Lipase catalyzed kinetic resolution of (±)-1-(1-naphthyl) ethanol under microwave irradiation

Article abstract of DOI:10.1016/j.molcatb.2012.05.007

1-(1-Naphthyl) ethanol is an important chiral building block for the synthesis of active pharmaceutical intermediates (APIs). In this work, lipase catalyzed kinetic resolution of (RS)-1-(1-naphthyl) ethanol via transesterification was studied under microwave irradiation with different acyl donors. Vinyl acetate was the most effective and hence used as the acyl donor. Three different commercially available immobilized lipases were used, among which Candida antarctica lipase B, immobilized on acrylic resin (Novozym 435) was found to be the best catalyst in n-heptane as solvent. The effect of various parameters was studied in a systematic manner. The reaction was intrinsically kinetically controlled. Maximum conversion of 47.74% with enantiometric excess of substrate (ee_s) of 90.05% and enantiomeric ratio (E) of 433.39 was obtained in 3 h using 30 mg of enzyme loading with equi-molar ratio of alcohol to ester at 60°C. From the progress curve analysis, the ping-pong bi-bi mechanism with inhibition by both substrates was found to fit the initial rates. Kinetic parameters were obtained by using non-linear regression. The process is efficient and easily scalable as compared to the chemical process.

Full text of DOI:10.1016/j.molcatb.2012.05.007

Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Lipase catalyzed kinetic resolution of ( )-1-(1-naphthyl) ethanol under
microwave irradiation
Ganapati D. Yadav∗, Saravanan Devendran
Department of Chemical Engineering, Institute of Chemical Technology, 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:
1-(1-Naphthyl) ethanol is an important chiral building block for the synthesis of active pharmaceutical
intermediates (APIs). In this work, lipase catalyzed kinetic resolution of (RS)-1-(1-naphthyl) ethanol via
transesterification was studied under microwave irradiation with different acyl donors. Vinyl acetate was
the most effective and hence used as the acyl donor. Three different commercially available immobilized
lipases were used, among which Candida antarctica lipase B, immobilized on acrylic resin (Novozym 435)
was found to be the best catalyst in n-heptane as solvent. The effect of various parameters was studied
in a systematic manner. The reaction was intrinsically kinetically controlled. Maximum conversion of
47.74% with enantiometric excess of substrate (ees) of 90.05% and enantiomeric ratio (E) of 433.39 was
obtained in 3 h using 30 mg of enzyme loading with equi-molar ratio of alcohol to ester at 60 ◦C. From the
progress curve analysis, the ping–pong bi–bi mechanism with inhibition by both substrates was found
to fit the initial rates. Kinetic parameters were obtained by using non-linear regression. The process is
efficient and easily scalable as compared to the chemical process.
Received 30 January 2012
Received in revised form 17 April 2012
Accepted 8 May 2012
Available online 17 May 2012
Keywords:
1-(1-Naphthyl) ethanol
Novozym 435
Transesterification
Kinetic resolution
Microwave irradiation
Ping–pong bi–bi mechanism
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
of mild reaction conditions. Above all, their easy availability from
different sources such as plant, bacteria and fungi makes them
Chiral secondary alcohols are used as important building blocks
and chiral auxiliaries for the synthesis of active pharmaceutical
intermediates (APIs) and/or in a variety of processes in chem-
ical, flavors, fine chemicals, pharmaceuticals and agrochemicals
industries [1]. In the past, enantiomeric pure alcohols were made
by several approaches such as use of chiral ligands, separation
by chiral chromatography and asymmetric reduction of prochiral
compounds using chiral metal complexes [2–4]. All these processes
have their own inherent drawbacks such as difficulty in opera-
tion, low enantiomeric excess (ee), generation of by-products and
exorbitant cost. Biotransformation is an excellent alternative to
chemical processes, in which microbes and enzymes are used as
catalysts due to their inherent advantages including easy adoptabil-
ity in non-aqueous media, requirement of mild reaction condition,
high regio- and stereo-selectivity and suppression of by-products
[5,6].
attractive biocatalysts [7]. Lipase catalyzed resolution of racemic
secondary alcohols through esterification and transesterification
in non-aqueous media is a more suitable approach as compared to
asymmetric reduction of prochiral compounds using oxidoreduc-
tases, which require expensive co-factor like NAD(P)H and have
less adoptability in non-aqueous condition [8].
Microwave irradiation has received much attention among the
organic chemists as clean and green alternative for both organic and
material synthesis vis-à-vis conventional heating. In the latter case,
heat is transferred to the reactor by means of conductive and con-
vective processes. Hence it requires longer equilibrium time and is
difficult to control. While in microwave heating, the heat is trans-
ferred by direct pairing of microwave and excited polar molecules
in the reaction mixture and this process is independent of ther-
mal conductivity of reaction vessel [9–11]. The synergism with
microwave is found to enhance the reaction rate in enzymatic reac-
tions. It has been reported that the rate of lipase catalyzed reaction
is increased to several folds when compared to conventional heat-
ing and also observed that lipase is quite stable under microwave
irradiation [12,13].
In recent years, lipases have been extensively used as biocata-
lysts for organic synthesis, because of their stability in non-aqueous
media, their ability to accept a broad range of substances and use
S-1-(1-Naphthyl)ethanol is used as an intermediate for the syn-
thesis of mevinic acid analogues, which acts as a potential inhibitor
of 3-hydroxy-3-methyl glutaryl co-enzyme A reductase (HMGR),
which catalyzes the conversion of HMG-CoA to mevalonate, a key
step in cholesterol synthesis [14]. There are a few studies reported
∗
Corresponding author. Tel.: +91 22 3361 1001/1111/2222;
fax: +91 22 3361 1002/1020.
E-mail addresses: gd.yadav@ictmumbai.edu.in,
gdyadav@yahoo.com (G.D. Yadav).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.05.007

G.D. Yadav, S. Devendran / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
59
2.2. Chemicals
Nomenclature
Iso-octane, n-heptane, toluene, cyclohexane, vinyl acetate, and
other analytical and HPLC grade reagents were purchased from M/s
S.D. Fine Chemicals Pvt. Ltd., Mumbai, India. ( )-1-(1-Naphthyl)
ethanol, vinyl butyrate and vinyl laurate were purchased from
Sigma–Aldrich India Pvt. Ltd., Bangalore, India. All chemicals and
enzymes were used without any further modification/purification.
A
B
initial concentration of vinyl acetate (mol dm−3
)
initial concentration of 1-(1-naphthyl) ethanol
(mol dm−3)
E
free enzyme
EA
EꢀB
F
FA
FB
KiA
KiB
enzyme–acyl complex with A
enzyme–alcohol dead end complex
modified enzyme
2.3. General experimental setup
modified enzyme–ester complex
modified enzyme–alcohol complex with B
2.3.1. Conventional heating
inhibition constant for vinyl acetate (mol dm−3
)
The experimental set-up consisted of a 3 cm i.d. mechanically
agitated glass reactor of 50 cm3 capacity, equipped with four baf-
fles and a six-bladed turbine impeller. The entire reactor assembly
was immersed in a thermostatic water bath, which was maintained
at a predetermined temperature with an accuracy of 1 ◦C. A typ-
ical reaction mixture consisted of 0.001 mol racemic alcohol and
0.001 mol vinyl ester diluted to 15 cm3 with n-heptane as a sol-
vent. The reaction mass was agitated at 60 ◦C for 15 min at a speed
of 400 rpm and then 30 mg of enzyme was added to initiate the
reaction.
inhibition constant for 1-(1-naphthyl) ethanol
(mol dm−3
Michaelis constant for vinyl acetate (mol dm−3
Michaelis constant for 1-(1-naphthyl) ethanol
(mol dm−3
)
KmA
KmB
)
)
P
Q
v
acetaldehyde
1-(1-naphthyl) ethyl acetate
rate of reaction (mol dm−3 s−1 g-enz−1
)
vm
maximum rate of reaction (mol dm−3 s−1 g-enz−1
)
ꢀR − SꢀH‡ differential activation enthalpy
ꢀR − SꢀS‡ differential activation entropy
ꢀR − SꢀG‡ difference in transition state energy of both
2.3.2. Microwave heating
The studies were carried out in a commercial microwave reactor
assembly (Discover, CEM-SP1245 model, CEM Corp., Matthews, NC,
USA) at appropriate temperature and pressure control. The reac-
tor was a 100 ml capacity, 4.5 cm i.d. cylindrical glass vessel with a
provision for mechanical stirring. A standard six-blade-pitched tur-
bine impeller of 1.5 cm diameter was used for agitation. However,
the actual reactor volume exposed to the microwave irradiation
was 45 ml with 5.5 cm height. The temperature in the reactor was
computer controlled.
enantiomers
T
R
E
ees
eep
c
temperature (K)
gas constant (J mol−1 K−1
enantiomeric ratio
)
enantiomeric excess for substrate
enantiomeric excess for product
conversion
The quantities of reactant and enzyme used for microwave
reaction procedure were identical to those used for conventional
heating.
on the synthesis of S-1-(1-naphthyl) ethanol by asymmetric reduc-
tion of prochiral ketone using microbes, asymmetric chemical
catalysis or lipase catalyzed kinetic/dynamic resolution under con-
ventional heating [15–19]. All these processes require longer time
and there is no information on kinetics, which is required for
reactor design and scale-up. In this work, microwave assisted
lipase catalyzed kinetic resolution of 1-(1-naphthyl) ethanol via
transesterification with vinyl acetate in non-aqueous medium is
reported. The effect of various parameters such as effect of dif-
ferent commercially available immobilized lipases, acyl donors,
solvents, agitation speed, temperature, catalyst loading, and acyl
donor concentration is studied systematically by varying one
parameter at a time. Finally mechanism and kinetics are also
developed.
2.4. Analysis
The concentration profiles and enantiomeric excess (ee) were
monitored by periodical withdrawal of the clear liquid samples
from the reaction mixture which were analyzed by high perfor-
mance liquid chromatography (HPLC) (1260 infinity series, Agilent
Technologies, CA, USA) equipped with chiralpak-IB analytical col-
umn (250 mm × 4.6 mm i.d.) (Daicel Corporation, Japan and particle
size 5 ␮m). Samples (10 ␮l) were injected via auto-sampler. The
mobile phase consisted of n-hexane and isopropyl alcohol (90:10)
and the flow rate was maintained at 1 ml min−1. A DAD detector
was used at a wavelength of 230 nm.
The enantioselectivity ratio (E) and racemate conversion (c, %),
were calculated from the enantiomeric excess of the substrate (ees,
%) and product (eep, %) based on the following equation:
2. Materials and methods
ln 1 − c 1 − ee
[(
) (
) (
s)]
s)]
E =
(1)
2.1. Enzymes
ln 1 − c 1 + ee
[(
where,
The following enzymes were received as gift samples from M/s
Novozymes A/S (Bagsvaerd, Denmark): (i) Novozym 435: Lipase
B from Candida antarctica, supported on a macroporous acrylic
resin with a water content of 1–2% (w/w) and enzyme activity
10,000 PLU/g, (ii) lipozyme RMIM: Lipase from Rhizomucor miehei,
supported on a macroporous anion exchange resin with a water
content of 2–3% (w/w) and enzyme activity 6 BAU/g, (iii) lipozyme
TLIM: Lipase from Thermomyces lanuginosus, supported on porous
silica granulates with water content 1–2% and enzyme activity
175 IU/g.
ees
ees + eep
c =
(2)
(3)
B(S) − B(R)
B(S) + B(R)
ees =
and
Q(S) − Q(R)
Q(S) + Q(R)
eep
=
(4)

60
G.D. Yadav, S. Devendran / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
H3C
OH
H3C
OH
O
O
Novozym 435
(S) -1-(1-naphthyl) ethanol
H
+
+
H2C
O
CH3 n-heptane, MW
H C
3
H3C
O
CH3
Acetaldehyde
(R/S) 1-(1-naphthyl) ethanol
Vinyl acetate
O
(R) -1-(1-naphthyl) ethyl acetate
Scheme 1. Lipase catalyzed kinetic resolution of (R/S)-1-(1-naphthyl) ethanol under microwave irradiation.
where, B(R), B(S), Q(R) and Q(S) denote area under the curve of
(R)-1-(1-naphthyl) ethanol, (S)-1-(1-naphthyl) ethanol and their
corresponding esters.
RMIM (R. miehei lipase) and lipozyme TLIM (T. lanuginosus lipase)
were evaluated for resolution of ( )-1-(1-naphthyl) ethanol. The
reaction was carried out by using 1:1 mole ratio of racemic alcohol
to vinyl acetate, with 30 mg enzyme loading at 60 ◦C in n-heptane
as solvent; the liquid phase volume was made up to 15 cm3 for
3 h under microwave irradiation Novozym 435 had the highest
activity among the different catalysts employed in this reaction
(Fig. 1). With Novozym 435, 47.74% conversion was obtained. While
lipozyme RMIM and lipozyme TLIM gave conversions of only 9.31%
and 5.2%, respectively. It is reported that both lipozyme RMIM and
lipozyme TLIM are mainly used to inter-esterify bulk molecules
such as fatty acid derivatives and they have less activity in the res-
olution of racemic molecules [22]. However, we wanted to find if
microwave irradiation could have some positive effect with these
enzymes. Hence, further study was carried out using Novozym 435
as the catalyst.
3. Results and discussion
3.1. Comparison of activity during microwave irradiation and
conventional heating
In order to compare the effect of microwave irradiation and
conventional heating on enzyme activity and ee, the trans-
esterification of ( )-1-(1-naphthyl) ethanol with vinyl acetate
as a model acyl donor was carried out using both modes of
heating under identical conditions as follows: 60 ◦C at agi-
tation speed of 400 rpm; equal moles of alcohol and ester
(0.001 mol), Novozym 435 (30 mg loading). It was found that
both conversion and reaction rate were increased substantially
under microwave irradiation as compared to conventional heat-
ing. The activity of enzyme under microwave irradiation and
conventional heating was 3.11 × 10−4 mol dm−3 s−1 g−1-enz and
2.44 × 10−4 mol dm−3 s−1 g−1-enz, respectively. Thus, there was an
enhancement of activity of 1.27 fold. The E and ees under microwave
irradiation increased by 2.66 and 1.67 fold, respectively. The final
conversion 47.74%, with E and ees of 433.39 and 90.05%, respec-
tively were obtained after 3 h under microwave irradiation. While
in conventional heating, a conversion of 39.5 with E and ees of 164
and 63.77%, respectively were obtained after 5 h. Under microwave
irradiation, the ions or dipole of the sample align in the applied elec-
tric field. As the applied field oscillates, the dipole or ion field tries to
realign itself with the alternating electric field and, in the process;
energy is lost in the form of heat through molecule collisions and
dielectric loss [20]. The amount of heat generated is directly related
to the ability of the matrix to align itself with the frequency of the
applied field. Microwave irradiation produces efficient in situ heat-
ing, resulting in even distribution of temperature throughout the
sample, as compared to conventional heating [21]. A control experi-
ment in the absence of enzyme did not show any conversion, which
indicated that there must have been a definite synergism between
enzyme catalysis and microwave irradiation. Thus, all further stud-
ies were carried out using microwave irradiation (Scheme 1).
3.3. Effect of acyl donors
Vinyl esters are well accepted as acyl donors for lipase cat-
alyzed kinetic resolution of chiral alcohols, because of their
100
90
80
70
60
50
40
30
20
10
0
CAL
B
RMIM
TLIM
Enzyme source
Fig. 1. Effect of type of catalysts on racemic conversion and enantiomeric excess
(ees) (reaction conditions: 1-(1-naphthyl) ethanol – 0.001 mol; vinyl acetate –
0.001 mol; n-heptane up to 15 cm3; speed of agitation – 400 rpm; catalyst loading
– 30 mg; temperature – 60 ◦C; ( ) conversion; ( ) ees; CAL-B – candida antarctica
lipase B; RMIM – Rhizomucor miehei lipase; TLIM – Thermomyces lanuginosus lipase).
3.2. Effect of nature catalysts
Activities of three different commercially available immobilized
lipases such as Novozym 435 (C. antarctica lipase B), lipozyme

G.D. Yadav, S. Devendran / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
61
50
45
40
35
30
25
20
15
10
5
100
90
80
70
60
50
40
30
20
10
0
vinyl acetate
vinyl butyrate
vinyl laurate
0
Acyl donor
0
15 30 45 60 75 90 105 120 135 150 165 180 195
Fig. 2. Effect of acyl donor on racemic conversion and enantiomeric excess (ees).
(reaction conditions: 1-(1-naphthyl) ethanol – 0.001 mol; acyl donor – 0.001 mol;
n-heptane up to 15 cm3; speed of agitation – 400 rpm; catalyst loading – 30 mg;
temperature – 60 ◦C, ( ) conversion; ( ) ees).
Time (min)
Fig. 3. Effect of reaction media on racemic conversion (reaction conditions: 1-(1-
naphthyl) ethanol – 0.001 mol; vinyl acetate – 0.001 mol; solvent up to 15 cm3;
speed of agitation 400 rpm; catalyst loading – 30 mg; temperature – 60 ◦C, (
)
toluene; ( ) n-hexane; ( ) cyclohexane; (×) n-heptane).
irreversibility in reaction. The chain length of acyl donors has
significant influences on the conversion and enantioselectivity
[23]. We studied the effect of chain length of different acyl
donors by employing different vinyl esters such as vinyl acetate,
butyrate and laurate. Increasing chain length has a negative effect
on initial rate (activity), enantioselectivity and conversion. The
highest conversion (47.74%) with ees of 90.05% and activity of
9.33 × 10−6 mol dm−3 s−1 were obtained with vinyl acetate as the
acyl donor whereas low conversion (34.61%) with ees of 51.6% and
activity of 2.86 × 10−6mol dm−3 s−1 were obtained with vinyl lau-
rate. Similar kind of result was reported in kinetic resolution of
mandelic acid using Pseudomonus sp. lipase as catalyst [24]. Thus,
further studies were carried using vinyl acetate as the acyl donor
(Fig. 2).
each and 30 mg Novozym 435 made up to 15 cm3 with n-heptane
at 60 ◦C (Fig. 4). Both the conversion and rate of reaction increased
with an increase of agitation speed from 100 to 400 rpm. There was
a marginal decrease in conversion at 500 rpm. It was observed that
at 500 rpm the enzyme particles were thrown out of reaction mix-
ture sticking to the reactor wall. This indicated that there was no
external mass transfer limitation above 400 rpm. Hence, 400 rpm
was chosen as speed of agitation for further studies.
3.6. Effect of temperature
It has been reported in most of the publications that temperature
has a significant influence on enzyme activity and enantioselectiv-
ity. Increase in temperature can reduce mixture viscosity; enhance
solubility and diffusion process of substrates, thus reducing exter-
nal mass transfer resistance and favoring interactions between
enzyme particles and substrates that lead to enhancement in
enzyme activity and rate of reaction. However, at high tempera-
ture, the conformation of protein molecule gets disturbed due to
3.4. Effect of reaction media
Proper selection of organic solvent is primarily the most impor-
tant criterion in any enzymatic reaction. It has been well reported
that solvent has the ability to alter the enzyme activity by changing
its conformational structure. Activity is also affected by microwave
irradiation [25]. The effect of four different solvents such as toluene
(log P = 2.5), n-heptane (log P = 4), hexane (log P = 3.6) and cyclohex-
ane (log P = 3.2) was studied under similar conditions (Fig. 3). The
highest conversion (47.74%) and enantiomeric excess (ees = 90.05%)
were observed when n-heptane employed as the solvent whereas
low conversion (6.73%) and enantiomeric excess (ees = 7.2%) were
observed in toluene, which has the lowest log P value among the
solvents used in this study. On the contrary, conversions of 45.8%
and 44.5% were obtained with hexane and cyclohexane, respec-
tively. Solvents having high log P are hydrophobic in nature but do
not strip the water layer around enzyme particle. Thus, they show
high enzyme activity. Further work was carried out using n-heptane
as the solvent.
50
45
40
35
30
25
20
15
10
5
3.5. Effect of speed of agitation
0
0
15 30 45 60 75 90 105 120 135 150 165 180 195
The magnitudes of external mass transfer resistance and intra-
particle diffusion limitation play a crucial role in immobilized-
enzyme catalyzed reactions and affect the intrinsic kinetics [26].
It can be overcome by choosing appropriate speed of agita-
tion. A number of experiments were performed in the range of
100–500 rpm by taking 0.001 mol racemic alcohol and vinyl acetate
Time (min)
Fig. 4. Effect of agitation speed on racemic conversion (reaction conditions: 1-(1-
naphthyl) ethanol – 0.001 mol; vinyl acetate – 0.001 mol; n-heptane up to 15 cm3;
speed of agitation – 100–500 rpm; catalyst loading – 30 mg; temperature – 60 ◦C,
(
) 100 rpm; ( ) 200 rpm; ( ) 300 rpm, (×) rpm; (+) 500 rpm).

62
G.D. Yadav, S. Devendran / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
6.4
6.3
6.2
6.1
6
50
45
40
35
30
25
20
15
10
5
5.9
5.8
y = 3070.x + 15.27
R² = 0.986
5.7
5.6
5.5
5.4
5.3
0.0029 0.00295 0.003 0.00305 0.0031 0.00315 0.0032 0.00325
1/T (K 1)
0
Fig. 7. Graphical determination of ꢀR−SꢀH1 and ꢀR−SꢀS1.
0
15 30 45 60 75 90 105 120 135 150 165 180 195
Time (min)
almost same and the lines are nearly parallel in the Arrhenius plot.
This would suggest that there is no change in mechanism due to
the mode of heating but the rate enhancement under microwave
is due to increase in collision frequency of reaction molecules in
consonance with published literature [29,11]. It was observed in
the pre-exponential factor in the Arrhenius equation and their
values are 1.139 and 0.546 dm−3 mol−1 s−1 for microwave and
conventional heating, respectively. The enhancement of 2.09 fold
in the pre-exponential factor was due to microwave irradiation.
As far as enantioselectivity is concerned, it was observed that the
E-value increases as temperature is increased from 40 to 70 ◦C. It
has been reported that enantioselectivity is dependent on reaction
temperature and is controlled by enthalpy and entropy [30]. The
following equation was used to relate E with enthalpy and entropy
of the system.
Fig. 5. Effect of temperature on racemic conversion. (reaction conditions: 1-(1-
naphthyl) ethanol – 0.001 mol; vinyl acetate – 0.001 mol; n-heptane up to 15 cm3;
speed of agitation – 400 rpm; catalyst loading – 30 mg; temperature – 40–70 ◦C; (
) 40 ◦C; ( ) 50 ◦C; ( ) 60 ◦C; (×) 70 ◦C).
denaturation of 3-D structure of protein [27]. So it important to
choose an appropriate operating temperature for maintaining the
activity of enzyme molecules. Several experiments were carried
in the range of 40–70 ◦C under similar conditions (Fig. 5). It was
observed that both conversion and rate of reaction increased
with increase in temperature up to 60 ◦C beyond which there
was no change in conversion. The enzyme either gets denatured
or intra-particle diffusion resistance sets in. Further, Arrhenius
plots were obtained by plotting the ln(initial rate) versus 1/T for
both modes of heating (Fig. 6). The activation energies obtained
for conventional and microwave heating were 31.15 kJ mol−1 and
32.44 kJ mol−1, respectively. These values are similar to those
reported for most of the enzymatic reactions [28]. The values of
activation energy for the conventional and microwave heating are
ꢀR − SꢀH‡
1
T
ꢀR − SꢀS‡
ln E = −
.
+
(5)
(6)
R
R
ꢀR − SꢀG‡ = −RT ln (E)
The differential activation enthalpy (ꢀR − SꢀH‡) and entropy
ꢀR − SꢀS‡ were calculated by plotting the ln(E) values against the
reciprocal reaction temperature (Fig. 7). The values of enthalpy and
11
entropy terms obtained were 25.52 kJ mol−1 and 127 J mol−1 K−1
,
11.2
respectively. The difference in transition state energy of both enan-
tiomers (e.g. ꢀR − SꢀG‡ = −16.77 kJ mol−1) is responsible for the
strong influence of temperature on enantiomeric ratio E. The tran-
sition state energy ꢀR − SꢀG‡ is dominated by the entropy terms
TꢀR − SꢀS‡ favors the maximum enantioselectivity at high tem-
perature [30]. Similar kind of relationship has been reported for
kinetic resolution of 2-octanol under microwave irradiation using
same catalyst [31]. Since E-value was found to be the highest at
60 ◦C while maintaining a higher enzyme activity, the temperature
of 60 ◦C was selected as the optimum. Thus further experiments
were conducted at 60 ◦C.
y = 3,901.84x + 0.13
R² = 0.98
11.4
11.6
11.8
12
12.2
12.4
12.6
12.8
y = 3,746.43x 0.61
R² = 0.97
3.7. Effect of enzyme loading
To select the appropriate amount of enzyme loading, a num-
ber of experiments were performed in the range from 20 to 35 mg
under similar conditions (Fig. 8). The conversion and rate of reac-
tion increased with increase in enzyme loading up to 30 mg, above
this loading value, there was no significant changes in conversion
(Fig. 9). The number of active sites is proportional to the amount of
enzyme. This plot shows that the initial rate increases linearly with
0.0029 0.00295 0.003 0.00305 0.0031 0.00315 0.0032 0.00325
1/T (K 1)
Fig. 6. Arrhenius plot. (reaction conditions: 1-(1-naphthyl) ethanol – 0.001 mol;
vinyl acetate – 0.001 mol; n-heptane up to 15 cm3; speed of agitation 400 rpm;
catalyst loading – 30 mg; temperature – 40–70 ◦C; ( ) microwave heating; (
conventional heating).
)

G.D. Yadav, S. Devendran / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
63
50
45
40
35
30
25
20
15
10
5
50
45
40
35
30
25
20
15
10
5
0
0
0
15 30 45 60 75 90 105 120 135 150 165 180 195
0
15 30 45 60 75 90 105 120 135 150 165 180 195
Time (min)
Time (min)
Fig. 8. Effect of enzyme loading on racemic conversion. (reaction conditions: 1-(1-
Fig. 10. Effect of acyl donor concentration (reaction conditions: 1-(1-naphthyl)
ethanol – 0.001 mol; vinyl acetate – 0.001–0.004 mol; n-heptane up to 15 cm3; speed
of agitation – 400 rpm; catalyst loading – 30 mg; temperature – 60 ◦C, ( ) 1:1; (
) 1:2; ( ) 1:3; ( ) 1:4).
naphthyl) ethanol – 0.001 mol; vinyl acetate – 0.001 mol; n-heptane up to 15 cm3;
speed of agitation 400 rpm; catalyst loading – 20–35 mg; temperature – 60 ◦C; (
20 mg; ( ) 25 mg; ( ) 30 mg; ( ) 35 mg).
)
enzyme loading. It clearly indicated that the reaction was kinet-
ically controlled. At loadings above 30 mg, the number of active
sites is more than the reacting molecules and therefore, there was
no further increase in rate of reaction or enantioselectivity. Thus
further experiments were carried out at 30 mg enzyme loading.
of vinyl acetate. At high concentration, the interaction between
acyl donor and enzyme active site increases that leads to reversible
competition with alcohol at enzyme active site.
3.9. Catalyst reusability
3.8. Effect of acyl donor concentration
Catalyst reusability is very important in order to make the pro-
cess is more economical at industrial level and also to determine
the stability of catalyst after each run. Initial experiments were car-
ried out using 30 mg of catalyst loading under similar conditions.
After completion of each run, the catalyst particles were filtered
using a membrane filter. Multiple washes were given to the cata-
lyst with fresh solvent-heptane, dried at room temperature for 12 h
and reused. It was found that there was a marginal decrease in con-
version from 47.74 to 44.8% after three reuses, which was due to
loss of enzyme during filtration and drying. No make-up quantity
was added (Fig. 11).
A number of experiments were carried out to analyze the effect
of acyl donor concentration on the rate and conversion in the reso-
lution of ( )-1-(1-naphthyl) ethanol at 60 ◦C using 30 mg of enzyme
loading in n-heptane as solvent. The concentration of ( )-1-(1-
naphthyl) ethanol was kept constant (0.001 mol), and vinyl acetate
concentration was varied from 0.001 to 0.004 mol; the mixture
volume was kept constant at 15 cm3 by adjusting the amount of n-
heptane. It was found that the conversion and rate of reaction had
negative relationship with concentration of vinyl acetate (Fig. 10).
The overall conversion decreased with increase in concentration
50
45
40
35
30
25
20
15
10
5
10
9
y = 0.268x
R² = 0.986
8
7
6
5
4
3
2
1
0
0
fresh
fresh
1st reuse
1st reuse
2nd reuse
2nd reuse 3rd reuse
3rd reuse
0
5
10
15
20
25
30
35
40
Enzyme loading (mg)
Fig. 9. Plot of initial rate of reaction versus enzyme loading (reaction conditions: 1-
(1-naphthyl) ethanol – 0.001 mol; vinyl acetate – 0.001 mol; n-heptane up to 15 cm3;
speed of agitation 400 rpm; catalyst loading – 20–35 mg; temperature – 60 ◦C).
Fig. 11. Catalyst reusability (reaction conditions: 1-(1-naphthyl) ethanol –
0.001 mol; vinyl acetate – 0.001 mol; n-heptane up to 15 cm3; speed of agitation
400 rpm; catalyst loading – 30 mg; temperature – 60 ◦C).

64
G.D. Yadav, S. Devendran / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
Table 1
6000
5000
4000
3000
2000
1000
0
Values of kinetic parameters for transesterification reaction.
Kinetic parameter
Value
Vmax (mol dm−3 s−1 g−1 of enzyme)
1.12 × 10−3
0.0125
0.0287
0.0817
0.210
KmA (mol dm−3
KmB (mol dm−3
)
)
KiA (mol dm−3
KiB (mol dm−3
)
)
Inhibition steps by 1-(1-naphthyl) ethanol
ꢀ
k
2
E + B ꢀ Eꢀ B
(14)
ꢀ
k
−2
Here, a possibility of inhibition by vinyl acetate at higher concen-
tration was also considered. From these observations we postulate
a ping–pong bi–bi mechanism with inhibition by both substrates.
These assumptions were used to design a reaction mechanism
which is depicted in Cleland’s notation, as shown below:
By analogy to the classical mechanism of lipase, it is assumed
that vinyl acetate (A) binds first to the free enzyme (E) and forms
a non-covalent enzyme acetate complex (EA), which releases the
first product, aldehyde (P) and (F) modified enzyme. The second
substrate alcohol (B) reacts with modified enzyme (F) to give the
complex (FB) which gives the product 1-(1-naphtyl) ethyl acetate
(Q) and free enzyme (E). Along with this, alcohol (B) also forms
the dead end complex (EꢀB) by binding to free enzyme (E) and vinyl
acetate (A) also forms the dead end complex with modified enzyme
(F) to give complex (FA).
0
5
10
15
20
25
30
35
1/Concentraꢀon of 1 (1 naphtyl) ethanol (mol 1.dm3)
Fig. 12. Lineweaver–Burk plot (reaction conditions: 1-(1-naphthyl) ethanol
–
0.0333–0.1333 M; vinyl acetate – 0.0333–0.1333 M mol; n-heptane up to 15 cm3;
speed of agitation 400 rpm; catalyst loading – 30 mg; temperature – 60 ◦C; (
0.0333 M; ( ) 0.0666 M; ( ) 0.0999 M; ( ) 0.1333 M).
)
3.10. Kinetic modeling
The kinetic model for transesterification of ( )-1-(1-naphthyl)
ethanol with vinyl acetate was developed by using initial rates
obtained under similar conditions. Number of reactions were
carried out by changing the concentrations of both alcohol
(0.0005–0.002 M) and vinyl acetate (0.0005–0.002 M) over a wide
range. Initial rates were calculated systematically from the linear
portion of the concentration–time profiles. The Lineweaver–Burk
plots of reciprocal rate versus reciprocal concentration of 1-(1-
naphthyl) ethanol were made (Fig. 12). From the plot, it is observed
that there are no crossings of lines which rules out the possibility
of sequential mechanism. The profile lines are parallel, suggest-
ing the ping pong bi–bi mechanism. Similar kind of mechanism
was observed for citronellyl acetate synthesis by the same catalyst
under microwave irradiation [32].
At low concentration of alcohol and vinyl acetate, the slope of the
lines is not influenced by the concentration of the fixed substrate.
This is indicative of a mechanism that requires the dissociation
of one product before the association of the second substrate to
the enzyme–substrate complex. The proposed mechanism is as
follows:
The rate equation obtained with the above mechanism is:
ꢁm[A][B]
ꢀ
ꢁ
ꢀ
ꢁ
ꢁ =
(15)
KmA[B] 1 + [B]/KiB + KmB[A] 1 + [A]/KiA + [A][B]
where v is the rate of reaction, vm the maximum rate of reaction,
[A] the initial concentration of vinyl acetate, [B] the initial con-
centration of 1-(1-naphthyl) ethanol, KmA the Michaelis constant
for vinyl acetate, KmB the Michaelis constant for 1-(1-naphthyl)
ethanol, KiA the inhibition constant for vinyl acetate, and KiB the
inhibition constant for 1-(1-naphthyl) ethanol. The initial rate
data were used to determine the kinetic parameters of above
mechanism by non-linear regression analysis using the software
package Polymath 5. 1 and their values are tabulated (Table 1). The
initial rates of reaction for different reactant concentration were
4
k
1
3.5
E + A ꢀ EA
(7)
y = 0.981x
R² = 0.986
k
−1
3
EA ⇔ FP
(8)
(9)
k
2
2.5
2
FP ꢀ F + P
k
−2
k
3
F + B ꢀ FB
(10)
1.5
1
k
−3
FB ⇔ EQ
(11)
(12)
k
4
0.5
0
EQ ꢀ E + Q
k
−4
Inhibition steps by vinyl acetate
0
0.5
1
1.5
2
2.5
3
3.5
4
ꢀ
k
1
Experimental rate × 10 4 (mol.dm 3.s .g 1 enz)
1
F + A ꢀ FA
(13)
ꢀ
k
−1
Fig. 13. Parity plot of experimental versus simulated rates.

G.D. Yadav, S. Devendran / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 58–65
65
simulated using the above equation to verify the proposed kinetic
model. The parity plot between simulated rate and experimental
rate suggested that the proposed mechanism was valid for this
reaction (Fig. 13).
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4. Conclusion
In this work, we have studied microwave assisted lipase
catalyzed kinetic resolution of 1-(1-naphthyl) ethanol via trans-
esterification with vinyl acetate in non-aqueous media. Among the
employed catalysts, Novozym 435 was found to be the best cata-
lyst in n-heptane as solvent. Both the reaction rate and conversion
were enhanced under microwave irradiation compared to conven-
tional heating. The maximum conversion of 47.74% with 90.05%
enantioselectivity were obtained in 3 h using 30 mg enzyme load-
ing with equimolar concentration of alcohol and ester at 60 ◦C
under microwave irradiation. From the progress curve analysis, it
was found that reaction followed the ping–pong bi–bi mechanism
with inhibition by both reactants. The kinetic parameters were
refined by using non-linear regression. There was a good agree-
ment between experimental and simulated rates. As compared to
any chemical process for preparation of chiral secondary alcohols,
lipase catalyzed kinetic resolution is more viable and easy to scale
up on industrial level.
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G.D.Y. received support from R. T. Mody Distinguished Professor
Endowment and J. C. Bose National Fellowship of Department of Sci-
ence and Technology, Government of India. S.D. received JRF from
UGC under its Meritorious Fellowship BSR program (CAS in Food
Engineering and Technology). The authors are greatly thankful to
Novo Nordisk, Denmark for providing lipases as gift sample.
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