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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
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).
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
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).
O
OH
+ H2C
OH
CH3
Novozyme 435
OAc
+ 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
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
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.
338
S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342
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.)
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
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
OH
OH
OH
OH
O
H2 C
OH
340
S.V. Pawar, G.D. Yadav / Journal of Industrial and Engineering Chemistry 31 (2015) 335342
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.
E
EBA
EPQ
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
Kinetic parameters
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.)
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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