Lipase-catalyzed kinetic resolution of (±)-1-(2-furyl) ethanol in nonaqueous media

Article abstract of DOI:10.1002/chir.22317

S-1-(2-Furyl) ethanol serves as an important chiral building block for the preparation of various natural products, fine chemicals, and is widely used in the chemical and pharmaceutical industries. In this work, lipase-catalyzed kinetic resolution of (R/S)-1-(2-furyl) ethanol using different acyl donors was investigated. Vinyl esters are good acyl donors vis-a-vis alkyl esters for kinetic resolution. Among them, vinyl acetate was found to be the best acyl donor. Different immobilized lipases such as Rhizomucor miehei lipase, Thermomyces lanuginosus lipase, and Candida antarctica lipase B were evaluated for this reaction, among which C. 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. Maximum conversion of 47% and enantiomeric excess of the substrate (ee_s) of 89% were obtained in 2 h using 5 mg of enzyme loading with an equimolar ratio of alcohol to vinyl acetate at 60C at a speed of 300 rpm in a batch reactor. From the analysis of progress curve and initial rate data, it was concluded that the reaction followed the ordered bi-bi mechanism with dead-end ester inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is more economical, green, and easily scalable than the chemical processes. Chirality 26:286-292, 2014. 2014 Wiley Periodicals, Inc.

Full text of DOI:10.1002/chir.22317

CHIRALITY (2014)
Lipase-Catalyzed Kinetic Resolution of ( )-1-(2-Furyl) Ethanol in
Nonaqueous Media
SARAVANAN DEVENDRAN AND GANAPATI D. YADAV
*
Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Mumbai-400019, India
ABSTRACT
S-1-(2-Furyl) ethanol serves as an important chiral building block for the
preparation of various natural products, fine chemicals, and is widely used in the chemical and phar-
maceutical industries. In this work, lipase-catalyzed kinetic resolution of (R/S)-1-(2-furyl) ethanol
using different acyl donors was investigated. Vinyl esters are good acyl donors vis-à-vis alkyl esters
for kinetic resolution. Among them, vinyl acetate was found to be the best acyl donor. Different
immobilized lipases such as Rhizomucor miehei lipase, Thermomyces lanuginosus lipase, and
Candida antarctica lipase B were evaluated for this reaction, among which C. antarctica lipase B,
immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n-heptane as sol-
vent. The effect of various parameters was studied in a systematic manner. Maximum conversion of
47% and enantiomeric excess of the substrate (ees) of 89% were obtained in 2 h using 5 mg of en-
zyme loading with an equimolar ratio of alcohol to vinyl acetate at 60°C at a speed of 300 rpm in a
batch reactor. From the analysis of progress curve and initial rate data, it was concluded that the
reaction followed the ordered bi–bi mechanism with dead-end ester inhibition. Kinetic parameters
were obtained by using nonlinear regression. This process is more economical, green, and easily
scalable than the chemical processes. Chirality 00:000-000, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: 1-(2-Furyl) ethanol; Candida antarctica lipase B; kinetic resolution; ordered bi–bi
mechanism; vinyl acetate
INTRODUCTION
reaction media for the lipase-catalyzed reaction widens the
scope, including greater solubility of organic molecules,
enhanced stability of enzymes, and ease of recovery of product
and catalyst as compared to aqueous media.12,13
The production of optically active single enantiomers has
not only gained great demand in pharmaceutical industries
but it is also required for the synthesis of fine chemicals in
fragrance, flavor, agro, and chemical industries.1,2 The
chemical methods used for the synthesis of optically active
molecules are beset with many drawbacks such as the
requirement for protection and deprotection steps, sluggish
reaction rates, and longer reaction time, low yield, and
byproduct formations.3 In this regard, biocatalytic processes
are favored for the synthesis of enantiomeric compounds, in
view of their incredible advantages such as high chemo-,
stereo-, regioselectivity, high efficacy, mild temperature
protocols, suppression of byproduct formation, ease of scale-
up, and low cost.4,5
Enantiomerically pure alcohols are one of the most attractive
building blocks for active pharmaceutical intermediate (API)
syntheses and are also used as chiral auxiliaries. In
biocatalysis, both whole cell and isolated enzymes are used as
catalysts.6 Highly pure enantiomer of secondary alcohol can
be prepared by adopting different methods such as resolution
of existing racemic mixtures via enzymatic kinetic resolution,
reduction of prochiral compounds by asymmetric reduction
using whole-cell catalysts, and C-C bond formation with
lyases.7–9 Among them, the enzymatic method has been
successfully applied. Especially, lipase-catalyzed kinetic
resolution has been well adapted for preparation of single
isomeric compounds such as secondary alcohol, acid,
and amine through esterification/transesterification and
hydrolytic reactions, respectively. Lipases offer high chemo-,
regio-, and stereoselectivity, accept a repertoire of compounds,
function without expensive cofactors, and are commercially
prepared from different species like microbes, animals, and
plants.9–11 Further, the use of organic and neoteric solvent as
© 2014 Wiley Periodicals, Inc.
The optically active S-enantiomer of 1-(2-furyl) ethanol is
used as an important building block for the synthesis of
various natural products such as flavonoids, polyketide antibi-
otics, and carbohydrate derivatives.14–16 Kobayashi et al.
reported the enantioselective synthesis of natural product
macrosphelides A and B, which act as anti-adhesion com-
pounds and effectively inhibit the adhesion of human
leukemia HL-60 cells to human-umbilical-vein endothelial
cells, using S-1-(2-furyl) ethanol as the starting precursor.17
There are a few studies reported on the synthesis of the
single enantiomer of 1-(2-furyl) ethanol by asymmetric reduc-
tion using baker’s yeast, asymmetric chemical catalysts, or
lipase-catalyzed kinetic resolution.18–21 All these reported pro-
cesses require a longer time and there is no information on
the kinetics of the reaction, which is required for reactor
design and to scale up of the process. In the current work,
lipase-catalyzed transesterification of (R/S)-1-(2-furyl) ethanol
with various acyl donors was conducted in nonaqueous media
(Scheme 1). The effect of various parameters such as differ-
ent commercially available immobilized lipases, acyl donors,
solvents, agitation speed, temperature, catalyst loading, and
acyl donor concentration was studied systematically by
varying one parameter at a time. Finally, the mechanism
*Correspondence to: G. D. Yadav, Department of Chemical Engineering, Insti-
tute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai,
India - 400 019. E-mail: gdyadav@yahoo.com; gd.yadav@ictmumbai.edu.in
Received for publication 2 November 2013; Accepted 3 February 2014
DOI: 10.1002/chir.22317
Published online in Wiley Online Library
(wileyonlinelibrary.com).

DEVENDRAN AND YADAV
Scheme 1. Lipase-catalyzed kinetic resolution of ( )-1-(2-furyl) ethanol.
QðSÞ ꢀ QðRÞ
QðSÞ þ QðRÞ
and kinetics were also developed. The results are new and
have potential for scale up and industrial application.
eep ¼
(4)
where,B ,B , B(R), B(S), Q(R) and Q(S) denote area under the curve of (R)-
1-(1-furyl) ethanol, (S)-1-(1-furyl) ethanol, and their corresponding esters.
ðR0Þ ðS0Þ
MATERIALS AND METHODS
Enzymes
The following enzymes were received as gift samples from M/s
Novozymes A/S (Bagsvaerd, Denmark): 1) 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; 2)
Lipozyme RM-IM: 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; 3) Lipozyme TL IM: Lipase from
Thermomyces lanuginosus, supported on porous silica granulates with
water content 1–2% and enzyme activity 175 IU/g.
RESULTS AND DISCUSSION
Effect of Catalyst
Lipases from different origins such as Candida antarctica
lipase B (Novozym 435), Rhizomucor miehei lipase (Lipozyme
RMIM), and Thermomyces lanuginosus lipase (Lipozyme
TLIM) were evaluated for resolution of ( )-1-(2-furyl) ethanol
under similar conditions. The reactions were carried out using
n-heptane as solvent. At the end of 2-h reaction time, lipase B
from C. antarctica (Novozym 435) had given the maximum
conversion compared to other immobilized catalysts. With
Novozym 435, conversion of 47% was obtained compared to
Lipozyme RMIM (1.8%) and Lipozyme TLIM (1.5%). The order
of activity was as follows: Novozym 435 > Lipozyme RMIM
Lipozyme TLIM. It is reported that both Lipozyme RMIM and
Lipozyme TLIM are mainly used to interesterify bulk molecules
such as fatty acid derivatives and they have less activity in the
resolution of racemic molecules.22 However, Novozym 435 is
a well-known and commercially available lipase for both chiral
resolution and synthesis of small molecules esters.23 Hence,
further study was carried out using Novozym 435 as catalyst.
Chemicals. Iso-octane, n-heptane, toluene, cyclohexane, vinyl acetate,
methyl acetate, acetic anhydride, ethyl acetate, and other analytical and
high-performance liquid chromatography (HPLC)-grade reagents were pur-
chased from M/s S.D. Fine Chemicals, Mumbai, India. ( )-1-(2-Furyl) etha-
nol, vinyl butyrate, and vinyl laurate were purchased from Sigma- Aldrich,
Bangalore, India. All chemicals and enzymes were used without any further
modification/purification.
Experimental Setup
The experimental setup consisted of a 3-cm i.d. mechanically agitated
glass reactor of 50cm3 capacity, equipped with four baffles and a six-bladed
turbine impeller. The entire reactor assembly was immersed in a thermo-
static water bath, which was maintained at a predetermined temperature
with an accuracy of 1°C. A typical reaction mixture consisted of 0.001 mol
racemic alcohol and 0.001mol vinyl ester diluted to 20 cm3 with n-heptane
as a solvent. The reaction mass was agitated at 60°C for 15 min at a speed
of 300 rpm and then 5 mg of enzyme was added to start the reaction.
Effect of Acyl Donor
The selection of a suitable acyl donor is primarily important
for the lipase-catalyzed kinetic resolution of secondary alcohols.
It has been well reported that the type of acyl donor and its
chain length can influence both the reaction rate and
enantioselectivity of enzyme in transesterification/interes-
terification reactions.24–27 In order to examine the effect of acyl
donor on kinetic resolution of ( )1-(2-furyl) ethanol, initially we
had taken different alkyl and vinyl esters such as acetic
anhydride, methyl acetate, ethyl acetate, and vinyl acetate as
model acylating agents (Fig. 1). The reactions were performed
under similar conditions. At the end of 2 h, it was observed that
vinyl acetate gave higher conversion (47%) and enantiomeric
purity (89% ee) with E > 200 compared to the other alkyl esters.
Among alkyl esters, acetic anhydride had given good conver-
sion (39%) and enantiomeric purity (65% ee) than other esters.
It has been reported that vinyl esters are better acylating agents
than alkyl esters.28 The liberated coproduct is vinyl alcohol
(enol form), which is unstable and easily tautomerized to acetal-
dehyde (aldehyde form) and it is no longer a substrate for li-
pase. So the reaction equilibrium shifts to the forward
direction, which leads to the formation of the chiral ester. Fur-
ther, the effect of the acyl chain length was evaluated using
three different vinyl esters such as vinyl acetate, butyrate, and
laurate. Increasing the chain length led to a decrease in
Analysis
The reaction progress and enantiomeric excess (ee) were monitored
by periodical withdrawal of clear liquid samples from the reaction mixture
which were analyzed by HPLC (1260 infinity series, Agilent Technolo-
gies, Palo Alto, CA) equipped with Chiralpak- IB analytical column (250
× 4.6 mm ID) (Daicel, Japan; particle size 5 μm). Samples (10 μl) were
injected via autosampler. The mobile phase consisted of n-hexane and iso-
propyl alcohol (99:1) and the flow rate was maintained at 0.75 ml.min-1. A
DAD detector was used at a wavelength of 230 nm. The retention time of
(R) and (S) alcohol were 23.1 and 23.9 min, respectively.
The enantioselectivity ratio (E) and conversion (c,%), were calculated
from the enantiomeric excess of the substrate (ees,%) and product (eep,%)
based on the following equation:
ln½ð1 ꢀ cÞð1 ꢀ eesÞꢁ
E ¼
(1)
ln½ð1 ꢀ cÞð1 þ eesÞꢁ
Where,
B
ðRÞ þ B
þ B
ðSÞ
c ¼ 1 ꢀ
(2)
(3)
B
ðR0Þ
ðS0Þ
BðSÞ ꢀ BðRÞ
BðSÞ þ BðRÞ
ees ¼
and
Chirality DOI 10.1002/chir

LIPASE-CATALYZED KINETIC RESOLUTION OF ( )-1-(2-FURYL) ETHANOL
(toluene to n-heptane). The highest conversion (47%) and ees
(89%) were obtained when n-heptane was employed as solvent,
whereas low conversion (22%) and ees (29%) were obtained with
toluene. Due to the low solubility of racemic alcohol in iso-oc-
tane, moderate conversion and enantiomeric purity were ob-
served. This clearly correlated with the reported literature
that solvents having high-log P are hydrophobic in nature but
do not strip the water layer around enzyme particle, which is es-
sential for maintaining the conformation structure.31 Thus, they
show high enzyme activity. Further work was carried out using
n-heptane as the solvent.
Effect of Speed of Agitation
In immobilized catalysts, reactants have to pass from the
bulk liquid phase to the enzyme particle’s surface, and then
diffuse from the external surface to the enzyme active site.
The external mass transfer resistance and intraparticle diffu-
sion rate play significant roles during the reaction. It must
be overcome to study the intrinsic kinetics of lipase-catalyzed
transesterification reaction. Several experiments were
performed in the range of 100 to 400 rpm by taking
0.001 mol racemic alcohol and vinyl acetate each at 60°C with
5 mg Novozym 435 and volume made up to 20 cm3 using
n-heptane as solvent. From the progress curve (Fig. 3), the
conversion is seen to increase with increase in agitation
speed from 100 to 300 rpm and beyond this value; there was
no significant difference in conversion. This implies that
there is no external mass transfer resistance.
Fig. 1. Effect of acyl donor. Reaction condition: 1-(2-furyl) ethanol,
0.001 mol; acyl donor, 0.001 mol; n-heptane up to 20 cm3; speed of agitation,
300 rpm; catalyst loading, 5 mg; temperature, 60°C,
ees,
Conversion,
AA, acetic anhydride; EA, ethyl acetate; MA, methyl acetate; VA, vinyl acetate;
VB, vinyl butyrate, VL, vinyl laurate.
enantioselectivity and conversion. The highest conversion
(47%) with ees of 89% was obtained with vinyl acetate as the acyl
donor, whereas low conversion (38%) with ees of 62% was
obtained with vinyl laurate. Hence, further study was carried
out using vinyl acetate as the acyl donor.
Effect of Organic Media
Solvent plays a significant role in any enzymatic reaction.
The important criteria for proper choice of organic media are
substrate solubility, product recovery, and enzyme stability.29
It has been reported in the literature that solvent can alter
the enzyme activity, reaction rate, and enantioselectivity by
modifying the aqueous layer around the enzyme particle or
changing the enzyme conformational structure.30 Several ex-
periments were performed to evaluate the effect of chosen sol-
vents such as iso-octane (logP – 4.5), n-heptane (logP – 4),
cyclohexane (logP – 3.2) and toluene (logP –2.5) on transes-
terification reaction (Fig. 2). The experiments were carried
out using 1:1 mole ratio of alcohol to ester (each 1 mmol), with
5 mg enzyme loading at 60°C and the liquid phase volume was
made up to 20cm3. Both conversion and enantioselectivity
were found to increase as logP value decreased from -2.5 to -4
The values of solid-liquid mass transfer coefficients for
both reactants with n-heptane as solvent were calculated
using the correlations developed for polymer supported
catalyst which took into account the effects of the Reynolds
and Schmidt numbers.
!
1=4
ꢀ
ꢁ
ed4p ρ3L
1=3
kSLdP
Deψ
μL
ρLDe
¼ 2 þ 0:4
(5)
μ3
L
= Power consumption and P ¼ NP N3DI5ρL,
P
Where, e ¼
ρLVL
NP, the power number was calculated from the correlation for
the impeller used in this work. Diffusivity of racemic alcohol
(De) at 60°C was calculated using the Wilke-Chang equation
32
as 4.837×10-5 cm2 sꢀ1
.
The effective diffusivity is given by
Fig. 2. Effect of solvent. Reaction condition: 1-(2-furyl) ethanol, 0.001; vinyl
Fig. 3. Effect of agitation speed. Reaction condition: 1-(2-furyl) ethanol,
0.001; vinyl acetate, 0.001 mol; n-heptane up to 20 cm3, 100–400 catalyst load-
acetate, 0.001 mol; solvent up to 20 cm3; speed of agitation, 300 rpm; catalyst
loading, 5 mg; temperature, 60°C,
Iso-octane.
Toluene,
Cyclohexane,
n-Heptane
ing, 5 mg; temperature, 60°C,
RPM.
100 RPM,
200 RPM, 300 RPM 400
Chirality DOI 10.1002/chir

DEVENDRAN AND YADAV
Dε
confirm the previous finding that the reaction is intrinsically
kinetically controlled. The elevated temperature reduces the
viscosity of the reaction mixture and increases the collusion
frequency between reactants and enzyme particles that lead
to increase in reaction rate. Both conversion and
enantioselectivity were found to be maximum at 60°C and
also maintained the enzyme activity. Therefore, the
temperature of 60°C was selected as the optimum and, fur-
ther experiments were conducted at 60°C. The Arrhenius
plot was made on the basis of ln(initial rates) vs. the recipro-
cal of temperature (Fig. 5). The apparent activation energy
was calculated from the observed initial rates at different tem-
peratures under otherwise similar conditions. The activation
energy was found to be 8.57 kcal/mol. This value lies within
the typical value for an enzyme catalytic reaction.36
De ¼ where the porosity (ε) and tortuoisity (τ) were taken
τ
as conservative values of 0.38 and 3, respectively. The
average diameter of the support particle (Dp) was taken as
0.06 cm, since the particle size ranged between 0.03 and
0.09 cm. The value of mass transfer coefficient of liquid phase
(kSL) was calculated as 5.2 × 10-3 cm.s-1 by using a standard
correlation for organic phase.31 From this value, the calculated
kSLDp
Sherwood number Sh ¼
was 640, indicating that there
was no external mass transDfer resistance above 300 rpm.33
Further, it is necessary to rule out the intraparticle diffu-
sion. This could be done by assessing the Thiele modulus
(φ), which is defined as the ratio of reaction rate to internal
diffusion rate. If the Thiele’s modulus value is less than 0.3,
then the effectiveness factor of the reaction approaches unity.
Then it can be concluded that the reaction rate would be
kinetically controlled and internal diffusion effect can be
neglected.33 Thiele modulus can be estimated using the
following equation.
Effect of Catalyst Loading
The addition of enzyme loading has a considerable effect
on reaction rate and enantioselectivity.12 In order to study the
effect of catalyst loading, several experiments were performed
by keeping the mole ratio constant while enzyme loading was
changed in the range from 1.75 × 10-4 to 4 × 10-4 g.cm-3 under
similar reaction conditions (Fig. 6). It was observed that the
reaction rate had a linear dependence on enzyme loading.
The overall conversion was increased with increase in
enzyme loading from 1.75 × 10-4 to 2.5 × 10-4 g.cm-3. Above this
value, there was no significant change in substrate conversion.
The increase of enzyme loading increases the number of active
sites proportionately and facilitates the interaction between
enzyme active sites and reactant that further enhance the
reaction rate and conversion. However, at high enzyme
concentration, the available enzyme sites are much larger
than the required amount for reaction and it will not fur-
ther improve the conversion. Thus, 2.5 × 10-4 g.cm-3 was
taken as optimum and further studies were carried out
with this loading.
ꢀ ꢁ
2
rðC0Þ
R
3
ϕ ¼
(6)
Deff C0
Both C0 and r (C0) were determined experimentally and
their values were 0.05 mol.dm-3 and 9.833 × 10-6 mol.dm-3.s-1.
The effective diffusion of racemic alcohol is 6.127 × 10-6. From
these values, the Thiele modulus of the reaction is 3.21 × 10-3
and this value is less than 0.3, suggesting that there was no
intraparticle diffusion limitation. Thus, the reaction was
controlled by intrinsic enzyme kinetics. Therefore, further
experiments were performed at a speed of 300 rpm.
Effect of Temperature
Temperature is one of the important parameters that affects
the reaction rate and enantioselectivity in lipase-catalyzed
kinetic resolution of secondary alcohols.34,35 In order to study
the effect of temperature, a number of experiments was
performed in the range of 40°C to 70°C under similar
conditions (Fig. 4). It was observed that reaction rate,
enantioselectivity, and conversion increased with an increase
in temperature up to 60°C. Above this value, there was no
significant change in conversion. It has been reported that
the enzyme is thermally stable at 60°C.9 This would further
Effect of Acyl Donor Concentration
In order to study the effect of acyl donor concentration on
reaction rate and conversion of kinetic resolution of ( )-1-(2-
furyl) ethanol, a number of experiments were performed at
60°C with 5 mg of enzyme loading in n-heptane as solvent.
The concentration of ( )-1-(2-furyl) ethanol was kept constant
(0.001 mol), and vinyl acetate concentration was varied from
Fig. 4. Effect of temperature. Reaction condition: 1-(2-furyl) ethanol, 0.001;
vinyl acetate, 0.001 mol; n-heptane up to 20 cm3; speed of agitation, 300 rpm;
catalyst loading, 5mg; temperature, 40–70°C, 40°C, 50°C, 60°C, 70°C.
Fig. 5. Arrhenius plot. Reaction condition: 1-(2-furyl) ethanol, 0.001; vinyl
acetate, 0.001 mol; n-heptane up to 20 cm3; speed of agitation, 300 rpm; catalyst
loading, 5 mg; temperature, 40–70°C.
Chirality DOI 10.1002/chir

LIPASE-CATALYZED KINETIC RESOLUTION OF ( )-1-(2-FURYL) ETHANOL
Kinetic Model
Two models have been proposed for two substrate-based
enzymatic reaction, the first is the random model, where
the order of binding of substrate to the enzyme active site is
random and the second model is the bi–bi model, where the
first step is formation of an acyl-enzyme complex.37 It has
been well reported that the first step of a lipase-catalyzed re-
action involves the formation of an acyl enzyme complex with
the acyl donor and will rules out a random mechanism.38 As a
result, it can only be the bi–bi model, wherein two mecha-
nisms have been proposed, namely, the ordered bi–bi mech-
anism or ternary complex mechanism and the ping–pong
bi–bi mechanism. In the former mechanism, both reactants
bind to the enzyme and form a ternary complex and subse-
quently the products are released. In the latter mechanism,
the product is released between the additions of substrates.38–40
An intricate kinetic analysis was carried out in order to
develop the appropriate mechanism for the kinetic resolu-
tion of ( )-1-(2-furyl) ethanol. A number of experiments were
performed to investigate the effect of substrate concentra-
tion on the rate of reaction over a wide range using 5 mg
Novozym 435 under similar conditions and the volume was
made up to 20 cm-3 with n-heptane. For determination of ini-
tial rates, the concentration of vinyl acetate (0.025–0.1 M)
was varied over a wide range of ( )-1-(2-furyl) ethanol
concentration (0.025–0.1 M).
The initial rates were calculated systematically from the lin-
ear portion of the concentration–time profiles. It was observed
that the rate increased with increasing concentration of alco-
hol. However, the rate of reaction decreased with increasing
concentration of vinyl acetate. Hara et al.41 carried out the
lipase-catalyzed kinetic resolution of furan-based alcohols in
diisopropyl ether as solvent. They observed the formation of
acetic acid / butyric acid as a byproduct which affected the
course of the kinetic resolution. The possible reason for this
behavior is that diisopropyl ether is polar solvent and has low
log P value (logP -1.4).41 It might have stripped out the essen-
tial (residual) water layer around the enzyme particles. In the
presence of released water molecules, lipase catalyze hydroly-
sis of acylating agent (vinyl acetate or vinyl butyrate), which
led to the formation of acids as byproducts. Contrary to this,
Fig. 6. Effect of catalyst loading. Reaction condition: 1-(2-furyl) ethanol,
0.001; vinyl acetate, 0.001 mol; n-heptane up to 20 cm3; speed of
agitation, 300 rpm; catalyst loading, 3.5–8 mg; temperature, 60°C,
3.5 mg,
5mg,
6.5 mg, 8mg.
0.001 to 0.004 mol; the mixture volume was kept constant at
15 cm3 by adjusting the addition of n-heptane (Fig. 7). It was
found that the conversion and rate of reaction had decreased
with an increase in the concentration of vinyl acetate. The
overall conversion decreased from 47% to 41.7%. At high
concentration, vinyl acetate acts as an inhibitor and this will
be explained later.
Catalyst Reusability
Catalyst reusability is very important in order to make the
process more viable at the industrial level and also to examine
the stability of the catalyst after each run. Initial experiments
were carried out using 5 mg of catalyst loading under similar
conditions. After completion of each run, the catalyst particles
were filtered by a membrane filter. Multiple washes were
given to the catalyst with fresh solvent-heptane, dried at room
temperature for 12 h, and reused. It was found that there was
a marginal decrease in conversion from 47 to 44.5% after three
reuses, which was due to loss of enzyme during filtration and
drying. No make-up quantity was added.
Fig. 7. Effect of acyl donor concentration. Reaction condition: 1-(2-furyl)
ethanol, 0.001; vinyl acetate, 0.001–0.004 mol; n-heptane up to 20 cm3; speed
Fig. 8. Lineweaver-Burk plot. Reaction condition: 1-(2-furyl) ethanol,
0.025- 0.1 M; vinyl acetate, 0.025–0.1 M; n-heptane up to 20 cm3; speed of
of agitation, 300 rpm; catalyst loading, 5 mg; temperature, 60°C,
1:2, 1:3, 1:4.
1:1,
agitation, 300 rpm; catalyst loading, 5 mg; temperature, 60°C,
0.025 M,
0.05 M,
0.075 M, 0.1 M.
Chirality DOI 10.1002/chir

DEVENDRAN AND YADAV
TABLE 1. Comparison with previous results
Temperature
(°C)
Enzyme loading
(mg)
Mole ratio
(alcohol: ester)
Time
(h)
Conversion
(%)
eep
(%)
Lipase
E
Ref.
Pesudomonas cepacia lipase
Porcine Pancreas lipase
Burkholderia cepacia lipase
Candida antarctica lipase B
40
R.T
R.T
60
100
100
50
1:2
1:5
1:2
1:1
21
24
24
2
48
53
53
47
>99
>99
>99
>99
>300
64
>200
21
41
42
5
>200 This work
we carried out the same reaction in n-heptane which has a high
log P value 4 and found that there is no formation of acetic acid
and loss of enzyme activity during the reaction. This indicated
that the residual water layer around the enzyme was intact.
Based on these observations, it is concluded that there would
be reversible competition between vinyl acetate and alcohol
at the active site of the enzyme which led to decreasing the
reaction rate at high concentration of vinyl acetate.
we achieved good conversion (47%) and enantiomeric purity
(eep>99.9) and E>200 with low enzyme loading in a very short
time.21,42,43 Considering the industrial implementation or scale up,
Novozym 435 catalyzed kinetic resolution of ( )-1-(2-furyl) ethanol
is the most suitable process compared to other reported lipases.
CONCLUSION
The Lineweaver–Burk plot (Fig. 8), with double reciprocal of
the initial rate and concentration of alcohol, shows that the
lines are not parallel, ruling out the possibility of a ping–pong
bi–bi mechanism. The slope and intercept changed linearly
with increased concentration of the acyl donor. The lines were
intersecting at certain points suggesting the ternary complex
mechanism. According to this, the lipase (E) will react with
acyl donor (A) to form a complex (EA). The second reactant
1-(2-furyl) ethanol (B) then reacts to form a ternary complex
(EAB). This ternary complex then isomerizes to another
ternary complex EPQ, which releases the product ester
(P) and vinyl group (Q) and frees the enzyme E. At higher
concentrations of vinyl acetate, a reversible deadend complex
is formed. A typical reaction sequence is shown below.
In this work, the kinetic resolution of ( )-1-(2-furyl) ethanol
was carried out using different immobilized catalysts. Among
which, C. antarctica lipase B (Novozym 435) was found to be
the best catalyst in n-heptane as solvent. The effect of the acyl
donor was compared by employing both alkyl and vinyl es-
ters. The highest conversion and initial rate were obtained
with vinyl acetate as the acyl donor. The effect of kinetic pa-
rameters on reaction conversion and initial rate was analyzed
systematically over a wide range. Maximum conversion of
47% was obtained in 2 h using 5 mg enzyme loading with equi-
molar concentration of alcohol and ester at 60°C. The prog-
ress curve and initial rate data were used to predict the
suitable model and then various kinetic parameters were esti-
mated using nonlinear regression. The ordered bi–bi mecha-
nism with acyl donor inhibition was found to fit the initial rate
data. This model was used to simulate the rate data, which
were in excellent agreement with the experimental values.
The optimized values and kinetic mechanism for resolution
of ( )-1-(2-furyl) ethanol are very useful and give new insight
for scaling up the enzymatic process at industrial level.
ACKNOWLEDGMENTS
S.D. received JRF from UGC under its Meritorious Fellowship
BSR programme (CAS in Food Engineering and Technology).
The authors thank Novo Nordisk, Denmark, for providing
lipases as a gift. G.D.Y. received support from an R. T. Mody
Distinguished Professor Endowment and J. C. Bose National
Fellowship of Department of Science and Technology, Govern-
ment of India.
The equation obtained with the above mechanism is:
vm½Aꢁ½Bꢁ
v ¼
(7)
KiAKmB þ KmA½Bꢁ þ KmB½Aꢁ þ ½Aꢁ½Bꢁ
where v is the rate of reaction, vm the maximum rate of reac-
tion, [A] the initial concentration of vinyl acetate, [B] the
initial concentration of 1-(2-furyl) ethanol, KmA the Michaelis
constant for vinyl acetate, KmB the Michaelis constant for
1-(2-furyl) ethanol, and KiA the inhibition constant for vinyl ace-
tate. The initial rate data were used to determine the kinetic
parameters of the above mechanism by nonlinear regression
analysis using the software package Polymath 5.1 and their
values are Vm - 4.67×10-3 mol.dm-3.s-1, KmA - 0.0261mol.dm-3,
KmB - 0.0313 mol.dm-3 and Ki - 0.0585 mol.dm-3. The rates of
reaction for different reactant concentrations were simulated
using the above equation to verify the proposed kinetic model.
The simulated and experimental rate had good agreement, which
suggested that the proposed mechanism was valid for this reac-
tion. As compared to previously reported processes (Table 1),
NOMENCLATURE
tr
td
C0
time constant for reaction, s
time constant for diffusion, s
initial concentration of limiting reactant, mol.dm-3
r(C0) initial rate of reaction, mol.dm-3.s-1
DS
kSL
dp
Rp
φ
diffusivity of limiting reactant, cm2. sꢀ1
liquid side mass transfer coefficient, m.sꢀ1
diameter of supported particle, cm
radius of supported particle, cm
phase volume ratio
a
interfacial area per unit volume of organic phase
initial concentration of vinyl acetate, mol.dm-3
initial concentration of 1-(2-furyl) ethanol, mol.dm-3
Enzyme
[A]
[B]
E
Chirality DOI 10.1002/chir

LIPASE-CATALYZED KINETIC RESOLUTION OF ( )-1-(2-FURYL) ETHANOL
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EAB ternary complex
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Ester
Acetaldehyde
rate of reaction, mol.dm-3.s-1.g-1-enz
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