Straightforward Enzyme-Catalyzed Asymmetric Synthesis of Caffeic

Jul 1, 2014 - ABSTRACT: Enantiopure caffeic acid esters were successfully ... The optimal synthesis parameters for caffeic acid ester synthesis were ...
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Straightforward Enzyme-Catalyzed Asymmetric Synthesis of Caffeic Acid Esters in Enantioenriched Form Peiliang Xiao,† Aijun Zhang,† Liangyu Zheng,*,† and Yanqiu Song*,‡ †

College of Life Sciences, Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, People’s Republic of China ‡ The First Hospital, Jilin University, Changchun 130012, People’s Republic of China S Supporting Information *

ABSTRACT: Enantiopure caffeic acid esters were successfully synthesized from caffeic acid and corresponding (R,S)-alcohol catalyzed by an immobilized lipase (Novozym 435) from Candida antarctica. Effects of the alcohol carbon chain length and the type of organic solvents were first investigated, and anhydrous isooctane was finally selected as the most suitable reaction medium. The optimal synthesis parameters for caffeic acid ester synthesis were evaluated using response surface methodology. The actual experimental conversions of 33.8 ± 1.8% and an E value of >100 for (R)-1-methylbutyl caffeate, achieved under optimized reaction conditions, indicated a good fit to the predictive model. Kinetic and thermodynamic analyses were conducted to determine the main factors affecting enantiomeric discrimination. Results from the batch reuse stability study show that the immobilized enzyme could be effectively reused. The present study is the first to use lipase as the chiral catalyst to prepare optically pure caffeic acid ester compounds.

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INTRODUCTION Caffeic acid, a naturally occurring phenolic acid, is widely distributed throughout the plant kingdom (in fruits, grains, coffee beans, and vegetables)1 and displays a broad spectrum of biological activities, such as antimicrobial,2 antitumor,3 and antioxidative activities.4 However, the low solubility of caffeic acid in nonpolar media limits its effectiveness in oil systems. Esterification of hydrophilic compounds with aliphatic molecules, such as fatty alcohols, can be employed to alter their solubility in oil-based formulas and emulsions, which widens the application range of caffeic acid esters in the food industry.5 In addition, compared with caffeic acid, caffeic acid esters have more obvious biological functions, such as antioxidative, antimicrobial, antiviral, and antineoplastic activities.6−8 Recently, chiral caffeic acid esters were found to exhibit the best antileishmanial activity with moderate cytotoxicity in murine macrophages.9 Leishmaniasis is a tropical disease caused by trypanosomatids of the genus Leishmania. This disease has spread to 88 countries in Africa, Asia, Europe, and America and exhibits a wide range of clinical symptoms. The World Health Organization has estimated two million new cases of this disease every year, and 350 million people are considered to be at risk.10 Although chiral caffeic acid esters can be isolated from leaves of Piper sanguineispicum, their extraction requires multiple purification steps yielding only small amounts, leading to high operating costs.11 Therefore, mastering efficient chiral caffeic acid ester synthesis in the laboratory renders it available for wider human use.12 Chemical synthesis of caffeic acid esters is difficult, as caffeic acid is heat-sensitive and susceptible to oxidation under certain pH conditions.13,14 In addition, caffeic acid has two OH groups on the benzene ring. This configuration is highly resonance stabilized, making it extremely difficult for the alcohols, © 2014 American Chemical Society

especially the increasing carbon numbers of aliphatic alcohols, to attack and form esters.6 Furthermore, it is difficult for the chemical method to control the stereogenic centers of the substrates. So far, only several chemical methods have been developed for the preparation of caffeic acid esters,7,15,16 and the synthesized caffeic acid esters are also limited to be achiral. As an alternative method, enzyme-catalyzed enantioselective esterification reaction was applied. Among various enzymes, lipase has been widely used for the enantioselective synthesis of bioactive compounds because of its wide substrate specificity and ability to recognize chirality. 17 Novozym 435, an immobilized lipase from Candida antarctica, is an efficient enzyme that can tolerate varied experimental conditions and catalyze many organic reactions, including large-scale commercial reactions.18,19 The immobilized enzyme can improve enzyme activity and stability against extreme conditions, such as high temperature and organic media. In addition, immobilized enzymes offer several advantages, including easy separation of the enzyme from the product, allowing repeated use of the catalyst, and possibility of continuous operation. Application of Novozym 435 in esterification of phenolic acid with achiral alcohols, such as phenethyl alcohol, 2-ethyl hexanol, ethanol, and so on, has been reported.13,14,20,21 However, synthesis of enantiopure caffeic acid esters from caffeic acid and corresponding (R,S)-alcohols catalyzed by Novozym 435 and investigation of the enzyme-catalyzed enantioselective mechanism have not been reported. The present study describes the enantioselective esterification of caffeic acid by (R,S)-2-pentanol, (R,S)-2-heptanol, or Received: Revised: Accepted: Published: 11638

March 11, 2014 June 30, 2014 July 1, 2014 July 1, 2014 dx.doi.org/10.1021/ie5010477 | Ind. Eng. Chem. Res. 2014, 53, 11638−11645

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Scheme 1. Novozym 435-Catalyzed Asymmetric Synthesis of (R)-Caffeic Acid Esters

(R,S)-2-octanol in the presence of Novozym 435 (Scheme 1). Organic solvents were chosen as the reaction media because of their advantages, such as the increase in solubility of substrates and easy separation of enzyme from product. Using response surface methodology (RSM) with a central composite rotatable design (CCRD), several reaction conditions affecting the catalytic activity and enantioselectivity of Novozym 435 were evaluated. The relationships between reaction conditions and response (percent conversion yield and enantiomeric excesses (e.e.p)) were studied. Kinetic studies and thermodynamic analyses were performed to investigate the enzyme-catalyzed enantioselective mechanism. The study of Novozym 435 recovery aimed to determine the number of cycles for which the enzymes can be used. The established enzyme-catalyzed asymmetric esterification method in the present study can be used to prepare other chiral phenolic acid esters.

esters were confirmed according to the chiral separation of the standard of (R, S)- caffeic acid esters (purity >99.5%). The absolute configuration of the enantiomers was established by comparison of the measured optical rotation with the literature data.9 Control reactions without enzymes were performed under the same conditions. (R)-1: 1H NMR (300 MHz, DMSO) δ 9.58 (s, 1H), 9.12 (s, 1H), 7.45 (d, J = 15.9 Hz, 1H), 7.10−6.91 (m, 2H), 6.75 (d, J = 8.1 Hz, 1H), 6.23 (d, J = 15.9 Hz, 1H), 4.98−4.86 (m, 1H), 1.63−1.45 (m, 2H), 1.32 (m, 2H), 1.21 (d, J = 6.3 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). (R)-2: 1 H NMR (300 MHz, DMSO) δ 9.60 (s, 1H), 9.17 (s, 1H), 7.48 (d, J = 15.8 Hz, 1H), 7.16−6.91 (m, 2H), 6.79 (d, J = 8.0 Hz, 1H), 6.27 (d, J = 15.9 Hz, 1H), 4.94 (dd, J = 12.3, 6.1 Hz, 1H), 1.62 (dd, J = 27.5, 6.4 Hz, 2H), 1.35−1.21 (m, 9H), 0.88 (d, J = 6.6 Hz, 3H). (R)-3: 1H NMR (300 MHz, DMSO) δ 9.58 (s, 1H), 9.12 (s, 1H), 7.44 (d, J = 15.9 Hz, 1H), 7.10−6.93 (m, 2H), 6.75 (d, J = 8.1 Hz, 1H), 6.23 (d, J = 15.9 Hz, 1H), 4.90 (dd, J = 12.5, 6.2 Hz, 1H), 1.55 (s, 2H), 1.31−1.17 (m, 11H), 0.84 (d, J = 6.4 Hz, 3H). Experimental Design. Based on the screening experiments for enzyme-catalyzed (R)-1 synthesis, four major independent parameters were identified: reaction temperature, reaction time, substrate molar ratio of caffeic acid to (R, S)-2-pentanol, and the enzyme additive amount. Conversion (%) and e.e.p (%) were used as dependent variables. The design matrix of the 5level-4-factor CCRD was employed to optimize the enzymecatalyzed reaction. According to the single parameter study, the four major factors and their levels, reaction temperature (50 to 90 °C), substrate molar ratio of caffeic acid to 2-pentanol (1:4− 1:36), reaction time (8−136 h) and the enzyme additive amount (10−130 mg) were designated as X1, X2, X3, and X4, respectively. Low, middle, and high levels of X1, X2, X3, and X4 were designated as −2, −1, 0, 1 and 2, respectively (Table S1 of the Supporting Information). Statistical Analysis. The response surface regression (RSREG) procedure of statistical analysis system (SAS) and Design-Expert 7.0 software were used to analyze the experimental data. Experimental data were fitted to a secondorder polynomial model and regression coefficients were obtained. The generalized second-order polynomial model used in the response surface analysis was as follows:

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MATERIALS AND METHODS Materials. Immobilized Candida antarctica lipase B (Novozym 435) was purchased from Novo Nordisk Industries, China. Caffeic acid, (R, S)-2-pentanol, (R, S)-2-heptanol and (R, S)-2-octanol were purchased from Aladdin Industrial Inc., China. All organic solvents were reagent grade and used without further purification. The authenticity of the organic compounds prepared during the study was confirmed by 300 MHz nuclear magnetic resonance (Mercury-300B, VARIAN, USA). Reactions were routinely monitored on silica gel plates (Qingdao Haiyang Chemical Co. Ltd., China) using UV light for spot detection. General Procedure for Novozym 435-Catalyzed Enantioselective Esterification. Before use, all solvents were dehydrated using molecular sieves (4 Å in diameter) for 24 h. Novozym 435 (40 mg) was added to an anhydrous isooctane solution (0.5 mL) containing caffeic acid (20 mM) and (R,S)-aliphatic alcohols (200 mM). The resulting mixture was stirred at 70 °C in a sealed flask. After termination of the reaction by adding acetone, the mixture was filtered to remove the enzyme; this mixture was then concentrated under reduced pressure. The residue was dissolved in minimal ethyl acetate and loaded onto a silica gel column. The products were monitored on silica gel plates using UV light for spot detection. All the purified products, (R)- and (S)-caffeic acid esters, were eluted with ethyl acetate: petroleum ether (1:1 v/v, containing acetic acid). After the samples were dissolved in 1 mL ethanol, a 10 μL aliquot of the sample was obtained and analyzed by highperformance liquid chromatography (HPLC, Acme 9000, Young Lin Instrument Co., Ltd.) using Daicel Chiralpack AD-H column. The elution solvent was a 90:10:0.05 mixtures of n-hexane, isopropanol, and trifluoroacetic acid. The flow rate was 0.8 mL/min, and detection was achieved under UV light at 254 nm. The retention time of (R) - and (S)-1-methylbutyl caffeate ((R)- and (S)-1), (R)- and (S)-1-methylhexyl caffeate ((R)- and (S)-2), and (R)- and (S)-1-methylheptyl caffeate ((R)- and (S)-3) were 9.2, 11.6, 8.6, 10.6, 8.7, and 10.5 min, respectively. The retention time of (R)- and (S)-caffeic acid

4

Y = b0 +

∑ i=1

4

biXi +

∑ i=1

3

biiXi2 +

4

∑ ∑ i=j

j=i+1

bijXij (1)

where Y is the predicted response value (percent of molar conversion or e.e.p); Xi and Xj are independent variables; b0 is a constant; and bi, bii, and bij are linear, quadratic, and interaction effect terms, respectively. The model goodness-of-fit was evaluated by the coefficient of determination (R2) and the analysis of variance (ANOVA). Quadratic polynomial equations were attained by holding one of the independent variables at a constant value and changing the level of the other variable. Analysis. Novozym 435 (40 mg), caffeic acid (20 mM) and (R,S)-aliphatic alcohols (200 mM) were added to an anhydrous 11639

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organic solvents with log P > 2.5, such as isooctane, n-heptane, n-hexane, and toluene, were selected as reaction medium. As shown in Table S2 of the Supporting Information, anhydrous isooctane with the high log P gives the highest conversions for the esterification of caffeic acid by all the three aliphatic alcohols, although the substrates had the lowest solubility in anhydrous isooctane. The highest conversions of (R)-caffeic acid esters were 21.1%, 10.2%, and 8.4% for (R, S)-2-pentanol, (R, S)-2-heptanol, and (R, S)-2-octanol as acyl acceptors, respectively. Therefore, we could conclude that the longer the carbon chain length in the molecular substrate alcohol, the lower the enzyme-catalyzed efficiency. This may be ascribed to the bulkiness and steric effects of the alcohol, making them inaccessible to the active enzyme site or causing obstruction in the enzyme interactions. In the hydrophobic organic media, the biocatalytic system showed the same stereopreference to produce (R)-caffeic acid esters, and the enantioselectivity of Novozym 435 was almost independent. e.e.p of 99% and the enantiomeric ratio (E value >100) were obtained for all alcohols from (R, S)-2-pentanol to (R, S)-2-octanol as substrates. Besides above organic solvents, the enzyme could catalyze the esterification reaction in isopropyl ether. The reaction rates and the enantioselectivity of Novozym 435 were dependent on alcohol structures, and both decreased with the increasing carbon chain lengths of aliphatic alcohols. The reaction rates (VR) were 3.8 mM·h−1, 3.2 mM·h−1, and 2.0 mM·h−1 with E values of 89.3, 25.6, and 24.1 for (R, S)-2-pentanol, (R, S)-2heptanol and (R, S)-2-octanol as substrates, respectively. In contrast, no product was detected in organic media, including trichloromethane, dichloromethane, methyl tert-butyl ether (MTBE), ether, and acetone when using (R, S)-2-heptanol and (R, S)-2-octanol as substrates. The performance of (R, S)2-pentanol was somewhat different. Esterification could be observed using trichloromethane, MTBE, and ether as solvents; however, the reaction rates were slow, and reaction conversions were only 0.4%, 1.4%, and 1.5%, respectively. Changes in the activity and enantioselectivity of Novozym 435 induced by the reaction medium may be attributed to conformational changes in enzyme molecules. In general, an enzyme requires an essential water layer to maintain the flexibility of the protein molecule necessary for catalysis. In hydrophobic solvents, the conformation of the active site remains stable, and, accordingly, the enzyme maintains its activity and stability. On the other hand, hydrophilic solvents provided lower conversion by stripping the essential water from the enzyme, distorting catalytic conformation and leading to enzyme inactivation.25,26 When tetrahydrofuran was selected as the reaction medium, the configuration of the products of caffeic acid esters was reversed from R to S independent of the alcohol used, although the E value was only 2.7, 4.2, and 3.8 for (R, S)-2-pentanol, (R, S)-2-heptanol, and (R, S)-2-octanol as substrates, respectively. The data confirmed that the reaction medium could have an important role in the enzyme-catalyzed enantioselective reaction and should be carefully optimized. We are planning a series of further experiments to improve the S-enantioselectivity of the enzyme and to understand why tetrahydrofuran can lead to enantioselectivity reversal. These results will be reported later. Considering environmental and economic factors, we also attempted to perform enzyme-catalyzed esterification without added solvent. Excess alcohol was used which enabled efficient stirring of the reaction medium. The data (Table S2 of the

isooctane solution (0.5 mL) at a temperature range of 60 to 80 °C with stirring. Samples were removed and injected onto the above HPLC system at different time intervals for analysis. The conversions of (R)- or (S)-caffeic acid esters were confirmed by external standard method using (R, S)-caffeic acid esters (purity >99.5%) as standard. From the slop of the linear plot of the conversions−time curve for appearance of product, where less than 1% conversion of reactant to product has taken place, the initial rates for both enantiomers (VR or Vs) and E-value defined as the ratio of the initial rates were estimated. Kinetic Model. Esterification of fatty acids with alcohols catalyzed by a lipase is often modeled using the Ping Pong Bi− Bi kinetic mechanism.22,23 So herein for this mechanism, a general steady-state scheme for this type of reactions was established (Scheme 2),22,24 B Km

A Km

k1

k2

k3

k4

E + A HooI EA → E′ + B HooI E′B → E k −1

(Scheme 2)

k −3

The initial rates V are expressed as follows: V=

VmaxCAC B αK mAC B

+ K mBCA + CAC B

where α = k4 /k 2 (2)

where V is the initial reaction rate; Vmax is the maximum reaction rate; CA and CB are the concentrations of caffeic acid and (R, S)-alcohols, respectively; KAm and KBm are apparent kinetic constants for caffeic acid and (R, S)-alcohols, respectively. KAm, KBm and Vmax can be estimated from the initial rate data coupled with eq 2 at fixed concentrations of substrate caffeic acid and different alcohol concentrations from 10 to 300 mM, or reverse. Thermodynamic Analysis. Transition state theory related the rate constants of an elementary step to the Gibbs free energy difference between the reactant’s ground state and the activated transition state. By assuming Michaelis−Menten kinetics for each substrate, an equation relating the E value to the Gibbs free energy differences ΔΔG at the transition state for both enantiomers, i.e., RT ln(E) = −ΔΔG = −ΔΔH + TΔΔS, was derived. From the variation of ln(E) with the inverse of absolute temperature, the activation enthalpy ΔΔH and activation entropy ΔΔS were estimated.

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RESULTS AND DISCUSSION Effects of Alcohol Type and Organic Solvent on Esterification. Using caffeic acid and (R,S)-aliphatic alcohols as substrates, the comparative analysis of lipase-catalyzed enantioselective esterification reactions in different organic solvents was investigated. The selected aliphatic alcohols of increasing carbon chain lengths included (R, S)-2-pentanol, (R, S)-2-heptanol, and (R, S)-2-octanol. Organic solvents were selected based on their log P (P as the partitioning coefficient of the solvent between n-octanol and water). The results have been summarized in Table S2 of the Supporting Information. Experimental data supported the idea that the activity and enantioselectivity of Novozym 435 in the enantioselective synthesis of chiral caffeic acid esters were dependent on the alcohol carbon chain length and the type of organic solvents. However, no correlations relating the initial rates (VR and VS) and E value with log P of organic solvent were found. Caffeic acid could be esterified, and excellent conversions could be obtained, independent of the alcohol used when hydrophobic 11640

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exerting a statistically significant overall effect (p < 0.0001) on the responding conversion of (R)-1. Enzyme additive amount (X4) in the experimental scale (10−130 mg) of the present study was the least important factor (p-value =0.3895) compared with the other reaction parameters. Similarly, the p-values of the factors X1, X3, and X4 were lower than 0.0001, which were the major factors and had significant effects on e.e.p. X2 was not the major factor (p-value =0.6429) and had no significant statistical significance. The optimal values of the selected variables were obtained by solving the regression models eq 3 and eq 4 with the leastsquares method using Design-Expert 7.0 software. According to this, the optimal reaction conditions (predicted values) were as follows: reaction time of 52 h, reaction temperature of 67 °C, substrate molar ratio of caffeic acid to 2-pentanol of 1:21, and enzyme additive amount of 50.8 mg. Under these conditions, the predicated e.e.p was over 99% and the conversion was 31%. The adequacy of this predictive model was examined through additional, independent experiments for (R)-1 synthesis under the suggested optimal synthesis conditions. The conversions of the predicted value and the actual experimental value were 31% and 33.8 ± 1.8%, respectively. All the actual e.e.p in synthesis reactions was over 99%. Typical experimental data were in agreement with theoretical predictions. The moderate conversions may be attributed to the existence of two OH groups on the benzene ring of the caffeic acid. The existence of the OH group induces the electron donating effect that intrinsically deactivates the electrophilic carbon center of the carboxylic group for the nucleophilic attack of alcohol.14 To better understand the effects of the two OH groups in caffeic acid, the cinnamic acid with no OH group on the phenyl cycle and 3, 4dimethoxycinnamic acid with two methyloxylated parahydroxyls on the phenyl cycle were selected as substrates for esterification with (R, S)-2-pentanol under the same operating conditions as that of caffeic acid. The conversions were 68% and 45%, respectively, which confirmed that the enzymecatalyzed esterification reaction conversions depended on the substrate structure. The presence of two OH groups on the benzene cycle of caffeic acid inhibited the catalytic action of enzyme. Similar results had also been reported by other groups.13,27 Based on the established RSM method, the reaction conditions for other chiral caffeic acid esters including (R)-2 and (R)-3 were also successfully optimized (data not shown). In conclusion, high optically pure caffeic acid esters with e.e.p >99% could be easily obtained by Novozym 435-catalyzed enantioselective esterification. It could be deduced that caffeic acid and the selected (R, S)-alcohols could be adequate substrates for the enzyme. The reaction can occur, which is in accordance with the observation of Chen and co-workers on the enzymatic esterification of caffeic acid and octanol with Novozym 435, and the experimental conversion was 90.34%.5 Twu and co-workers investigated Novozym 435 to catalyze the direct esterification of hydroxyphenylpropionic acid and octanol in a solvent-free system, with 95.9% conversion.28 However, the above-reported product esters were achiral. As the different organic acid and alcohol substrates could significant influence the esterification rate in lipase-catalyzed esterification reactions,29 it was reasonable for us to obtain chiral caffeic acid ester (R)-1 with 33.8% conversion (a maximum theoretical conversion of 50%). The excellent enantiopurities of caffeic acid esters observed here had a close relationship with the structure of the racemic secondary

Supporting Information) showed the reaction could occur in solvent-free systems, but the (R)- and (S)-alcohols had the same reaction rate: 6.3 × 10−2 mM·h−1 for (R)- and (S)-2pentanol, 5.8 × 10−2 mM·h−1 for (R)- and (S)-2-heptanol, and 4.4 × 10−2 mM·h−1 for (R)- and (S)-2-octanol as substrates. It could be concluded that enzyme-catalyzed esterification had no enantioselective preference in solvent-free systems. Therefore, enzyme-catalyzed esterification without added solvent was infeasible. Comprehensively considering the catalytic activity and enantioselectivity of the enzyme, anhydrous isooctane was finally selected as the most suitable reaction medium in the present study. Optimizing the Reaction Conditions of LipaseCatalyzed Ester Synthesis. In the process of enzymatic resolution, the reaction temperature, substrate molar ratio of caffeic acid to alcohol, reaction time, and enzyme additive amount were important parameters worthy of careful investigation because they may influence the sufficient expression of enzyme activity. In comparison with the onefactor-at-a time design, RSM employed in the present study, is more efficient at reducing the experimental runs and time for optimal enantiopure caffeic acid ester synthesis. It could also be used to better understand the relationships between the variables of caffeic acid ester synthesis reaction conditions and the conversion percentage and e.e.p. Here, the enzymecatalyzed enantioselective synthesis of (R)-1 was selected as the model reaction to optimize the reaction conditions. Independent variables, their levels, and real values for enzyme-catalyzed (R)-1 synthesis in anhydrous isooctane have been presented in Table S3 of the Supporting Information. To obtain a model for (R)-1 synthesis, the results from the 5-level-4-factor CCRD (Table S3 of the Supporting Information) were used. The RSREG procedure and DesignExpert 7.0 software were employed to match the second-order polynomial eq 1 to the responses, percentage conversion Y1, and e.e.p Y2. Equation 3 and eq 4 were thus generated and given below: Y1 = 40.67 + 11.96X1 + 3.96X 2 + 8.21X3 − 0.63X4 + 0.94X1X 2 + 3.06X1X3 + 2.06X1X4 − 0.19X 2X3 − 0.44X 2X4 − 1.06X3X4 − 3.76X12 − 1.01X 22 − 0.51X32 − 5.01X42

(3)

Y2 = 96.00 − 5.08X1 − 0.083X 2 − 2.08X3 − 1.58X4 + 1.25X1X 2 − 1.37X1X3 + 0.50X1X4 − 0.87X 2X3 + 0.25X 2X4 + 0.88X3X4 − 3.77X12 − 4.02X 22 − 0.40X32 − 0.77X42

(4)

Analysis of these two polynomial models by ANOVA indicated that the second-order polynomial model was highly significant and adequate to represent the actual relationships between the responses and the significant variables, with a satisfactory coefficient of determination (R2) of 0.9738 and 0.9931 for percentage conversion and e.e.p, respectively (Table S4 of the Supporting Information). Table S5 of the Supporting Information lists further analysis of the effects of various factors on percentage conversion and e.e.p (%). The statistical analysis revealed that reaction temperature (X1), substrate molar ratio (X2), and reaction time (X3) were the most important variables, 11641

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alcohols. Novozym 435 displays a very high enantioselectivity toward most secondary alcohols when one of the substituents is an ethyl group or smaller and the other is a propyl group or larger.30,31 The selected alcohols in this paper, (R,S)-2pentanol, (R, S)-2-heptanol or (R,S)-2-octanol, containing one methyl group and the other propyl group, amyl group and hexyl group, respectively, which is in accordance with the substrate requirement for the enzyme. So, all the alcohols had a good accommodation and orientation in the active section of the enzyme, thus leading a very good enantioselectivity for Novozym 435 toward the selected (R, S)-alcohols. The results are fully comparable to the following papers published previously by using Novozym 435 for the enantioselective alcohol esterification. Päiviö and co-workers reported Novozym 435-catalyzed DKR of a wide range of structurally varying secondary alcohols by using the acylation with isopropenyl acetate in toluene, and the highly enantiopure (R)-acetates (mostly e.e.p >99%) were obtained.32 Sontakke and co-workers studied systematically enzymatic kinetic resolution of (R, S)-2pentanol using vinyl acetate as an acyl donor, and Novozyme 435 was found to be the most efficient catalyst among different catalysts studied.33 Jacobsen and co-workers reported that derivatives of 1-phenoxy-2-alkanols were kinetically resolved by esterification with acyl donors using Novozyme 435 as a catalyst, and found the size limitation for the secondary alcohol in the stereospecificity pocket of the enzyme is a propyl group connected to the stereocenter.34 Thermodynamic Analysis. Novozym 435-catalyzed esterification reactions between caffeic acid and (R, S)-2-pentanol, (R, S)-2-heptanol, and (R, S)-2-octanol in anhydrous isooctane at temperatures ranging from 60 to 80 °C were investigated. Figures 1a, b, and c illustrate the effects of temperature on the time-course conversions of XR, XS, and e.e.p, respectively. Based on the time-course, it was observed that the esterification reaction between caffeic acid and (R)-alcohol was more prone to occur than that between caffeic acid and (S)-alcohol. As expected, an increase of the reaction temperature led to an increase in the conversions of XR and XS. This could be due to both the increase in enzyme activity and the improvement of caffeic acid solubility. The enantioselectivity of Novozym 435catalyzed esterification decreased with increasing temperature and prolonged reaction time. The high e.e.p of Novozym 435 (>99%) was achieved at 60 °C. At 80 °C, e.e.p significantly reduced to 83%, 85.3%, and 87.3% for (R, S)-2-pentanol, (R, S)-2-heptanol, and (R, S)-2-octanol, respectively, as reaction substrates. This was attributed to the temperature effects. At relatively high temperature, the reaction could also be under thermodynamic control which allows “enzyme-(S)-enantiomer” complex to easily reach the transition state and thus increase the rate of Vs, but not VR. In addition, the enantioselective recognition of enzymes toward (R) or (S)-alcohol could also be altered because of enzymatic thermal denaturation. Similar results had been reported by other groups.35−37 Thermodynamic analysis was performed to investigate the effects of the acyl acceptors (alcohols) on the temperature dependence of enantioselectivity in Novozym 435-catalyzed esterification in anhydrous isooctane. The logarithm of enantiomeric ratio (E value) varied with the inverse of absolute temperature. ln(E) = −ΔΔH/RT + ΔΔS/R was employed for estimating activation enthalpy (i.e., ΔΔH = ΔΔHR − ΔΔHS) and activation entropy (i.e., ΔΔS = ΔSR − ΔSS) for the transition states of both enantiomers. Figure 2 shows that a good linear relationship of ln(E) = 14461/T − 36.033 (r2 =

Figure 1. Temperature effects on the time-course. (a) XR of (R)-1, (R)-2, and (R)-3; (b) XS of (R)-1, (R)-2, and (R)-3; (c) e.e.P of (R)-1, (R)-2, and (R)-3.

0.98), ln(E) = 12303/T − 30.19 (r2 = 0.97), and ln(E) = 7957/ T − 17.549 (r2 = 0.98) was obtained for (R,S)-2-pentanol, (R,S)-2-heptanol, and (R,S)-2-octanol, respectively, as reaction substrates. With the linear relationship, we obtained −ΔΔH = 120.23 kJ/mol and −ΔΔS = 299.58 J/mol for the esterification of caffeic acid by (R,S)-2-pentanol. These results indicated that both −ΔΔH and −ΔΔS were important for enantiomer discrimination, and −ΔΔH was dominant in the reaction 11642

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Figure 2. Variations of ln (E) with the inverse of temperature for Novozym 435: (blue circles) Novozym 435-catalyzed asymmetric synthesis of (R)-1; (red circles) Novozym 435-catalyzed asymmetric synthesis of (R)-2; (green circles) Novozym 435-catalyzed asymmetric synthesis of (R)-3.

Figure 3. Initial esterification rates as a function of the substrate concentration of caffeic acid, (R)- and (S)- 2-pentanol, (R)- and (S)-2heptanol, and (R)- and (S)-2-octanol: (blue squares) caffeic acid concentration varies, (R,S)-2-pentanol: 200 mM; (blue triangles) (R)2-pentanol concentration varies, caffeic acid: 20 mM; (blue circles) (S)-2-pentanol concentration varies, caffeic acid: 20 mM; (red squares) caffeic acid concentration varies, (R,S)-2-heptanol: 200 mM; (red triangles) (R)-2-heptanol concentration varies, caffeic acid: 20 mM; (red circles) (S)-2-heptanol concentration varies, caffeic acid: 20 mM; (black squares) caffeic acid concentration varies, (R,S)-2octanol: 200 mM; (black triangles) (R)-2-octanol concentration varies, caffeic acid: 20 mM; (black circles) (S)-2-octanol concentration varies, caffeic acid: 20 mM.

systems. It could be concluded that a temperature-dependent inversion of stereochemical configuration might occur. Similar results were observed frequently in enzymatic reaction systems.35,38−42 This was also valid for the esterification of caffeic acid by (R,S)-2-heptanol and (R,S)-2-octanol. However, the replacement of substrate alcohols with the extension of carbon chain length led to some changes of the thermodynamic parameters. −ΔΔH and −ΔΔS decreased with the extension of alcohol carbon chains, in which −ΔΔH = 102.29 kJ/mol and −ΔΔS = 250.99 J/mol for the esterification of caffeic acid by (R,S)-2-heptanol and −ΔΔH = 66.15 kJ/mol and −ΔΔS = 145.9 J/mol for the esterification of caffeic acid by (R, S)-2octanol. These results demonstrated that the carbon chain length of alcohols may play an important role in decreasing the enthalpy and entropy contribution in the transient state. Kinetic Studies. To determine the kinetic constants in the Novozym 435-catalyzed esterification reaction, reaction rates were determined and analyzed using different starting mole fractions of the substrates caffeic acid, (R)-2-pentanol, (S)-2pentanol, (R)-2-heptanol, (S)-2-heptanol, (R)-2-octanol, and (S)-2-octanol at 67 °C. The target esters (R)-1, (R)-2, and (R)3 were formed as the main products. Figure 3 illustrates the progress curves of the initial esterification rates where the reaction rates increased with increasing substrate concentration. The reaction rate reached the balance point when the substrate concentration reached 80 mM for all substrates, indicating that Novozym 435 may be inhibited by substrates or products. Novozym 435 exhibited high reaction rates when the concentration of caffeic acid was varied and the concentration of (R,S)-pentanol was constant, whereas slightly high reaction rates of Novozym 435 were observed when the concentrations of (R,S)-heptanol and (R,S)-octanol were constant. The results indicated that caffeic acid esterification rates in the presence of Novozym 435 were based on the structure of another substrate (R,S)-alcohol. The results are not surprising because Novozym 435 is a lipase with a biological function to catalyze the hydrolysis of triglycerides to fatty acids and glycerol. Therefore, the hydrophobic active site of the lipase has a preference for both carboxylic acids and alcohols in the esterification reaction. Similar results were reported by Pelt and co-workers.43

The apparent kinetic constants KAm, KBm and maximum reaction rate Vmax were estimated from the Lineweaver−Burk plots (Figure 4). Novozym 435 had a low affinity for caffeic acid (KAm was 11.2, 11.7, and 10.7 mM) when (R)-2-pentanol, (R)-2heptanol, or (R)-2-octanol, respectively, were used as substrates, and a substantially high affinity for (R)-2-pentanol, (R)-2-heptanol, or (R)-2-octanol (KBm was 7.5, 5.8, and 4.8 mM, respectively) (Table 1). Decreased Km correlated to extended alcohol chain length, which indicated that variation of alcohol structure had a great effect on the affinity of Novozym 435. Table 1 shows the calculated KBm and kcat values for (R)- and (S)-alcohols were also different. It indicated that the affinity of (R)-alcohols to the active site of the enzyme was high and Novozym 435 showed a stronger specificity toward (R)-alcohol. Thus, the (R)-caffeic acid esters were the main products. The enantioselective ratio (E value), which depends on the secondorder rate constant of kcat/Km for each enantiomer, was over 100. These results demonstrated that the method established in the present study for the asymmetric synthesis of chiral caffeic acid esters was feasible and that high optically pure chiral caffeic acid esters can be easily obtained. Recovery and Reusability of the Immobilized Lipase. The present study aimed to determine the number of times the enzymes can be used and to ascertain loss of enzyme activity from batch to batch. This will be extremely important for analyzing the economical aspects of the process. After each run of Novozym 435-catalyzed esterification at 70 °C, the beads were separated, washed with acetone, and reused with fresh substrates in anhydrous isooctane. The reaction rates (VR and VS), reaction conversions (XR and XS), e.e.p, and enantioselective ratio (E value) after recycling seven times have been shown in Table S6 of the Supporting Information. The results 11643

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CONCLUSION We successfully synthesized chiral caffeic acid esters using enzyme-catalyzed stereoselective esterification. The catalytic properties of Novozym 435 were strongly dependent on the alcohol carbon chain length and the type of organic solvents. The experimental (R)-1 conversion was 33.8 ± 1.8% with an E value of >100 under the optimal synthesis conditions, which was in agreement with the theoretical predictions. The kinetic and thermodynamic analyses indicated that the enantiomer discrimination was mainly driven by kcat/Km and −ΔΔH. Recycling experiments showed the immobilized enzyme could be reused several times effectively. This method was useful for preparing other chiral phenolic acid esters.

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ASSOCIATED CONTENT

* Supporting Information S

Details of experimental parameters, and experimental data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Figure 4. Variations of the inverse of initial rate with the inverse of substrate concentration of caffeic acid, (R)-2-pentanol, (R)-2-heptanol, and (R)-2-octanol: (blue squares) caffeic acid concentration varies, (R,S)-2-pentanol: 200 mM; (blue triangles) (R)-2-pentanol concentration varies, caffeic acid: 20 mM; (red squares) caffeic acid concentration varies, (R,S)-2-heptanol: 200 mM; (red triangles) (R)2-heptanol concentration varies, caffeic acid: 20 mM; (black squares) caffeic acid concentration varies, (R,S)-2-octanol: 200 mM; (black triangles) (R)-2-octanol concentration varies, caffeic acid: 20 mM.

AUTHOR INFORMATION

Corresponding Authors

*(L.Z.) Tel.: +86 431 85155252. Fax: +86 431 85155252. Email: [email protected]. *(Y.S.) Tel.: +86 431 85155252. Fax: +86 431 85155252. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support provided by the National Natural Science Foundation of China (Nos. 21372098 and 20802025), Jilin Provincial Science and Technology Sustentation Program (Nos. 201215033 and 20110436), Basic Operating Expenses, and 985 Project at Jilin University.

show that the catalytic activity of Novozym 435 decreased gradually from the first to the seventh cycle, although the decrease was not obvious. The VR values in the first run were 8.9, 4.0, and 3.6 mM·h−1 for Novozym 435-catalyzed synthesis of (R)-1, (R)-2, and (R)-3, respectively. In the seventh run, the VR values were 6.3, 2.9, and 2.5 mM·h−1, respectively. For Novozym 435-catalyzed synthesis of (R)-2 and (R)-3, the enzyme showed excellent enantioselectivity (E value > 100) in each cycle. However, enantioselective loss of enzyme was observed after four cycles for Novozym 435-catalyzed synthesis of (R)-1, and the E value decreased from over 100 in the first cycle to 84 in the seventh cycle, which was closely related to the improvement of VS. The decreased activity of the immobilized enzyme may be caused by the existence of alcohol compounds, (R, S)-2-pentanol, (R, S)-2-heptanol, and (R, S)-2-octanol, probably near the immobilized enzyme particle. These alcohol compounds could distort the essential water layer necessary for enzyme activity. Lipase leakage from the support caused by the mechanical loss during the reaction and recovery for recycling could also lead to enzyme activity loss.

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REFERENCES

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Table 1. Kinetic Parameters and E Values for Novozym 435-Catalyzed Esterification of Caffeic Acid and (R,S)-Alcohola

Vmax × 10−2 (mM·h−1) Km (mM) kcat × 10−3 (mmol·h−1·g−1) (kcat/Km) × 10−4(L·h−1·g−1)

(R)-2pentanol

(S)-2pentanol

caffeicb acid

(R)-2heptanol

(S)-2heptanol

caffeicc acid

(R)-2octanol

(S)-2octanol

caffeicd acid

10.4 7.5 2.6 3.5

0.27 23.7 0.07 0.03

16.1 11.2 4.0 3.6

4.2 5.8 1.0 1.7

0.02 124.9 0.005 0.0004

5.5 11.7 1.4 1.2

3.7 4.8 0.9 1.9

0.01 224.5 0.0025 0.0001

4.5 10.7 1.1 1.0

a

E value > 100. [E = (kcatR/KmR)/(kcatS/KmS)]. bCaffeic acid concentration varies, (R, S)-2-pentanol: 200 mM. cCaffeic acid concentration varies, (R, S)-2-heptanol: 200 mM. dCaffeic acid concentration varies, (R, S)-2-octanol: 200 mM. 11644

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