Kinetics and mechanism of regioselective monoacetylation of 3-aryloxy-1,2-propandiols using immobilized Candida antarctica lipase

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JIEC 2590 1–8 Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx

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Kinetics and mechanism of regioselective monoacetylation of 3-aryloxy-1,2-propandiols using immobilized Candida antarctica lipase Q1 Sandip

V. Pawar, Ganapati D. Yadav *

Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400 019, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 March 2015 Received in revised form 1 July 2015 Accepted 1 July 2015 Available online xxx

Optically pure 3-aryloxy-1,2-propanediols are important intermediates in the synthesis of various pharmaceutical compounds. This study reports the kinetics and mechanism of lipase catalyzed regioselective monoacetylation of 3-aryloxy-1,2-propandiols. Regioselective monoacetylation of 3-(2methylphenoxy) propane-1,2-diol was chosen as model reaction and the various commercially available lipases were screened for this reaction. Candida antarctica lipase B (Novozyme 435) was found to be the best catalyst among all the screened biocatalysts considering the yield and excess of monoacetylated product. The important reaction parameters were optimized systematically to improve the rate of reaction and the conversion viz., speed of agitation, reaction solvent, catalyst loading, reaction temperature and mole ratio. The study of initial rate and progress curve demonstrated that reaction obeys ternary complex mechanism and 3-(2-methylphenoxy) propane-1,2-diol inhibits the reaction at higher concentrations. Theoretical predictions and experimental data match very well based on nonlinear regression analysis. Under optimized reaction conditions the study was further extended to variety of 3-(aryloxy)-1,2-propanediols. ß 2015 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.

Keywords: Immobilized lipase 3-Aryloxy-1,2-propanediol Kinetic study Enzyme catalysis Regioselective monoacetylation

7 8

Introduction

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

The prevalent application of biocatalysis in non-aqueous media has included stereo-selective synthesis of enantiopure drugs over the past two decades [1–4]. The production of enantiomerically pure compounds has gained significant importance in chemical and pharmaceutical industry. Biocatalysts have predominance due to their high chemo-, regio, enantio-selectivity and exhibit high stability in organic reaction medium [5–7]. Biocatalytic asymmetric synthesis offers great potential as an alternative tool to chemical synthesis and provides a major path for the synthesis of enantiomerically pure compounds through kinetic resolution of racemic substrates or asymmetrization of prochiral compounds [8–14]. The regioselective acetylation of polyhydroxy compounds has been an interesting area of research in chemistry for many years. The selective manipulation of hydroxyl groups poses many challenges in synthesis of active

* Corresponding author. Tel.: +91 22 3361 1001; fax: +91 22 3361 1020. E-mail addresses: [email protected], [email protected] (G.D. Yadav).

pharmaceutical intermediates [15–17]. The conventional chemical methods produce mixture of monoacetylated and diacetylated products. Lipase catalyzed monoacetylation reactions are superior to conventional chemical methods owing to high selectivity, product purity, mild reaction conditions, and also omits the use of toxic catalysts [18]. The enzymatic regioselective synthesis plays vital role in pursuing asymmetric synthesis of active chiral compounds. Lipase catalyzed regioselective monoacetylation has been established as a suitable approach to obtain the monoacetylated products using various diols [19–22]. Monoacetylated derivatives of 1,2-diols can be subsequently used for synthesis of enantiomerically pure diols via sequential kinetic resolution [19]. Higher yield of enantioselective product is obtained in final step when pure monoacetylated product is used as substrate. Lipases are the most important group of biocatalysts in nonaqueous enzymology; they do not need cofactors for assisting the reaction, are capable of differentiating the chiral centers and have extensive substrate specificity [23–32]. Lipases are widely present in different sources which include bacteria and fungi and hence can be readily available for biotransformation of interest. Lipases are suitable catalyst in terms of catalytic activity and selectivity [33–41].

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46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67

The compounds containing 1,2-diol functional groups are of great interest in the synthesis of pharmaceutical intermediates. Optically pure 3-aryloxy-1,2-propanediols are considered as important precursors in the synthesis of pharmaceuticals including muscle relaxants [42], b-receptor blockers, antifungal and antibacterial agents [43] and for other synthetic purpose such as building blocks for crown ethers as well as chiral ligands for transition metals complexes [44]. 3-Aryloxy-1,2propanediols are also extensively used as preservatives in cosmetic and food products, are also used in dyes and nonspreading lubricants, resins, pigmented ink, polyesters and optics [22,45]. There have been no report on kinetics and mechanism of regioslective monoacetylation of 3-aryloxy-1,2-propanediols using lipase as catalyst. The present study reports an environment friendly lipase catalyzed regioselective monoacetylation process and optimization of reaction parameters in non-aqueous medium under mild conditions. The reaction mechanism and kinetic constants for the reaction were determined by using non-linear regression analysis. The study was also further extended to different 3-aryloxy-1,2-propanediols under otherwise similar conditions.

68

Experimental

69

Enzymes

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

The Novozyme 435, Lipozyme RM IM and Lipozyme TL IM were procured as gift samples from Novo Nordisk, Denmark. Lipase AYS ‘‘Amano’’, Lipase AS ‘‘Amano’’, Lipase AK ‘‘Amano’’ and Lipase PS ‘‘Amano’’ were procured as gift samples from Amano Enzyme Inc. Japan. Novozyme 435 is Candida antarctica lipase immobilized on a macroporous polyacrylic resin beads (bead size 0.3–0.6 mm, bulk density 0.430 g cm3, water content 3%, activity of 7000 PLU/g); Lipozyme RM IM is Mucor miehei immobilized on an ionic resin with activity of 5–6 BAUN while Lipozyme TL IM is Thermomyces lanuginosus immobilized on silica. Lipase AYS ‘‘Amano’’ is Candida rugosa lipase in form of lyophilized powder (activity  30,000 m/ g); Lipase PS ‘‘Amano’’ is Burkholderia cepacia lipase immobilized on diatomaceous earth (activity  500 m/g); Lipase AK ‘‘Amano’’ is powdered Pseudomonas fluoroscens lipase (activity  20,000 m/g); Lipase AS ‘‘Amano is Aspergillus niger lipase in powder form (activity of 12,000–15,000 m/g).

86

Chemicals

87 88 89 90 91 92 93 94 95

All chemicals used were of AR grade and obtained from the firms of repute. Tetrahydrofuran, 1, 4-dioxane, acetonitrile, ethanol, isopropyl alcohol, diisopropyl ether, tert-butanol, xylene, acetone, tert-butyl methyl ether, cyclohexanone, toluene, n-decane (S.d. Fine Chemicals Pvt. Ltd., Mumbai, India). 3-(Prop-2-en-1lyloxy) propane-1,2-diol was procured from Sigma Aldrich, India and 3-aryloxy-1,2-propanediols were synthesized using the procedure reported by Egri et al. and further purified by recrystallization to achieve 98% purity [46].

OH O

R

Experimental set up

96

It consists of a 3 cm i.d. glass reactor of 50 ml capacity; fully baffled and mechanically agitated, and included a six bladed pitched-turbine impeller. The thermostatic water bath was maintained at the desired temperature with an accuracy of 1 8C and the entire reactor assembly was immersed in it. The reaction mixture in a typical experiment consists 4 mmol of 3aryloxy-1,2-propanediol and 6 mmol of vinyl acetate diluted to 15 ml with toluene as a reaction solvent. The resulting reaction mixture was then agitated at 50 8C at 300 rpm for 15 min. The reaction the samples were taken regularly and the reaction analysis was carried out using gas chromatography.

97 98 99 100

Analytical method

108

The concentration of reaction components were determined by Ceres 800, a high resolution GC equipped with a FID. A 30 m  0.32 mm BPX-5 capillary column packed with 5% phenyl polysilphenylene-siloxane was used for analysis. The product confirmation was done by using GCMS. The GC chromatogram of reaction mixture (Fig. 1 Supplementary information) and GC–MS Chromatogram (Fig. 2 Supplementary information) are provided in supplementary information. 1H NMR was obtained with Bruker DPX 300 (1H 300 MHz) spectrometer using CDCl3 as solvent. Chemical shifts are expressed in parts per million (ppm), with tetramethylsilane as an internal standard (Fig. 3 Supplementary information).

109 110 111 112 113 114 115 116 117 118 119 120

Results and discussion

121

Effect of various biocatalysts

122

A variety of 3-(aryloxy)-1,2-propanediols with different substitution on the aromatic ring were used as substrate for regioselective monoacetylation using lipase (Scheme 1). Biocatalytic regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol was chosen as model reaction. Initially, all available lipases were screened for regioselective monoacetylation to obtain the desired monoacetylated product. As shown in Table 1, almost all lipases were able to catalyze the reaction to produce the desired compound except lipase Amano AS. However, Novozyme 435 and Lipase Amano PS showed high catalytic activity in comparison to all other screened lipases (Table 1, entries 1 and 4). The conversion with Novozyme 435 and Lipase Amano PS Amano was 99 and 86.6%, respectively and the monoacetylated excess was above 95%. This study reports improved conversion for monoacetylated product using C. antarctica lipase compared to the of recent report by Meena and Banerjee [22]. C. antarctica lipase B (CALB), a serine hydrolase, is the most widely used biocatalyst for organic synthesis in lab scale and industrial scale. Being thermostable, CALB is a robust lipase. It catalyzes diversity of reactions and it exhibits very high substrate selectivity both with respect to regio- and enantioselectivity. It has been extensively used as region-selective

123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144

O OH

+ H2C

O

OH CH3 Novozyme 435

O

OAc

R + CH3CHO

Scheme 1. Regioselective monoacetylation of 3-aryloxy-1, 2-propandiol using immobilized lipase.

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Table 1 Effect of various biocatalysts on regioselective monoacetylation 3-(2-methylphenoxy) propane-1,2-diol. Sr. no.

Biocatalyst

Conversion (%)

ME (%)

1 2 3 4 5 6 7

Novozyme 435 (Candida antarctica) Lipozyme RMIM (Rhizomucor miehei) Lipozyme TMIM (Thermomyces lanuginosus) Lipase Amano PS (Burkholderia cepacia) Lipase Amano AYS (Candida rugosa) Lipase Amano AS (Aspergillus niger) Lipase Amano AK (Pseudomonas fluorescens)

99  0.68 11.1  0.94 16.2  0.81 86.6  1.08 7.7  1.14 No reaction 49.8  0.92

95.15  0.96 100 100 96.6  0.84 100 – 98.4  1.01

Reaction conditions 3-(2-methylphenoxy)propane-1,2-diol—4 mmol; vinyl acetate—6 mmol; biocatalyst—0.2 wt%; temperature 50 8C; h i speed of % monoacetylated% diacetylatedÞ agitation 300 rpm; toluene up to 15 ml, reaction time: 60 min. ME % ðMonoacetylation excess %Þ ¼ ðð%  100. monoacetylatedþ% diacetylatedÞ

145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165

catalyst for many reactions and specifically shows enantioselectivity towards secondary alcohols [47–50]. CALB belongs to the a/b-hydrolase-fold superfamily of enzymes and is built of 317 amino acids and has molecular weight of 33 kDa. The active site of CALB contains catalytic triad of Ser105-His224-Asp187 amino acids, common in all serine hydrolases. CALB possesses oxyanion hole, an arrangement of three hydrogen bond donors in its active site which stabilizes the transition state of reaction as well as the reaction intermediate. The active site also has stereospecificity pocket in which secondary alcohols orient during catalysis and this explains the high enantio-selectivity of CALB towards secondary alcohols [51–54]. Between the two hydroxyl functional groups present in 3-aryloxy-1,2-propanediol, the primary hydroxyl group is presumably more reactive and acetylated faster regioselectively than the secondary one [34]. Owing to the efficiency of Novozyme 435 to catalyze regioselective monoacetlyation of 3-(2-methylphenoxy) propane-1,2-diol with higher conversion and monoacetylated excess; it was adopted as effective biocatalyst in all further studies. The control experiment with absence of CALB did not show any conversion.

166

Effect of speed of agitation

167 168

Optimum speed of agitation and low enzyme loading of the optimum size are important to reduce the limitations of external

mass transfer and internal diffusion. The reactants diffuse from the bulk liquid to the external surface of the catalyst particle and from there into the interior pores of the immobilized biocatalyst. The effect of agitation speed was studied on reaction rate and conversion, and reactions were carried out by varying agitation speed from 100 rpm to 400 rpm (Fig. 1). The initial rates were obtained from the time vs. concentration profile and it was observed that there was increase in the conversion and rate of reaction up to 300 rpm. The graph of speed of agitation (rpm) vs. initial rate clearly shows plateau reached around 300 rpm (Fig. 4 Supplementary information). However, no significant change in reaction rate and conversion was observed above 300 rpm this indicates that reaction is not mass transfer controlled. Thus, the optimum speed of agitation for this reaction was found to be 300 rpm and was adopted for further experiments.

169 170 171 172 173 174 175 176 177 178 179 180 181 182 183

Effect of different solvents

184

The enzyme activity is strongly affected by the varied functional groups as well as molecular structure of the solvent [55]. The selection of a suitable reaction solvent for the biocatalytic reaction is important because most of non-aqueous solvents are known to denature the enzymes. It has been reported that enzymes are more stable in non-polar solvents that have low solubility for water than in polar solvents [56]. The enzymes work efficiently in organic solvents and water layer remain bound to enzyme molecule to preserve its biological activity. Water functions as lubricant and promotes the conformational stability which is required for the optimal catalysis. Table 2 shows a number of solvents used for regioselective monoacetylation of 3-(2-methylphenoxy) propane1,2-diol. Lower conversion of 3-(2-methylphenoxy) propane-1,2diol was observed with ethanol and isopropyl alcohol, whereas 1, 4-dioxane, t-butanol and cyclohexanone showed moderate conversion of 69.1%, 58.2% and 41.1%, respectively. The other solvents

185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200

Table 2 Effect of different solvents on regioselective monoacetylation 3-(2-methylphenoxy) propane-1,2-diol.

Fig. 1. Effect of speed of agitation on regioselective monoacetylation 3-(2methylphenoxy) propane-1,2-diol. Reaction conditions: 3-(2-methylphenoxy) propane-1,2-diol—4 mmol; vinyl acetate—6 mmol; Novozyme 435—0.2%; temperature 50 8C; toluene up to 15 ml, 100 RPM, 200 rpm, 300 rpm, 400 rpm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Sr. no.

Solvent

log p

Conversion (%)

ME (%)

1 2 3 4 5 6 7 8 9 10

Tetrahydrofuran Acetonitrile 1,4-Dioxane Ethanol Isopropyl alcohol Diisopropyl ether tert-Butanol Xylene Acetone tert-Butyl methyl ether Cyclohexanone Toluene

0.49 0.33 1.1 0.24 0.8 1.9 0.58 3.1 0.23 1.35

84.8  0.93 89  0.49 69.1  1.04 6.8  1.54 14.3  0.44 85.9  0.75 58.2  0.05 89.8  0.81 97.2  0.66 88.7  1.15

98.8  0.44 94.6  1.12 98.8  0.89 100 100 92.2  1.68 96.8  0.95 81  1.04 87  0.59 88.2  0.81

0.96 2.5

41.4  0.74 99.4  0.43

100 98.8  0.21

11 12

Reaction conditions: 3-(2-methylphenoxy)propane-1,2-diol—4 mmol; vinyl acetate—6 mmol; Novozyme 435—0.2 wt%; temperature 50 8C; speed of agitation— 300 rpm; solvent up to 15 ml, reaction time—60 min.

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201 202 203 204 205 206 207 208 209 210 211 212 213

screened for the reaction showed higher conversion among which toluene was found to give complete conversion with high percentage of monoacetylated excess. With change of environment of reaction medium from hydrophilic to hydrophobic, the overall enzyme efficiency changes dramatically. Hydrophilic solvents easily penetrate into active site of enzyme and induce the structural changes, also hydrophilic solvents have higher tendency to eliminate the essential water layer around active site of enzyme which results in loss of enzyme activity. However, the nonpolar organic solvents does not disrupt the structural integrity of enzymes keeping essential water layer intact around active site [57]. All further studies were carried out using toluene as a reaction solvent.

214

Effect of catalyst loading

215 216 217 218 219 220 221 222 223 224 225 226 227

The effect of catalyst loading was studied for regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol by changing the concentration of catalyst from 0.067 to 0.26 (weight %) and molar ratios of the reactants were kept constant. It was observed that the rate of reaction and conversion increased as the catalyst loading increased up to 0.2 wt% and beyond 0.2 wt% catalyst loading there was marginal increase in conversion and reaction rate. Overall increase in conversion was observed from 72% to 99% (Fig. 2). The graph of catalyst loading (wt%) vs. initial rate clearly shows linear increase in rate of reaction, this indicates that the reaction is kinetically controlled (Fig. 5 Supplementary information). The enzyme loading of 0.2 wt% was found to be optimum and it was adopted in further experiments.

228

Effect of temperature

229 230 231 232 233 234 235

The effect of temperature on the activity of Novozyme 435 was studied from 30 8C to 60 8C for regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol. It was observed that the initial rate increased from 4.1  103 to 8.4  103 mol/l min and also the conversion increased from 74 to 99.8% as temperature was increased from 30 to 60 8C (Fig. 3). Arrhenius plot of logarithmic value of initial rate vs. the inverse temperature, 1/T was made to

Fig. 2. Effect of catalyst loading on regioselective monoacetylation 3-(2methylphenoxy) propane-1,2-diol. Reaction conditions: 3-(2-methylphenoxy) propane-1,2-diol—4 mmol; vinyl acetate- 6 mmol; temperature 50 8C; toluene up to 15 ml, 0.067% 0.13%, 0.2%, X 0.26%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Effect of temperature on regioselective monoacetylation 3-(2methylphenoxy) propane-1,2-diol. Reaction conditions: 3-(2methylphenoxy)propane-1,2-diol—4 mmol; vinyl acetate—6 mmol; Novozyme 435—0.2%; toluene up to 15 ml, 30 8C, 40 8C, 50 8C, 60 8C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

obtain the activation energy (Fig. 4). The value of activation energy 5.2 kcal/mol obtained from Arrhenius plot was found to be reasonable for enzymatic reaction in absence of diffusion limitation.

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Effect of concentration of substrate

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The effect of concentration of 3-(2-methylphenoxy) propane1,2-diol was studied in the range of 4 to 10 mmol, keeping concentrations of the other components of reaction mixture constant: vinyl acetate (4 mmol), Novozyme 435 (0.2%), toluene (up to 15 ml). The increasing concentration of 3-(2-methylphenoxy) propane-1,2-diol after certain limit caused decrease in the rate of reaction and conversion (Fig. 5). The result of varying molar concentration of vinyl acetate was studied by changing its concentration in the range of 4 to 10 mmol under otherwise

241 242 243 244 245 246 247 248 249

Fig. 4. Arrhenius plot.

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Table 4 Substrate study. Sr. no

Substrate

1. O

Conversion (%)

ME (%)

99.5  0.004

98.8  0.06

98.4  0.17

91.6  0.96

98.1  0.03

95.8  0.19

51.6  1.67

69.2  0.98

64.7  1.24

63.6  1.01

73.4  0.56

75.2  1.09

96.8  0.93

95.8  0.81

OH

CH3

OH

2. O

OH

H3C

OH

3. H3C

O

OH OH

4. O

OH

OH

5. Fig. 5. Effect of substrate concentration on regioselective monoacetylation 3-(2 methylphenoxy) propane-1,2-diol. Reaction conditions: 3-(2methylphenoxy)propane-1, 2-diol—4 to 10 mmol; vinyl acetate—4 to 10 mmol; Novozyme 435—0.2%; temperature 50 8C; toluene up to 15 ml, reaction time— 60 min. Vinyl acetate, 3-(2-methylphenoxy) propane-1,2-diol. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Cl O

Cl

OH

6. Cl

O Cl

7.

250 251 252 253 254 255 256 257 258 259 260

similar conditions: 3-(2-methylphenoxy) propane-1,2-diol (4 mmol), Novozyme 435 (0.2%), toluene (up to 15 ml). It was found that with increase in concentration of vinyl acetate, the reaction rate and overall conversion also increased at all concentrations studied (Fig. 5). For many enzymatic reaction, the velocity curves rise to maximum and then decline with increasing substrate concentration, this is referred as substrate inhibition. The substrate inhibition of enzyme by excess substrate may result from an interaction of substrate at a secondary binding site on the enzyme which induces a conformational change at the active site resulting the reduced enzyme activity [58,59].

261

Reusability of Novozyme 435

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The stability of enzyme was determined by carrying out reusability study of biocatalyst. The biocatalyst was filtered and washed with reaction solvent and it was dried at room temperature after each use. As shown in Table 3 Novozyme 435 was not denatured or deactivated by repeated use up to three cycles; whereas a slight decrease in activity of enzyme was observed after three cycles of use for regioselective monoacetylation of 3-(aryloxy)-1,2-propanediols. This might be due to the loss of biocatalyst in reaction medium due to mechanical agitation and during handling. The experiments were carried out in duplicate and no make-up quantity was added. These results suggest that Table 3 Effect of reusability of Novozyme 435 on regioselective monoacetylation 3-(2-methylphenoxy) propane-1,2-diol. Reusability of Novozyme 435

Conversion (%)

Fresh First reuse Second reuse Third reuse

99.03  0.94 96.59  1.45 89.72  1.78 81.4  1.69

Reaction conditions: 3-(2-methylphenoxy)propane-1,2-diol—4 mmol; vinyl acetate—6 mmol; Novozyme 435—0.2 wt%; temperature 50 8C; toluene up to 15 ml, reaction time—60 min; fresh enzyme, first reuse, second reuse, third reuse.

OH

OH OH

OH O H2 C

OH

Reaction conditions 3-aryloxy-1,2-propanediol—4 mmol; vinyl acetate—6 mmol; Novozyme 435—0.2 wt%; temperature 50 8C; toluene up to 15 ml, reaction time— 60 min.

Novozyme 435 is the stable and reusable catalyst for enzymatic regioselective monoacetylation.

273 274

Effect of different 3-aryloxy-1,2-propanediols

275

Under the optimized conditions 3-(aryloxy)-1,2-propanediol derivatives with different substitution on the aromatic ring were used as substrate for regioselective monoacetylation. It was found that the method was applicable for regioselective monoacetylation of 3-(aryloxy)-1,2-propanediols. As shown in Table 4 the derivatives with the substituents in -ortho, -meta, -para position of the aromatic ring showed good regioselectivity with excellent conversion and monoacetylated excess. However, 3-(naphthalen1-yloxy)propane-1,2-diol, and the corresponding derivatives with chlorine substitution on aromatic ring at different positions reduced the overall conversion with decrease in monoacetylated excess which has ultimate effect on regioselectivity. In contrast, the replacement of aryloxy by an alkyl substituent (Table 4, entry 7) showed no sign of change in regioselectivity; it gave excellent conversion with high percentage of monoacetylated excess.

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Kinetic resolution of monoacetylated product

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1,2-Diols are acetyted in two steps, in the first step acetylation of the primary hydroxyl group generates the racemic monoacetlyated product. Once the primary hydroxy group is completely acetylated, subsequent acetylation at secondary hydroxyl group results in kinetic resolution of racemic monoacetylated product. The two step acetylation was studied using 3-(2-methylphenoxy) propane-1,2-diol as model compound and it was observed that two

292 293 294 295 296 297 298

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step acetylation generates the 49% of diacetlyated product R-()3-(2-methylphenoxt)propane-1,2-diyl diacetate, with enantiomeric excess of 97%.

302

Kinetic study

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The detailed kinetic study was carried out in order to investigate the reaction mechanism of this particular reaction. The effect of concentration of both reactants 3-(2-methylphenoxy) propane-1,2-diol and vinyl acetate were studied systematically over a wide range. To determine the initial rates of the reaction, two sets of studies were carried out by using 0.2 wt% of Novozyme 435 with appropriate quantities of 3-(2-methylphenoxy) propane1,2-diol and vinyl acetate and the total volume of the reaction was made up to 15 ml with reaction solvent toluene. In first set of experiment, concentration of 3-(2-methylphenoxy) propane-1,2diol was varied from 4 to 10 mmol keeping fixed concentration of vinyl acetate. In another part, concentration of vinyl acetate was changed from 4 to 10 mmol at fixed concentration of 3-(2methylphenoxy) propane-1,2-diol. The conversions were quantified and initial rates of the reaction were determined from the progress curves. The initial rate was calculated by multiple point method and it was determined form plot of concentration of limiting reactant vs. time. From the initial rates (r0) measurement, it was found that, when concentration of 3-(2-methylphenoxy) propane-1,2-diol [A] increased by keeping the concentration of vinyl acetate [B] constant, initial rates of reaction increased up to certain concentration and further increase in concentration of 3-(2-methylphenoxy) propane-1,2-diol leads to decreased initial rate. However, in other case by changing concentration of vinyl acetate [B] under otherwise similar conditions resulted in the

B

Fig. 6. Lineweaver–Burk plot. Reaction conditions: 3-(2-methylphenoxy)propane1,2-diol–4 mmol to 10 mmol; vinyl acetate—4–10 mmol; Novozyme 435—0.2%; temperature 50 8C; toluene up to 15 ml, reaction time—60 min 0.27 M, 0.4 M, 0.53 M, 0.67 M.

the dead end binary complex between 3-(2-methylphenoxy) propane-1,2-diol and enzyme is formed instead of enzyme and vinyl acetate and this results in inhibition of reaction, which can be observed by reduced initial rates at higher concentration of 3-(2methylphenoxy) propane-1,2-diol. The reaction mechanism is represented as follows:

A

P

E

increase of reaction rate and overall conversion at all concentrations studied and there was no evidence of inhibition by vinyl acetate. It can be seen in the Lineweaver–Burk plot of 1/r0 vs. 1/[A], the pattern of lines are not parallel and thus it rules out the possibility of ping-pong bi–bi mechanism (Fig. 6). In most of lipase catalyzed reactions, it is reported that the enzyme first forms the acyl complex with acyl donors ruling out the possibility of random mechanism [60]. As a result, there is only the possibility of ordered bi–bi mechanism. As per the ordered bi– bi mechanism, acyl donor vinyl acetate [B] binds with enzyme [E] in first attempt and forms complex enzyme acyl complex [EB]. The other reactant 3-(2-methylphenoxy) propane-1,2-diol [A] then combines with vinyl acetate-enzyme complex [EB] to form the ternary complex [EBA]. The resulting ternary complex then undergoes isomerization to form yet another ternary complex which results in the release of the first product as vinyl alcohol. The vinyl alcohol immediately tautomerizes to binary complex and acetaldehyde due to its high instability. The binary complex subsequently releases 2-hydoxy-3(2-methylphenoxy) propyl acetate, desired product. However, at higher concentration of 3-(2-methylphenoxy) propane-1,2-diol

Q

E EBA

329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349

350 351 352 353 354 355

EPQ

where E—enzyme, A—3-(2-methylphenoxy) propane-1,2-diol, B— vinyl acetate, Q—acetaldehyde, P—2-hydoxy-3-(2-methylphenoxy) propyl acetate, EBA—ternary complex and EPQ is the isomer of EBA. The rate equation for the ternary complex mechanism, for initial conditions is as follows:

356 357 358 359 360 361 362

r0 ½ A½B ¼ rmax K ið AÞ K mðBÞ þ K mð AÞ ½B þ K mðBÞ ½ A þ ½ A½B where r0 = initial rate of reaction, rmax = maximum rate of reaction, [A] = initial concentration of 3-(2-methylphenoxy) propane-1,2diol, [B] = initial concentration of vinyl acetate, Km(A) = Michaelis constant for 3-(2-methylphenoxy) propane-1,2-diol, Km(B) = Michaelis constant for vinyl acetate (mol/l), Ki = inhibition constant of 3-(2-methylphenoxy) propane-1,2-diol. To validate the application of ternary complex mechanism, the data were analyzed by non-linear regression analysis using polymath. The initial rate of reaction were obtained from the concentration–time profile and kinetic parameters were obtained from the polymath 6.0 (Table 5).

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363 364 365 366 367 368 369 370 371 372 373 374

G Model

JIEC 2590 1–8 S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx Table 5 Kinetic parameters for regioselective monoacetylation of 3-(2methylphenoxy) propane-1,2-diol. Kinetic parameters

Values by polymath 6.0

rmax (mol/l min) KmA (mol/l) KmB (mol/l) KiA

1.04  102 11.69 0.432 10.08

Fig. 7. Parity plot. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

375 376 377 378 379

The plot of simulated vs. experimental rate resulted shows straight line passing through origin with an excellent correlation coefficient (Fig. 7). This shows that the proposed model of ternary complex mechanism is valid for the regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol.

380

Conclusion

381 382 383 384 385 386 387 388 389 390 391 392 393 394

Regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol was carried out by employing different immobilized lipases and Novozyme 435 was the suitable catalyst. Effects of various reaction parameters including agitation speed, solvent, loading of catalyst, reaction temperature and mole ratio of reactants were studied systematically. From the quantified data, a kinetic model was proposed for regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol. The ternary complex mechanism with inhibition of 3-(2-methylphenoxy) propane-1,2diol provides support for the mechanism. The mechanism was found to fit the data well using non-linear regression analysis for enzymatic regioselective monoacetylation. The study was further extended to variety of 3-(aryloxy)-1,2-propanediols under optimized conditions.

395

Acknowledgements

396 397 398 399 400

SVP thanks to the University Grant Commission for an award of Q2 fellowship under BSR scheme. GDY received support from R.T. Q3 Mody Distinguished Professor Endowment and J. C. Bose National

Fellowship of Department of Science and Technology, Government of India.

7

Appendix A. Supplementary data

401

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jiec.2015.07.007.

402 403

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