Lipase-Catalyzed Production of (S)-Carprofen Enhanced by Hydroxyethyl-β-cyclodextrins: Experiment and Optimization

Article abstract of DOI:10.1021/acs.oprd.9b00009

Stereoselective resolution of (R,S)-carprofen methyl ester (CPOMe) by lipase-catalyzed hydrolysis to (S)-carprofen (CP) was investigated in an aqueous medium. With the highest catalytic activity, Candida antarctica Lipase A (CALA) was selected as catalyst compared with eight other lipases. Hydroxyethyl-β-cyclodextrin (HE-β-CD) was added to enhance the solubility of (R,S)-CPOMe, which significantly raised the conversion of substrate from 11.12% to 30.84%. Response surface methodology (RSM) was adopted to evaluate the influence of factors on the substrate conversion (c) and enantiomeric excess of product (ee_p), such as pH, concentrations of enzyme and HE-β-CD, temperature, substrate loading, and reaction time. The optimal conditions were obtained, including pH 6.0, 40 mg/mL CALA, 0.05 mmol substrate, 35 mmol/L HE-β-CD, agitation speed of 600 rpm, temperature of 76 °C, and reaction time of 30 h. Under the above conditions, (S)-CP as the desired product was obtained with an enantiomeric excess of 96.24% and overall conversion of 46.07%.

Full text of DOI:10.1021/acs.oprd.9b00009

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Full Paper
Lipase-catalyzed Production of (S)-Carprofen Enhanced by
Hydroxyethyl-#-cyclodextrins: Experiment and Optimization
Xin Yuan, Guangyong Liu, Pan-Liang Zhang, Weifeng Xu, and Ke-Wen Tang
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00009 • Publication Date (Web): 03 Apr 2019
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Lipase-catalyzed Production of (S)-Carprofen Enhanced by
Hydroxyethyl-β-cyclodextrins: Experiment and Optimization
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Xin Yuan, Guangyong Liu, Panliang Zhang*, Weifeng Xu, Kewen Tang*
Department of Chemistry and Chemical Engineering, Hunan Institute of Science and
Technology, Yueyang 414006, Hunan, China
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ABSTRACT
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Stereoselective resolution of (R,S)-carprofen methyl ester (CPOMe) by
lipase-catalyzed hydrolysis to (S)-carprofen (CP) was investigated in an aqueous
medium. With the highest catalytic activity, Candida antarctica lipase A (CALA) was
selected as catalyst compared to eight other lipases. Hydroxyethyl-β-cyclodextrin
(HE-β-CD) was added to enhance the solubility of (R,S)-CPOMe, which significantly
raised the conversion of substrate from 11.12% to 30.84%. Response surface
methodology (RSM) was adopted to evaluate the influence of factors on the substrate
conversion (c) and enantiomeric excess of product (eep), such as pH, concentrations of
enzyme and HE-β-CD, temperature, substrate loading and reaction time. The optimal
conditions were obtained, including pH 6.0, 40 mg/mL CALA, 0.05 mmol substrate,
35 mmol/L HE-β-CD, agitation speed of 600 rpm, temperature of 76 °C, and reaction
time of 30 h. Under above conditions, (S)-CP as the desired product was obtained
with enantiomeric excess of 96.24% and overall conversion of 46.07%.
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Keywords:
(S)-carprofen;
response
surface
methodology;
hydrolysis;
hydroxyethyl-β-cyclodextrin; Candida antarctica lipase A
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INTRODUCTION
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The enantiomers of chiral drugs are substantially identical in physical and
chemical properties, but they often show significant differences in biological activity1.
The life system is a chiral environment, so different properties and effects of
pharmacological as well as different pharmacokinetic properties will be exhibited
with taking racemic drugs2,3. On the contrary, optically pure drugs have the
advantages of reducing the dose and metabolic burden of the body, which can reduce
the risk of side effects caused by the undesired enantiomer4,5. Therefore, it is highly
preferable to use enantiomerically pure compounds in pharmaceutical field6.
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Generally, methods used to obtain monoenantiotropic drugs are chemical
synthesis and racemic resolution. Chemical synthesis is easy to obtain single
enantiomer, but the development of this method would be time consuming and
expensive7. Racemic resolution is simple, easy to industrial production, low cost and
short development time8 Therefore, it has been dominant in industrial applications.
.
In general, racemic separation techniques include crystallization9, simulated moving
bed chromatography (SMB)10, membrane11, enantioselective liquid–liquid extraction
(ELLE)12-14 and so on. However, some disadvantages limit the further development of
above methods. For example, chromatography method is low rate of recovery and
difficulty in scale-up15. Membrane separation method is poor stability and low
transport rate16. The enzymatic kinetic resolution has been an interesting method17-23
with its high stereoselectivity and regioselectivity. It may avoid some problems such
as producing a large number of ineffective or even harmful enantiomers to the
environment caused by chemical synthesis. Enzyme-catalyzed kinetic resolution can
be carried out by esterification17,18, transesterification19,20, hydrolysis21-23, etc.
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Carprofen (CP, 6-chloro-α-methyl-carbazole-2-acetic acid) is an important
non-steroidal anti-inflammatory possessing antipyretic and analgesic effects, which is
often used to treat rheumatoid arthritis, rheumatic spondylitis, postpartum
postoperative pain and other diseases5. The activity of (S)-enantiomer is higher
28-fold than the corresponding (R)-enantiomer24. Therefore, obtaining the optically
pure (S)-CP is of great significance. At present, several methods to obtain carprofen
enantiomers have been reported in the literature, including high performance liquid
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chromatography25 and asymmetric hydrogenation26,27
.
In this paper, enzymatic hydrolysis was employed for resolution of
(R,S)-carprofen methyl ester to obtain (S)-CP in an aqueous system. However, the
solubility of (R,S)-CPOMe in aqueous solution was very poor, which resulted in low
enzymatic reaction rate and low conversion rate of substrate28,29. Herein, adding
β-cyclodextrin derivatives to enhance the solubility of (R,S)-CPOMe and the
hydrolysis reaction rate. The effects of influencing factors had been investigated,
including pH, concentrations of enzyme and HE-β-CD, temperature, substrate loading,
reaction time and so on. To achieve the optimal separation effects, response surface
methodology (RSM) was applied to optimize reaction conditions.
EXPERIMENTAL
Materials
Lipase AK “Amano”, lipase PS and lipase AY were obtained from Amano
pharmaceutical Co., Ltd. (Nagoya, Japan). Novozym 435, Candida antarctica Lipase
A (CALA), Novozym40086, lipozyme RM IM and lipozyme TL IM were purchased
from Novozymes Biopharma DK A/S (Denmark). PPL was purchased from
Sigma-Aldrich (USA). (R,S)-CP was obtained from Xianju Pharmaceutical Co., Ltd.
(Zhejiang,
China).
Hydroxyethyl-β-cyclodextrin
(HE-β-CD)
and
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Hydroxypropyl-β-cyclodextrin (HP-β-CD) were purchased from Shandong New Fine
Chemical Co., Ltd. (Shandong, China). Methylated-β-cyclodextrin (Me-β-CD),
Sulfobutylether-β-cyclodextrin (SBE-β-CD) and Carboxymethyl-β-cyclodextrin
(CM-β-CD) were purchased from Shandong Zhiyuan Biotechnology Co., Ltd.
(Shandong, China). (R,S)-CPOMe was prepared in the laboratory. Methanol and
acetonitrile were chromatographic grades. Other reagents were analytical grade and
supplied by different commercial company.
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Aalytical Methods
The concentrations of (R,S)-CP enantiomers were analyzed on ODS-2 column
(150 mm × 4.6mm i.d., 5μm) by HPLC (waters e2695, Waters Corporation, USA) at a
wavelength of 273 nm. The mobile phase was composed of methanol and aqueous
solution (containing 20 mmol/L sodium dihydrogen phosphate and 25 mmol/L
HP-β-CD) at the ratio of 35:65 (v/v) (pH = 5.50, adjusted with sodium hydroxide).
The column temperature was maintained at 37 °C. The flow rate was kept at 1
mL/min. The injection volume was 10 µL. The retention time of (R)-CP and (S)-CP
were 53.7 min and 60.5 min, respectively. This analytical method was learned from
the literature25.
The enantiomeric excess (eep) express the purity of (S)-CP in Eq. (1).
[S][R]
[S][R]
(1)
eeP 
100%
Conversion (c) of (R,S)-CPOMe was calculated as follows:
([S][R])V
nCPME,0
(2)
c 
100%
The enantioselectivity was calculated by the following Eq. (3).
ln[1 c(1 eeP )]
E 
(3)
ln[1 c(1 eeP )]
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where, [S] and [R] are the concentrations of (S)-CP and (R)-CP in reaction mixture,
respectively; V represents the volume of the reaction mixture; nCPME,0 is initial amount
of (R,S)-CPOMe (mmol).
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Synthesis of (R, S)-CPOMe
(R, S)-CPOMe was synthesized with commercially available (R, S)-CP (purity,
98%) by esterification reaction. (R, S)-CP (17.4 mmol, 5.0 g) was dissolved in excess
methanol (0.17 L), then 3% (w/v) sulfuric acid was added as catalyst. This reaction
was performed in an oil bath at 70 °C for 6 hours. The reaction keep monitoring with
TLC plate until the esterification reaction was terminated for six hours. The saturated
sodium bicarbonate solution was used to wash reactive mixture, which was
precipitated as a solid by washing with deionized water to neutral. The precipitated
solid was recrystallized once by methanol and deionized water, and the mixture was
filtered and the resulting solid was dried in oven. Finally, (R, S)-CPOMe was obtained.
The yield was greater than 86%, and the purity of (R, S)-CPOMe was higher than
98% determined by HPLC.
Lipase-catalyzed Hydrolysis of (R, S)-CPOMe
The lipase-catalyzed hydrolysis reaction was operated in a 25 mL schlenk tube.
A certain amount of (R, S)-CPOMe and lipase were added to 1 mL of 0.1 mol/L
sodium phosphate buffer containing β-CDs to initialize the reaction. The agitation
speed was kept at 600 rpm. Figure 1 show lipase-catalyzed hydrolysis reaction. After
the termination of the reaction, 1-2 mL of acetonitrile was added to the suspension,
which dissolved all insoluble substrates and products. Then, the reaction solution was
filtered via a 0.45 µm microporous membrane, and product concentration was
determined by HPLC. In this paper, all experiments were repeated three times under
same conditions and the accuracy of the duplicate values was within ± 3%.
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Figure 1. Lipase catalyzed hydrolysis of (R,S)-CPOMe in buffer median.
Design of Experiments and Statistical Analysis
RSM was employed to predict the process of hydrolysis of (R,S)-CPOMe by
enzyme catalyzed in a phosphate buffer solution. The effect of important factors on c
and eep were investigated, including temperature (A), pH (B), lipase concentration (C),
cyclodextrin concentration (D), substrate loading (E) and reaction time (F). In RSM
equations, parameters were acquired by regression analysis of the experimental data.
The independent variables were reaction temperature (A, 40-100 °C), pH (B,
4-9), lipase concentration (C, 10-50 mg/mL), HE-β-CD concentration (D, 0-50
mmol/L), substrate loading (E, 0.01-0.06 mmol) and reaction time (F, 2-36 h). It is
found that the quadratic model of response surface for optimization is suitable and
effective by experimental data analysis. The second-order polynomial equations for c
and eep are shown in Eqs. (4) and (5).
k
k
k
c     x 
 x x   x2
(4)
(5)

 

0
i
i
ij
i
j
ii i
i1
i1 ji1
i1
k
k
k
ee  b  b x 
b x x  b x2

 

p
0
i
i
ij
i
j
ii i
i1
i1 ji1
i1
where, c and eep represent conversion and enantiomeric excess, respectively; α0 and b0
are the constant coefficient; αi and bi, αij and bij, αii and bii are the linear coefficient,
squared coefficient, and cross-product coefficient, respectively; k is the number of
factors; xi and xj are the independent variable. All the coefficients in Eqs. (4) and (5)
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were calculated by analysis of variance (ANVOVA). Design Expert (version 8.0.5b)
can be employed to acquire all coefficient of model and, optimize multiple responses.
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RESULT AND DISCUSSION
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Selection of various Enzymes
Stereoselective hydrolysis of (R,S)-CPOMe enantiomers was carried out through
enzymatic in aqueous medium. Different enzymes show various selectivity and
catalytic activity. Herein, the effect of nine enzymes on conversion and enantiomeric
excess were investigated for stereoselective hydrolysis of (R,S)-CPOMe (Table 1).
Among them, CALA, Lipase AY, Lipase AK as well as Lipase PS were able to be
dissolved in the buffer solution, while other enzymes were insoluble or slightly
soluble. Soluble enzymes increase the substrate contact with the enzyme to enhance
the rate of hydrolysis. The results show the highest E of 30.48 is obtained with CALA.
When the catalyst is CALA, the total conversion and product purity are relatively high,
which can reach up to 19.90% and 92.12%, respectively. Compared with other lipases,
CALA has stronger association with the oil-water interface to expose the active site,
so that the binding ability between substrate and lipase becomes stronger. Meanwhile,
high interfacial activation can increase the hydrophobicity of the catalytic site, leading
to reorientation of the α-helix, thereby exposing the active site to enhance catalytic
activity. Therefore, CALA is chosen as the optimal catalyst for further experiments.
Table 1. Results of hydrolysis of (R,S)-CPOMe catalyzed by different lipases
lipase
CALA
source
eep (%)
92.12 ± 0.08
48.44 ± 0.24
--
c (%)
19.90 ± 0.35
4.30 ± 2.32
--
E
Candida antarctica A
Candida antarctica B
Candida rugosa
30.48 ± 1.11
2.94 ± 0.37
--
Novozym 435
Lipase AY
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Lipase PS
Lipase AK
Burkholderia cepacia
89.02 ± 0.17
0.05 ± 3.00
17.22 ± 1.56
Pseudomonas
Aspergillus oryzae
Rhizomucor miehei
Candida cylindracea
Porcine pancreatic
--
--
--
Novozym40086
Lipozyme RM
Lipozyme TL
PPL
86.92 ± 0.20
0.45 ± 2.55
14.35 ± 1.62
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91.18 ± 0.11
0.45 ± 0.32
21.77 ± 0.94
--
--
--
--
--
--
Conditions: 20 mg/mL lipase, 0.02 mmol (R,S)-CPOMe, reaction temperature 40 °C,
pH 6.0, and reaction volume 1 mL.
Selection of Additive Types
As shown in Table 1, with CALA as the biocatalyst, high enantioselectivity is
achieved, while the total conversion of (R,S)-CPOMe is relatively low. Therefore, it is
necessary to improve the substrate conversion. The solubility of (R,S)-CPOMe in
aqueous solution is very poor. Low solubility of ester will result in low total
conversion in enzymatic hydrolysis reaction system30,31. Adding the additives into
aqueous phase can improve the solubility of substrate and the hydrolysis reaction rate
32,33
.
Nine additives are employed to improve stereoselective hydrolysis of
(R,S)-CPOMe (Table 2). The eeP is slightly enhanced with additive added. It is
satisfied that the total conversion and enantioselectivity are greatly increased by the
addition of Tween 80, HP-β-CD, Me-β-CD and HE-β-CD. This may be because
tween 80 acts as an emulsifier to enhance the solubility of the insoluble substrate,
thereby increasing the rate of hydrolysis reaction. β-CD derivative has a special
chemical structure with an internal hydrophobic cavity and external hydrophilic
surface. Hydrophilic β-CD derivative and CPOMe can form a water-soluble inclusion
complex, which can enhance the dissolution of the substrate. Meanwhile, β-CD
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derivatives as chiral additives can selectively identify (R) and (S)-enantiomers. The
selectivity is enhanced by the synergistic effect of CALA and β-CD derivatives.
Meanwhile, HE-β-CD is considered as an excellent solubilizer because it is
pollution-free, not toxic, economical and easy to be separated from products. The total
conversion rate with the addition of tween 80 was increased by 5% compared with
that of HE-β-CD, while the enantioselectivity was relatively low. What’s more, tween
80 as surfactant generally would cause a complex purification process and high
production cost. Thus, HE-β-CD is chosen as the optimal additive for enzymatic
hydrolysis of (R,S)-CPOMe.
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Table 2. Effects of different additives on lipase-catalyzed hydrolysis of (R,S)-CPOMe
Additives
None
eep (%)
95.44 ± 0.08
97.69 ± 0.06
97.34 ± 0.05
96.76 ± 0.05
97.93 ± 0.07
96.94 ± 0.09
96.38 ± 0.06
97.36 ± 0.08
97.48 ± 0.05
95.92 ± 0.07
c (%)
E
11.12 ± 0.31
15.27 ± 0.23
18.01 ± 0.45
35.47 ± 0.18
16.14 ± 0.43
27.26 ± 0.27
10.48 ± 0.62
30.56 ± 0.20
30.89 ± 0.24
10.80 ± 0.69
48.20 ± 1.85
102.00 ± 2.87
91.67 ± 2.23
103.69 ± 1.61
114.94 ± 3.46
91.89 ± 3.26
60.62 ± 1.91
114.08 ± 3.07
120.49 ± 2.17
53.83 ± 1.97
Span 60
Tween 60
Tween 80
Macrogol 400
HP-β-CD
SBE-β-CD
Me-β-CD
HE-β-CD
CM-β-CD
Conditions: 40 mg/mL CALA, 0.05 mmol (R,S)-CPOMe, 20 mmol/L additives,
reaction temperature 80 °C, pH 6.0, reaction time 14 h, and reaction volume 1 mL.
Regression Model and Statistical Analysis
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The effect of six independent variables, including temperature, CALA
concentration, pH, HE-β-CD concentration, substrate loading and reaction time on
resolution performance were investigated. The c and eep of different experiment
conditions are listed in Table 3.
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Table 3. Central composite design matrix of the independent variables with their
corresponding response
run Temperature pH
(°C )
CALA
HE-β-CD
Substrate
(×10-3 mmol)
Reaction
time (h)
c (%) eep (%)
(mg/mL)
(mmol/L)
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80
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80
80
80
6
20
20
20
20
20
40
45
40
40
40
40
40
40
40
40
40
40
40
40
20
20
20
20
20
20
20
15
20
25
40
40
40
40
40
0
0
20
20
20
20
20
20
20
50
50
40
45
50
60
50
50
50
50
50
50
20
20
20
20
20
20
20
20
20
20
15
20
25
30
35
4
4
3.8
75.78
85.72
90.04
91.68
92.76
95.06
94.42
93.9
2
6
5.7
3
6
0
4
10.59
13.26
15.29
19.74
18.28
10.68
14.31
46.17
46.26
46.07
39.91
31.57
38.53
42.09
43.95
46.07
46.27
13.03
16.56
7.31
10
11
21
22
23
24
38
39
40
41
42
43
44
45
46
47
4
5
0
4
5.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6
0
4
0
4
0
4
0
4
5
4
94.23
95.94
96.02
95.92
95.67
95.5
40
40
40
40
40
40
40
40
40
40
0
30
30
30
30
6
12
19
24
30
36
4
95.64
95.22
95.08
95.94
95.09
92.59
94.16
91.3
6
6
0
4
7
6
0
4
8
6
0
4
1.25
87.7
9
4
0
4
4.8
88.5
14
15
16
17
18
33
34
35
36
37
7
0
4
15.67
2.3
92.79
73.8
9
0
4
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
0
4
9.07
93.8
0
4
12.52
15.34
47.4
94.47
94.56
87.1
0
4
40
40
40
40
40
30
30
30
30
30
46.9
91.5
46.42
46.25
46.58
94.02
95.08
95.78
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80
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80
80
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80
80
80
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6
40
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40
40
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40
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30
35
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15
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25
30
35
40
40
0
50
50
50
50
50
50
50
10
20
20
20
20
4
4
18.57
22.22
25.76
24.21
26.74
29.11
26.19
48.2
94.58
95.34
95.42
95.52
95.88
95.92
95.94
83.16
94.16
94.55
95.14
93.94
4
4
4
4
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4
30
4
16.66
17.6
6.5
6.5
6.5
0
4
0
4
16.03
17.45
0
4
To obtain the coefficients, the experimental data was employed to fit both Eqs. (4)
and (5) polynomial equation. The multiple quadratic regression models for eep and c
could be expressed by the following second order polynomial Eqs. (6) and (7).
c(%)  42.50+6.2A 1.69B+4.62C+6.24D 3.83E+9.44F+2.91DE
18.46A2 9.99B2  4.9C2  7.55D2  2.36E2  6.79F2
(6)
eeP (%)  94.35+11.27A  6.36B+1.12C 1.33D+2.71E+0.31F+3.45DE
16.08A2 12.72B2  0.94C2 1.14D2  6.57E2 +0.13F2
(7)
When p-value is more than 0.0500, it implies that the model coefficients are
insignificant. The p-value of c and eep models are less than 0.0001, indicating two
models are significant (Table 4). The corresponding correlation coefficients R2 of c
and eep are 0.9884 and 0.9745, respectively, indicating that the model has high
reliability and fitting well with the experimental data.
Table 4. ANOVA for response surface reduced quadratic model
c
eep
mean
F
p-value
mean
p-value
F value
source
source
square
value
square
Prob
>
Prob >
Model 830.47 216.52 < 0.0001 significant Model
79.71
97.17
< 0.0001 significant
αA
αB
89.28
7.25
23.28
1.89
< 0.0001
0.1786
bA
bB
295.01
359.61
< 0.0001
< 0.0001
102.55
125.01
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αC
αD
56.14
62.72
85.91
14.64
16.35
22.40
0.0006
0.0003
bC
bD
3.33
2.85
42.83
0.47
--
4.06
3.48
52.21
0.57
--
0.0522
0.0711
αE
< 0.0001
bE
< 0.0001
αF
431.57 112.52 < 0.0001
bF
0.4567
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αAB
αAC
αAD
αAE
αAF
αBC
αBD
αBE
αBF
αCD
αCE
αCF
αDE
αDF
αEF
αAA
αBB
αCC
ɑDD
αEE
αFF
--
--
--
--
--
bAB
bAC
bAD
bAE
bAF
bBC
bBD
bBE
bBF
bCD
bCE
bCF
bDE
bDF
bEF
bAA
bBB
bCC
bDD
bEE
bFF
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
25.15
--
6.56
--
0.0152
--
35.21
--
42.93
--
< 0.0001
--
--
--
--
--
--
--
267.56
164.8
7.89
32.95
7.2
41.48
69.76
42.97
2.06
8.59
1.88
10.82
< 0.0001
< 0.0001
0.161
0.0061
0.1798
0.0024
202.96
267.19
0.29
0.75
56.1
0.016
247.41
325.71
0.36
0.91
68.39
0.02
< 0.0001
< 0.0001
0.5542
0.3467
< 0.0001
0.8895
Influence of Reaction Temperature and pH
It is well known that enzymes are proteins with biocatalytic activity, whose
activity is usually affected by temperature. The reaction rate will be reduced if
temperature is too low. However, excess temperature result in the irreversible protein
denaturation of enzyme and the decrease of enzymatic activity. Therefore, a suitable
temperature should be selected for enzymatic hydrolysis reaction. In addition, the
enzyme contains both amino and carboxyl groups, which have effect on the activity of
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enzyme owning to pH of solution. According to the experimental results, RSM model
is used to simulate the above influence. As shown in Figure 2, the effect of
temperature and pH on c and eep is similar. From Figure 2a, the c increases firstly as
temperature increases, then reaches a maximum at 76 °C. The c is decreased with the
further increase of temperature (≥ 76 °C). This result shows that CALA is a
heat-resistant lipase with good temperature tolerance, benefiting for catalytic reaction
at a high temperature34,35. Likewise, the CALA has the optimal pH of 6.0. From
Figure 2b, the maximum of eep is observed at temperature of 76 °C and pH of 6.0. In
order to obtain high c and eep, the reaction temperature and pH are set at 76 °C and
6.0, respectively.
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Figure 2. Effects of reaction temperature and pH on c and eep. Conditions: 40 mg/mL
CALA, 0.05 mmol (R, S)-CPOMe, 35 mmol/L HE-β-CD, and reaction time 30 h.
Influence of Concentration of CALA and the Loading of Substrate
Influence of the concentration of CALA and the loading of substrate on c and
eep was investigated, fixing the total volume of 1 mL (Figure 3). As shown in Figure
3a, with the concentration of CALA increase, the c value increases rapidly (≤ 40
mg/mL) and decrease slightly (≥ 40 mg/mL). However, the c increases slowly with
the increase of the substrate loading before 0.02 mmol, then decreases dramatically
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with further increase. From Figure 3b, the eep is slightly increased by increasing the
concentration of CALA, while increasing substrate loading can greatly influence the
eep. With the increase of substrate loading, the eep increases, then reaches maximum
at about 0.05 mmol (R, S)-CPOMe. With substrate loading further increase, eep
decrease. To acquire higher product purity and yield, the concentration of CALA
should be kept being 40 mg/mL.
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Figure 3. Effects of the loading of substrate and the concentration of CALA on c and
eep. Conditions: 35 mmol/L HE-β-CD, reaction temperature 76 °C, pH 6.0, and
reaction time 30 h.
Influence of pH and HE-β-CD Concentration
With the addition of HE-β-CD, c can be obviously increased while eep is not
affected. In addition, pH may affect the dissociation of hydroxyl groups in HE-β-CD,
resulting in changes in the existing form of HE-β-CD. Therefore, the effects of pH
and HE-β-CD concentration on c and eep are necessary to be studied. From Figure 4,
as the concentration of HE-β-CD increases, the c can be greatly enhanced. Increasing
HE-β-CD concentration at any tested pH, c increases fastly, and then increases slowly
when HE-β-CD concentration is above 35 mmol/L. Meawhile, there is slightly
influence of HE-β-CD concentration on eep. However, in any HE-β-CD concentration,
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both c and eep rise rapidly with increasing pH, reaching maximum at pH around 6.0.
Continuing to increase the pH, c and eep obviously reduced, which is consistent with
the above trend (Figure 2). The β-CD derivatives are able to include hydrophobic
molecules inside their cavity to form inclusion complexes that are soluble in water 36.
Meanwhile, the inclusion equilibrium constants between HE-β-CD and the reactant
(CPOMe) is evaluated as 11937.85 L/mol by using phase solubility method (Figure
S1), which indicates that HE-β-CD has a stronger inclusion ability for (S)-CPOMe
than that for (R)-CPOMe. The concentration of fast-reaction enantiomer in water was
increased by adding β-CD derivatives, which led to the increase of enantioselectivity.
To achieve high c and eep, 35 mmol/L HE-β-CD and pH 6.0 are selected.
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Figure 4. The effects of pH and HE-β-CD concentration on c and eep. Conditions: 40
mg/mL CALA, 0.05 mmol (R, S)-CPOMe, temperature 76 °C, and reaction time 30 h.
Influence of HE-β-CD Concentration and Substrate Loading
By adding HE-β-CD, the substrate conversion can be improved, which is
accustomed to the inclusion interaction between HE-β-CD and the substrate.
Therefore, the effect of HE-β-CD concentration and substrate loading on c and eep is
necessary to be investigated. As shown in Figure 5, the trend of the influence of
substrate loading on c and eep is the same as that in Figure 3. And it is also observed
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that the effect of HE-β-CD concentration on c and eep is consistent with the trends of
Figure 4. It can be concluded that 35 mmol/L HE-β-CD and 0.05 mmol (R,S)-CPOMe
are preferential to achieve high c and eep in the reaction system.
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Figure 5. Effects of the loading of substrate and HE-β-CD concentration on c and eep.
Conditions: 40 mg/mL CALA, temperature 76 °C, pH 6.0, and reaction time 30 h.
Influence of Temperature and Reaction Time
Temperature has an important influence on enzymatic activity. The rate of the
enzymatic hydrolysis is affected by temperature, thereby changing the time of the
entire reaction. Thus, the relationships among temperature, reaction time, c as well as
eep were studied (Figure 6). From Figure 6, c and eep increase significantly as the
temperature increases (≤ 76 °C), and they reduced sharply with temperature further
increased (≥ 76 °C). The maximum of c and eep can be reached at 76 °C, The trends is
coincided with the effect of temperature in Figure 2. There is a rapid increase of c
when reaction time is before 30 h and then a slow increase is observed. It is obvious
that c increases greatly with reaction time increases, c increases slowly after 30 h.
However, there is nearly no influence of reaction time on eep. Therefore, 30 h is
enough to obtain large conversion and high product purity.
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Figure 6. Effects of reaction temperature and time on conversion c and eep,
Conditions: 40 mg/mL CALA, 0.05 mmol (R, S)-CPOMe, 35 mmol/L HE-β-CD, and
pH 6.0.
APPLICATION AND VALIDATION OF THE MODEL
The optimal reaction conditions can be obtained according to the above results,
including pH 6.0, 40 mg/mL CALA, 0.05 mmol substrate, 35 mmol/L HE-β-CD, 76
°C for temperature, and 30 h for reaction time. The enzymatic hydrolysis reaction was
repeated three times under optimal conditions. Product purity was analyzed through
HPLC (Figure 7). The chromatograms of racemic CP and the product are shown in
Figure 7a and b, respectively. The experimental results and predicted values of RSM
under the optimal conditions are displayed in Table 5. Relative deviation indicates
that the experimental values are coincided with the model prediction very well.
Therefore, the lipase-catalyzed hydrolysis process can be simulated and optimized to
obtain optimal process parameters through the RSM. The results can provide the
theoretical basis for process design and operation of large-scale production. However,
more factors, such as reactor type and size, economic benefits and safety, should be
considered in applying these process parameters to industrial-scale production.
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Table 5. Predicted and observed values for the response variables based on optimal
conditions.
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8
9
Response variable Predicted value Experimental value Relative deviation
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c (i)
eep (i)
c (ii)
45.76%
97.02%
44.68%
96.87%
44.43%
92.36%
46.07%
96.24%
43.86%
95.38%
42.91%
94.34%
0.68%
0.80%
1.84%
1.54%
3.42%
2.14%
eep (ii)
c (iii)
eep (iii)
Figure 7. Chromatograms of (R, S)-CP and product by HPLC. (a) CP sample
(racemic), (b) product of lipase catalyzed stereoselective hydrolysis (R,S)-CPOMe.
CONCLUSION
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In this paper, an efficient reaction system of stereoselective resolution of (R,
S)-CPOMe by lipase-catalyzed hydrolysis was constructed to obtain (S)-CP.
Candida antarctica lipase A was selected as the most efficient catalyst. With the
addition of HE-β-CD into the reaction system, c was increased from 11.12% to
30.84%, while eeP value was changed slightly. The RSM model was applied to
simulate and optimize the reaction system to acquire the optimal reaction conditions.
The results show that eeP depends primarily on temperature, pH and substrate loading.
Other conditions such as HE-β-CD concentration and time, have small effect on eeP,
but have great influence on substrate conversion. Experimental results show that eep
and c under the optimal reaction conditions can reach 96.24% and 46.07%,
respectively. These results are coincided well with the model predicted values, which
shows that the RSM of quadratic model was a powerful tool to optimize the
lipase-catalyzed hydrolysis of (R, S)-CPOMe process. This work provide important
guiding significance for the large-scale production.
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ASSOCIATED CONTENT
Supporting Information
Phase solubility diagram of CPOMe in presence of HE-β-CD (Figure S1).
AUTHOR INFORMATION
* Corresponding author.
E-mail address: tangkewen@sina.com (K.W. Tang).
Tel.: +86 13762003936
Fax: +86 730 8640921
E-mail address: qpanny@163.com (P.L. Zhang).
Tel.: +86 13786023146
Fax: +86 730 8640921
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
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This work was supported by the National Natural Science Foundation of China
(grant number. 21676077).
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