- Email: [email protected]
Kinetic model of resolution of 4-methoxymandelic acid enantiomers by lipase-catalyzed transesterification reaction
Applied Catalysis A, General 587 (2019) 117274
Contents lists available at ScienceDirect
Applied Catalysis A, General journal homepage: www.elsevier.com/locate/apcata
Kinetic model of resolution of 4-methoxymandelic acid enantiomers by lipase-catalyzed transesterification reaction
T
⁎
Xin Yuan, Panliang Zhang, Weifeng Xu, Kewen Tang
Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang, 414006, Hunan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipase AK (RS)-4-methoxymandelic acid Transesterification Kinetic resolution Modeling
This paper reports the kinetics resolution of (R,S)-4-methoxymandelic acid (4-MMA) by lipase AK-catalyzed transesterification reaction, using vinyl acetate (VA) as acyl donor in methyl tert-butyl ether (MTBE). Considering the Ping-Pong bi-bi mechanism with 4-methoxymandelic acid inhibition, the kinetic model was established. Kinetic parameters were obtained by fitting time-concentration curves for different initial 4methoxymandelic acid concentrations. The average relative error below 3.0% indicated that the model predictions were in good agreement with the experimental data. Furthermore, the kinetic model was employed to predict the effects of enzyme loading, initial vinyl acetate concentration and reaction time on enantiomeric excess (ees) and conversion of substrate (c), and to optimize the conditions. The excellent results with high conversion of 4-MMA (50.2%) and large enantiomeric excess of substrate (98.6%) were achieved under the optimal conditions.
1. Introduction Chiral drugs play dominant role in the global pharmaceutical market. At present, the total number of drugs used in the world is about 1900, of which more than 50% are chiral drugs. Among the 200 drugs commonly used in clinical practice, there are as many as 114 chiral drugs [1,2]. Therefore, the separation and purification of drug enantiomers are of great scientific significance and economic value in the rapidly growing market demand. Currently, several methods have been developed to obtain optically pure compounds, such as high performance liquid chromatography [3], crystallization [4], liquid-liquid extraction [5,6], membrane separation [7], and enzymatic resolution [8,9]. High optical purity of single enantiomer can be obtained by HPLC and crystallization method, but it has the disadvantages of low yield and low economic benefit. Liquid-liquid extraction can realize continuous production with high yield, but it is very difficult to screen out a chiral extractant with high selectivity and versatility. Compared with the other separation methods, enzymatic resolution method has mild reaction conditions, high selectivity, environmental protection, wide applicability and is easy to achieve industrial production. So, enzymatic resolution method has become a research hotspot [10]. Lipase (triacylglycerols acyl esters hydrolases; EC 3.1.1.3) is a sort of proteins with biological activity and multiple catalytic capabilities. Lipases are also one of the important industrial enzyme, which can
⁎
catalyze hydrolysis [11], transesterification [12], esterification [13] and other reactions [14,15], and is widely used in oil processing, food, medicine, daily chemical and other industries. Lately, lipases of different sources as biocatalysts are widely applied to the kinetic resolution of chiral drugs due to their excellent chemical-, stereo- and enantio- selectivity, such as lipase from Rhizomucor miehei [16], Pseudomonas fluorescens [17], Candida rugosa [18], Candida antarctica B [19]. Lipases selectively catalyze the faster conversion of one enantiomer to the target product due to the high selectivity, thereby achieving the purpose of separating the racemic mixture. Mandlic acid with their derivatives are very important to chiral building blocks, organic synthesis intermediates and auxiliaries in pharmaceutical and fine chemicals industries [20,21]. Besides, they are also optical resolving agents that have been applied for the separation of racemic alcohols and amines [22]. 4-Methoxymandelic acid (2-Hydroxy-2-(4-methoxyphenyl)ethanoic acid) has important applications in many fields, such as pharmaceutical production and asymmetric synthesis. Currently, it was widely used to synthesize optically pure amino acids, angiotensin converting enzyme inhibitors and coenzyme A, etc [23]. The methods of racemic separation used to obtain single enantiomers of mandelic acid and its derivatives have been reported in the literature [24–28]. However, the resolution of the 4-methoxymandelic acid enantiomers by lipase catalyzed has not been reported. The mathematical model of multifactor optimization is rarely found in
Corresponding author. E-mail address: [email protected] (K. Tang).
https://doi.org/10.1016/j.apcata.2019.117274 Received 29 July 2019; Received in revised form 20 September 2019; Accepted 21 September 2019 Available online 23 September 2019 0926-860X/ © 2019 Elsevier B.V. All rights reserved.
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Scheme 1. Kinetic resolution of (R,S)-4-methoxymandelic acid by enzymatic transesterification.
breakdown of E’B complex. Forth, since there is only little water (0.10% v/v), the hydrolysis reaction is neglected. Additionally, the vinyl alcohol of highly unstable is immediately tautomerism to form acetaldehyde, so this reaction is irreversible (k-2 = 0). The concentrations of various forms of enzymes can be expressed by the following pseudo-steady state equilibrium relationship.
the literature. In this paper, an efficient reactive system was established for kinetic resolution of 4-methoxymandelic acid enantiomers (4-MMA) by lipase AK-catalyzed transesterification in organic system (Scheme 1). Both of the purity and conversion are varied with the reaction progress. Therefore, prediction of reaction progress is crucial for process design and operation of enzymatic kinetic resolutions. An quantitative mathematical model based on the ping-pong bi-bi mechanism with 4-MMA inhibitor was constructed to describe the kinetic behavior of the transesterification reaction and obtain the optimal reaction conditions. Meanwhile, the effects of enzyme loading, vinyl acetate concentration and reaction time on conversion rate and ees were simulated and optimized using the kinetic model. This model has important guiding significance for controlling the reaction process to obtain the best industrial parameters. 2. Model development 2.1. Kinetic analysis
d[EA] = k1 [E][A] − (k−1 + k2)[EA] = 0 dt
(1)
d[E'BS] = k3S [E'][BS] − (k −3S + k 4S)[E'BS] = 0 dt
(2)
d[E'BR ] = k3R [E'][BR ] − (k −3R + k 4R)[E'BR ] = 0 dt
(3)
d[EBS] = k5S [E][BS] − k −5S [EBS] = 0 dt
(4)
d[EBR ] = k5R [E][BR ] − k −5R [EBR ] = 0 dt
(5)
d[E'] = k2 [EA] + k −3S [E'BS] − k3S[E'][BS] + k −3R [E'BR ] − k3R [E'][BR ] dt (6) =0
The enzymatic transesterification reaction follows an irreversible Ping-Pong bi-bi mechanism with dead-end inhibition of 4-MMA. This reaction usually involves the following steps:
Substituting Eqs. (2) and (3) into (6) can be obtained: 1 The Enzyme-Acyl complex (EA) is formed through the interaction between free lipase (E) and vinyl acetate (A). 2 The nucleophilic serine (Ser105) residue in the EA interacts with carbonyl group of vinyl acetate, forming the first intermediate complex (E’P). E’P releases the first product, vinyl alcohol (P) and modified lipase (E’). 3 The lipase-4-MMA complex (E’B) is formed by combining the hydroxyl group of 4-MMA (B) with the modified lipase (E’). The E’B is rapidly isomerized to form the second intermediate complex EQ, which subsequently releases product ester (Q) and regenerating enzyme (E). 4 4-MMA (B), which competes with vinyl acetate (A), combines with free lipase (E) to form dead-end complex (EiB).
k [B ] k [B ] K [E] = ⎛ 4S S + 4R R ⎞ mA [E'] KmBR ⎠ k2 [A] ⎝ KmBS ⎜
⎟
(7)
The mass balance equation for the total enzyme concentration [E]T is expressed as follows:
[E]T = [E] + [EA] + [E'] + [E'BR ] + [E'BS] + [EBR ] + [EBS]
(8)
The rate equation for (R)-4-MMA and (S)-4-MMA can be defined as:
−
d[BR ] 1 [E'] = VR = k 4R [E'BR ] = k 4R [BR ]K −mB,R dt
−
d[BS] 1 [E'] = VS = k 4S [E'BS] = k 4S [BS]K −mB,S dt
(9)
(10)
Substituting Eq. (7) into Eqs. (9) and (10), Eqs. (11) and (12) are obtained:
These steps of transesterification reaction are represented by Scheme 2: where k1 and k-1, k3 and k-3, k2 and k4 are the rate constant for the intermediate EA, E’B, product of P and Q, respectively; KiB,S and KiB,R are the inhibition constant of (S)-4-MMA and (R)-4-MMA on lipase. For the kinetic model, some assumptions are made to derive the initial rate equation. First, all complexes are assumed to be in a pseudosteady state, because the substrate concentration is much more than enzyme concentration and the concentrations of complexes are approximately constant during the reaction. Second, the formation and dissociation of all complexes are very fast, which is assumed to be close to equilibrium. Third, the overall reaction rate is limited by the
VR =
VS =
1 Vmax,R [BR ][A]K −mB,R 1 −1 [A] + [BR ]K −mB,R {[A] + αR KmA (1 + [BR ]K -1 iB,R + [BS]K iB,S)} 1 −1 {[A] + α SKmA (1 + [BR ]K -1 + [BS]K−mB,S iB,R + [BS]K iB,S)}
(11)
1 Vmax,S [BS][A]K −mB,S −1 −1 [A] + [BR ]K mB,R {[A] + αR KmA (1 + [BR ]K -1 iB,R + [BS]K iB,S)} -1 −1 −1 + [BS]K mB,S {[A] + α SKmA (1 + [BR ]K iB,R + [BS]K iB,S)}
(12)
where, αR=k4Rk2 2
−1
and
αS=k4Sk2-1.
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Scheme 2. Mechanism of enzymatic transesterification reaction with 4-MMA inhibition.
Vmax,i = k 4i × [E]T
(13)
nives lipase (referred as RNL, SD) was bought from Sigma-Aldrich Co., Ltd. (USA). (R,S)-4-methoxymandelic acid (purity ≥98%) was obtained from Aladdin Industrial Corporation (Shanghai, China). Some important solvent were purchased as folllows: vinyl acetate (> 98%), isopropenyl acetate (> 99%), ethyl acetate, butyl acetate and isobutyl acetate (> 98%) from Adamas Reagent Co., Ltd. (Basel, Switzerland); vinyl propionate (> 98%) and vinyl butyrate (> 98%) from MacklinReagent Co., Ltd. (Shanghai, China); methyl tert-butyl ether (> 98%, MTBE) from Titanchem Co., Ltd. (Shanghai, China). Other organic solvent were of analytical grade and purchased from different companies.
where, i is R or S. The differential equation of the reaction rate is solved using the fourth-order Runge-Kutta method, and parameters of the mathematical model are obtained by nonlinear fitting of the experimental data. 2.2. Thermodynamic analysis The effects of temperature on lipase activity can be explained by the Arrhenius or Eyring theory [29,30]. An increase in temperature accelerates the rate of the catalytic reaction, however, excessive temperature leads to inactivation of lipase. Meanwhile, the enantioselectivity is dominated by the difference in the activation free energy of the transient state of the fast-reacting enantiomer (S) and the slow-reacting enantiomer (R) [31]. Gibbs free energy of transition state formation can be shown as follows:
kcat , j ⎞ ΔGj = −RT ln ⎛⎜ ⎟ ⎝ K m, j ⎠
3.2. Enzymatic transesterification of (R,S)-4-MMA The enzymatic transesterification of 4-MMA enantiomers with vinyl acetate was carried out in 25 mL schlenk tube equipped with a heating and stirrer reactor (IKA RCT basic, Germany). 4-MMA enantiomers (50 mmol/L) and various concentrations of vinyl acetate (250–500 mmol/L) were dissolved in anhydrous MTBE at a constant volume of 25 mL. The lipase was added into a schlenk tube containing 2 mL of the mixed reaction solution, once it heated to the expected temperature. The stirring speed was maintained at 500 rpm. The water content was kept at 0.10% of reaction mixture solution. After reaction, the lipase was filtered off to obtain a sample. The sample was pretreated by drying the solvent, then diluted with methanol and detected by HPLC. The effects of different reaction conditions on the catalytic activity and enantioselectivity were investigated as follows: reaction temperature, varied from 35 to 65 °C; the concentration of vinyl acetate, from 100 to 500 mmol/L; the enzyme loading, evaluated from 10 to 30 mg/ mL. To analyze reaction kinetics and thermodynamic behavior, the effects of different initial concentrations of 4-MMA (25–80 mmol/L) and vinyl acetate (250–450 mmol/L) as well as reaction temperature on the initial rate were investigated.
(14)
where kcat,j is ; Km,j is affinity constant of 4-MMA (mmol/L); R is universal gas constant (8.314 J/(mol·K)); i is R or S. The overall reaction rate constant is defined as Vmax,j/ET, where ET is the total concentration of enzyme (mg/L). Therefore, Eq. (14) can be written as Eq. (15).
Vmax,j ⎞ ΔGj = −RTln ⎜⎛ ⎟ ⎝ E T Km,j ⎠
(15)
Eq. (16) is derived based on the difference of Gibbs free energy of transition state between (R)-enantiomer and (S)-enantiomer.
Vmax,R/KmB,R ⎞ ΔΔG = −RTln ⎜⎛ ⎟ ⎝ Vmax,S/KmB,S ⎠
(16)
3. Experimental
3.3. HPLC analysis
3.1. Materials
The analysis of 4-MMA enantiomers was carried out by High Performance Liquid Chromatography (Waters e2596, U.S.A), using Daicel Chiralcel OJ-RH column (150 mm × 4.6 mm I.D., Tokyo, Japan). The wavelength of photodiode array detector (waters 2998, U.S.A) was 225 nm. The composition of the mobile phase is as follows: 20% anhydrous ethanol and 80% aqueous solution (pH 3.0, adjusted with glacial acetic acid). In sample analysis, the volume of 10 μL was injected. The flow rate was kept at 0.6 mL/min. The column temperature
Lipase AYS (SD), Lipase AK (SD), Lipase PS (SD) were obtained from Amano Enzyme Inc. (Nagoya, Japan). Lipozyme TL (IM) and Lipozyme RM (IM), Novozym 40086 (IM), Novozym 435 (IM) and Candida antarctica Lipase A (referred as CALA, L) were provided by Novozymes (Denmark). Porcine pancreatic lipase (referred as PPL, SD) was provided by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Rhizopus 3
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
was maintained at 25.0 °C. The retention time of (S)-4-MMA, (R)-4MMA and 4-MMA ester were about 24.50 min, 30.65 min and 50.44 min, respectively. The enantiomeric excess of substrate (ees, %), conversion rate of (R)-4-MMA (cR, %) and conversion rate of (S)-4-MMA (cS, %) were defined as:
Table 2 The influence of various acyl donors on c, eeS and E value. Acyl donors
ees (%)
cS (%)
Vinyl acetate Vinyl propionate Vinyl butyrate Ethyl acetate Butyl acetate Isobutyl acetate Butyl acrylate
99.1 ± 0.1 98.0 ± 0.8 50.7 ± 0.5 2.3 ± 0.5 0 0 0
99.6 99.0 74.9 34.6 28.5 18.9 22.1
± ± ± ± ± ± ±
0.1 0.4 1.1 1.6 0.5 1.7 0.6
cR (%)
E
3.1 ± 0.3 6.4 ± 0.4 23.3 ± 0.7 31.5 ± 1.1 28.1 ± 0.7 17.9 ± 0.8 21.0 ± 0.9
169.5 ± 4.9 69.9 ± 2.9 5.2 ± 0.2 1.1 ± 0.1 0 0 0
ees =
[R] − [S ] × 100% [S ] + [R]
(17)
cS =
[S ]0 − [S ] × 100% [S ]0
(18)
[R]0 − [R] × 100% [R]0
Conditions: 50 mmol/L of (R,S)4-MMA; 300 mmol/L of acyl donors; 50 mg of lipase AK; 2 mL of MTBE; T = 50 °C; t = 11 h.
(19)
4. Results and discussion
donor types on c, eeS and E value were investigated (Table 2). The best results are obtained using the vinyl acetate as acyl donor, exhibiting the highest c (cS, 99.6%; cR, 3.2%), eeS (99.1%) and E value (164.5) in the kinetic resolution of (R,S)-4-MMA. The c and E decrease with increasing the alkyl chain length of acyl donors. Moreover, Ethyl acetate, butyl acetate, isobutyl acetate and butyl acrylate act as acly donor with good conversion, while enantioselectivity of enzyme are very small in transesterification reaction. Low enantioselectivity may be caused by the same ability of the nucleophilic attack of (R)- and (S)-alcohol [33]. Vinyl acetate prefers to react with (S)-4-MMA in the lipase mediated reaction compared with other enol esters. Considering the above results, vinyl acetate was screened as acyl donor in further experiments.
4.1. Selection of lipase
4.3. Selection of organic solvents
There were different catalytic structures for different lipases, and the structure determines stereoselectivity, stability and activity of lipase. In this study, the lipase-catalyzed kinetic resolution of (R,S)-4MMA was carried out in organic solvent. The effects of nine lipases on conversion rate, enantiomeric excess and enantioselectivity were investigated for the kinetic resolution of (R,S)-4-MMA (Table 1). The results show Lipozyme TL, Lipase PS, Novozym 40086, Novozym 435 and lipase AK possess good catalytic activity and enantioselectivity. Among them, Lipase AK resolves (R,S)-4-MMA to the corresponding (S)ester and (R)-4-MMA, possessing the highest conversion and enantioselectivity as well as the largest enantiomeric excess. Because substrate specificity of lipase makes lipase difficult to combine with the substrate to form a complex, the catalytic activity of Lipase AYS and RNL are low. Then, this phenomenon of rapid enantioselective conversion of Lipase AK towards (S)-enantiomer may be attributed to the favorable interactions between the amino acid residues in the active site and the ligands [32]. Therefore, Lipase AK was selected as the optimal catalyst based on excellent conversion and enantiomeric excess.
Organic solvents with different polarities affect not only activity and enantioselectivity of enzyme, but also distribution of substrates and products. To obtain the maximum conversion and ees, screening of proper solvent was essential for lipase-catalyzed enantioseletive conversion of (R,S)-4-MMA. Based on the solubility of the substrate, the organic solvents with log P values in the range of 0.49–2.5 were studied, such as toluene (TL, 2.50), diisopropyl ether (DIPE, 1.90), dichloromethane (DCM, 0.60), tetrahydrofuran (THF, 0.49), methyl tertbutyl ether (MTBE, 1.40), Acetonitrile (ACN, -0.33) and 1,2-dichloroethane (DCE, 1.46). From Fig. 1, the excellent results are obtained using MTBE as reaction medium with the highest conversion rate (51.6%), E value (126.7) and enantiomeric excess (eeS 98.7%). It indicates that the catalytic triad of Lipase AK has high structural stability in MBTE, leading to the preferential recognition and transformation of (S)-enantiomers. In general, the strong polar organic solvents (DCM, THF and ACN) tend to peel off the essential water layers that bind tightly to the surface of enzyme molecules, damaging the hydrogen bond network of the enzyme molecules to reduce enzyme activity [34]. Although toluene has high catalytic activity, its enantioselectivity is very low. The active center of the enzyme has a binding site and a catalytic site that determine selectivity and catalytic efficiency, respectively. The structural similarity of 4-MMA and toluene results in the pi-pi electron interaction on the benzene ring of toluene and more likely
cR =
Conversion rate (c) was defined as Eq. (20). Enantioselectivity (E) was calculated by using total conversion (c) and enantiomeric excess (ees).
[S ] + [R] ⎞ c = ⎛1 − × 100% [ S ]0 + [R]0 ⎠ ⎝
(20)
ln{(1 − c)(1 − ees)} ln{(1 − c)(1 + ees)}
(21)
⎜
E=
⎟
4.2. Selection of acyl donors Various acyl donors have significant effects on the reaction rate and enantioselectivity of enzymatic reactions. Hence, the influence of acyl Table 1 Effect of various lipases on the catalytic activity and enantioselectivity. Lipase
Source
ees (%)
cS (%)
cR (%)
E
Lipozyme TL Lipozyme RM Novozym 435 Novozym 40086 lipase AK Lipase PS Lipase AYS CALA RNL
Thermomyces lanuginosus Aspergillus oryzae Candida Antarctica B Rhizomucor miehei Pseudomonas fluorescens Burkholderia cepacia Candida rugosa Candida Antarctica A Rhizopus nives
91.7 ± 1.5 7.1 ± 2.1 57.1 ± 0.7 57.7 ± 0.9 99.1 ± 0.1 69.1 ± 0.6 2.5 ± 0.5 15.0 ± 0.4 0
97.5 ± 0.8 38.2 ± 0.6 77.5 ± 0.7 73.6 ± 0.6 99.6 ± 0.1 82.0 ± 0.4 8.9 ± 0.5 27.5 ± 0.4 0
42.3 ± 0.7 28.8 ± 0.5 17.5 ± 0.7 1.6 ± 0.2 3.1 ± 0.3 1.5 ± 0.1 13.4 ± 0.5 1.7 ± 0.2 0
6.7 ± 0.2 1.4 ± 0.1 7.7 ± 0.2 80.0 ± 3.3 169.5 ± 4.9 111.9 ± 4.4 1.5 ± 0.1 18.1 ± 0.6 0
Conditions: 50 mmol/L of (R,S)4-MMA; 300 mmol/L of vinyl acetate; 50 mg of lipase; 2 mL of MTBE; T = 50 °C; t = 11 h. 4
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
conversion is due to less collision between the substrate and the enzyme molecule at lower temperatures. The effect of temperature on the enzymatic reaction is consistent with that reported by Surbhi M. Gupta et al [36]. In addition, the difference of Gibbs free energy of transition state between (S)-enantiomer and (R)-enantiomer was calculated by simulating kinetic parameters at 50 ℃. The ΔΔG for transesterification reaction is calculated as 9.671 kJ/mol, indicates that lipase AK catalyzed resolution of 4-MMA enantiomers has relatively high enantioselectivity at 50 ℃. 4.5. parameters estimation
attracts the phenyl group of 4-MMA. The substrate molecule can normally bind to the catalytic site in the active center, thus exhibiting high catalytic activity. The tight binding of toluene and substrate results in an increase in the space resistance of the substrate into the enantiomerrecognition pocket. Lipase AK has good catalytic activity in other organic solvents, while its enantioselectivity is very low. Maybe different ability of various solvents to maintain the essential water layer causes varied conversion and ee. Therefore, MTBE was chosen as the optimal reaction medium.
The lipase-catalyzed transesterification reactions with various initial concentrations of 4-MMA were performed to determine kinetic parameters. Based on the mass conservation principle, namely, v=-d [B]/dt, integration of Eqs. (11) and (12) give the relation between concentration of 4-MMAR and time and the relation between concentration of 4-MMAS and reaction time. Then, the differential equations of the reaction rate were solved by using the fourth-order RungeKutta method. The concentration-time curves date were used to simulate the kinetic parameters by Matlab program (MATLAB 2013A). The kinetic parameters were obtained as follows: 52.07 mmol/(L.h) of Vmax,S, 0.43 mmol/(L.h) of Vmax,R, 15.06 mmol/L of KmB,S, 4.5510 mmol/L of KmB,R, 165.09 mmol/L of αSKmA, 6.02 mmol/L of αRKmA, 247.56 mmol/L of KiB,S and 1.58 mmol/L of KiB,R. The comparison of the simulated values with the experimental data was shown in Fig. 3. The experimental values agree well with the simulated values, indicating the validity of the kinetic model.
4.4. Effect of temperature
4.6. Model prediction
Temperature as an important parameter has a large effect on the activity and enantioselectivity of enzyme. As Fig. 2 shows, the effects of temperature on conversion rate and enantiomeric excess of substrate were carried out in the range of 35–65 ℃. The eeS increased with the increasing of temperature (≤ 50℃), however, further increase of temperature lead to the ees decreased. The variation trend of eeS is consistent with the results of Phillips [35]. The conversion rate also increases rapidly as the temperature increases below 50 ℃. However, continued increase in temperature resulted in decreased both conversion rate and ees, indicating that higher temperatures have negative impact on enzyme activity. The active site of the lipase is a triplet structure composed of Ser, Asp and His residues. The reaction rate and selectivity are determined by the combination of the catalytic triad and the substrate to form tetrahedral transition state. Maybe the higher temperature destroys the three-dimensional structure of lipase and the hydrogen bonds, resulting in decreasing enzyme activity. The lower
4.6.1. Effects of the enzyme loading Based on Eqs. (11)–(13), the kinetic model was applied to evaluation of the effect of enzyme loading on the enzymatic resolution reaction, and experiments were conducted at 50 ℃ with vinyl acetate to 4MMA molar ratio of 6:1. As is shown in Fig. 4, the scatter dots present the experimental data of conversion rate and ees, and the line presents the simulation values. It is clear from Fig. 4 that the conversion rate increases rapidly with the increase of enzyme loading. When the enzyme loading reaches 17.5 mg/mL, the total conversion rate is close to 50%. Meanwhile, with enzyme loading is increased from 10 to 17.5 mg/mL, ees values increase from 57% to 98%, but little improvement of the ees is observed with further increase of enzyme loading. This phenomenon can be explained by the increase of enzyme load in the reaction mixture, which increases the active sites available for substrate binding and facilitates the interaction between the enzyme and substrate. Additionally, higher enzyme loading may enhance the external mass transfer resistance to affect the reaction rate, resulting in slow increase in conversion. Excess enzyme granules at high enzyme dosages may form enzyme aggregates even under appropriate agitation, which reduces the interaction of the enzyme with the substrate. This result is consistent with that has been reported in the literature [8]. Considering all the results in terms of conversion rate and ees, 17.5 mg/mL of enzyme loading was selected as the optimal value for further runs.
Fig. 1. Influence of various solvents on c, eeS and E-value. Conditions: 50 mmol/L of (R,S)-4-MMA; 300 mmol/L of vinyl acetate; 50 mg of lipase; 2 mL of solvents; T = 50 °C; t = 11 h.
4.6.2. Effects of the substrate ratio To investigate the effect of vinyl acetate concentrations on conversion rate and ees, sets of experiments were carried out from 100 to 500 mmol/L by keeping the concentration of (R,S)-4-MMA constant. Based on the kinetic model, the conversion rate and ees are predicted as a function of vinyl acetate concentration. With the concentration of vinyl acetate increased, the conversion and ees values show an upward trend as shown in Fig. 5. Vinyl acetate concentration of 300 mmol/L give an optimal conversion rate of 50.6%, and higher enantiomeric excess with 98.3% is obtained. Further increasing of vinyl acetate
Fig. 2. Effect of temperature on conversion rate and ees. Conditions: 50 mmol/L of (R,S)-4-MMA; 300 mmol/L of vinyl acetate; 50 mg of lipase; 2 mL of solvents; t = 11 h. 5
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Fig. 3. Comparison of simulated values with the experimental data of (S)-4-MMA (a) and (R)-4-MMA (b).
concentration can’t increase the reaction rate obviously. Additionally, even though the concentration of vinyl acetate is increased, it does not show any inhibitory effect on lipase activity. The possible reason is that increasing the concentration of acyl donor makes it easier to form enzyme and the acyl donor complex, which leads to increase in the conversion and ees. However, the active site is limited at a certain amount of enzyme. After the active sites are all occupied, increasing the acyl donor concentration does not significantly increase the conversion. Similar results have been reported in enzymatic transesterification of (R,S)-2-octanol using Novozyme 435 [37].
courses of the lipase-catalyzed transesterification of 4-MMA and vinyl aetate in MTBE at 50 ℃. The conversion rate increased with the increase of the reaction time (≤ 13 h), and the conversion rate became very slow after 13 h of reaction. The ees also exhibits the similar tendency and is up to 98% at 13 h, as shown in Fig. 7. It might be because the reaction is highly enantioselective and the reaction rate of fast-reacting enantiomers is much greater than that of slow-reacting enantiomers. At around 50% conversion rate, the reaction gets slower because there is little amount of faster reacting enantiomer in the mixture.
4.6.3. Effects of reaction time The influence of reaction time on conversion and enantiomeric excess can be predicted through the kinetic model. Fig. 6 presents the time
5. Conclusion Lipase AK-catalyzed transesterification of (R,S)-4-methoxymandelic 6
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Fig. 4. Effect of enzyme loading on lipase catalyzed transesterification of 4-MMA in MTBE. Experimental conditions: 4-MMA concentration of 50 mmol/L, VA concentration of 300 mmol/L, 50 ℃, 13 h.
Fig. 5. Effect of vinyl acetate concentration on lipase catalyzed transesterification of 4-MMA in MTBE. Experimental conditions: 4-MMA concentration of 50 mmol/L, enzyme loading of 17.5 mg/mL, 50 ℃, 13 h.
Fig. 6. Effect of reaction time on conversion rate and ees. Experimental conditions: 4-MMA concentration of 50 mmol/L, VA concentration of 300 mmol/L, enzyme loading of 17.5 mg/mL, 50 ℃.
transesterification system to achieve high conversion rate (50.2%) and large enantiomer excess of substrate (98.6%). As a tool for process optimization, this model provides theoretical reference for industrial applications.
acid in methyl tert-butyl ether was investigated for the preparation of (R)-4-methoxymandelic acid and (S)-4-methoxymandelic acid ester. By varying the acyl donor, vinyl acetate was selected as the best acyl donor. Based on Ping-Pong bi-bi mechanism with 4-MMA competitive inhibition, the kinetic model was developed for enzymatic resolution of 4-methoxymandelic acid enantiomers. The mathematic model, which was applied to describe the tranesterification reaction, successfully predicted the effects of enzyme loading, vinyl acetate concentrations and reaction time on conversion as well as enantiomeric excess. The experimental results were in good agreement with the model predictions. The optimized parameters were used for the enzymatic
Declaration of Competing Interest All authors declare that they have no conflict of interest.
7
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
[6] K.W. Tang, P.L. Zhang, C. Pan, AIChE J. 57 (2011) 3027–3036. [7] N. Sunsandee, N. Leepipatpiboon, P. Ramakul, U. Pancharoena, Chem. Eng. J. 180 (2012) 299–308. [8] M.P. Kamble, S.A. Chaudhari, R.S. Singhal, et al., Biochem. Eng. J. 117 (2017) 121–128. [9] P.L. Zhang, Q. Cheng, W.F. Xu, et al., Ind. Eng. Chem. Res. 57 (2018) 11246–11256. [10] A.S.D. Miranda, L.S.M. Miranda, R.O.M.A. DSouza, Biotechnology Adv. 33 (2015) 372–393. [11] O. Sahin, S. Erdemir, A. Uyanik, et al., Appl. Catal. A Gen. 369 (2009) 36–41. [12] E.N. López, A.R. Medina, P.A. GMoreno, et al., Bioresour. Technol. Rep. 203 (2016) 236–244. [13] M. Hümmer, S. Kara, A. Liese, et al., Mol. Cata. 458 (2018) 67–72. [14] Y.Z. Qin, Y.M. Li, M.H. Zong, et al., Green Chem. 17 (2015) 3718–3722. [15] S.L. Cao, X. Deng, P. Xu, et al., J. Agr. Food Chem. 65 (2017) 2084–2088. [16] Q. Cheng, G.Y. Liu, P.L. Zhang, et al., Biotechnol. Progr. 34 (2018) 1355–1362. [17] B.P. Dwivedee, J. Bhaumik, S.K. Rai, et al., Bioresour. Technol. Rep. 239 (2017) 464–471. [18] S. Park, T.T.N. Doan, Y.M. Koo, et al., Mol. Catal. 459 (2018) 113–118. [19] K. Zhang, Z. Pan, Z. Diao, et al., Enzyme Microb. Tech. 110 (2018) 8–13. [20] G.D. Yadav, P. Sivakumar, Biochem. Eng. J. 19 (2004) 101–107. [21] Z.J. Zhang, J. Pan, J.F. Liu, et al., J. Biotechnol. 152 (2011) 24–29. [22] K. Kibara, K. Sakai, Y. Hashimoto, H. Nohira, K. Saigo, Tetrahedron Asymmetry 7 (1996) 1539–1542. [23] W. Zhe, B. La, J.M. Fortunak, X.J. Meng, G.W. Kabalka, Tetrahedron Lett. 39 (1998) 5501–5504. [24] R. Aneja, P.M. Luthra, S. Ahuja, Chirality. 22 (2010) 479–485. [25] Y.P. Xue, S.Z. Xu, Z.Q. Liu, et al., J. Ind. Microbiol. Biot. 38 (2011) 337–345. [26] R. Lu, Q. He, C. Feng, et al., Chirality 29 (2017) 708–715. [27] T. Miyazawa, S. Kurita, S. Ueji, T. Yamada, S. Kuwata, J. Chem. Soc. Perkin Trans. I 1 (1992) 2253–2255. [28] M. Poterała, M. Dranka, P. Borowiecki, Eur. J. Org. Chem. 2017 (2017) 2290–2304. [29] M.F. Peterson, R.M. Daniel, M.J. Danson, R. Eisenthal, Biochem. J. 402 (2007) 331–337. [30] K. Dabkowska, K.W. Szewczyk, Biochem. Eng. J. 46 (2009) 147–153. [31] A.B. Canelas, C. Ras, A. ten Pierick, et al., Metab. Eng. 13 (2011) 294–306. [32] S. Soni, B.P. Dwivedee, V.K. Sharma, et al., RSC Adv. 7 (2017) 36566–36574. [33] M. Paravidino, U. Hanefeld, Green Chem. 43 (2015) 2651–2657. [34] J. da Silva Crespo, N. Queiroz, M. da Graça Nascimento, et al., Process Biochem. 40 (2005) 401–409. [35] R.S. Phillips, Trends Biotechnol. 14 (1996) 13–16. [36] S.M. Gupta, M.P. Kamble, G.D. Yadav, Mol. Catal. 440 (2017) 50–56. [37] G.D. Yadav, S.V. Pawar, Appl. Microb. Biotechnol. 96 (2012) 69–79.
Fig. 7. Chromatograms of (R,S)-4-MMA and the product after reaction: (a) racemic 4-MMA and (b) the product after reaction under optimal conditions.
Acknowledgments This work was supported by the National Natural Science Foundation of China (grant number, 21676077), Supported by Hunan Provincial Innovation Foundation for Postgraduate (CX2018B772). References [1] [2] [3] [4]
H. Caner, E. Groner, L. Levy, et al., Drug Discov. Today 9 (2004) 105–110. L.A. Nguyen, H. He, C. Pham-Huy, Int. J. Biomed. Sci. 2 (2006) 85–100. R. Kramell, G. Schneider, O. Miersch, Phytochem. Anal. 7 (2015) 209–212. S. Wacharineantar, G. Levilain, V. Dupray, et al., Org. Process Res. Dev. 14 (2014) 1358–1363. [5] K.W. Tang, H. Zhang, Y. Liu, AIChE J. 59 (2013) 2594–2602.
8
Contents lists available at ScienceDirect
Applied Catalysis A, General journal homepage: www.elsevier.com/locate/apcata
Kinetic model of resolution of 4-methoxymandelic acid enantiomers by lipase-catalyzed transesterification reaction
T
⁎
Xin Yuan, Panliang Zhang, Weifeng Xu, Kewen Tang
Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang, 414006, Hunan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipase AK (RS)-4-methoxymandelic acid Transesterification Kinetic resolution Modeling
This paper reports the kinetics resolution of (R,S)-4-methoxymandelic acid (4-MMA) by lipase AK-catalyzed transesterification reaction, using vinyl acetate (VA) as acyl donor in methyl tert-butyl ether (MTBE). Considering the Ping-Pong bi-bi mechanism with 4-methoxymandelic acid inhibition, the kinetic model was established. Kinetic parameters were obtained by fitting time-concentration curves for different initial 4methoxymandelic acid concentrations. The average relative error below 3.0% indicated that the model predictions were in good agreement with the experimental data. Furthermore, the kinetic model was employed to predict the effects of enzyme loading, initial vinyl acetate concentration and reaction time on enantiomeric excess (ees) and conversion of substrate (c), and to optimize the conditions. The excellent results with high conversion of 4-MMA (50.2%) and large enantiomeric excess of substrate (98.6%) were achieved under the optimal conditions.
1. Introduction Chiral drugs play dominant role in the global pharmaceutical market. At present, the total number of drugs used in the world is about 1900, of which more than 50% are chiral drugs. Among the 200 drugs commonly used in clinical practice, there are as many as 114 chiral drugs [1,2]. Therefore, the separation and purification of drug enantiomers are of great scientific significance and economic value in the rapidly growing market demand. Currently, several methods have been developed to obtain optically pure compounds, such as high performance liquid chromatography [3], crystallization [4], liquid-liquid extraction [5,6], membrane separation [7], and enzymatic resolution [8,9]. High optical purity of single enantiomer can be obtained by HPLC and crystallization method, but it has the disadvantages of low yield and low economic benefit. Liquid-liquid extraction can realize continuous production with high yield, but it is very difficult to screen out a chiral extractant with high selectivity and versatility. Compared with the other separation methods, enzymatic resolution method has mild reaction conditions, high selectivity, environmental protection, wide applicability and is easy to achieve industrial production. So, enzymatic resolution method has become a research hotspot [10]. Lipase (triacylglycerols acyl esters hydrolases; EC 3.1.1.3) is a sort of proteins with biological activity and multiple catalytic capabilities. Lipases are also one of the important industrial enzyme, which can
⁎
catalyze hydrolysis [11], transesterification [12], esterification [13] and other reactions [14,15], and is widely used in oil processing, food, medicine, daily chemical and other industries. Lately, lipases of different sources as biocatalysts are widely applied to the kinetic resolution of chiral drugs due to their excellent chemical-, stereo- and enantio- selectivity, such as lipase from Rhizomucor miehei [16], Pseudomonas fluorescens [17], Candida rugosa [18], Candida antarctica B [19]. Lipases selectively catalyze the faster conversion of one enantiomer to the target product due to the high selectivity, thereby achieving the purpose of separating the racemic mixture. Mandlic acid with their derivatives are very important to chiral building blocks, organic synthesis intermediates and auxiliaries in pharmaceutical and fine chemicals industries [20,21]. Besides, they are also optical resolving agents that have been applied for the separation of racemic alcohols and amines [22]. 4-Methoxymandelic acid (2-Hydroxy-2-(4-methoxyphenyl)ethanoic acid) has important applications in many fields, such as pharmaceutical production and asymmetric synthesis. Currently, it was widely used to synthesize optically pure amino acids, angiotensin converting enzyme inhibitors and coenzyme A, etc [23]. The methods of racemic separation used to obtain single enantiomers of mandelic acid and its derivatives have been reported in the literature [24–28]. However, the resolution of the 4-methoxymandelic acid enantiomers by lipase catalyzed has not been reported. The mathematical model of multifactor optimization is rarely found in
Corresponding author. E-mail address: [email protected] (K. Tang).
https://doi.org/10.1016/j.apcata.2019.117274 Received 29 July 2019; Received in revised form 20 September 2019; Accepted 21 September 2019 Available online 23 September 2019 0926-860X/ © 2019 Elsevier B.V. All rights reserved.
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Scheme 1. Kinetic resolution of (R,S)-4-methoxymandelic acid by enzymatic transesterification.
breakdown of E’B complex. Forth, since there is only little water (0.10% v/v), the hydrolysis reaction is neglected. Additionally, the vinyl alcohol of highly unstable is immediately tautomerism to form acetaldehyde, so this reaction is irreversible (k-2 = 0). The concentrations of various forms of enzymes can be expressed by the following pseudo-steady state equilibrium relationship.
the literature. In this paper, an efficient reactive system was established for kinetic resolution of 4-methoxymandelic acid enantiomers (4-MMA) by lipase AK-catalyzed transesterification in organic system (Scheme 1). Both of the purity and conversion are varied with the reaction progress. Therefore, prediction of reaction progress is crucial for process design and operation of enzymatic kinetic resolutions. An quantitative mathematical model based on the ping-pong bi-bi mechanism with 4-MMA inhibitor was constructed to describe the kinetic behavior of the transesterification reaction and obtain the optimal reaction conditions. Meanwhile, the effects of enzyme loading, vinyl acetate concentration and reaction time on conversion rate and ees were simulated and optimized using the kinetic model. This model has important guiding significance for controlling the reaction process to obtain the best industrial parameters. 2. Model development 2.1. Kinetic analysis
d[EA] = k1 [E][A] − (k−1 + k2)[EA] = 0 dt
(1)
d[E'BS] = k3S [E'][BS] − (k −3S + k 4S)[E'BS] = 0 dt
(2)
d[E'BR ] = k3R [E'][BR ] − (k −3R + k 4R)[E'BR ] = 0 dt
(3)
d[EBS] = k5S [E][BS] − k −5S [EBS] = 0 dt
(4)
d[EBR ] = k5R [E][BR ] − k −5R [EBR ] = 0 dt
(5)
d[E'] = k2 [EA] + k −3S [E'BS] − k3S[E'][BS] + k −3R [E'BR ] − k3R [E'][BR ] dt (6) =0
The enzymatic transesterification reaction follows an irreversible Ping-Pong bi-bi mechanism with dead-end inhibition of 4-MMA. This reaction usually involves the following steps:
Substituting Eqs. (2) and (3) into (6) can be obtained: 1 The Enzyme-Acyl complex (EA) is formed through the interaction between free lipase (E) and vinyl acetate (A). 2 The nucleophilic serine (Ser105) residue in the EA interacts with carbonyl group of vinyl acetate, forming the first intermediate complex (E’P). E’P releases the first product, vinyl alcohol (P) and modified lipase (E’). 3 The lipase-4-MMA complex (E’B) is formed by combining the hydroxyl group of 4-MMA (B) with the modified lipase (E’). The E’B is rapidly isomerized to form the second intermediate complex EQ, which subsequently releases product ester (Q) and regenerating enzyme (E). 4 4-MMA (B), which competes with vinyl acetate (A), combines with free lipase (E) to form dead-end complex (EiB).
k [B ] k [B ] K [E] = ⎛ 4S S + 4R R ⎞ mA [E'] KmBR ⎠ k2 [A] ⎝ KmBS ⎜
⎟
(7)
The mass balance equation for the total enzyme concentration [E]T is expressed as follows:
[E]T = [E] + [EA] + [E'] + [E'BR ] + [E'BS] + [EBR ] + [EBS]
(8)
The rate equation for (R)-4-MMA and (S)-4-MMA can be defined as:
−
d[BR ] 1 [E'] = VR = k 4R [E'BR ] = k 4R [BR ]K −mB,R dt
−
d[BS] 1 [E'] = VS = k 4S [E'BS] = k 4S [BS]K −mB,S dt
(9)
(10)
Substituting Eq. (7) into Eqs. (9) and (10), Eqs. (11) and (12) are obtained:
These steps of transesterification reaction are represented by Scheme 2: where k1 and k-1, k3 and k-3, k2 and k4 are the rate constant for the intermediate EA, E’B, product of P and Q, respectively; KiB,S and KiB,R are the inhibition constant of (S)-4-MMA and (R)-4-MMA on lipase. For the kinetic model, some assumptions are made to derive the initial rate equation. First, all complexes are assumed to be in a pseudosteady state, because the substrate concentration is much more than enzyme concentration and the concentrations of complexes are approximately constant during the reaction. Second, the formation and dissociation of all complexes are very fast, which is assumed to be close to equilibrium. Third, the overall reaction rate is limited by the
VR =
VS =
1 Vmax,R [BR ][A]K −mB,R 1 −1 [A] + [BR ]K −mB,R {[A] + αR KmA (1 + [BR ]K -1 iB,R + [BS]K iB,S)} 1 −1 {[A] + α SKmA (1 + [BR ]K -1 + [BS]K−mB,S iB,R + [BS]K iB,S)}
(11)
1 Vmax,S [BS][A]K −mB,S −1 −1 [A] + [BR ]K mB,R {[A] + αR KmA (1 + [BR ]K -1 iB,R + [BS]K iB,S)} -1 −1 −1 + [BS]K mB,S {[A] + α SKmA (1 + [BR ]K iB,R + [BS]K iB,S)}
(12)
where, αR=k4Rk2 2
−1
and
αS=k4Sk2-1.
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Scheme 2. Mechanism of enzymatic transesterification reaction with 4-MMA inhibition.
Vmax,i = k 4i × [E]T
(13)
nives lipase (referred as RNL, SD) was bought from Sigma-Aldrich Co., Ltd. (USA). (R,S)-4-methoxymandelic acid (purity ≥98%) was obtained from Aladdin Industrial Corporation (Shanghai, China). Some important solvent were purchased as folllows: vinyl acetate (> 98%), isopropenyl acetate (> 99%), ethyl acetate, butyl acetate and isobutyl acetate (> 98%) from Adamas Reagent Co., Ltd. (Basel, Switzerland); vinyl propionate (> 98%) and vinyl butyrate (> 98%) from MacklinReagent Co., Ltd. (Shanghai, China); methyl tert-butyl ether (> 98%, MTBE) from Titanchem Co., Ltd. (Shanghai, China). Other organic solvent were of analytical grade and purchased from different companies.
where, i is R or S. The differential equation of the reaction rate is solved using the fourth-order Runge-Kutta method, and parameters of the mathematical model are obtained by nonlinear fitting of the experimental data. 2.2. Thermodynamic analysis The effects of temperature on lipase activity can be explained by the Arrhenius or Eyring theory [29,30]. An increase in temperature accelerates the rate of the catalytic reaction, however, excessive temperature leads to inactivation of lipase. Meanwhile, the enantioselectivity is dominated by the difference in the activation free energy of the transient state of the fast-reacting enantiomer (S) and the slow-reacting enantiomer (R) [31]. Gibbs free energy of transition state formation can be shown as follows:
kcat , j ⎞ ΔGj = −RT ln ⎛⎜ ⎟ ⎝ K m, j ⎠
3.2. Enzymatic transesterification of (R,S)-4-MMA The enzymatic transesterification of 4-MMA enantiomers with vinyl acetate was carried out in 25 mL schlenk tube equipped with a heating and stirrer reactor (IKA RCT basic, Germany). 4-MMA enantiomers (50 mmol/L) and various concentrations of vinyl acetate (250–500 mmol/L) were dissolved in anhydrous MTBE at a constant volume of 25 mL. The lipase was added into a schlenk tube containing 2 mL of the mixed reaction solution, once it heated to the expected temperature. The stirring speed was maintained at 500 rpm. The water content was kept at 0.10% of reaction mixture solution. After reaction, the lipase was filtered off to obtain a sample. The sample was pretreated by drying the solvent, then diluted with methanol and detected by HPLC. The effects of different reaction conditions on the catalytic activity and enantioselectivity were investigated as follows: reaction temperature, varied from 35 to 65 °C; the concentration of vinyl acetate, from 100 to 500 mmol/L; the enzyme loading, evaluated from 10 to 30 mg/ mL. To analyze reaction kinetics and thermodynamic behavior, the effects of different initial concentrations of 4-MMA (25–80 mmol/L) and vinyl acetate (250–450 mmol/L) as well as reaction temperature on the initial rate were investigated.
(14)
where kcat,j is ; Km,j is affinity constant of 4-MMA (mmol/L); R is universal gas constant (8.314 J/(mol·K)); i is R or S. The overall reaction rate constant is defined as Vmax,j/ET, where ET is the total concentration of enzyme (mg/L). Therefore, Eq. (14) can be written as Eq. (15).
Vmax,j ⎞ ΔGj = −RTln ⎜⎛ ⎟ ⎝ E T Km,j ⎠
(15)
Eq. (16) is derived based on the difference of Gibbs free energy of transition state between (R)-enantiomer and (S)-enantiomer.
Vmax,R/KmB,R ⎞ ΔΔG = −RTln ⎜⎛ ⎟ ⎝ Vmax,S/KmB,S ⎠
(16)
3. Experimental
3.3. HPLC analysis
3.1. Materials
The analysis of 4-MMA enantiomers was carried out by High Performance Liquid Chromatography (Waters e2596, U.S.A), using Daicel Chiralcel OJ-RH column (150 mm × 4.6 mm I.D., Tokyo, Japan). The wavelength of photodiode array detector (waters 2998, U.S.A) was 225 nm. The composition of the mobile phase is as follows: 20% anhydrous ethanol and 80% aqueous solution (pH 3.0, adjusted with glacial acetic acid). In sample analysis, the volume of 10 μL was injected. The flow rate was kept at 0.6 mL/min. The column temperature
Lipase AYS (SD), Lipase AK (SD), Lipase PS (SD) were obtained from Amano Enzyme Inc. (Nagoya, Japan). Lipozyme TL (IM) and Lipozyme RM (IM), Novozym 40086 (IM), Novozym 435 (IM) and Candida antarctica Lipase A (referred as CALA, L) were provided by Novozymes (Denmark). Porcine pancreatic lipase (referred as PPL, SD) was provided by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Rhizopus 3
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
was maintained at 25.0 °C. The retention time of (S)-4-MMA, (R)-4MMA and 4-MMA ester were about 24.50 min, 30.65 min and 50.44 min, respectively. The enantiomeric excess of substrate (ees, %), conversion rate of (R)-4-MMA (cR, %) and conversion rate of (S)-4-MMA (cS, %) were defined as:
Table 2 The influence of various acyl donors on c, eeS and E value. Acyl donors
ees (%)
cS (%)
Vinyl acetate Vinyl propionate Vinyl butyrate Ethyl acetate Butyl acetate Isobutyl acetate Butyl acrylate
99.1 ± 0.1 98.0 ± 0.8 50.7 ± 0.5 2.3 ± 0.5 0 0 0
99.6 99.0 74.9 34.6 28.5 18.9 22.1
± ± ± ± ± ± ±
0.1 0.4 1.1 1.6 0.5 1.7 0.6
cR (%)
E
3.1 ± 0.3 6.4 ± 0.4 23.3 ± 0.7 31.5 ± 1.1 28.1 ± 0.7 17.9 ± 0.8 21.0 ± 0.9
169.5 ± 4.9 69.9 ± 2.9 5.2 ± 0.2 1.1 ± 0.1 0 0 0
ees =
[R] − [S ] × 100% [S ] + [R]
(17)
cS =
[S ]0 − [S ] × 100% [S ]0
(18)
[R]0 − [R] × 100% [R]0
Conditions: 50 mmol/L of (R,S)4-MMA; 300 mmol/L of acyl donors; 50 mg of lipase AK; 2 mL of MTBE; T = 50 °C; t = 11 h.
(19)
4. Results and discussion
donor types on c, eeS and E value were investigated (Table 2). The best results are obtained using the vinyl acetate as acyl donor, exhibiting the highest c (cS, 99.6%; cR, 3.2%), eeS (99.1%) and E value (164.5) in the kinetic resolution of (R,S)-4-MMA. The c and E decrease with increasing the alkyl chain length of acyl donors. Moreover, Ethyl acetate, butyl acetate, isobutyl acetate and butyl acrylate act as acly donor with good conversion, while enantioselectivity of enzyme are very small in transesterification reaction. Low enantioselectivity may be caused by the same ability of the nucleophilic attack of (R)- and (S)-alcohol [33]. Vinyl acetate prefers to react with (S)-4-MMA in the lipase mediated reaction compared with other enol esters. Considering the above results, vinyl acetate was screened as acyl donor in further experiments.
4.1. Selection of lipase
4.3. Selection of organic solvents
There were different catalytic structures for different lipases, and the structure determines stereoselectivity, stability and activity of lipase. In this study, the lipase-catalyzed kinetic resolution of (R,S)-4MMA was carried out in organic solvent. The effects of nine lipases on conversion rate, enantiomeric excess and enantioselectivity were investigated for the kinetic resolution of (R,S)-4-MMA (Table 1). The results show Lipozyme TL, Lipase PS, Novozym 40086, Novozym 435 and lipase AK possess good catalytic activity and enantioselectivity. Among them, Lipase AK resolves (R,S)-4-MMA to the corresponding (S)ester and (R)-4-MMA, possessing the highest conversion and enantioselectivity as well as the largest enantiomeric excess. Because substrate specificity of lipase makes lipase difficult to combine with the substrate to form a complex, the catalytic activity of Lipase AYS and RNL are low. Then, this phenomenon of rapid enantioselective conversion of Lipase AK towards (S)-enantiomer may be attributed to the favorable interactions between the amino acid residues in the active site and the ligands [32]. Therefore, Lipase AK was selected as the optimal catalyst based on excellent conversion and enantiomeric excess.
Organic solvents with different polarities affect not only activity and enantioselectivity of enzyme, but also distribution of substrates and products. To obtain the maximum conversion and ees, screening of proper solvent was essential for lipase-catalyzed enantioseletive conversion of (R,S)-4-MMA. Based on the solubility of the substrate, the organic solvents with log P values in the range of 0.49–2.5 were studied, such as toluene (TL, 2.50), diisopropyl ether (DIPE, 1.90), dichloromethane (DCM, 0.60), tetrahydrofuran (THF, 0.49), methyl tertbutyl ether (MTBE, 1.40), Acetonitrile (ACN, -0.33) and 1,2-dichloroethane (DCE, 1.46). From Fig. 1, the excellent results are obtained using MTBE as reaction medium with the highest conversion rate (51.6%), E value (126.7) and enantiomeric excess (eeS 98.7%). It indicates that the catalytic triad of Lipase AK has high structural stability in MBTE, leading to the preferential recognition and transformation of (S)-enantiomers. In general, the strong polar organic solvents (DCM, THF and ACN) tend to peel off the essential water layers that bind tightly to the surface of enzyme molecules, damaging the hydrogen bond network of the enzyme molecules to reduce enzyme activity [34]. Although toluene has high catalytic activity, its enantioselectivity is very low. The active center of the enzyme has a binding site and a catalytic site that determine selectivity and catalytic efficiency, respectively. The structural similarity of 4-MMA and toluene results in the pi-pi electron interaction on the benzene ring of toluene and more likely
cR =
Conversion rate (c) was defined as Eq. (20). Enantioselectivity (E) was calculated by using total conversion (c) and enantiomeric excess (ees).
[S ] + [R] ⎞ c = ⎛1 − × 100% [ S ]0 + [R]0 ⎠ ⎝
(20)
ln{(1 − c)(1 − ees)} ln{(1 − c)(1 + ees)}
(21)
⎜
E=
⎟
4.2. Selection of acyl donors Various acyl donors have significant effects on the reaction rate and enantioselectivity of enzymatic reactions. Hence, the influence of acyl Table 1 Effect of various lipases on the catalytic activity and enantioselectivity. Lipase
Source
ees (%)
cS (%)
cR (%)
E
Lipozyme TL Lipozyme RM Novozym 435 Novozym 40086 lipase AK Lipase PS Lipase AYS CALA RNL
Thermomyces lanuginosus Aspergillus oryzae Candida Antarctica B Rhizomucor miehei Pseudomonas fluorescens Burkholderia cepacia Candida rugosa Candida Antarctica A Rhizopus nives
91.7 ± 1.5 7.1 ± 2.1 57.1 ± 0.7 57.7 ± 0.9 99.1 ± 0.1 69.1 ± 0.6 2.5 ± 0.5 15.0 ± 0.4 0
97.5 ± 0.8 38.2 ± 0.6 77.5 ± 0.7 73.6 ± 0.6 99.6 ± 0.1 82.0 ± 0.4 8.9 ± 0.5 27.5 ± 0.4 0
42.3 ± 0.7 28.8 ± 0.5 17.5 ± 0.7 1.6 ± 0.2 3.1 ± 0.3 1.5 ± 0.1 13.4 ± 0.5 1.7 ± 0.2 0
6.7 ± 0.2 1.4 ± 0.1 7.7 ± 0.2 80.0 ± 3.3 169.5 ± 4.9 111.9 ± 4.4 1.5 ± 0.1 18.1 ± 0.6 0
Conditions: 50 mmol/L of (R,S)4-MMA; 300 mmol/L of vinyl acetate; 50 mg of lipase; 2 mL of MTBE; T = 50 °C; t = 11 h. 4
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
conversion is due to less collision between the substrate and the enzyme molecule at lower temperatures. The effect of temperature on the enzymatic reaction is consistent with that reported by Surbhi M. Gupta et al [36]. In addition, the difference of Gibbs free energy of transition state between (S)-enantiomer and (R)-enantiomer was calculated by simulating kinetic parameters at 50 ℃. The ΔΔG for transesterification reaction is calculated as 9.671 kJ/mol, indicates that lipase AK catalyzed resolution of 4-MMA enantiomers has relatively high enantioselectivity at 50 ℃. 4.5. parameters estimation
attracts the phenyl group of 4-MMA. The substrate molecule can normally bind to the catalytic site in the active center, thus exhibiting high catalytic activity. The tight binding of toluene and substrate results in an increase in the space resistance of the substrate into the enantiomerrecognition pocket. Lipase AK has good catalytic activity in other organic solvents, while its enantioselectivity is very low. Maybe different ability of various solvents to maintain the essential water layer causes varied conversion and ee. Therefore, MTBE was chosen as the optimal reaction medium.
The lipase-catalyzed transesterification reactions with various initial concentrations of 4-MMA were performed to determine kinetic parameters. Based on the mass conservation principle, namely, v=-d [B]/dt, integration of Eqs. (11) and (12) give the relation between concentration of 4-MMAR and time and the relation between concentration of 4-MMAS and reaction time. Then, the differential equations of the reaction rate were solved by using the fourth-order RungeKutta method. The concentration-time curves date were used to simulate the kinetic parameters by Matlab program (MATLAB 2013A). The kinetic parameters were obtained as follows: 52.07 mmol/(L.h) of Vmax,S, 0.43 mmol/(L.h) of Vmax,R, 15.06 mmol/L of KmB,S, 4.5510 mmol/L of KmB,R, 165.09 mmol/L of αSKmA, 6.02 mmol/L of αRKmA, 247.56 mmol/L of KiB,S and 1.58 mmol/L of KiB,R. The comparison of the simulated values with the experimental data was shown in Fig. 3. The experimental values agree well with the simulated values, indicating the validity of the kinetic model.
4.4. Effect of temperature
4.6. Model prediction
Temperature as an important parameter has a large effect on the activity and enantioselectivity of enzyme. As Fig. 2 shows, the effects of temperature on conversion rate and enantiomeric excess of substrate were carried out in the range of 35–65 ℃. The eeS increased with the increasing of temperature (≤ 50℃), however, further increase of temperature lead to the ees decreased. The variation trend of eeS is consistent with the results of Phillips [35]. The conversion rate also increases rapidly as the temperature increases below 50 ℃. However, continued increase in temperature resulted in decreased both conversion rate and ees, indicating that higher temperatures have negative impact on enzyme activity. The active site of the lipase is a triplet structure composed of Ser, Asp and His residues. The reaction rate and selectivity are determined by the combination of the catalytic triad and the substrate to form tetrahedral transition state. Maybe the higher temperature destroys the three-dimensional structure of lipase and the hydrogen bonds, resulting in decreasing enzyme activity. The lower
4.6.1. Effects of the enzyme loading Based on Eqs. (11)–(13), the kinetic model was applied to evaluation of the effect of enzyme loading on the enzymatic resolution reaction, and experiments were conducted at 50 ℃ with vinyl acetate to 4MMA molar ratio of 6:1. As is shown in Fig. 4, the scatter dots present the experimental data of conversion rate and ees, and the line presents the simulation values. It is clear from Fig. 4 that the conversion rate increases rapidly with the increase of enzyme loading. When the enzyme loading reaches 17.5 mg/mL, the total conversion rate is close to 50%. Meanwhile, with enzyme loading is increased from 10 to 17.5 mg/mL, ees values increase from 57% to 98%, but little improvement of the ees is observed with further increase of enzyme loading. This phenomenon can be explained by the increase of enzyme load in the reaction mixture, which increases the active sites available for substrate binding and facilitates the interaction between the enzyme and substrate. Additionally, higher enzyme loading may enhance the external mass transfer resistance to affect the reaction rate, resulting in slow increase in conversion. Excess enzyme granules at high enzyme dosages may form enzyme aggregates even under appropriate agitation, which reduces the interaction of the enzyme with the substrate. This result is consistent with that has been reported in the literature [8]. Considering all the results in terms of conversion rate and ees, 17.5 mg/mL of enzyme loading was selected as the optimal value for further runs.
Fig. 1. Influence of various solvents on c, eeS and E-value. Conditions: 50 mmol/L of (R,S)-4-MMA; 300 mmol/L of vinyl acetate; 50 mg of lipase; 2 mL of solvents; T = 50 °C; t = 11 h.
4.6.2. Effects of the substrate ratio To investigate the effect of vinyl acetate concentrations on conversion rate and ees, sets of experiments were carried out from 100 to 500 mmol/L by keeping the concentration of (R,S)-4-MMA constant. Based on the kinetic model, the conversion rate and ees are predicted as a function of vinyl acetate concentration. With the concentration of vinyl acetate increased, the conversion and ees values show an upward trend as shown in Fig. 5. Vinyl acetate concentration of 300 mmol/L give an optimal conversion rate of 50.6%, and higher enantiomeric excess with 98.3% is obtained. Further increasing of vinyl acetate
Fig. 2. Effect of temperature on conversion rate and ees. Conditions: 50 mmol/L of (R,S)-4-MMA; 300 mmol/L of vinyl acetate; 50 mg of lipase; 2 mL of solvents; t = 11 h. 5
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Fig. 3. Comparison of simulated values with the experimental data of (S)-4-MMA (a) and (R)-4-MMA (b).
concentration can’t increase the reaction rate obviously. Additionally, even though the concentration of vinyl acetate is increased, it does not show any inhibitory effect on lipase activity. The possible reason is that increasing the concentration of acyl donor makes it easier to form enzyme and the acyl donor complex, which leads to increase in the conversion and ees. However, the active site is limited at a certain amount of enzyme. After the active sites are all occupied, increasing the acyl donor concentration does not significantly increase the conversion. Similar results have been reported in enzymatic transesterification of (R,S)-2-octanol using Novozyme 435 [37].
courses of the lipase-catalyzed transesterification of 4-MMA and vinyl aetate in MTBE at 50 ℃. The conversion rate increased with the increase of the reaction time (≤ 13 h), and the conversion rate became very slow after 13 h of reaction. The ees also exhibits the similar tendency and is up to 98% at 13 h, as shown in Fig. 7. It might be because the reaction is highly enantioselective and the reaction rate of fast-reacting enantiomers is much greater than that of slow-reacting enantiomers. At around 50% conversion rate, the reaction gets slower because there is little amount of faster reacting enantiomer in the mixture.
4.6.3. Effects of reaction time The influence of reaction time on conversion and enantiomeric excess can be predicted through the kinetic model. Fig. 6 presents the time
5. Conclusion Lipase AK-catalyzed transesterification of (R,S)-4-methoxymandelic 6
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
Fig. 4. Effect of enzyme loading on lipase catalyzed transesterification of 4-MMA in MTBE. Experimental conditions: 4-MMA concentration of 50 mmol/L, VA concentration of 300 mmol/L, 50 ℃, 13 h.
Fig. 5. Effect of vinyl acetate concentration on lipase catalyzed transesterification of 4-MMA in MTBE. Experimental conditions: 4-MMA concentration of 50 mmol/L, enzyme loading of 17.5 mg/mL, 50 ℃, 13 h.
Fig. 6. Effect of reaction time on conversion rate and ees. Experimental conditions: 4-MMA concentration of 50 mmol/L, VA concentration of 300 mmol/L, enzyme loading of 17.5 mg/mL, 50 ℃.
transesterification system to achieve high conversion rate (50.2%) and large enantiomer excess of substrate (98.6%). As a tool for process optimization, this model provides theoretical reference for industrial applications.
acid in methyl tert-butyl ether was investigated for the preparation of (R)-4-methoxymandelic acid and (S)-4-methoxymandelic acid ester. By varying the acyl donor, vinyl acetate was selected as the best acyl donor. Based on Ping-Pong bi-bi mechanism with 4-MMA competitive inhibition, the kinetic model was developed for enzymatic resolution of 4-methoxymandelic acid enantiomers. The mathematic model, which was applied to describe the tranesterification reaction, successfully predicted the effects of enzyme loading, vinyl acetate concentrations and reaction time on conversion as well as enantiomeric excess. The experimental results were in good agreement with the model predictions. The optimized parameters were used for the enzymatic
Declaration of Competing Interest All authors declare that they have no conflict of interest.
7
Applied Catalysis A, General 587 (2019) 117274
X. Yuan, et al.
[6] K.W. Tang, P.L. Zhang, C. Pan, AIChE J. 57 (2011) 3027–3036. [7] N. Sunsandee, N. Leepipatpiboon, P. Ramakul, U. Pancharoena, Chem. Eng. J. 180 (2012) 299–308. [8] M.P. Kamble, S.A. Chaudhari, R.S. Singhal, et al., Biochem. Eng. J. 117 (2017) 121–128. [9] P.L. Zhang, Q. Cheng, W.F. Xu, et al., Ind. Eng. Chem. Res. 57 (2018) 11246–11256. [10] A.S.D. Miranda, L.S.M. Miranda, R.O.M.A. DSouza, Biotechnology Adv. 33 (2015) 372–393. [11] O. Sahin, S. Erdemir, A. Uyanik, et al., Appl. Catal. A Gen. 369 (2009) 36–41. [12] E.N. López, A.R. Medina, P.A. GMoreno, et al., Bioresour. Technol. Rep. 203 (2016) 236–244. [13] M. Hümmer, S. Kara, A. Liese, et al., Mol. Cata. 458 (2018) 67–72. [14] Y.Z. Qin, Y.M. Li, M.H. Zong, et al., Green Chem. 17 (2015) 3718–3722. [15] S.L. Cao, X. Deng, P. Xu, et al., J. Agr. Food Chem. 65 (2017) 2084–2088. [16] Q. Cheng, G.Y. Liu, P.L. Zhang, et al., Biotechnol. Progr. 34 (2018) 1355–1362. [17] B.P. Dwivedee, J. Bhaumik, S.K. Rai, et al., Bioresour. Technol. Rep. 239 (2017) 464–471. [18] S. Park, T.T.N. Doan, Y.M. Koo, et al., Mol. Catal. 459 (2018) 113–118. [19] K. Zhang, Z. Pan, Z. Diao, et al., Enzyme Microb. Tech. 110 (2018) 8–13. [20] G.D. Yadav, P. Sivakumar, Biochem. Eng. J. 19 (2004) 101–107. [21] Z.J. Zhang, J. Pan, J.F. Liu, et al., J. Biotechnol. 152 (2011) 24–29. [22] K. Kibara, K. Sakai, Y. Hashimoto, H. Nohira, K. Saigo, Tetrahedron Asymmetry 7 (1996) 1539–1542. [23] W. Zhe, B. La, J.M. Fortunak, X.J. Meng, G.W. Kabalka, Tetrahedron Lett. 39 (1998) 5501–5504. [24] R. Aneja, P.M. Luthra, S. Ahuja, Chirality. 22 (2010) 479–485. [25] Y.P. Xue, S.Z. Xu, Z.Q. Liu, et al., J. Ind. Microbiol. Biot. 38 (2011) 337–345. [26] R. Lu, Q. He, C. Feng, et al., Chirality 29 (2017) 708–715. [27] T. Miyazawa, S. Kurita, S. Ueji, T. Yamada, S. Kuwata, J. Chem. Soc. Perkin Trans. I 1 (1992) 2253–2255. [28] M. Poterała, M. Dranka, P. Borowiecki, Eur. J. Org. Chem. 2017 (2017) 2290–2304. [29] M.F. Peterson, R.M. Daniel, M.J. Danson, R. Eisenthal, Biochem. J. 402 (2007) 331–337. [30] K. Dabkowska, K.W. Szewczyk, Biochem. Eng. J. 46 (2009) 147–153. [31] A.B. Canelas, C. Ras, A. ten Pierick, et al., Metab. Eng. 13 (2011) 294–306. [32] S. Soni, B.P. Dwivedee, V.K. Sharma, et al., RSC Adv. 7 (2017) 36566–36574. [33] M. Paravidino, U. Hanefeld, Green Chem. 43 (2015) 2651–2657. [34] J. da Silva Crespo, N. Queiroz, M. da Graça Nascimento, et al., Process Biochem. 40 (2005) 401–409. [35] R.S. Phillips, Trends Biotechnol. 14 (1996) 13–16. [36] S.M. Gupta, M.P. Kamble, G.D. Yadav, Mol. Catal. 440 (2017) 50–56. [37] G.D. Yadav, S.V. Pawar, Appl. Microb. Biotechnol. 96 (2012) 69–79.
Fig. 7. Chromatograms of (R,S)-4-MMA and the product after reaction: (a) racemic 4-MMA and (b) the product after reaction under optimal conditions.
Acknowledgments This work was supported by the National Natural Science Foundation of China (grant number, 21676077), Supported by Hunan Provincial Innovation Foundation for Postgraduate (CX2018B772). References [1] [2] [3] [4]
H. Caner, E. Groner, L. Levy, et al., Drug Discov. Today 9 (2004) 105–110. L.A. Nguyen, H. He, C. Pham-Huy, Int. J. Biomed. Sci. 2 (2006) 85–100. R. Kramell, G. Schneider, O. Miersch, Phytochem. Anal. 7 (2015) 209–212. S. Wacharineantar, G. Levilain, V. Dupray, et al., Org. Process Res. Dev. 14 (2014) 1358–1363. [5] K.W. Tang, H. Zhang, Y. Liu, AIChE J. 59 (2013) 2594–2602.
8