Microwave-assisted resolution of α-lipoic acid catalyzed by an ionic liquid co-lyophilized lipase

Article abstract of DOI:10.3390/molecules20069949

The combination of the ionic liquid co-lyophilized lipase and microwave irradiation was used to improve enzyme performance in enantioselective esterification of α-lipoic acid. Effects of various reaction conditions on enzyme activity and enantioselectivity were investigated. Under optimal condition, the highest enantioselectivity (E = 41.2) was observed with a high enzyme activity (178.1 μmol/h/mg) when using the ionic liquid co-lyophilized lipase with microwave assistance. Furthermore, the ionic liquid co-lyophilized lipase exhibited excellent reusability under low power microwave.

Full text of DOI:10.3390/molecules20069949

Molecules 2015, 20, 9949-9960; doi:10.3390/molecules20069949
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Microwave-Assisted Resolution of α-Lipoic Acid Catalyzed by
an Ionic Liquid Co-Lyophilized Lipase
1
2
2
2
2
2
3,
Ning Liu , Lei Wang , Zhi Wang , Liyan Jiang , Zhuofu Wu , Hong Yue and Xiaona Xie *
1
Central Laboratory, The Second Hospital of Jilin University, Changchun 130021, China;
E-Mail: liu_ning@jlu.edu.cn
2
Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education,
College of Life Science, Jilin University, Changchun 130012, China;
E-Mails: w_lei@jlu.edu.cn (L.W.); wangzhi@jlu.edu.cn (Z.W.); jiangliyan@jlu.edu.cn (L.J.);
wuzf06@mails.jlu.edu.cn (Z.W.); xinqingtianlan@sina.cn (H.Y.)
3
The First Hospital of Jilin University, Changchun 130021, China
*
Author to whom correspondence should be addressed; E-Mail: xiexn@jlu.edu.cn;
Tel.: +86-431-8515-5247; Fax: +86-431-8515-5243.
Academic Editor: Raquel P. Herrera
Received: 17 April 2015 / Accepted: 19 May 2015 / Published: 29 May 2015
Abstract: The combination of the ionic liquid co-lyophilized lipase and microwave irradiation
was used to improve enzyme performance in enantioselective esterification of α-lipoic acid.
Effects of various reaction conditions on enzyme activity and enantioselectivity were
investigated. Under optimal condition, the highest enantioselectivity (E = 41.2) was observed
with a high enzyme activity (178.1 μmol/h/mg) when using the ionic liquid co-lyophilized
lipase with microwave assistance. Furthermore, the ionic liquid co-lyophilized lipase exhibited
excellent reusability under low power microwave.
Keywords: ionic liquid co-lyophilized lipase; α-lipoic acid; enantioselectivity; enzyme
activity; esterification; microwave
1
. Introduction
As an important cofactor involved in many enzyme-catalyzed reactions [1], α-lipoic acid has already
been used to treat many diseases [2–4]. Since (R)-α-lipoic acid exhibits higher biological activity than

Molecules 2015, 20
9950
its (S)-enantiomer [5], great attention has been focused on the stereoselective synthesis of its
R)-enantiomer. There are two main routes to synthesize (R)-α-lipoic acid, including the chemical
(
synthesis and biocatalytic process. The chemical synthesis is usually conducted using asymmetric
synthesis [6,7] or synthesis from a chiral starting material [8]. Due to the interest in a product with high
enantiomeric purity, several alternative biocatalytic processes have been developed recently, including
reductase catalysis [9–12], Bakers’ yeast reduction [13], and lipase-catalyzed kinetic resolution [14–17].
Among these processes, lipase-catalyzed kinetic resolution is one of the most attractive methods. Zhou
and his group had successfully prepared the chiral chlorohydrin precursor of (R)-α-lipoic acid via lipase
catalyzed enantioselective transesterification [17]. In 1997, Fadnavis firstly reported that the
commercially available Candida rugosa lipase can catalyze the enzymatic resolution of α-lipoic acid
directly [14]. Yan and his co-workers have reported that the lipase from Aspergillus oryzae WZ007 is
another potential candidate for enzymatic resolution of α-lipoic acid [15,16]. However, all these lipases
exhibited poor enzyme performance (low enzyme activity or unsatisfied enantioselectivity), which may
increase the reaction time and reduce the yield of the enantiomerically pure product. Thus, novel enzymes
and new techniques are warranted to improve the performance of enantioselective esterification.
Microwave irradiation (MW) and ionic liquid (IL) are two important and rapid developing
technologies in green chemistry [18–25]. The combination of MW and IL has already attracted a great
deal of attention for enzymatic reactions in recent years [26–28]. In reports, IL was used as reaction
media due to its excellent microwave-absorbing ability. However, the high viscosity of IL may have an
adverse effect on the mass transfer of the substrates and worsen enzyme performance [29]. It is well
known that a small amount of IL can induce dramatic changes in the overall dielectric properties of
the reaction medium under microwave irradiation [30,31]. Furthermore, lyophilization of enzyme in
the presence of a small amount of IL can improve the activity, stability, and enantioselectivity of
enzymes [32–37]. All of the above reports may provide a new breakthrough to improve the activity and
enantioselectivity of enzyme during biocatalytic synthesis.
In the present work, the combination of the ionic liquid co-lyophilized lipase and microwave
irradiation was used to improve the enzyme performance in the enantioselective esterification of
α-lipoic acid. The enzyme has been firstly co-lyophilized with a small amount of ionic liquid and then
used as catalyst to carry out the enantioselective esterification of α-lipoic acid under microwave
irradiation (Scheme 1). The effects of reaction conditions on the activity and enantioselectivity were
investigated and the reusability of the ionic liquid co-lyophilized lipase under microwave was also studied.
COOH
ionic liquid co-lyophilized
lipase
COOH
COOR
+
ROH
+
S
S
microwave
S
S
S
S
(S)
(R)
Scheme 1. Enantioselective esterification of α-lipoic acid catalyzed by ionic liquid
co-lyophilized lipase under microwave.

Molecules 2015, 20
9951
2
. Results and Discussion
2
.1. Effect of Lipase Source
Four commercially available lipases were selected for enantioselective esterification of α-lipoic acid
and the results were listed in Table 1. All selected lipases could catalyze the enantioselective
esterification of α-lipoic acid. Moreover, all the enzymes favor the (S)-enantiomer of α-lipoic acid, but
exhibited poor enantioselectivity. Among the tested lipases, CLL (Candida lipolytic lipase) showed the
highest enantioselectivity (E = 3.2) and a higher enzyme activity (3.7 μmol/h/mg). Thus, CLL was
selected as the catalyst for further study.
Table 1. Effect of lipase source on the enantioselective esterification of α-lipoic acid.
Reaction
Conversion
(%)
ee
s
Enzyme Activity
Enantioselectivity
Lipase
Stereoselectivity
Time (h)
(%)
8.4
(μmol/h/mg)
(E value)
2.2
Candida cylindracea A.Y. lipase (AYL)
Mucor miehei lipase (MML)
12
20
30
12
19.8
3.3
2.4
1.1
3.7
S
S
S
S
24.0
6.3
1.6
Porcine pancreatic lipase (PPL)
Candida lipolytic lipase (CLL)
16.5
5.8
1.9
22.2
13.6
3.2
Reaction conditions: (R,S)-α-lipoic acid (2 mmol), lipase (10 mg), n-octanol (5 mmol) and heptane (10 mL)
were performed at 30 °C and 150 rpm.
2
.2. Effect of Organic Media
Organic media can alter the solubility of substrates and affect enzyme performance [38]. In this study,
organic solvents with different log P were selected to investigate the effect of the reaction medium; log P
is the most frequently used parameter to denote the polarity or hydrophobicity of a solvent [39]. The
results shown in Table 2 indicate that the best catalytic performance of CLL was observed when
cyclohexane was used as the reaction media. When polar solvents were used as the reaction media, they
could disrupt the functional structure of the enzyme by stripping off the essential water from the protein
and decrease the enzyme activity and the enantioselectivity [40,41].
Other reaction conditions were also investigated (data not shown). Our experimental results indicated
that n-octanol was the most suitable acyl-acceptor. The optimal substrate molar ratio (α-lipoic acid/n-octanol)
was 1:2.5 and the enzyme dosage in this reaction system was 10 mg.
Table 2. Effect of organic solvents on the enantioselective esterification of α-lipoic acid.
Reaction
Time (h)
Enzyme Activity Enantioselectivity
Solvent
Log P
Conversion (%) ee
s
(%)
(μmol/h/mg)
(E value)
1.2
Isooctane
Heptane
Cyclohexane
Toluene
Acetonitrile
,4-Dioxane
4.5
4.0
3.2
2.5
−0.33
−1.1
12
12
12
15
28
60
25.2
3.0
4.2
3.7
3.6
2.1
1.4
0.9
22.2
21.6
15.8
19.6
26.8
13.6
17.5
8.5
3.2
5.3
2.9
2.4
1.3
9.3
4.2
1
Reaction conditions: (R,S)-α-lipoic acid (2 mmol), CLL (10 mg), n-octanol (5 mmol) and organic solvent
10 mL) were performed at 30 °C and 150 rpm.
(

Molecules 2015, 20
.3. Effect of ILs Used for the Ionic Liquid Co-Lyophilized Enzyme
Lyophilization of enzyme in the presence of a suitable ionic liquid is a simple method to modify the
9952
2
enzyme and can improve enzyme performance in non-aqueous media [32–37]. The ionic liquid
co-lyophilized enzyme, prepared by different hydrophilic ionic liquids (15%, wt %, IL/enzyme), was
used to catalyze the enantioselective esterification of α-lipoic acid. As shown in Table 3, CLL showed
poor enzyme performance when CLL was co-lyophilized with [Bmim] Cl or [Bmim] Ac. This may be
due to the high hydrogen-bond basicity of these two ionic liquids destroying the enzyme conformation
and decreasing the activity and the enantioselectivity. Among the different ionic liquids, [Bmim] BF
was selected as the most suitable ionic liquid for further study since the best catalytic performance was
observed when CLL was co-lyophilized with [Bmim] BF . The activation effect of co-lyophilization might
4
4
be due to the active-site preservation or molecular flexibility increase in organic environments or a
combination of both [36,38].
Table 3. Effect of ionic liquids on the enantioselective esterification of α-lipoic acid.
Reaction
Enzyme Activity Enantioselectivity
Ionic Liquid
Conversion (%)
ee
s
(%)
Time (h)
(μmol/h/mg)
(E value)
2.3
[Bmim] Cl
48
30
5
16.8
21.0
26.5
21.6
7.4
0.7
1.4
[Bmim] Ac
11.7
27.0
17.5
2.9
[Bmim] BF
4
10.6
3.6
9.1
None
12
5.3
Reaction conditions: Reaction conditions: (R,S)-α-lipoic acid (2 mmol), CLL (10 mg) co-lyophilized with
different ionic liquid (15%, wt %, IL/enzyme), n-octanol (5 mmol) and cyclohexane (10 mL) were performed
at 30 °C and 150 rpm.
When the ionic liquid co-lyophilized enzyme catalyzed the enantioselective esterification, the ionic
liquid content may be an important influencing factor. In this study, the correlation between the content
of [Bmim] BF
that both the enzyme activity and the enantioselectivity exhibited bell-shaped curves when changing the
content of [Bmim] BF , which meant a suitable content of [Bmim] BF was necessary for enzyme
lyophilized preparation. The best enzyme performance was obtained at a content of [Bmim] BF (20%,
wt %, IL/enzyme) with an enzyme activity of 9.8 μmol/h/mg and an enantioselectivity (E value) of 12.5.
4
(0%–40%, wt %, IL/enzyme) and enzyme performance was investigated. Figure 1 showed
4
4
4
2
.4. Microwave Irradiation vs. Conventional Heating
Compared with the results obtained from conventional heating, microwave irradiation can enhance
both the enzyme activity and enantioselectivity of the free CLL or the ionic liquid co-lyophilized CLL
Table 4). Moreover, the improvement of enzyme performance for the ionic liquid co-lyophilized CLL
(
under MW was much greater than that of the free enzyme. The dramatic enhancement of enzyme
performance might be that microwaves can directly act on the ionic liquid co-lyophilized enzyme
because of the excellent microwave-absorbing ability of IL [42] and change the protein conformation
of CLL.

Molecules 2015, 20
9953
1
1
1
4
2
0
8
6
4
2
0
14
12
10
8
6
4
Enzyme activity
E value
2
0
0
5
10
15
20
25
30
35
40
Content of [Bmim] BF (wt%, IL/enzyme)
4
Figure 1. Effects of [Bmim] BF content during lyophilized preparation on the enzyme
4
activity (μmol/h/mg, ○) and the enantioselectivity (E value, ●) in enantioselective esterification
of α-lipoic acid. Reaction conditions: (R,S)-α-lipoic acid (2 mmol), CLL (10 mg)
co-lyophilized with different contents of [Bmim] BF
4
(wt %, IL/enzyme), n-octanol
(
5 mmol) and cyclohexane (10 mL) were performed at 30 °C and 150 rpm.
Table 4. Effect of microwaves on the enantioselective esterification of α-lipoic acid.
Conventional Heating
Microwave
Enzyme Activity Enantioselectivity Enzyme Activity Enantioselectivity
(
μmol/h/mg)
(E value)
(μmol/h/mg)
(E value)
Free CLL
3.6
5.3
5.1
6.7
Ionic liquid
9
.8
12.5
170.4
38.6
co-lyophilized CLL
Reaction conditions: (R,S)-α-lipoic acid (2 mmol), free CLL (10 mg) or CLL (10 mg) co-lyophilized with 20%
of [Bmim] BF (wt %, IL/enzyme), n-octanol (5 mmol) and cyclohexane (10 mL) were performed at
0 °C. The microwave was carried out in a microwave bath (480 W). Conventional shaking was performed
at 150 rpm.
4
3
2
.5. Effect of Microwave Power
The effect of microwave power on the enzyme performance of the ionic liquid co-lyophilized CLL
was examined in the range of 0–640 W. As shown in Figure 2, ionic liquid co-lyophilized CLL exhibited
the highest enantioselectivity (E value, 38.6) and a higher enzyme activity (170.4 μmol/h/mg) when the
microwave power was 480 W. Generally, the higher the microwave power was set, the faster the dipole
reorientated under the MW, which might make the functional groups obtain a higher reactivity [28,43]. Since
all of the ionic liquid co-lyophilized enzyme and substrate used in this study had significant dipole
moments, microwave could significantly impact theses molecules and activate enzyme and substrate. At
high microwave power, the enzyme activity and the enantioselectivity decreased, possibly due to the
denaturation of enzymes, caused by the quick change of temperature at the very beginning of the
reaction. Based on the experimental results, 480 W was chosen as the optimal microwave power.

Molecules 2015, 20
9954
200
160
120
40
3
3
6
2
28
2
2
4
0
8
4
0
0
0
16
Enzyme activity
E value
1
2
1
60
240
320
400
480
560
640
Microwave(W)
Figure 2. Effect of microwave power on the enzyme activity (μmol/mg/h, ○) and
enantioselectivity (E value, ●) of ionic liquid co-lyophilized CLL in enantioselective
esterification of α-lipoic acid. Reaction conditions: (R,S)-α-lipoic acid (2 mmol), CLL
(
10 mg) co-lyophilized with 20% of [Bmim] BF (wt %, IL/enzyme), n-octanol (5 mmol)
4
and cyclohexane (10 mL) were performed at 30 °C under microwave with various power (W).
2
.6. Effect of Microwave Temperature
The effect of temperature was examined in the range of 15–50 °C under microwave irradiation. The
results in Figure 3 demonstrated that the highest enzyme activity (178.1 μmol/h/mg) was obtained, with
a high enantioselectivity (E = 41.2), at 25 °C for the ionic liquid co-lyophilized lipase. Therefore, 25 °C
was selected as the optimal microwave temperature. Under optimal conditions, a preparative reaction on
a twenty-fold-larger scale gave (R)-α-lipoic acid with about 41% yield and 99% ee.
2
2
1
1
40
00
60
20
45
4
3
0
5
30
2
2
5
0
8
4
0
0
0
Enzyme activity
E value
15
1
5
0
0
1
0
15
20
25
30
35
40
45
50
o
Microwave temperature ( C)
Figure 3. Effect of microwave temperature on the enzyme activity (μmol/mg/h, ○) and
enantioselectivity (E value, ●) of ionic liquid co-lyophilized CLL in enantioselective
esterification of α-lipoic acid. Reaction conditions: (R,S)-α-lipoic acid (2 mmol), CLL (10 mg)
co-lyophilized with 20% of [Bmim] BF
4
(wt %, IL/enzyme), n-octanol (5 mmol) and
cyclohexane (10 mL) were performed at different temperature under microwaves (480 W).

Molecules 2015, 20
.7. Reusability of the Ionic Liquid Co-Lyophilized Enzyme under Microwave Irradiation
The ionic liquid co-lyophilized lipase exhibited excellent reusability under low power microwaves.
9955
2
As shown in Table 5, the ionic liquid co-lyophilized CLL could still keep 95.2% of its original activity
and 94.4% of its original enantioselectivity, even after six reaction cycles.
Table 5. Reusability of ionic liquid co-lyophilized CLL under microwaves.
Reaction Cycle
Relative Activity (%)
E value
41.2
40.9
40.6
40.3
1
2
3
4
5
6
100
99.2
98.5
97.7
96.4
95.2
39.5
38.9
Reaction conditions: (R,S)-α-lipoic acid (2 mmol), CLL (10 mg) co-lyophilized with 20% of [Bmim] BF
4
(
wt %, IL/enzyme), n-octanol (5 mmol) and cyclohexane (10 mL) were performed at 25 °C under
microwaves (480 W).
. Experimental Section
.1. Materials
3
3
D-phenylalanine, α-lipoic acid and o-phthalaldehyde (OPA) were purchased from Sigma–Aldrich
St. Louis, MO, USA). 11-Mercaptoundecanoic acid was also purchased from Sigma–Aldrich and used
as the internal standard. All the ionic liquids (1-butyl-3-methylimidazolium chloride, [Bmim] Cl;
-butyl-3-methylimidazolium acetate [Bmim] Ac; 1-butyl-3-methylimidazolium tetrafluoroborate,
Bmim] BF ) were purchased from Shanghai Chengjie Chemical Co., Ltd. (Shanghai, China). Candida
(
1
[
4
cylindracea A.Y. lipase (AYL) was purchased from Amano Pharmaceutical Co., Ltd. (Nagoya, Japan).
Mucor miehei lipase (MML) was purchased from Novo (Bagsvaerd, Denmark). Porcine pancreatic
lipase (PPL) was purchased from Shanghai Dongfeng Biochemical Reagent Co., Ltd. (Shanghai, China).
Candida lipolytic lipase (CLL) was provided by Wuxi enzyme preparation plant (Wuxi, China). All the
other chemicals were obtained from commercial sources and were of analytical reagent grade.
The OPA-solution (25 mM) was prepared by dissolving OPA (33.5 mg) in 0.5 mL of methanol and
diluted by 10 mL of a potassium borate buffer (0.4 M, pH 9.9). The internal standard solution was
prepared by dissolving 10 mg 11-mercaptoundecanoic acid in 10 mL methanol.
3
.2. Microwave Equipment
Reactions were carried out in a commercial multimode microwave reactor (MCR-3, Shanghai JieSi
Microwave Chemistry Corporation). The machine consisted of a continuous focused microwave power
delivery system and an operator selectable power output from 0 to 800 W. The temperature of the
reaction mixture was monitored and kept constant (±1 °C) by using a contact Teflon platinum resistance
temperature transducer inserted directly into the reaction mixture. The content of the vessel was stirred
by a rotating magnetic plate located below the base of the microwave cavity and a Teflon-coated
magnetic stir bar in the vessel.

Molecules 2015, 20
.3. Preparation of the Co-Lyophilized Lipase
Lipase powder (1 g) was dispersed in phosphate buffer (50 mL, pH 8.0, 0.1 M) at 4 °C for 2 h under
9956
3
stirring, and the insoluble impurity was removed by centrifugation (8000 rpm, 5 min). The supernatant
was lyophilized for 48 h. The lyophilized enzyme powder (200 mg) was dissolved again in the above
phosphate buffer (10 mL). To prepare the co-lyophilized lipase, [Bmim] BF
4
(40 mg) was added to the
enzyme solution and the solution was lyophilized for 12 h.
3
.4. Resolution of α-Lipoic Acid
The reaction mixture containing cyclohexane (10 mL), (R,S)-α-lipoic acid (2 mmol) and n-octanol
5 mmol) was incubated in a microwave oven (480 W, 25 °C, 150 rpm). The reaction was initiated by
addition of CLL (10 mg) co-lyophilized with [Bmim] BF (20%, wt %, IL/enzyme). The reaction mixture
100 μL) was withdrawn periodically and extracted by the solution of sodium bicarbonate
25 mM, 200 μL) to obtain the un-reacted α-lipoic acid aqueous solution for High Performance Liquid
(
4
(
(
Chromatography (HPLC) detection.
3.5. Reusability of the Co-Lyophilized Lipase
After each round of reaction, the co-lyophilized lipase was recycled by centrifugation (10,000 rpm,
min, 4 °C) and washed with cyclohexane for three times. Then, the enzyme was repeatedly used in the
5
next reaction. The residue activity of the recycled enzyme was compared with the enzyme activity of the
first cycle (100%).
3
.6. High Performance Liquid Chromatography (HPLC) Analysis
α-Lipoic acid was determined using OPA derivatization and fluorescence detection, based on the
previous report [44], with a minor modification.
The extracted un-reacted α-lipoic acid solution (50 μL) was mixed with 50 μL of the internal standard
solution and 25 μL of a 0.05 M aqueous solution of NaOH containing 0.5 mg/mL of NaBH and incubated
4
at 60 °C for 10 min to obtain the reduced α-lipoic acid before OPA derivatization. The OPA derivatizations
were carried out by mixing the reduced sample solution (25 μL), the D-phenylalanine solution (25 μL,
2
5 mM) with the OPA-solution (50 μL, 25 mM) for 60 min, standing at room temperature. The reaction
was terminated by acidification with 100 µL of phosphate buffer (0.02 M, pH 5.8) before injection.
The sample analysis was performed on an Agilent 1200 HPLC equipped with a 250 × 4 mm
LiChrospher 100 RP-18 (5 μm) reversed-phase column (Merck, Darmstadt, Germany). The mobile phase
was composed of 55% (v/v) phosphate buffer (0.02 M, pH 5.8) and 45% (v/v) acetonitrile/methanol (v/v,
1
:1). A flow rate of 1.8 mL/min was used. Detection was carried out by excitation at 230 nm and emission
over 418 nm. The injection of samples (20 μL) was performed using an auto-sampler (Jasco 851-AS).
The enantiomeric excess of the un-reacted α-lipoic acid (ee) was determined by calculating the peak
areas of the two enantiomers, and the conversion (C) was determined based on the decrease of the
substrate. The enzyme activity (μmol/h/mg) was defined as the amount (in micromoles) of the decreased
substrate per hour per milligram of enzyme and was detected while the conversion was controlled in the

Molecules 2015, 20
9957
range of the 15%~30%. The enantiomeric ratio (E value) was calculated according to Chen et al. [45].
All experiments were carried out in triplicate and all data were obtained based on the average values.
ln [(1 - C)(1 -ee) ]
E =
(
1)
ln [(1 - C)(1 + ee) ]
4
. Conclusions
In this study, we take the advantage of the synergistic effect between ionic liquid co-lyophilized lipase
and microwave irradiation to enhance the enzyme performance in enantioselective esterification of
α-lipoic acid. Under the optimal reaction conditions, the ionic liquid co-lyophilized lipase exhibited a
satisfied enzyme performance (E value, 41.2; enzyme activity, 178.1 μmol/h/mg). Furthermore, high
reusability of the ionic liquid co-lyophilized lipase under low power MW conditions make it more
attractive for resolution of (R,S)-α-lipoic acid in industry.
Acknowledgments
The authors are grateful for the financial support from National Natural Science Foundation of China
(
No. 21172093, 21175055, 31070708, 81472030, and 21072075), the Special Fund for Basic Scientific
Research of Jilin University (2013), the Scientific Research Project of Liaoning Educational Committee
general project No. L2012431), Natural Science Foundation of Jilin Province (Nos. 20140101141JC,
01115038 and 20150204001YY), and Norman Bethune Program of Jilin University (2012210).
(
2
Author Contributions
Xiaona Xie conceived and designed the experiments; Ning Liu, Lei Wang, and Zhi Wang performed
the experiments and analyzed the data; Ning Liu, Lei Wang, Zhi Wang, Liyan Jiang, Zhuofu Wu,
Hong Yue, and Xiaona Xie wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1.
2.
3.
Reed, L.J.; Debusk, B.G.; Gunsalus, I.C.; Hornberger, C.S., Jr. Crystalline α-lipoic acid: A catalytic
agent associated with pyruvate dehydrogenase. Science 1951, 114, 93–94.
Packer, L.; Witt, E.H.; Tritschler, H.J. Alpha-lipoic acid as a biological antioxidant. Free Radic.
Biol. Med. 1995, 19, 227–250.
Cho, Y.S.; Lee, J.; Lee, T.H.; Lee, E.Y.; Lee, K.U.; Park, J.Y.; Moon, H.B.J. α-Lipoic acid inhibits
airway inflammation and hyperresponsiveness in a mouse model of asthma. Allergy Clin. Immunol.
2
004, 114, 429–435.
4
.
Baur, A.; Harrer, T.; Peukert, M.; Jahn, G.; Kalden, J.R.; Fleckenstein, B. Alpha-lipoic acid is
an effective inhibitor of human immuno-deficiency virus (HIV-1) replication. Wien. Klin. Wochenschr.
1
991, 69, 722–724.

Molecules 2015, 20
9958
5
6
.
.
Brookes, M.H.; Golding, B.T.; Howes, D.A.; Hudson, A.T. Proof that the absolute configuration of
natural α-lipoic acid is R by the synthesis of its enantiomer [(S)-(–)-α-lipoic acid] from (S)-malic
acid. J. Chem. Soc. Chem. Commun. 1983, 19, 1051–1053.
Upadhya, T.T.; Nikalje, M.D.; Sudalai, A. Asymmetric dihydroxylation and hydrogenation
approaches to the enantioselective synthesis of R-(+)-α-lipoic acid. Tetrahedron Lett. 2001, 42,
4
891–4893.
Zhang, S.; Chen, X.; Zhang, J.; Wang, W.; Duan, W. An enantioselective formal synthesis of
+)-(R)-α-lipoic acid by an l-proline-catalyzed aldol reaction. Synthesis 2008, 3, 383–386.
7.
8.
9.
(
Chavan, S.P.; Praveen, C.; Ramakrishna, G.; Kalkote, U.R. Enantioselective synthesis of R-(+)-α
and S-(−)-α-lipoic acid. Tetrahedron Lett. 2004, 45, 6027–6028.
Zhang, Y.J.; Zhang, W.X.; Zheng, G.W.; Xu, J.H. Identification of an ε-Keto ester reductase
for the efficient synthesis of an (R)-α-lipoic acid precursor. Adv. Synth. Catal. 2015,
doi:10.1002/adsc.201500001.
1
1
0. Olbrich, M.; Gewald, R. Process for the Enantioselective Reduction of 8-Chloro-6-oxo-octanoic
Acid Alkyl Esters. U.S. Patent 7,135,328 [P], 14 November 2006.
1. Müller, M.; Sauer, W.; Laban, G. From 8-Chloro-6-oxooctanoic Acid Alkyl Esters, Using Alcohol
Dehydrogenases or Carbonyl Reductases in the Presence of NADH or NADPH as Cofactor;
Intermediates to Alpha-Lipoic Acid. U.S. Patent 7,157,253[P], 2 January 2007.
2. Adger, B.; Bes, M.T.; Grogan, G.; McCague, R.; Pedragosa-Moreau, S.; Roberts, S.M.; Villa, R.;
Wan, P.W.H.; Willetts, A.J. The synthesis of (R)-(+)-lipoic acid using a monooxygenase-catalysed
biotransformation as the key step. Bioorganic Med. Chem. 1997, 5, 253–261.
1
1
1
1
1
1
3. Gopalan, A.S.; Jacobs, H.K. Bakers’ yeast reduction of alkyl 6-chloro-3-oxohexanoates: Synthesis
of (R)-(+)-α-lipoic acid. J. Chem. Soc. Perkin. Trans. 1990, 1, 1897–1900.
4. Fadnavis, N.W.; Koteshwar, K. Remote control of stereoselectivity: Lipase catalyzed enantioselective
esterification of racemic α-lipoic acid. Tetrahedron Asymmetry 1997, 8, 337–339.
5. Yan, H.D.; Zhang, Y.J.; Shen, L.J.; Wang, Z. Lipase-catalysed sequential kinetic resolution of
α-lipoic acid. Maejo Int. J. Sci. Technol. 2012, 6, 152–158.
6. Yan, H.D.; Wang, Z.; Chen, L.J. Kinetic resolution of α-lipoic acid via enzymatic differentiation of
a remote stereocenter. J. Ind. Microbiol. Biotechnol. 2009, 36, 643–648.
7. Zhou, W.J.; Ni, Y.; Zheng, G.W.; Chen, H.H.; Zhu, Z.R.; Xu, J.H. Enzymatic resolution of a chiral
chlorohydrin precursor for (R)-α-lipoic acid synthesis via lipase catalyzed enantioselective
transacylation with vinyl acetate. J. Mol. Catal. B Enzym. 2014, 99, 102–107.
1
1
2
2
2
8. Motasemi, F.; Ani, F.N. A review on microwave-assisted production of biodiesel. Renew. Sustain.
Energy Rev. 2012, 16, 4719–4733.
9. Oghbaei, M.; Mirzaee, O. Microwave versus conventional sintering: A review of fundamentals,
advantages and applications. J. Alloys Compd. 2010, 494, 175–189.
0. Lidström, P.; Tierney, J.; Wathey, B.; Westman, J. Microwave assisted organic chemistry—A review.
Tetrahedron 2001, 57, 9225–9283.
1. Strauss, C.R.; Rooney, D.W. Accounting for clean, fast and high yielding reactions under microwave
conditions. Green Chem. 2010, 12, 1340–1344.
2. Zhang, Q.; Zhang, S.; Deng, Y. Recent advances in ionic liquid catalysis. Green Chem. 2011, 13,
2
619–2637.

Molecules 2015, 20
9959
2
3. Tian, R.; Yang, C.H.; Wei, X.F.; Xun, E.N.; Wang, R.; Cao, S.G.; Wang, Z.; Wang, L. Optimization of
APE1547-catalyzed enantioselective transesterification of (R/S)-2-methyl-1-butanol in an ionic
liquid. Biotechnol. Bioprocess Eng. 2011, 16, 337–342.
2
2
2
2
2
2
3
3
3
4. Sheldon, R.A. Green solvents for sustainable organic synthesis: State of the art. Green Chem. 2005, 7,
2
67–278.
5. Yang, Z.; Pan, W. Ionic liquids: Green solvents for nonaqueous biocatalysis. Enzym. Microb.
Technol. 2005, 37, 19–28.
6. Martínez-Palou, R. Ionic liquid and microwave-assisted organic synthesis: A “green” and synergic
couple. J. Mex. Chem. Soc. 2007, 51, 252–264.
7. Yu, D.; Wang, Y.; Wang, C.; Ma, D.; Fang, X. Combination use of microwave irradiation and ionic
liquid in enzymatic isomerization of xylose to xylulose. J. Mol. Catal. B Enzym. 2012, 79, 8–14.
8. Yu, D.; Ma, D.; Wang, Z.; Wang, Y.; Pan, Y.; Fang, X. Microwave-assisted enzymatic resolution
of (R,S)-2-octanol in ionic liquid. Process Biochem. 2012, 47, 479–484.
9. Park, S.; Kazlauskas, R.J. Biocatalysis in ionic liquids-advantages beyond green technology. Curr.
Opin. Biotechnol. 2003, 14, 432–437.
0. Kappe, C.O. Microwave dielectric heating in synthetic organic chemistry. Chem. Soc. Rev. 2008, 37,
1
127–1139.
1. Mallakpour, S.; Rafiee, Z. New developments in polymer science and technology using combination
of ionic liquids and microwave irradiation. Prog. Polym. Sci. 2011, 36, 1754–1765.
2. Itoh, T.; Matsushita, Y.; Abe, Y.; Han, S.H.; Wada, S.; Hayase, S.; Kawatsura, M.; Takai, S.;
Morimoto, M.; Hirose, Y. Increased enantioselectivity and remarkable acceleration of lipase-catalyzed
transesterification by using an imidazolium PEG-alkyl sulfate ionic liquid. Chem. Eur. J. 2006, 12,
9
228–9237.
3
3
3
3. Abe, Y.; Hirakawa, T.; Nakajima, S.; Okano, N.; Hayase, S.; Kawatsura, M.; Hirose, Y.; Itoh, T.
Remarkable activation of an enzyme by (R)-oyrrolidine-substituted imidazolium alkyl PEG sulfate.
Adv. Synth. Catal. 2008, 350, 1954–1958.
4. Abe, Y.; Yoshiyama, K.; Yagi, Y.; Hayase, S.; Kawatsura, M.; Itoh, T. A rational design of
phosphonium salt type ionic liquids for ionic liquid coated-lipase catalyzed reaction. Green Chem.
2
010, 12, 1976–1980.
5. Abe, Y.; Yagi, Y.; Hayase, S.; Kawatsura, M.; Itoh, T. Ionic liquid engineering for
lipase-mediated optical resolution of secondary alcohols: Design of ionic liquids applicable to ionic
liquid coated-lipase catalyzed reaction. Ind. Eng. Chem. Res. 2012, 51, 9952–9958.
6. Lee, J.K.; Kim M.J. Ionic liquid co-lyophilized enzyme for biocatalysis in organic solvent:
Remarkably enhanced activity and enantioselectivity. J. Mol. Catal. B Enzym. 2011, 68, 275–278.
7. Yoshiyama, K.; Abe, Y.; Hayse, S.; Nokami, T.; Itoh, T. Synergetic activation of lipase by an amino
acid with alkyl-PEG-sulfate ionic liquid. Chem. Lett. 2013, 42, 663–665.
3
3
3
3
4
8. Hudson, E.P.; Eppler, R.K.; Clark, D.S. Biocatalysis in semi-aqueous and nearly anhydrous conditions.
Curr. Opin. Biotechnol. 2005, 16, 637–643.
9. Carrea, G.; Otto, L.G.; Riva, S. Role of solvents in the control of enzyme selectivity in organic
media. Trends Biotechnol. 1995, 13, 63–70.
0. Gorman, L.A.S.; Dordick, J.S. Organic solvents strip water off enzymes. Biotechnol. Bioeng. 1992, 39,
3
92–397.

Molecules 2015, 20
9960
4
4
4
4
4
1. Wang, L.; Tai, J.D.; Wang, R.; Xun, E.N.; Wei, X.F.; Wang, L.; Wang, Z. Enantioselective
transesterification of glycidol catalysed by a novel lipase expressed from Bacillus subtilis.
Biotechnol. Appl. Biochem. 2010, 56, 1–6.
2. Chen, C.; Sun, L.D.; Li, Z.X.; Li, L.L.; Zhang, J.; Zhang, Y.W.; Yan, C.H. Ionic liquid-based route
to spherical NaYF nanoclusters with the assistance of microwave radiation and their multicolor
4
upconversion luminescence. Langmuir 2010, 26, 8797–8803.
3. Yang, Z.C.; Niu, X.D.; Fang, X.D.; Chen, G.; Zhang, H.; Yue, H.; Wang, L.; Zhao D.T.; Wang, Z.
Enantioselective esterification of ibuprofen under microwave irradiation. Molecules 2013, 18,
5
472–5481.
4. Niebch, G.; Büchele, B.; Blome, J.; Grieb, S.; Brandt, G.; Kampa, P.; Raffel, H.H.; Locher, M.;
Borbe, H.O.; Nubert, I.; et al. Enantioselective high-performance liquid chromatography assay of
(
+)R- and (−)S-α-lipoic acid in human plasma. Chirality 1997, 9, 32–36.
5. 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.
Sample Availability: Samples of the compounds ((R,S)-α-lipoic acid and (R)-α-lipoic acid) are available
from the authors.
©
2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(