Journal of Industrial and Engineering Chemistry 31 (2015) 335342

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Journal of Industrial and Engineering Chemistry

journal homepage: www.elsevier.com/locate/jiec

Kinetics and mechanism of regioselective monoacetylation of

3-aryloxy-1,2-propandiols using immobilized Candida antarctica
lipase
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

Article history:
Received 29 March 2015
Received in revised form 1 July 2015
Accepted 1 July 2015
Available online 16 July 2015
Keywords:
Immobilized lipase
3-Aryloxy-1,2-propanediol
Kinetic study
Enzyme catalysis
Regioselective monoacetylation
Ternary complex mechanism

A B S T R A C T

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 a model reaction and various commercially available
lipases were screened as catalysts. Candida antarctica lipase B (Novozyme 435) was found to be the best
catalyst among all considering the yield and excess of monoacetylated product. Important reaction
parameters were optimized systematically to improve the rate of reaction and conversion, viz., speed of
agitation, reaction solvent, catalyst loading, reaction temperature and mole ratio. The study of initial rate
and progress curve demonstrated that the reaction obeys the ternary complex mechanism and 3-(2methylphenoxy) propane-1,2-diol inhibits the reaction at higher concentrations. Theoretical predictions
and experimental rates match very well based on non-linear regression analysis. Under optimized
reaction conditions the study was further extended to regioselective monoacetylation of a variety of 3(aryloxy)-1,2-propanediols.
2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
reserved.

Introduction
The prevalent application of biocatalysis in non-aqueous
media has included stereo-selective synthesis of enantiopure
drugs over the past two decades [14]. The production of
enantiomerically pure compounds has gained signicant importance in chemical and pharmaceutical industry. Biocatalysts have
predominance over chemical catalysts due to their high chemo-,
regio, enantio-selectivity and exhibit high stability in organic
reaction medium [57]. Biocatalytic asymmetric synthesis offers
a 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 [814]. 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

* Corresponding author. Tel.: +91 22 3361 1001; fax: +91 22 3361 1020.
E-mail addresses: gdyadav@yahoo.com, gd.yadav@ictmumbai.edu.in
(G.D. Yadav).

in synthesis of active pharmaceutical intermediates [1517]. The

conventional chemical methods produce a 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 they omit the use of toxic catalysts [18]. The
enzymatic regioselective synthesis plays a vital role in pursuing
asymmetric synthesis of active chiral compounds. Lipase catalyzed regioselective monoacetylation has been established as a
suitable approach to obtain monoacetylated products using
various diols [1922]. 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 the nal step when pure
monoacetylated product is used as substrate. Lipases are the most
important group of biocatalysts in non-aqueous enzymology;
they do not need cofactors for assisting the reaction, are capable of
differentiating the chiral centers and have extensive substrate
specicity [2332]. 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
catalysts in terms of catalytic activity and selectivity [3341].

http://dx.doi.org/10.1016/j.jiec.2015.07.007
1226-086X/ 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342

336

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 purposes such as
building blocks for crown ethers as well as chiral ligands for
transition metals complexes [44]. 3-Aryloxy-1,2-propanediols are
also extensively used as preservatives in cosmetic and food
products, and in dyes and nonspreading lubricants, resins,
pigmented ink, polyesters and optics [22,45].
There have been no reports 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
regioslective monoacetylation of different 3-aryloxy-1,2-propanediols under otherwise similar conditions.

Experimental
Enzymes
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 (CAL B)
immobilized on macroporous polyacrylic resin beads (bead size
0.30.6 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 56 BAUN while Lipozyme TL IM is
Thermomyces lanuginosus immobilized on silica. Lipase AYS
Amano is Candida rugosa lipase in the 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 uoroscens lipase (activity  20,000 m/g); Lipase AS Amano is
Aspergillus niger lipase in powder form (activity of 12,000
15,000 m/g).
Chemicals
All chemicals used in this work were of AR grade and obtained
from rms 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 other 3-aryloxy-1,2-propanediols were synthesized using the
procedure reported by Egri et al. and further puried by
recrystallization to achieve 98% purity [46].

OH
O

Experimental set up
The reaction assembly consisted of a 3 cm i.d. glass reactor of
50 ml capacity; fully bafed 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 consisted of 4 mmol
of 3-aryloxy-1,2-propanediol and 6 mmol of vinyl acetate diluted to
15 ml with toluene as solvent. The resulting reaction mixture was
then agitated at 50 8C at 300 rpm for 15 min. Reaction samples were
taken out regularly and analysis was carried out using gas
chromatography.
Analytical method
The concentrations of reaction components were determined
by Ceres 800, a high resolution GC equipped with FID. A
30 m  0.32 mm BPX-5 capillary column packed with 5% phenyl
polysilphenylene-siloxane was used for analysis. The product
conrmation was done by using GCMS (Perkin Elmer Instrument,
Clarus 500 with BP-1 capillary column (0.25 mm i.d., 30 m length)
and EI mode of MS). The GC chromatogram of reaction mixture
(Fig. 1 Supplementary information) and GCMS 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).

Results and discussion

Effect of various biocatalysts
A variety of 3-(aryloxy)-1,2-propanediols with different substitution on the aromatic ring were used as substrates for
regioselective monoacetylation using different lipases (Scheme
1). Biocatalytic regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol was chosen as the 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. 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 on lab scale and industrial scale. Being
thermostable, CALB is a robust lipase. It catalyzes diversity of
reactions and exhibits very high substrate selectivity both with
respect to regio- and enantio-selectivity. It has been extensively

O
OH

+ H2C

OH
CH3
Novozyme 435

OAc

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

+ CH3CHO

S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342

337

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

Biocatalyst

Conversion (%)

ME (%)

1
2
3
4
5
6
7

Novozyme 435 (Candida antarctica CALB)

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 uorescens)

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-diol4 mmol; vinyl acetate6 mmol; biocatalyst0.2

wt%; temperature 50 8C;
h
i speed of
% monoacetylated% diacetylated
agitation 300 rpm; toluene up to 15 ml, reaction time: 60 min. ME % Monoacetylation excess % %
 100.
monoacetylated% diacetylated

used as regio-selective catalyst for many reactions and specically

shows enantio-selectivity towards secondary alcohols [4750].
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-His224Asp187 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 stereo-specicity pocket in which secondary alcohols orient
during catalysis which explains the high enantio-selectivity of
CALB towards secondary alcohols [5154]. Between the two
hydroxyl functional groups present in 3-aryloxy-1,2-propanediol,
the primary hydroxyl group is presumably more reactive getting
acetylated faster regioselectively than the secondary one [34].
Owing to the efciency of Novozyme 435 to catalyze regioselective
monoacetlyation of 3-(2-methylphenoxy) propane-1,2-diol with
higher conversion and monoacetylated excess, it was employed as
the most active biocatalyst in all further studies. The control
experiment in the absence of CALB did not show any conversion.
Effect of speed of agitation
Minimum 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 phase 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, from 100 to 400 rpm (Fig. 1). The initial rates were
obtained from the concentration vs. time prole and it was
observed that there was an increase in the conversion and rate of
reaction up to 300 rpm. The effect of speed of agitation (rpm) vs.
initial rate clearly shows a plateau at around 300 rpm (Fig. 4
Supplementary information). However, no signicant change in
reaction rate and conversion was observed above 300 rpm which
indicated that reaction was not mass transfer controlled. Thus, the
minimum speed of agitation for this reaction was found to be
300 rpm which was adopted for further experiments.
Effect of different solvents
The enzyme activity is strongly affected by the various
functional groups attached to the substrate as well as molecular
structure of the solvent [55]. Selection of a suitable reaction solvent
for the biocatalytic reaction is important because most of nonaqueous 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 efciently in organic solvents and water layer
remains bound to the enzyme molecule to preserve its biological
activity. Water functions as lubricant and promotes the conformational stability which is required for catalysis. Table 2 shows
a number of solvents used for regioselective monoacetylation of
3-(2-methylphenoxy) propane-1,2-diol. Lower conversion of 3(2-methylphenoxy) propane-1,2-diol was observed with ethanol
and isopropyl alcohol, whereas 1, 4-dioxane, t-butanol and
cyclohexanone showed moderate conversion of 69.1%, 58.2% and

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-diol4 mmol; vinyl acetate6 mmol; Novozyme 4350.2%;
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 gure legend, the
reader is referred to the web version of this article.)

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-diol4 mmol; vinyl acetate6 mmol; Novozyme 4350.2 wt%; temperature 50 8C; speed of agitation
300 rpm; solvent up to 15 ml, reaction time60 min.

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S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342

41.1%, respectively. The other solvents 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 activity changes
dramatically. Hydrophilic solvents easily penetrate into active site
of enzyme and induce 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, nonpolar organic solvents do 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 the reaction solvent.
Effect of catalyst loading
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 (wt%)
and the molar ratio of the reactants was kept constant. It was
observed that the rate of reaction and conversion increased as the
catalyst loading increased up to 0.2 wt% beyond which there was a
marginal increase in conversion and reaction rate. Overall increase
in conversion was observed from 72% to 99% (Fig. 2). The effect of
catalyst loading (wt%) vs. initial rate clearly shows a linear increase
in the rate of reaction, which indicates that the reaction is
kinetically controlled (Fig. 5 Supplementary information). The
enzyme loading of 0.2 wt% was found to be optimum which was
used in further experiments.
Effect of temperature
The effect of temperature on the activity of Novozyme 435 was
studied from 30 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-diol4 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 gure 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-diol4 mmol; vinyl acetate6 mmol; Novozyme
4350.2%; toluene up to 15 ml,
30 8C,
40 8C,
50 8C, 60 8C. (For
interpretation of the references to color in this gure legend, the reader is
referred to the web version of this article.)

obtain the activation energy (Fig. 4). The value of activation energy
of 5.2 kcal/mol obtained from Arrhenius plot was found to be
reasonable for enzymatic reaction in the absence of diffusion
limitation.
Effect of concentration of substrate
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 wt%),
toluene (up to 15 ml). The increasing concentration of 3-(2methylphenoxy) 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

Fig. 4. Arrhenius plot.

S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342

339

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

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-diol4 to 10 mmol; vinyl acetate4 to 10 mmol;
Novozyme 4350.2%; 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 gure legend, the reader is
referred to the web version of this article.)

Cl
O

Cl

OH

6.
Cl

O
Cl

7.

otherwise similar conditions: 3-(2-methylphenoxy) propane-1,2diol (4 mmol), Novozyme 435 (0.2 wt%), 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 reactions,
the velocity curves rise to maximum and then decline with
increasing substrate concentration which is referred to 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 in to the reduced enzyme
activity [58,59].
Reusability of Novozyme 435
The stability of enzyme was determined by carrying out
reusability study. The biocatalyst was ltered, washed with
reaction solvent and 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 attrition and loss of enzyme
during ltration and handling. The experiments were carried out in
duplicate and no make-up quantity was added. These results
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-diol4 mmol;

vinyl acetate6 mmol; Novozyme 4350.2 wt%; temperature 50 8C;
toluene up to 15 ml, reaction time60 min;
fresh enzyme,
rst
reuse,
second reuse,
third reuse.

OH

OH
OH

OH
O
H2 C

OH

Reaction conditions 3-aryloxy-1,2-propanediol4 mmol; vinyl acetate6 mmol;

Novozyme 4350.2 wt%; temperature 50 8C; toluene up to 15 ml, reaction time
60 min.

suggest that Novozyme 435 is a stable and reusable catalyst for

enzymatic regioselective monoacetylation.
Effect of different 3-aryloxy-1,2-propanediols
Under the optimized conditions 3-(aryloxy)-1,2-propanediol
derivatives with different substitution on the aromatic ring were
used as substrates 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.
Kinetic resolution of monoacetylated product
1,2-Diols are acetyted in two steps: in the rst 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-(2methylphenoxy) propane-1,2-diol as the model compound and

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S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342

it was observed that it generates 49% of the diacetlyated product

R-()-3-(2-methylphenoxt)propane-1,2-diyl diacetate, with enantiomeric excess of 97%.
Kinetic study
A 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) propane1,2-diol and vinyl acetate was 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) propane-1,2-diol
and vinyl acetate and the total volume of the reaction was made up
to 15 ml with reaction solvent toluene. In rst set of experiment,
concentration of 3-(2-methylphenoxy) propane-1,2-diol was
varied from 4 to 10 mmol keeping xed concentration of vinyl
acetate. In another part, concentration of vinyl acetate was
changed from 4 to 10 mmol at xed concentration of 3-(2methylphenoxy) propane-1,2-diol. The conversions were quantied 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 from the 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] was
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-(2methylphenoxy) propane-1,2-diol led to decreased initial rate.
On the contrary, by changing concentration of vinyl acetate [B]
under otherwise similar conditions resulted in the increase of

Fig. 6. LineweaverBurk plot. Reaction conditions: 3-(2-methylphenoxy)propane1,2-diol4 mmol to 10 mmol; vinyl acetate410 mmol; Novozyme 4350.2%;
temperature 50 8C; toluene up to 15 ml, reaction time60 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 it 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:

E
EBA

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 LineweaverBurk plot of 1/r0 vs. 1/[A], the pattern of
lines are not parallel and thus it rules out the possibility of pingpong bibi mechanism (Fig. 6).
In most of lipase catalyzed reactions, it is reported that the
enzyme rst forms acyl complex with acyl donors ruling out the
possibility of random mechanism [60]. As a result, there is only the
possibility of ordered bibi mechanism. As per the ordered bibi
mechanism, acyl donor vinyl acetate [B] binds with enzyme [E] in
rst 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 rst 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, the desired product. However,
at higher concentration of 3-(2-methylphenoxy) propane-1,2-diol,

EPQ

where Eenzyme, A3-(2-methylphenoxy) propane-1,2-diol, B

vinyl acetate, Qacetaldehyde, P2-hydoxy-3-(2-methylphenoxy) propyl acetate, EBAternary complex and EPQ is the isomer
of EBA.
The rate equation for the ternary complex mechanism, for
initial conditions is as follows:
r0
AB

rmax K i A K mB K m A B K mB A AB
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 software. The initial rates of reaction were obtained
from the concentrationtime prole and kinetic parameters were
obtained from Polymath 6.0 (Table 5).

S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342
Table 5
Kinetic parameters for regioselective monoacetylation of 3-(2methylphenoxy) propane-1,2-diol.

341

Appendix A. Supplementary data

Kinetic parameters

Values by polymath 6.0

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.jiec.2015.07.007.

rmax (mol/l min)

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

1.04  102
11.69
0.432
10.08

References

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

The plot of simulated vs. experimental rate shows a straight line

passing through origin with an excellent correlation coefcient
(Fig. 7). Thus, it conrms that the proposed model of ternary
complex mechanism is valid for the regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol.

Conclusion
Regioselective monoacetylation of 3-(2-methylphenoxy) propane-1,2-diol was carried out by employing different immobilized lipases and Novozyme 435 was found to be the best catalyst.
Effects of various process parameters including agitation speed,
solvent, loading of catalyst, temperature and mole ratio of
reactants were studied systematically. From the quantied 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 t the experimental and simulated rate data well.
The study was further extended to regioselective monoacetylation of a variety of 3-(aryloxy)-1,2-propanediols under optimized
conditions.
Acknowledgements
SVP thanks the University Grant Commission for an award of
fellowship under BSR scheme. GDY received support from R.T.
Mody Distinguished Professor Endowment and J. C. Bose National
Fellowship of Department of Science and Technology, Government
of India.

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