg of the EST10 enzyme, 25 mg of racemic ibuprofen ethyl ester, and 0.7 ˝mL of
50 mM sodium acetate buffer (pH 5.5). The resulting mixture was shaken (180 rpm) at 65 C for

Molecules 2016, 21, 905
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10 h. These conditions were used for all experiments, except when otherwise stated. The effects of
IL concentration, reaction time, reaction temperature, and EST concentration on the production of
(S)-ibuprofen were investigated.
3.3. HPLC Analysis
Reactants and products were analyzed by HPLC using a chiracel QJ-Hcolumn (25 cm ˆ 4.6 cm
Daicel Chemical Industries Ltd., Tokyo, Japan). The samples were eluted with n-hexane:
2-propanol:acetic acid (90:10:0.5, v/v/v) at a flow rate of 1.0 mL/min and detected at 254 nm. Racemic
ibuprofen ethyl ester, (R)-ibuprofen, and (S)-ibuprofen were detected at retention times of 4.9, 13.0, and
18.6 min, respectively. The E was calculated from the eep and X according to the equations described
,
by Chen et al. [37]:
rpSq-ibuprofens ` rpRq-ibuprofens
X p%q “
ˆ 100%
ˆ 100%
(1)
(2)
(3)
initial ibuprofen ethyl ester
rpSq-ibuprofens ´ rpRq-ibuprofens
rpSq-ibuprofens ` rpRq-ibuprofens
eep p%q “
lnr1 ´ Xp1 ` eepqs
E “
lnr1 ´ Xp1 ´ eepqs
3.4. CD Spectroscopy
CD spectra (190–250 nm) of the EST enzyme˝were recorded on a Chirascan spectropolarimeter
(Applied Photophysics, Leatherhead, UK) at 25 C using a 0.1-cm quartz cuvette. Measurements
were conducted using 0.2 mg/mL solutions with or without an IL (0.1 M). The results of six spectra
were average for each sample. The scan speed, response time, and bandwidth were 200 nm ,
min´1
¨
0.5 s, and 1 nm, respectively. The sample spectra were obtained by subtracting the spectrum of the
appropriate blank media without EST from the experimental spectra. The secondary structures of
EST determined from the CD spectra were analyzed using the K2D2 web server provided by EMBL
(Heidelberg, Germany).
4. Conclusions
In this study, we investigated the enzymatic synthesis of (S)-ibuprofen in EST-catalyzed reaction
systems, using 19 different ILs as cosolvents. A [OmPy][BF4]-buffer (30:70, v/v) system was used as
the reaction medium for the enzymatic production of (S)-ibuprofen by the EST-catalyzed resolution
of racemic ibuprofen ethyl ester. A high X (47.4%), eep of (S)-ibuprofen (96.6%), and E of EST10
(177.0) were achieved under the following optimum conditions: EST10 concentration 15 mg/mL,
racemic ibuprofen ethyl ester concentration 150 mM, temperature 75 ˝C, with a reaction time of 10 h.
The reaction time needed to achieve the highest yield of (S)-ibuprofen was decreased from 24 h to
10 h. These results show that [OmPy][BF4] is a promising cosolvent for the enzymatic production of
(S)-ibuprofen via biotransformation.
Acknowledgments: This work was supported by grants from the National Natural Science Foundation of
China (Grant Nos. 21406210 and 21476216), the Innovation Scientists and Technicians Troop Construction
Projects of the Henan Province (Grant No. 144200510011), and the Henan Province Foreign Cooperation Projects
(Grant No. 152106000058).
Author Contributions: Tao Wei designed and performed the experiments, analyzed the data and prepared
the manuscript. Kunpeng Yang, Bing Bai, and Xuan Yu assisted in data analysis. Duobin Mao performed the
experiments, analyzed the data and assisted in manuscript preparation. All authors read and approved the
final manuscript.
Conflicts of Interest: The authors report no conflict of interest. The funding sponsors had no role in the design of
the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision
to publish the results.

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9 of 10
References
1.
Shanbhag, V.; Crider, R.; Gokhale, A.M.; Harpalani, R.; Dick, R.M. Ester and amide prodrugs of ibuprofen
and naproxen: Synthesis, anti-inflammatory activity, and gastrointestinal toxicity. J. Pharmaceut. Sci. 1992, 81,
149–154. [CrossRef]
2.
Sánchez, A.; Valero, F.; Lafuente, J.; Solá, C. Highly enantioselective esterification of racemic ibuprofen in
a packed bed reactor using immobilized Rhizomucor miehei lipase. Enzyme Microb. Technol. 2000, 27, 157–166.
[CrossRef]
3.
4.
5.
6.
7.
8.
9.
Kato, K.; Gong, Y.; Saito, T.; Kimoto, H. Efficient preparation of optically active ketoprofen by Mucor javanicus
lipase immobilized on an inorganic suppor. J. Biosci. Bioeng. 2000, 90, 157–166. [CrossRef]
Chávez-Flores, D.; Salvador, J.M. Commercially viable resolution of ibuprofen. Biotechnol. J. 2009, 4,
1222–1224. [CrossRef] [PubMed]
Yousefi, M.; Mohammadi, M.; Habibi, Z. Enantioselective resolution of racemic ibuprofen esters using
different lipases immobilized on octyl sepharose. J. Mol. Catal. B Enzym. 2014, 104, 87–94. [CrossRef]
Mustranta, A. Use of lipase in the resolution of racemic ibuprofen. Appl. Microbiol. Biotechnol. 1992, 38, 61–66.
[CrossRef] [PubMed]
Siódmiak, T.; Ziegler-Borowska, M.; Marszałł, M.P. Lipase-immobilized magnetic chitosan nanoparticles for
kinetic resolution of (R,S)-ibuprofen. J. Mol. Catal. B Enzym. 2013, 94, 7–14. [CrossRef]
Quijano, G.; Couvert, A.; Amrane, A. Ionic liquids: Applications and future trends in bioreactor technology.
Bioresour. Technol. 2010, 101, 8923–8930. [CrossRef] [PubMed]
Bekhouche, M.; Blum, L.J.; Doumeche, B. Contribution of dynamic and static quenchers for the study of
protein conformation in ionic liquids by steady-state fluorescence spectroscopy. J. Phys. Chem. B. 2012, 116,
413–423. [CrossRef] [PubMed]
10. Wang, S.H.; Meng, X.H.; Zhou, H.; Liu, Y.; Secundo, F.; Liu, Y. Enzyme Stability and Activity in Non-Aqueous
Reaction Systems: A Mini Review. Catalysts 2016, 32. [CrossRef]
11. Patel, R.; Kumari, M.; Khan, A.B. Recent advances in the applications of ionic liquids in protein stability and
activity: A review. Appl. Biochem. Biotechnol. 2014, 172, 3701–3720. [CrossRef] [PubMed]
12. Zhao, H. Methods for stabilizing and activating enzymes in ionic liquids—A review. J. Chem.
Technol. Biotechnol. 2010, 85, 891–907. [CrossRef]
13. Madeira Lau, R.; Van Rantwijk, F.; Seddon, K.R.; Sheldon, R.A. Lipase-catalyzed reactions in ionic liquids.
Org. Lett. 2000, 2, 4189–4191. [CrossRef] [PubMed]
14. Kim, K.W.; Song, B.; Choi, M.Y.; Kim, M.J. Biocatalysis in ionic liquids: Markedly enhanced enantioselectivity
of lipase. Org. Lett. 2001, 3, 1507–1509. [CrossRef] [PubMed]
15. Husum, T.L.; Jorgensen, C.T.; Christensen, M.W.; Kirk, O. Enzyme catalysed synthesis in ambient temperature
ionic liquids. Biocatal. Biotrans. 2001, 19, 331–338. [CrossRef]
16. Park, S.; Kazlauskas, R.J. Improved preparation and use of room-temperature ionic liquids in lipase-catalyzed
enantio- and regioselective acylations. J. Org. Chem. 2001, 66, 8395–8840. [CrossRef] [PubMed]
17. Wang, J.; Sun, G.X.; Yu, L.; Wu, F.A.; Guo, X.J. Enhancement of the selective enzymatic biotransformation of
rutin to isoquercitrin using an ionic liquid as a co-solvent. Bioresour. Technol. 2013, 128, 156–163. [CrossRef]
[PubMed]
18. Mutschler, J.; Rausis, T.; Bourgeois, J.M.; Bastian, C.; Zufferey, D.; Mohrenz, I.V.; Fischer, F. Ionic liquid-coated
immobilized lipase for the synthesis of methylglucose fatty acid esters. Green Chem. 2009, 11, 1793–1800.
[CrossRef]
19. Zhao, D.T.; Xun, N.E.; Wang, J.X.; Wang, R.; Wei, X.F. Enantioselective esterification of ibuprofen by a novel
thermophilic biocatalyst: APE1547. Biotechnol. Bioproc. E 2011, 16, 638–644. [CrossRef]
20. Wei, T.; Feng, S.X.; Mao, D.B.; Yu, X.; Du, C.C.; Wang, X.H. Characterization of a new thermophilic and acid
tolerant esterase from Thermotoga maritima capable of hydrolytic resolution of racemic ketoprofen ethyl ester.
J. Mol. Catal. B Enzym. 2013, 85, 23–30.
21. Anderson, J.L.; Ding, J.; Welton, T.; Armstrong, D.W. Characterizing ionic liquids on the basis of multiple
solvation interactions. J. Am. Chem. Soc. 2002, 124, 14247–14254. [CrossRef] [PubMed]
22. Park, S.; Kazlauskas, R.J. Biocatalysis in ionic liquids—Advantages beyond green technology.
Curr. Opin. Biotechnol. 2003, 14, 432–437. [CrossRef]

Molecules 2016, 21, 905
10 of 10
23. Sheldon, R.A.; Lau, R.M.; Sorgedrager, M.J.; van Rantwijk, F.; Seddon, K.R. Biocatalysis in ionic liquids.
Green Chem. 2002, 4, 147–151. [CrossRef]
24. Zhang, D.H.; Bai, S.; Ren, M.Y.; Sun, Y. Optimization of lipase-catalyzed enantioselective esterification of
(˘)-menthol in ionic liquid. Food Chem. 2008, 109, 72–80. [CrossRef] [PubMed]
25. Sun, S.D.; Qin, F.; Bi, Y.L.; Chen, J.N.; Yang, G.L.; Liu, W. Enhanced transesterification of ethyl ferulate with
glycerol for preparing glyceryl diferulate using a lipase in ionic liquids as reaction medium. Biotechnol. Lett.
2013, 35, 1449–1454. [CrossRef] [PubMed]
26. Liu, Y.; Chen, D.W.; Yan, Y.J.; Peng, C.; Xu, L. Biodiesel synthesis and conformation of lipase from
Burkholderia cepacia in room temperature ionic liquids and organic solvents. Bioresour. Technol. 2011, 102,
10414–10418. [CrossRef] [PubMed]
27. Liu, Y.; Chen, D.W.; Yan, Y.J. Effect of ionic liquids, organic solvents and supercritical CO2 pretreatment on
the conformation and catalytic properties of Candida rugosa lipase. J. Mol. Catal. B Enzym. 2013, 90, 123–127.
[CrossRef]
28. Wei, T.; Jia, W.W.; Yu, X.; Mao, D.B. Enhancement of enzymatic synthesis of sucrose 6-acetate with
Aspergillus oryzae fructosyltransferase using ionic liquid as a cosolvent. J. Mol. Catal. B Enzym. 2016, 123,
100–106. [CrossRef]
29. Silva, W.S.D.; Lapis, A.A.M.; Suarez, P.A.Z. Enzyme-mediated epoxidation of methyl oleate supported by
imidazolium-based ionic liquids. J. Mol. Catal. B Enzym. 2011, 68, 98–103. [CrossRef]
30. Pavlidis, I.V.; Gournis, D.; Papadopoulos, G.K. Lipases in water-in-ionic liquid microemulsions: Structural
and activity studies. J. Mol. Catal. B Enzym. 2009, 60, 50–56. [CrossRef]
31. Lou, W.; Zong, M.; Liu, Y.; Wang, J. Efficient enantioselective hydrolysis of D,L-phenylglycine methyl
ester catalyzed by immobilized Candida Antarctica lipase B in ionic liquid containing systems. J. Biotechnol.
2006, 125, 64–74. [CrossRef] [PubMed]
32. Manimaran, T.; Harkins, A.E. Process for Producing High Purity Ketones by Friedel-Crafts Acylation at Low
Temperature. U.S. W.O. 2007044270, 19 April 2004.
33. Chaudhari, RV.; Majeed, S.A.; Seayad, J. Process for the Preparation of Ibuprofen. U.S. 60933847, 25 July 2000
.
34. Marszałł, M.P.; Siódmiak, T. Immobilization of Candida rugosa lipase onto magnetic beads for kinetic
resolution of (R,S)-ibuprofen. Catal. Commun. 2012, 24, 80–84. [CrossRef]
35. Habibi, Z.; Mohammadi, M.; Yousefi, M. Enzymatic hydrolysis of racemic ibuprofen esters using
Rhizomucor miehei lipase immobilized on different supports. Process Biochem. 2013, 48, 669–676. [CrossRef]
36. Yang, Z.; Pan, W.B. Ionic liquids: Green solvents for nonaqueous biocatalysis. Enzyme Microb. Technol.
2005, 37, 19–28. [CrossRef]
37. Chen, C.S.; Fujimoto, Y.; Girdaukas, G.; Sih, C.J. Quantitative analyses of biochemical kinetic resolutions of
enantiomers. J. Am. Chem. Soc. 1982, 104, 7294–7299. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).