Enzymatic resolution of a chiral chlorohydrin precursor for (R)-α-lipoic acid synthesis via lipase catalyzed enantioselective transacylation with vinyl acetate

Article abstract of DOI:10.1016/j.molcatb.2013.11.005

A new and efficient process was developed by lipase-catalyzed transacylation to resolve ethyl 8-chloro-6-hydroxy octanoate (ECHO) to produce an important chiral precursor for the synthesis of (R)-α-lipoic acid. After optimization of biocatalyst, solvent, acyl donor, temperature and enzyme loading, (S)-O-acetylated ECHO was achieved in 94% ee, 35% isolated yield and 38 g L^-1 d^-1 space-time yield using Novozym 435 as biocatalyst. Subsequently, the enzymatic resolution reaction was successfully repeated for 7 batches, retaining over 40% conversions.

Full text of DOI:10.1016/j.molcatb.2013.11.005

Journal of Molecular Catalysis B: Enzymatic 99 (2014) 102–107
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzymatic resolution of a chiral chlorohydrin precursor for
(R)-␣-lipoic acid synthesis via lipase catalyzed enantioselective
transacylation with vinyl acetate
Wei-Jia Zhoua, Yan Nia, Gao-Wei Zhenga,∗, Huan-Hui Chenb, Zhi-Rong Zhub,
Jian-He Xua,∗
a Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
130 Meilong Road, Shanghai 200237, PR China
b Department of Chemistry, Tongji University, 1239 Siping Road, Shanghai 200092, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 June 2013
Received in revised form 6 November 2013
Accepted 7 November 2013
Available online 16 November 2013
A new and efficient process was developed by lipase-catalyzed transacylation to resolve ethyl 8-chloro-6-
hydroxy octanoate (ECHO) to produce an important chiral precursor for the synthesis of (R)-␣-lipoic acid.
After optimization of biocatalyst, solvent, acyl donor, temperature and enzyme loading, (S)-O-acetylated
ECHO was achieved in 94% ee, 35% isolated yield and 38 g L−1 d−1 space-time yield using Novozym 435
as biocatalyst. Subsequently, the enzymatic resolution reaction was successfully repeated for 7 batches,
retaining over 40% conversions.
Keywords:
© 2013 Elsevier B.V. All rights reserved.
Ethyl 8-chloro-6-hydroxy octanoate
(R)-␣-Lipoic acid
Transacylation
Novozym 435
1. Introduction
been paid to the stereoselective synthesis of optically pure (R)
or (S)-enantiomer, usually via asymmetric synthesis from its pre-
␣-Lipoic acid, an excellent antioxidant widely used in pharma-
ceuticals, health products and skin conditioners, has been
recognized as a B-family vitamin involved in the biochemical decar-
boxylation of ␣-keto acids and as a growth factor for certain
microorganisms. Despite generally commercialized as racemate,
the anti-oxidation activity of ␣-lipoic acid was evidenced to reside
in its (R)-enantiomer [1] which occurs naturally in mitochondrial
complexes. With the physiological function of regulating neuronal
calcium homeostasis [2] and pro-inflammatory cytokines as well
as altering the expression of “toxic genes”, (R)-␣-lipoic acid has
been widely used to treat diabetes [3,4], liver diseases [5], radi-
ation injury [6] and recommended as a “neuroprotective agent”
[7].
cursor [9]. Enzyme-catalyzed organic reactions have provided a
great impetus to organic synthesis these years [10–13] for sev-
eral advantages, such as mild and environmentally benign reaction
conditions and remarkable chemo-, regio-, and stereoselectivities.
(R)-␣-Lipoic acid has been obtained through kinetic resolution
of racemate by enzymatic esterification of the carboxylic group
that is 4 carbon atoms away from the stereogenic center [14,15].
However, enzymatic resolution involving a remote chiral center
represents a tough task in practice with a poor enantioselectivity
(E < 5).
In this study, we proposed an alternative route for enzymatic
resolution of racemic ethyl 8-chloro-6-hydroxy octanoate (ECHO),
a key chlorohydrin precursor for (R)-␣-lipoic acid (Scheme 1)
[16]. Lipases are well known for their low cost and great tol-
erance against various substrates and reaction solvents, and
lipase-catalyzed acylation of the hydroxyl group represents an
important class of enzymatic transformations in organic synthe-
sis. Considering these merits, a new method for resolving racemic
ECHO is reported herein through direct transacylation of the chi-
ral hydroxyl group, instead of esterifying the remote carboxylic
group of ECHO, using a commercially available immobilized lipase,
Novozym 435, under the optimal conditions which are described in
details.
Since the first isolation and identification of ␣-lipoic acid from
natural sources, several routes for its synthesis by chemical meth-
ods have been developed, e.g., from adipate, cyclohexanone and
their derivants [8]. With the growing demand for enantiomeri-
cally pure active pharmaceutical ingredients, great attention has
∗
Corresponding author. Fax: +86 21 6425 0840.
E-mail addresses: gaoweizheng@ecust.edu.cn (G.-W. Zheng),
jianhexu@ecust.edu.cn (J.-H. Xu).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.11.005

W.-J. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 102–107
103
O
OC2H5
Cl
OH
O
O
Lipase
CH3CHO
O
O
+
OC2H5
OC2H5
Cl
OH
Cl
OAc
KOH, CH3OH
O
SOCl2, pyridine
OC2H5
Cl
OH
O
CH3SO2Cl, Et3N
OC2H5
Cl
Cl
O
OC2H5
Cl
O
S
1. Na2S, S
O2
2. KOH, H2O
1. Na2S, S
2. KOH, H2O
O
COOH
OC2H5
S S
SH
SH
Scheme 1. The synthetic route of (R)-␣-lipoic acid from racemic ethyl 8-chloro-6-hydroxy octanoate.
2. Experimental
fluorescens, Pseudomonas putida and Bacillus subtilis were obtained
from our laboratory. Racemic ethyl 8-chloro-6-hydroxyoctanoate
was kindly provided by Fushilai Medicine & Chemical Co., Ltd.
(Changshu, Jiangsu, China). All other chemicals used in this work
were of analytical grade from local supplies, and were dried with
2.1. Enzymes and reagents
Novozym 435 (lipase B from Candida antarctica) and Lipozym
TLIM (from Thermomyceslanuginosus) were purchased from
Novozymes Co. (Denmark). Lipase CRL (from Candida rugosa) and
lipase PPL (from porcine pancreas, Type II) were purchased from
Sigma (St. Louis, MO). Lipase AYS, lipase AH, lipase PS-C, lipase
PS-D, lipase AK, lipase AS, lipase D and lipase OF were purchased
from Amano Enzymes Co. (Japan). Lipases from Pseudomonas
˚
molecular sieve 3 A before using.
2.2. General procedure for lipase-catalyzed transacylation
To optimize the reaction conditions, enzymatic reactions were
usually performed in shake flasks on a shaker. In a typical reaction,

104
W.-J. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 102–107
Table 1
a certain amount of (R,S)-ECHO and proportional amount of vinyl
acetate were added into 10 ml solvent contained in a 100-ml shake
flask. After the enzyme was added, the mixture was sealed and
shaken at 200 rpm in an incubator. The detailed conditions were
listed in the caption of figures or footnote of tables.
Screening of enzymes for transacylation of racemic ECHO with vinyl acetate.a
Entry
Enzyme
Time [h]
Conv. (%)
eep (%)
Ed
1
2
3
4
5
6
7
8
Novozym 435
Lipase MY
Lipase CRL
Lipase AYS
Lipase AK
Lipozym TLIM
Lipase PS-C
Lipase PS-D
Lipase AH
Lipase AS
Lipase D
Lipase OF
P. putida lipase
B. subtilis lipB
P. fluorescens lipase
48
72
72
72
24
72
12
48
72
72
72
72
72
72
72
49
30
26
22
48
28
54
48
12
n.a.b
n.a.
n.a.
8
86 (S)
66 (R)
67 (R)
59 (R)
44 (S)
29 (S)
19 (S)
20 (S)
15 (R)
n.d.c
n.d.
n.d.
59 (S)
49 (S)
29 (S)
34
6
6
5
4
2
2
2
1
n.d.
n.d.
n.d.
4
3
2
2.3. Repeated batch reactions
Racemic ECHO (50 mM), vinyl acetate (60 mM) and diisopropyl
ether (DIPE, 50 ml) were added into a 100-ml three-necked flask,
and then preheated to 40 ◦C. After 0.5 g of carrier-bound lipase
(Novozym 435) was added, the mixture was mechanically stirred
(100 rpm) at 40 ◦C. The reaction was terminated at about 42%
conversion by filtration of the immobilized enzyme. The filtered
enzyme was washed with DIPE (25 ml × 2) after each use, and then
added into a fresh reaction mixture for the next batch reaction.
9
10
11
12
13
14
15
13
33
a
Reactions were performed at 30 ◦C with 10 mM racemic ECHO, 50 mg enzymes
in 10 ml vinyl acetate.
2.4. Enzymatic transacylation in a preparative scale
b
No activity.
Not determined.
c
A mixture of ECHO (50 mM), vinyl acetate (60 mM) and DIPE (1 L)
was added into a 2-L three-necked flask fitted with reflux condenser
and mechanical stirrer (100 rpm), and then preheated to 40oC with
a temperature-controlled water bath. The reaction was initiated by
addition of 10 g Novozym 435 and incubation at 40 ◦C. The reaction
was terminated when the conversion reached 42%. After remov-
ing the enzyme from the reaction mixture by filtration, substrate
and product were separated by column chromatography on silica
gel using petroleum ether/ethyl acetate (10:1, v/v) as eluent, and
recovered by removal of the solvent under reduced pressure and
dried under vacuum. The product was characterized by 1H NMR,
13C NMR and mass spectrum. The optical purity of product was
determined by chiral gas chromatography and polarimetry.
d
Enantioselectivity (E) = ln[1 − c(1 + eep)]/ln[1 − c(1-eep)], which was calculated
according to Chen et al. [17].
acetate as both acyl donor and reaction medium (Table 1). Among
the lipases tested, Novozym 435 exhibited relatively high transacy-
lation ability and the highest enantioselectivity (E = 34) towards the
rac-ECHO, and the E value of the reaction under arbitrarily chosen
reaction conditions was much higher compared to previous reports
about direct resolution of ␣-lipoic acid via esterification with alco-
hol (E < 5) [15]. Thus, Novozym 435, a commercial immobilized
lipase, was chosen as the preferable catalyst for further study.
3.2. Effect of acyl donor
2.5. Analysis methods
It has been demonstrated that acyl donor has great influence on
both enantioselectivity and reaction rate [18]. Preliminary experi-
ment showed that no esterification products were detected when
acetic acid was used as an acyl donor. Therefore, a variety of acyl
donors including esters and anhydrides were examined and sum-
marized in Table 2. Obviously, transacylation of ECHO using esters
as acyl donor gave higher conversion and better stability than anhy-
drides. The highest reactivity and stability were achieved in the
transacylation of ECHO using vinyl acetate as acyl donor. In case of
enol ester as an acyl donor, the lipase-catalyzed transacylation with
a secondary alcohol proceeded more easily, affording the corre-
sponding ester of the chiral alcohol and vinyl alcohol. The latter can
escape from the reaction system in the form of acetaldehyde after
isomerization, therefore making the reaction irreversible. Although
8-chloro-6-hydroxy octanoate has an acyl group as well as a
hydroxyl group, no side reaction, such as intramolecular cycliza-
tion and polymerization, was observed in all the cases, based on
the GC spectra of reaction samples. Hence, vinyl acetate was used
as the acyl donor for the subsequent experiments
Enantiomeric excess of substrate (ees) was measured by
gas chromatography with chiral column after pre-derivation as
described below. The pre-derivation of samples (500 ␮l) was per-
formed by evaporating the solvent (DIPE) and then reacting with
100 ␮l propionic andydride and 20 ␮l pyridine for 2 h in boiling
water bath. The mixture subsequently was washed with 2 M H2SO4
(300 ␮l) and water (200 ␮l × 3), diluted with 400 ␮l ethyl acetate,
followed by drying over anhydrous Na2SO4. Enantiomeric excess of
product (eep) and ees were analyzed by gas chromatography with
chiral column directly, and conversion of substrate was calculated
with ees and eep.
Gas chromatography was performed on Shimadzu
GC-14C (Kyoto, Japan), equipped with FID detector and
high-resolution CP-ChiralSil-DEX CB chiral column (Varian,
25 m × 0.25 mm × 0.25 ␮m) using N2 as carrier gas. The column,
injector and detector temperatures were set at 155, 280 and
280 ◦C. Retention times: (R)-O-acetylated ECHO, 16.7 min; (S)-O-
acetylated ECHO, 17.4 min; (R)-O-propionylated ECHO, 24.9 min;
(S)-O-propionylated ECHO, 25.7 min.
3.3. Effect of solvent
3. Results and discussion
For enzymatic resolution of racemic ECHO in an organic sol-
vent system, a careful investigation on the reaction medium was
conducted since it has been demonstrated that the activity and
enantioselectivity of lipases are greatly affected by the nature of
the non-aqueous solvents used when catalysis occurs in a nearly
anhydrous environment [19]. In this work, effect on the reaction
of six hydrophobic solvents was evaluated, and the highest con-
version (43%) was observed in DIPE (Table 3). The poor solubility
of ECHO in isooctane and heptane was suspected to account for
the moderate conversion and enantioselectivity. A suitable organic
3.1. Screening of biocatalysts
During the last few decades, lipases have been applied as power-
ful tools in numerous of organic synthesis because of their excellent
activity and robustness. More importantly, they could smoothly
catalyze reactions without addition of expensive cofactor and could
be reused after immobilization, which makes them commercially
available at competitive prices. Initially, several lipases were there-
fore investigated for the resolution of racemic ECHO using vinyl

W.-J. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 102–107
105
Table 2
Effect of acyl donor on enzymatic transacylation of ECHO.a
Acyl donor
1st batch
Conv. (%)
2nd batch
Conv. (%)
Stability (%)b
eep (%)
eep (%)
Ester
Vinyl acetate
Vinyl propionate
Vinyl n-butyrate
43
29
34
94
96
97
41
25
24
95
96
98
95
86
71
Acetic anhydride
19
18
25
17
13
94
81
99
>99
>99
15
9
14
9
93
59
>99
>99
>99
79
50
56
53
92
Anhydride
Propionic andydride
Butyric anhydride
Isobutyric anhydride
Pentanoic anhydride
12
a
Reactions were performed at 40 ◦C for 7 h with 50 mM racemic ECHO, 60 mM acyl donors and 50 mg Novozym 435 in 10 ml DIPE.
Ratio of ECHO conversion in the 2nd batch reaction to that in the 1st batch.
b
Table 3
Effect of solvent on the enzymatic transacylation of ECHO with vinyl acetate.a
Solvent
1st batch
Conv. (%)
2nd batch
Conv. (%)
Stability (%)b
eep (%)
eep (%)
Diisopropyl ether
Methyl tertiary butyl ether
Isooctane
n-Heptane
Toluene
43
38
35
28
15
11
94
97
98
93
99
94
41
34
22
19
13
10
95
99
98
93
97
91
95
89
63
68
87
91
Butyl acetate
a
Reactions were performed at 40 ◦C for 7 h with 50 mM rac-ECHO, 60 mM vinyl acetate and 50 mg Novozym 435 in 10 ml various solvents.
Ratio of ECHO conversion in the 2nd batch reaction to that of the 1st batch.
b
solvent should dissolve enough substrates for the lipase-catalyzed
40 ◦C) would result in a relatively rapid loss of enzyme stability.
Indeed, above a certain temperature, enzyme inactivation occurs,
and the stability decreases. Comparing the results of 40 and 50 ◦C,
we found that the conversion at 50 ◦C was higher than that at 40 ◦C,
however, after 2 batches use, its relative conversion attained 95% at
40oC, while 73% at 50 ◦C. This was because high temperature (50 ◦C)
could deactivate the Novozym 435. Considering the activity and
stability of Novozym 435, we adopted 40 ◦C for further experiments
in this work.
transacylation, and the solvent should not affect the lipase activity
and stability significantly. The results showed that Novozym 435
generally exhibited high conversion in non-polar solvents but no
conversion was observed in hydrophilic solvents such as acetoni-
trile, DMSO and alcohols (data not shown) which might alter or
denature enzymes by stripping off the so-called “essential water”
from the enzyme [20], and alter the native conformation of the
lipase by disrupting hydrogen bonding and hydrophobic interac-
tions, thereby leading to very low activity which greatly limits the
application of enzymatic procedures in this area. The reaction gave
higher eep in MTBE than DIPE, but lower conversion and worse sta-
bility. DIPE therefore was finally considered as the best medium
for the biocatalytic resolution of racemic ECHO, with an increased
E value of this reaction from 35 to 69.
3.5. Effect of enzyme loading
The amount of enzyme loading is a crucial economical fac-
tor for successful practical application. Therefore, the effect of
enzyme loading on the initial rate of transacylation was exam-
ined with varied amounts of enzyme ranging from 1 to 25 g L−1
using DIPE as the reaction medium at 40 ◦C. As shown in Fig. 1,
the initial rates of enzymatic transacylation of ECHO depended lin-
early on the amount of enzyme applied, but further increase of
enzyme loading from 10 g L−1 to 25 g L−1 resulted in only slight
improvement. It demonstrated the saturation of the combina-
tion between the enzyme and substrate, and extra addition of
enzyme will not lead to notable acceleration of reaction further.
Due to the obvious adsorption of substrate by the immobilization
3.4. Effect of reaction temperature
Temperature is one of the important factors which influence
enzyme activity. The effect of reaction temperature on the enzy-
matic transacylation of ECHO was investigated over a range from
20 ◦C to 50 ◦C. As shown in Table 4, it is demonstrated that the
reaction rate increased with the rising of temperature while enan-
tioselectivity varied slightly. However, higher temperature (over
Table 4
Effect of temperature on enzymatic transacylation of ECHO.a
Temperature (oC)
1st batch
Conv. (%)
2nd batch
Conv. (%)
Stability (%)b
eep (%)
eep (%)
20
30
40
50
24
28
43
60
98
97
94
67
22
25
41
44
98
94
95
96
92
89
95
73
a
Reactions were performed at different temperatures for 7 h with 50 mM rac-ECHO, 60 mM vinyl acetate and 50 mg Novozym 435 in 10 ml DIPE.
Ratio of ECHO conversion in the 2nd batch reaction to that of the 1st batch.
b

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W.-J. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 102–107
100
12
10
8
80
60
40
20
0
6
4
2
1
2
3
4
5
6
7
8
9
10
Batch number
0
0
5
10
15
20
25
30
Fig. 2. Repeated batch reactions of enzymatic transacylation of ECHO. Reactions
were performed at 40◦ C with 50 mM racemic ECHO, 60 mM vinyl acetate and 0.5 g
Novozym 435 in 50 ml DIPE until the conversion reached 40%. Symbols: (solid bar)
conversion (solid circle) eep.
Enzyme load (g/l)
Fig. 1. Initial rate of the enzymatic transacylation of ECHO with vinyl acetate at var-
ied enzyme loads. Reactions were performed at 40◦ C for 0.5 h with 50 mM racemic
ECHO, 60 mM vinyl acetate and Novozym 435 of varied loadings in 10 ml DIPE.
Although the space-time yield of (S)-O-acetylated ECHO decreased
slightly to 23.3 g L−1 d−1 because of the enzyme inactivation during
the last 3 batches, the ratio of substrate to catalyst (S/C, g/g) was
markedly enhanced from 1 to 11.
carriers of enzyme in the presence of over-dose of Novozym 435
(data not shown) and from the economic point of view, an enzyme
loading of 10 g L−1 was adopted in the following repeated-batch
reactions.
4. Conclusions
A new route for synthesis of a chiral chlorohydrin precursor
for (R)-␣-lipoic acid was established by enzymatic enantiose-
lective transacylation of ECHO. After determining Novozym 435
as the most suitable catalyst, the biocatalytic reaction system
was carefully optimized for optical resolution, resulting in vinyl
acetate as acyl donor, DIPE as solvent, reaction temperature of
40 ◦C and enzyme loading of 10 g L−1. The space-time yield of (S)-
O-acetylated ECHO reached 38 g L−1 d−1 with eep of 94% for the
gram-scale synthesis. Under the optimized conditions, Novozym
435 could be reused for 7 batches with conversions of over
40% which led to an increase of S/C from 1 to 11. This study
suggests the potential applicability of enzymatic resolution for
the preparation of (R)-␣-lipoic acid precursor in terms of envi-
ronmental friendliness and operational simplicity in a technical
scale.
3.6. Gram scale synthesis of (S)-O-acetylated ECHO
The enzymatic transacylation of ECHO (50 mM) was prelimi-
narily scaled up by 100-fold to a reaction volume of 1 L in a 2-L
three-necked flask with mechanic agitation. The reaction was mon-
itored by GC analysis, and stopped at 3.5 h with a conversion of
42%. The space-time yield of (S)-acetylated ECHO was 38 g L−1 d−1
.
After isolation and purification by silica gel column and normal
work-up, 4.9 g of product was obtained with 94% ee and 35% iso-
lated yield; [a]D25: + 21.7 (c1.01, CHCl3) [16]. 1H NMR (500 MHz,
CDCl3) ␦ 5.04-4.99 (m, 1H),␦ 4.14-4.08 (q, 2H, J = 7.1Hz), ␦ 3.56-
3.47 (m, 2H), ␦ 2.30-2.27 (t, 2H, 7.4Hz), ␦ 2.04 (s, 3H), ␦ 2.02-1.96
(m, 2H), ␦ 1.69-1.52 (m, 4H), ␦ 1.40-1.29 (m, 2H), ␦ 1.26-1.23 (t, 3H,
7.2 Hz); 13C NMR (125 MHz, CDCl3) ␦ = 174.09 (C O), 171.26 (C O),
72.04 (CH), 60.93 (CH2), 41.41 (CH2), 37.79 (CH2), 34.78 (CH2), 34.45
(CH2), 25.38 (CH2), 25.33 (CH2), 21.75 (CH3), 14.91 (CH3); MS (ESI):
m/z = 265.12 (M++1), 207.09, 205.09, 169.12.
Acknowledgements
Thanks to the financial supports from National Natural Sci-
ence Foundation of China (Nos. 31200050 & 21276082), Ministry
of Science and Technology, PR China (Nos. 2011AA02A210 &
2011CB710800), and the Fundamental Research Funds for the Cen-
tral Universities, Ministry of Education, PR China.
3.7. Operational stability
One of the most important characteristics to evaluate feasibility
of an immobilized enzyme is its operational stability and reusability
over an extended period of time in practical application. From the
viewpoint of process economics, the more the number of batches
an enzyme is used, the more economical the process is. An optimal
solvent system for lipase-catalyzed transacylation should not only
increase the lipase activity toward ECHO but also not impair the sta-
bility of lipase during repeated use. Experiments were performed
to examine the stability of Novozym 435. Therefore, the operational
stability of Novozym 435 was evaluated by reusing in the transacy-
lation of ECHO with vinyl acetate. As shown in Fig. 2, the enzyme
retained its high activity for 7 batches of transacylation at an ECHO
concentration of 50 mM. Although the conversion still kept 40%, the
reaction time was lengthened after 8 batches reaction, indicating
that the enzyme activity started to decline. During the 10 batches of
reaction, the conversions were kept over 40% and (S)-O-acetylated
ECHO was produced with an improved eep from 91% to 96%. To the
best of our knowledge, this is the first time that bioresolution reac-
tion of ␣-lipoic acid precursor was achieved in repeated operation.
References
[1] I. Gunsalus, L.S. Barton, W. Gruber, J. Am. Chem. Soc. 78 (8) (1956)
1763–1766.
[2] L. Packer, H.J. Tritschler, K. Wessel, Free Radic. Biol. Med. 22 (1) (1997)
359–378.
[3] A. Faust, V. Burkart, H. Ulrich, C.H. Weischer, H. Kolb, Int. J. Immunopharmacol.
16 (1) (1994) 61–66.
[4] P. Ou, H.J. Tritschler, S.P. Wolff, Biochem. Pharmacol. 50 (1) (1995) 123–126.
[5] A. Marshall, R. Graul, M.Y. Morgan, S. Sherlock, Gut 23 (12) (1982)
1088–1093.
[6] N. Ramakrishnan, W.W. Wolfe, G.N. Catravas, Radiat. Res. 130 (3) (1992)
360–365.
[7] S. Stoll, H. Hartmann, S. Cohen, W. Müller, Pharmacol. Biochem. Behav. 46 (4)
(1993) 799–805.
[8] D.S. Acker. (1957) US Patent 2792406.
[9] R. Zimmer, U. Hain, M. Berndt, R. Gewald, H.U. Reissig, Tetrahedron: Asymm.
11 (4) (2000) 879–887.
[10] A.M. Klibanov, Nature 409 (6817) (2001) 241–246.

W.-J. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 102–107
107
[11] K. Griebenow, A.M. Klibanov, J. Am. Chem. Soc. 118 (47) (1996) 11695–11700.
[12] A.M. Klibanov, Trends Biochem. Sci. 14 (4) (1989) 141–144.
[13] A.M. Klibanov, Trends Biotechnol. 15 (3) (1997) 97–101.
[14] N. Fadnavis, K. Koteshwar, Tetrahedron: Asymm. 8 (2) (1997) 337–339.
[15] H. Yan, Z. Wang, L. Chen, J. Ind. Microbiol. Biotechnol. 36 (5) (2009) 643–648.
[16] G. Rainer, L. Gunter, B. Thomas. (1998) US5731448.
[17] C.S. Chen, Y.F. Fujimoto, G. Girdaukas, C.J. Sih, J. Am. Chem. Soc 104 (1982)
7294–7299.
[18] J. Xu, T. Kawamoto, A. Tanaka, Appl. Microbiol. Biotechnol. 43 (3) (1995)
402–407.
[19] S. Tawaki, A.M. Klibanov, J. Am. Chem. Soc. 114 (5) (1992) 1882–1884.
[20] A. Zaks, A.M. Klibanov, J. Biol. Chem. 263 (7) (1988) 3194–3201.