Enantioselective resolution of (R,S)-1-phenylethanol catalyzed by lipases immobilized in starch films

Article abstract of DOI:10.1590/S0103-50532011000800021

Lipases form different sources and two mycelium-bound lipases, in a free or immobilized form, in ginger starch film were screened as biocatalysts in the reaction of (R,S)-1-phenylethanol (1) with vinyl acetate and other acylating agents. The effect of various reaction parameters in the resolution of (1) catalyzed by lipase form Burkholderia cepacia (BCL) immobilized in ginger starch film was evaluated (acyl donor type, alcohol: acyl donor molar ratio, temperature and organic solvent). The catalytic efficiency of BCL immobilized in polymeric blends of ginger starch and polyethylene oxide (PEO), in different compositions, was also studied. Vinyl acetate and iso-propenyl acetate furnished the highest conversion (9percent) and enantiomeric excess (< 99percent) of the (R)-ester. The alcohol: acyl donor molar ratio and temperature optimum were 1: 1 and 28 °C, respectively. The mixture of n-hexane/glycerol (9: 1 v: v) was the most adequate for this reaction (conversion 23percent, E > 200). The ginger starch/PEO (7: 3 m/m) blend was successfully reused six times consecutively.

Full text of DOI:10.1590/S0103-50532011000800021

J. Braz. Chem. Soc., Vol. 22, No. 8, 1559-1567, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
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Article
A
Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases
Immobilized in Starch Films
Isabel Hoffmann, Vanessa D. Silva and Maria da G. Nascimento*
Departamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil
Lipases de diferentes fontes e dois micélios, na forma livre ou imobilizados em filme de amido
de gengibre, foram utilizados como catalisadores na reação do (R,S)-1-feniletanol (1) com acetato
de vinila e outros agentes acilantes. Os efeitos de vários parâmetros na resolução de (1) catalisada
pela lipase de Burkholderia cepacia (LBC) imobilizada em filme de amido de gengibre (tipo do
doador acila, razão molar álcool:doador acila, temperatura e solvente orgânico) foram avaliados.
A eficiência catalítica da LBC imobilizada em blendas de amido de gengibre e poli (oxido de etileno)
(PEO), em diferentes composições, também foi estudada. Os acetato de vinila e o de iso-propenila
forneceram as maiores conversões (9%) e excessos enantioméricos em (R)-éster (> 99%). A razão
molar álcool:doador acila e temperatura ótima foram de 1:1 e 28 °C, respectivamente. A mistura
de n-hexano/glicerol (9:1 v:v) foi a mais adequada para esta reação (conversão 23%, E > 200).
A blenda de amido de gengibre/PEO (7:3 m/m) foi reutilizada por seis vezes consecutivas.
Lipases from different sources and two mycelium-bound lipases, in a free or immobilized form,
in ginger starch film were screened as biocatalysts in the reaction of (R,S)-1-phenylethanol (1) with
vinyl acetate and other acylating agents. The effect of various reaction parameters in the resolution
of (1) catalyzed by lipase from Burkholderia cepacia (BCL) immobilized in ginger starch film was
evaluated (acyl donor type, alcohol:acyl donor molar ratio, temperature and organic solvent). The
catalytic efficiency of BCL immobilized in polymeric blends of ginger starch and polyethylene
oxide (PEO), in different compositions, was also studied. Vinyl acetate and iso-propenyl acetate
furnished the highest conversion (9%) and enantiomeric excess (> 99%) of the (R)-ester. The
alcohol:acyl donor molar ratio and temperature optimum were 1:1 and 28 °C, respectively. The
mixture of n-hexane/glycerol (9:1 v:v) was the most adequate for this reaction (conversion 23%,
E > 200). The ginger starch/PEO (7:3 m/m) blend was successfully reused six times consecutively.
Keywords: starch film, immobilization, lipases, enzymatic resolution
Introduction
applications, such as in detergents, food processing,
biotransformations, waste treatment and bioremediation.
They have been used by the pharmaceutical, cosmetics,
textile, leather and paper industries and also in medical
In recent years, the application of enzymes and
microorganisms as biocatalysts in organic synthesis has
1,2
5-7
become a well-known technique. Enzymatic procedures
to obtain a wide range of products under mild and
environmentaly-friendly conditions have become attractive
and promising due to the excellent stereoselectivity of
applications as drugs or biosensors. Lipases can
catalyze the resolution of several racemic compounds
8
9
10
11
including alcohols, amines, acids, a-hydroxy selenides
1
2
and tellurides. Due to the poor solubility in water of
most of these compounds, the enzymatic resolution is
usually performed in non-aqueous media, but enzymes
can denature in such media. A practical technique to
improve the enzyme efficiency, operational stability and
performance in organic solvents is the use of biocatalyst
immobilization. Besides the stabilizing effect, the quality
of the products can be improved by avoiding by-products or
unwanted intermediates and easy separation. Furthermore,
3
enzymes. Lipases (triacylglycerol hydrolases EC 3.1.1.3)
are the most widely applied enzymes for regio-, chemo-
and enantioselective biotransformations, because they also
possess wide substrate specificity, have an excellent ability
to recognize chirality, and do not require cofactors. They
have found wide usage in a growing range of industrial
1,4
*e-mail: graca@qmc.ufsc.br

1
560
Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases
J. Braz. Chem. Soc.
immobilized enzymes can be reused, making the process
However, currently one of the main uses is to produce
bioplastics with native or modified starch, or in combination
with natural or synthetic molecules. These are used in
specific industrial applications such as composting bags
13-16
economically feasible.
Severalimmobilizationmethodshavebeenreported,such
13
14
as covalent binding to a support, magnetic nanoparticles
26,27
and lipase entrapment in porous polymeric matrices and
and sacks, fast food service ware, packaging and others.
6
in gels. Amongst the various immobilization techniques
In this study, starch obtained from ginger was used as a
support for lipase immobilization and these systems were
employed in the resolution of the (R,S)-1-phenylethanol (1).
Several reaction parameters were evaluated including the
type and amount of lipase, alcohol:acyl donor molar ratio,
use of different organic solvents and temperature, as well
as the influence of lipase immobilization in starch film or
in polymeric blends formed of ginger starch/polyethylene
oxide (PEO). The recyclability of the immobilized catalyst
was also evaluated (Scheme 1).
employed, adsorption may have a higher commercial
potential because this method is less complex and expensive
1
7
than other techniques. Physical characteristics, such
as surface area, porosity and hydrophobic-hydrophilic
balance, are important parameters of the support. They
are crucial for enzyme immobilization and will influence
1
5,18
the enzyme loading as well as the catalytic behavior.
Recently, various biopolymers, such as dextran,
1
6
1
9,20
17
21
chitosan,
agarose and cellulose, have been used as a
support for enzyme immobilization.
Starch is a semi-crystalline biopolymer and is the most
abundant storage reserve carbohydrate inplants. Itisfoundin
manydifferentplantorgansincludingseeds, fruits, tubersand
roots. Many of these starch-storing organs, for example, the
grains of maize and rice or the tubers of cassava and potatoes,
Experimental
Materials and methods
All chemicals were commercially available and
were of reagent grade and used without further
purification. (R,S)-1-phenylethanol (98%) and 1-butyl-
3-methylimidazolium hexafluorophosphate [BMIm][PF6]
(98%) were purchased from Sigma-Aldrich. Polyethylene
oxide (PEO) (MM = 300000 daltons) was supplied byAcros
Organics.Acetic anhydride (97%), sodium bicarbonate and
glycerol (99.5%) were provided from Vetec. Vinyl acetate
(99%) and iso-propenyl acetate (99%) were obtained from
Fluka Chemika. Sulfuric acid was purchased from Quimex.
Anhydrous magnesium sulfate and ethyl acetate (99%)
were provided by Nuclear. The commercially-available
enzyme preparation, lipase from Candida antarctica B
22
are staple foodstuffs in the human diet. Starch granules
are composed of two types glucose polymers, amylose and
amylopectin, which represent approximately 98-99% of the
dry weight.The ratio between the two polysaccharides varies
23
according to the botanical origin of the starch. Amylose is
a relatively long, linear polymer containing 99% a-1-4 and
1
.0% a-1-6 glycoside bonds.Amylopectin contains 95% of
24
short a-1-4 linear chains and 5% a-1-6 linked side chains.
Starch has a broad range of applications both in the
food and non-food sectors. For example, it is an important
ingredient for the food industry, being used to improve
2
2-25
the nutritional and sensory quality.
Other important
-1
fields of starch application include textiles, paper, mining,
(CALB, 10000 PLU g ), was donated by NovozymesA/S.
Lipase from Burkholderia cepacia previously known as
2
6
cosmetics, pharmaceuticals, construction and paints.
O
OH
O
O
OH
R
lipases / support
organic solvent
+
O
+
(
R,S)-1
2a-d
(R)-3
(S)-1
c: 1-56%
eep: 85.9-99%
s
ee : 2
99%
O
2
a: R = CH2CH3
2b: R =
2c: R =
2d: R =
Scheme 1. Enzymatic resolution of (R,S)-1-phenylethanol (1) in organic media.

Vol. 22, No. 8, 2011
Hoffmann et al.
1561
-
1
Pseudomonas cepacia (BCL, 30000 U g ) was donated by
Amano Enzymes Inc., and lipase from Chromobacterium
R-(+)-1-phenylethanol were 6.1 and 5.8 min, and for the
corresponding S-(–) and R-(+)-acetyl esters they were
5.0 and 5.4 min, respectively. The enantiomeric excess
of the reagent (ee ) and product (ee ) was defined as
-1
viscosum (CVL, 5138.7 U mg ) was provided by Genzyme
Biochemicals.
s
p
Native lipases from Aspergillus niger AC-54 (ANL,
9.4 U mL ) and Rhizopus oligosporus (ROL, 14.9 U mL )
[(S) – (R)]/[(S) + (R)] × 100%, where (R) and (S) are the
peak areas of (R) and (S) enantiomers, respectively.
-1
-1
1
were kindly donated by Professor Patrícia de Oliveira
Carvalho from Universidade São Francisco (USF, Bragança
Paulista-SP, Brazil), and were isolated and purified by
microorganisms from the region of Bueno Brandão-MG,
Immobilization of lipases in starch film and ginger
starch/PEO blends
2
8
Brazil. The mycelium-bound lipases isolated from the
Amazonian fungi UEA_53 (Astrocaryum aculeatum) and
UEA_115 (Colletotrichum sp) were kindly donated by
Professor Sandra Patrícia Zanotto from the Universidade
Enzyme immobilization on ginger starch film was
performed by dissolving 1.0 g of ginger starch in 25 mL
of distilled water (pH 5.6). This solution was maintained
under constant magnetic stirring and mild heating for
1 h until complete dissolution of the polymer. After this
period, 0.3 mL of glycerol and 60 mg of lipases (BCL,
CVL, ROL or ANL) were added into the solution and the
mixture was stirred for more than 20 min. The solvent was
evaporated (30-40 °C) forming a stable film, which was then
cut into small pieces and maintained in organic solvent.
The ginger starch/PEO blends were prepared by varying
the composition of the ginger starch polymer and PEO,
ranging from 0 to 100% (m/m), the total mass being 1.0 g.
The lipase mass was the same, as previously described.
29
Estadual do Amazonas (UEA, Manaus-AM, Brazil).
The starch (S) was obtained from ginger (G, Zingiber
officiale) and was extracted and characterized using
an Olympus BX40 optical microscope, coupled with a
cybernetics cool SNAP-PRO cf color digital camera.
The equipment belongs to Laboratório de Microscopia
do Departamento de Ciência e Tecnologia de Alimentos,
Universidade Federal de Santa Catarina (UFSC,
Florianópolis-SC, Brazil), a collaboration with Professor
Elisa H. S. Moecke. The grains presented oval or circular
30
shape with diameters ranging from 15-30 mm.
Deuterium chloroform (99.8%) was purchased from
Cambridge Isotope Laboratories.All organic solvents were
obtained from commercial sources and were of analytical
grade.
General procedure for lipase-catalyzed resolution of (R,S)-
1-phenylethanol (1)
The lipase-catalyzed resolution of the (R,S)-1-
phenylethanol in organic media was performed in a 250 mL
shake flask. The reaction mixture contained 0.6-6.0 mL
(5-50 mmol) of (R,S)-1, 0.5-4.6 mL (5-50 mmol) of vinyl
acetate, 0.6 mL (5 mmol) of iso-propenyl acetate, 0.5 mL
(5 mmol) of acetic anhydride or 0.5 mL (5 mmol) of ethyl
acetate and the lipases (20 or 60 mg) free or immobilized
in ginger starch film or in ginger starch/PEO blends. A
similar procedure was used for the reactions catalyzed by
the mycelium-bound lipases (60 mg). The reaction media
was incubated in a shaker using n-hexane as a solvent,
except in the solvent screening studies, at 22-40 ºC with
constant stirring.Aliquots were withdrawn at specified time
intervals from the reaction mixture, and analyzed by chiral
chromatographytoevaluatethepercentageofconversionand
the enantiomeric excess of reactant and products. Substrate
and product peak areas were compared, and the sum of
the two was considered as 100%. The enantiomeric ratio
(E-value) was calculated from the enantiomeric excess of
Infrared spectra were acquired with an ABB Boomer
FTLA 2000-100 spectrometer using KBr for solid samples
-
1
1
13
(
range 4000-400 cm ). H and C NMR spectra were
recorded at 400 MHz, with a Varian 400 Mercury Plus
spectrometer using CDCl as the solvent and tetramethyl
3
silane (TMS) as the internal standard at Central Analítica,
Departamento de Química, UFSC (Florianópolis-SC,
Brazil). The specific rotations were determined using a
Schmidt + Haensch polarimeter. The pH of the water was
measured with a Marte MB10 pH meter.
The progress of the transesterification reaction was
followed by thin-layer chromatography (TLC) using
n-hexane:ethyl acetate (7:3 v/v) as the eluent. The
conversion, as well as the enantiomeric excesses of the
formed products was determined with a gas chromatograph
(
(
GC-14B Shimadzu) equipped with a chiral column
RT – BetaDex – SM, 30 m × 0.32 mm × 0.25 µm,
Restek) and H was used as the carrier gas with a pressure
2
of 75 kPa. The injector and detector temperature was
the product (ee ), and the conversion degree (c) according
to the method described by Chen et al.
After purification using silica gel column
chromatography(n-hexaneandethylacetate9:1v:v)thepure
p
31
2
2
30 ºC. Initial and final column temperatures were 100 and
00 ºC, respectively, and the temperature was increased
-1
by 3 ºC min . The retention times observed for S-(–) and

1
562
Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases
J. Braz. Chem. Soc.
(
R)-1-phenylethyl acetate was obtained. This compound
lower conversions into (R)-3 and higher ee and E-values
were obtained in comparison with free lipases (Table 1,
entries 8-11). The conversion degrees ranged from 1-10%,
p
1
was also analyzed by H NMR, IR, chiral GC and specific
rotation.
resulting in ee > 99% and E-values > 200.
p
Results and discussion
Using the fungi UEA_53 and UEA_115, lower
conversion degrees to the product were obtained, these
being 1 and 4%, respectively. However, these Amazonian
fungi showed a promising enantioselective capacity, since
Screening of enzymes
In the first step of this study, three commercially available
lipases (from C. antarctica B, B. cepacia and C. viscosum),
two natives lipases (from R. oligosporus and A. niger) free or
immobilized in ginger starch film (GS) and two mycelium-
bound lipases isolated from the Amazonian fungi UEA_53
good ee (> 99%) and E (> 200) values were obtained
p
29
(Table 1, entries 6-7). Zanotto et al. have also studied
the resolution of (R,S)-2-octanol via transesterification
reactions with vinyl acetate catalyzed by these fungi.
Owing to some advantages of using immobilized
lipases, like the simple recovery of the products and
reactants, easy separation of enzyme from product and the
possibility of continuous and repeated use of the catalyst,
the BCL/GS system was chosen as the biocatalyst for the
further investigations.
(A. aculeatum) and UEA_115 (Colletotrichum sp ) were
tested for the resolution of the (R,S)-1-phenylethanol (1)
with vinyl acetate in n-hexane at 35 °C (Table 1). In all
transesterification reactions catalyzed by free or immobilized
lipases, the stereopreference was towards the (R)-enantiomer.
These data followed the empirical rule proposed by
Kazluaskasetal.andareinagreementwithdatareportedinthe
literature which showed that CAL-B and other lipases have a
Effect of acyl donor
32,33
highly pronounced catalytic preference for R-enantiomer.
It is generally described that the acyl group from
the acylating agents can affect the reaction rate and the
enantioselectivity in a lipase-catalyzed transesterification
The acetylation reaction performed in the absence of the
biocatalyst led to the recovery of the starting material.
3
4
reaction. Thus, in order to examine the effect of acyl
donors, different acyl donors including vinyl esters
Table 1. Resolution of (R,S)-1-phenylethanol with vinyl acetate catalyzed
by various free and immobilized lipases
(vinyl acetate and the iso-propenyl acetate), ethyl acetate
a
entry
Biocatalyst
ROL
c / %
14
53
56
55
51
1
ee / %
ee / %
E-value
> 200
39
and acetic anhydride were used in the resolution of the
(R,S)-1-phenylethanol catalyzed by B. cepacia lipase
immobilized in the ginger starch film in n-hexane. The
influence of the reaction time was previously evaluated
from 8 to 72 h at 35 °C, and no significant change in the
conversion degrees was observed (9-10%). These results
may be analyzed considering that for the time studied the
conversion degree was relatively low. However, it is worth
mentioning that the enantioselectivity of the process was
s
p
1
2
3
4
5
6
7
8
9
14
89
> 99
95
97
2
> 99
86
CVL
ANL
98
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
BCL
99
CALB
99
UEA_53
UEA_115
CVL/GS
ROL/GS
ANL/GS
BCL/GS
> 99
> 99
> 99
> 99
> 99
> 99
4
6
6
3
1
4
maintained, forming the (R)-ester 3 with ee > 99% and
p
1
1
0
1
10
9
8
E-values > 200.Thus, a reaction time of 24 h was selected for
the subsequent studies and the results are shown in Table 2.
As observed in Table 2, the selectivity and conversion
degrees reached after 24 h reaction were greatly influenced
by the nature of the acyl donor. The highest conversion
degree (9%) was obtained when vinyl acetate and
iso-propenyl acetate were used, respectively, forming the
13
Reaction conditions: (R,S)-1 (0.6 mL, 5 mmol), vinyl acetate (0.5 mL,
mmol), CVL (20 mg g of starch), BCL, ANL, ROL, CALB, UEA_53
and UEA_115 (60 mg g of starch), n-hexane (25 mL), 35 °C, 72 h;
-
1
5
-
1
a
calculated as described in reference 31.
As shown in Table 1, both free and immobilized
lipases were able to convert (R,S)-1 to (R)-1-phenylethyl
acetate (3). The highest conversion degrees to (R)-3 were
achieved when the lipases CVL, ANL, BCL and CALB
were employed in a free form, after 72 h of reaction,
(R)-ester 3 with ee > 99% (Table 2, entries 1-2). Using ethyl
p
acetate and acetic anhydride, the values for the conversion
degree to product were 5 and 4%, with ee > 99% and 38%,
p
respectively (Table 2, entries 3-4).
these being in the range of 51-56% with the ee values of
These results are also in agreement with those reported
in the literature for enzyme-catalyzed esterification and
transesterification reactions in organic media, where vinyl
p
86-99% (Table 1, entries 2-5). When the lipases CVL, ROL,
ANL and BCL were immobilized in ginger starch film,

Vol. 22, No. 8, 2011
Hoffmann et al.
1563
Table 2. Effect of acyl donor on the transesterification of (R,S)-1 with vinyl acetate catalyzed by the BCL/GS system
a
entry
Acyl donor
vinyl acetate
c / %
ee / %
ee / %
E
s
p
1
2
3
4
9
5
9
4
13
13
17
13
> 99
> 99
> 99
38
> 200
> 200
> 200
2.5
ethyl acetate
iso-propenyl acetate
acetic anhydride
-1
a
Reaction conditions: (R,S)-1 (0.6 mL, 5 mmol), acyl donors (5 mmol), BCL (60 mg g of starch), n-hexane (25 mL), 35 °C, 24 h; calculated as described
in reference 31.
acetate is considered to be a good choice for the acyl donor.
In this case, the leaving group is an enol that immediately
tautomerizes to a carbonyl compound (acetaldehyde),
(10:1, 2:1) the conversion degree to the product ranged from
2 to 6% with an ee value of > 99%. These results show the
p
effect of substrate inhibition caused by the action of the
alcohol on the catalyst, causing a decrease in the conversion
18,35
thereby driving the reaction to completion.
33
36
degree. Ong et al. and Trubiano et al. reported a similar
behavior for the enantioselective esterification of racemic
ketoprofen in organic medium and ethyloleate synthesis in
a solvent-free system, respectively, both using a commercial
immobilized lipase from C. antarctica B (Novozyme 435).
Considering these results, an alcohol:acyl donor molar ratio
of 1:1 was selected for the subsequent experiments.
Effect of alcohol:acyl donor molar ratio
To evaluate the influence of the (R,S)-
1-phenylethanol:vinyl acetate molar ratio in the resolution
of the (R,S)-1-phenylethanol mediated by lipase from B.
cepacia (BCL) immobilized in ginger starch film (GS) in
n-hexane, the alcohol:acyl donor molar ratio was varied
from 1:1 to 1:10 (Figure 1).
Effect of temperature
Another key factor which affects the rate of a reaction
catalyzed by an enzyme is the temperature. The temperature
influences the activity, selectivity and stability of the
37
biocatalyst as well as the reaction equilibrium. To evaluate
the influence of the temperature in the resolution of the
(R,S)-1-phenylethanol with vinyl acetate catalyzed by the
system BCL/GS in n-hexane, the reaction temperature was
varied within the range of 22 to 40 °C. Figure 2 shows the
time course for (R)-3 ester formation at various temperatures.
Figure 1. The effect of the (R,S)-1-phenylethanol/vinyl acetate molar ratio
on the production of (R)-1-phenylethyl acetate (3) using the BCL/GS as
catalytic system. Filled and open symbols (¢, £) represent the conversion
degrees and enantiomeric excesses (ee ), respectively. Reaction conditions:
p
(
R,S)-1 (0.6-6.0 mL, 5-50 mmol), vinyl acetate (0.5-4.6 mL, 5-50 mmol),
-1
BCL (60 mg g starch), n-hexane (25 mL), 35 °C, 24 h.
It is apparent from Figure 1 that using the catalytic
system LBC/GS, the conversion to (R)-3 increased as the
(R,S)-1-phenylethanol/vinyl acetate molar ratio increased.
The highest conversion degrees obtained were 24% using
the molar ratio of 1:8. However, there was a small decrease
in the enantiomeric excess (ca. 95%) with the use of this
molar ratio.When the transesterification reactions catalyzed
by BCL/GS were carried out with an excess of alcohol
Figure 2. The influence of the temperature on the enzymatic resolution
of (R,S)-1-phenylethanol mediated by BCL/GS. Filled and open symbols
(¢, £) represent the conversion and enantiomeric excesses, respectively.
Reaction conditions: (R,S)-1 (0.6 mL, 5 mmol), vinyl acetate (0.5 mL,
-1
5 mmol), BCL (60 mg g of starch), n-hexane (25 mL), 24 h.

1
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Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases
J. Braz. Chem. Soc.
The results showed a significant variation in the
selectivity was achieved (E = 27). When dichloromethane,
ethyl ether, acetonitrile and 1,4-dioxane were used, the
conversion degrees were low (< 5%), but the selectivity
was high (E > 200) (Table 3, entries 3-6). This data is
in agreement with those reported in the literature, where
high hydrophobic solvents with logP values > 4 are
considered the most suitable solvents for use in biocatalytic
processes. The solvents with logP values between 2 and
4 are moderately effective. Polar solvents with logP < 2
conversion to (R)-ester 3 when the reaction was performed
at different temperatures. As the temperature increased
from 22 to 28 ºC, the conversion degree increased and
a value of 14% was reached at 28 °C in 24 h reaction. A
further increase in temperature led to a small decrease
in the conversion to (R)-3. The enantioselectivity of the
immobilized LBC was not affected by the change in
temperature in the range of 22-40 °C, with ee being > 99%.
p
39,40
Based on these results, a temperature of 28 ºC was
considered as appropriate to evaluate the effect of the
organic solvent as well as the BCL immobilization in ginger
starch/PEO blends.
are often ineffective,
and this may be attributed to the
minimum distortion of the hydration layer around the
enzyme. However, such solvents, due to their high affinity
for water, might strip the essential hydration layer around
the enzyme which preserve its conformation flexibility to
39,40
Effect of organic solvent
some extent, thus decreasing the enzyme activity.
Theeffectofusingco-solvents(ionicliquidandglycerol)
in the resolution of (R,S)-phenylethanol was also studied.
The results showed that when the reaction was performed
using a solvent mixture of n-hexane/[BMIm][PF6]
(9:1 v:v), the conversion to (R)-3 ester was lower (6%)
than that obtained using pure n-hexane (14%), while an
excellent enantioselectivity was maintained. These results
The most important criteria for solvent selection in
biocatalysis are a high substrate and product recovery
capacity and biocompatibility, although other characteristics
such as chemical and thermal stability, nonbiodegradability,
a nonhazardous nature and low market price are also
38
desirable. The most commonly used parameter to classify
solvents in terms of biocompatibility is the logP value,
which is defined as being the partition coefficient of a given
showed that the n-hexane/[BMIm][PF ] (9:1 v:v) mixture
6
has a negative influence on the transesterification reaction
of (R,S)-1 with vinyl acetate compared to the reaction using
pure organic solvents (Table 3, entry 7).
39
compound in a two-phase n-octanol and water system.
Thus, in order to study the effect of organic solvents
in the resolution of (R,S)-phenylethanol catalyzed by
B. cepacia lipase immobilized in ginger starch film, a
series of solvents was chosen covering a wide range of
logP values (Table 3).
As observed from these results, the percentage of
ester formation and selectivity were strongly dependent
on the solvent. When the less polar solvents such as
n-heptane and n-hexane (logP ≥ 3.5) were employed in
the resolution of (R,S)-1, moderate conversion degrees
were obtained, these being 12 and 14%, respectively
41
Hara et al. obtained similar results in the acylation
of (R,S)-1-phenylethanol and other racemic alcohols with
vinyl acetate catalyzed by B. cepacia immobilized in a
sol-gel (BCLxero) and as a CLEA (BCL-CLEA) in organic
media and/or in mixtures of organic solvent/ionic liquids.
Using BCL-CLEA, the conversion to (R)-ester slightly
decreased in a mixture of [BMIM][PF ]/toluene (2:1) (15%)
6
and [BMIM][PF ]/toluene (1:2) (20%) compared to the
6
pure toluene (50%).
When the solvent mixture of n-hexane/glycerol (9:1 v:v)
was used, the degree of conversion to the (R)-3 ester was
(Table 3, entries 1-2). However, on using n-heptane a lower
Table 3. Effect of organic media on the conversion to (R)-3 ester using the BCL/GS catalytic system
a
b
entry
Solvents
n-heptane
n-hexane
CH Cl
logP
4.0
c / %
12
14
2
ee / %
E
p
1
2
3
4
5
6
7
8
91
27
3.5
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 200
> 200
> 200
> 200
> 200
> 200
> 200
1.5
2
2
diethyl ether
acetonitrile
0.83
−0.33
−1.1
−
2
5
1,4-dioxane
4
c
n-hexane/[BMIm][PF6]
6
c
n-hexane/glycerol
−
23
-
1
a
Reaction conditions: (R,S)-1 (0.6 mL, 5 mmol), vinyl acetate (0.5 mL, 5 mmol), BCL (60 mg g of starch), 25 mL organic solvent; from reference 39;
b
c
calculated as described in reference 31; 10 mL n-hexane:LI or n-hexane:gycerol (9:1 v/v), 28 °C, 24 h.

Vol. 22, No. 8, 2011
Hoffmann et al.
1565
much better, being 23% with an ee > 99% (Table 3,
It is evident from Figure 3 that the conversion to
(R)-ester 3 was influenced by the proportion of PEO in
ginger starch/PEO blends and the reaction medium. Using
n-hexane, when the proportion of PEO increased from 0 to
90%, an increase in the conversion to (R)-3 ester from 14
to 25% was observed. The highest conversion was obtained
when BCL was immobilized in a film formed only with
PEO, this being of 56%. However, using a mixture of
n-hexane/glycerol (9:1 v/v), better conversion degrees were
achieved regardless of the blend composition, presenting
values of 22-40%. When BCL was immobilized in a film
formed with PEO, the conversion degree was 40%, that is
lower than that obtained when the solvent was n-hexane,
probably due to the presence of glycerol.
p
42
entry 8). Andrade et al. also observed an improvement
in the enzymatic performance of Aspergillus terreus and
Rhizopus oryzae in the enantioselective bioreduction of
haloacetophenones using glycerol as a co-solvent. In most
cases, glycerol has demonstrated its potential to improve
the conversion (up to > 99%) and enantioselectivity
(up to > 99%) when compared to reactions in aqueous or
other aqueous-organic media (THF, diethyl ether, toluene,
DMSO and acetonitrile).
Thus, glycerol was shown to be an excellent alternative
for use as a co-solvent in the resolution of (R,S)-
phenylethanol catalyzed by B. cepacia lipase immobilized
in ginger starch film. Moreover, the choice of this co-solvent
is reinforced by its increasing availability on the market,
Regardless of the blend composition or the reaction
42
since it is a by-product of biodiesel production.
media, the ee values in all studies were > 99%, which
p
indicated an excellent enantioselectivity of the process.
This increase in the product conversion may be related to
both the film porosity and the organic media. Recently,
Effect of BCL immobilization in ginger starch/PEO blends
47
As previously mentioned, the immobilization of lipases
in solid supports has been a topic of active research for
several decades.A variety of supports, including inorganic
Hara et al. reported the resolution of (R,S)-1 catalyzed
by BCL immobilized in different supports, such as active
carbon coal and alumina, among others. The conversion
degrees and E-values were highly dependent on the support
and ranged from 1-50% with E-values from 7 > 200,
1
8,43
20,44
45
materials,
natural
or synthetic polymers and
4
6
polymeric blends, has been explored with a view to
their use as supports for enzyme immobilization in order
to improve the catalytic efficiency and reduce the cost of
the process.
48
respectively. Similar results were obtained by Shah et al.,
showing the great influence of the support specific surface
area and the immobilization technique.
Thus, hereinthecatalyticefficiencyofBCLimmobilized
in polymeric ginger starch/PEO blends using different
compositions was evaluated in the resolution of (R,S)-
phenylethanol with vinyl acetate in n-hexane and in a
mixture of n-hexane/glycerol (9:1 v/v) at 28 °C (Figure 3).
In this study, the SEM micrographs revealed an increase
in the porosity as the PEO composition increased (results
not presented). Thus, a higher porosity seems to favor the
diffusion of reactants and products, thereby favoring the
enzymatic catalysis, being in agreement with data reported
15,47,49
in the literature.
Reusability of the LBC immobilized on ginger starch/PEO
blend
From an economic point of view, the reuse of an enzyme
constitutes one of the main advantages of the biocatalyst
immobilization process. Thus, BCL was immobilized in
the ginger starch/PEO blend (7:3 m/m) and this system
was reused in the resolution of the (R,S)-1-phenylethanol
with vinyl acetate in n-hexane at 28 °C. After 24 h for
each run, the immobilized lipase was collected, washed
with n-hexane in order to remove any substrate or product
retained in the support, and reused for 6 successive cycles.
The results are shown in Figure 4.
Figure 3. The effect of the PEO content in the ginger starch/PEO blends
on the enzymatic resolution of (R,S)-1-phenylethanol (1) with vinyl acetate
in n-hexane (¢, £) and n-hexane/glycerol (9:1 v:v) (, ). Filled (¢, )
and open symbols (£, ) represent the conversion and enantiomeric
excess, respectively. Reaction conditions: (R,S)-1 (0.6 mL, 5 mmol), vinyl
As can be seen in Figure 4, the degree of conversion to
(
R)-3 ester ranged from 26-16%, presenting an excellent
-
1
selectivity with ee values of > 99% (E > 200) during the
acetate (0.5 mL, 5 mmol), BCL (60 mg g of ginger starch/PEO blends),
p
n-hexane (25 mL) or n-hexane:gycerol (9:1 v/v) (10 mL), 28 °C, 24 h.
reuse. These results can be attributed to BCL, maintaining

1
566
Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases
J. Braz. Chem. Soc.
e Tecnologia (INCT-Catalysis) which provided financial
support and scholarships (M. G. N, I. H. and V. D. S.).
We also thank Amano Pharmaceutical Co. (Japan),
Novozymes (Brazil) and Professor Patrícia O. Carvalho
from Universidade de São Francisco (USF, Brazil) for the
donation of native lipases, and also Professor Sandra P.
Zanotto from Universidade Estadual do Amazonas (UEA,
Brazil) for the donation of mycelia.
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2
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Figure 4. Reusability of immobilized B. cepacia lipase in the resolution of
R,S)-1 to afford the (R)- 3 ester. Filled and open symbols (¢, £) represent
(
the conversion degrees and enantiomeric excesses (ee ), respectively.
p
3. Csajági, C.; Szatzker, G.; Toke, E. R.; Ürge, L.; Darvas, F.;
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Reaction conditions: (R,S)-1 (0.6 mL, 5 mmol), vinyl acetate (0.5 mL,
-
1
5
mmol), BCL (60 mg g of ginger starch/PEO blend 7:3 m/m), n-hexane
(
25 mL), 28 °C, 24 h.
its catalytic activity when immobilized in this blend,
which represents, as expected, another advantage of this
process.
5
. Sharma, R.; Chisti,Y.; Barnerjee, U. C.; Biotechonol. Adv. 2001,
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Lipases from different sources were immobilized on
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(
(
R,S)-phenylethanol. Lipase from Burkolderia cepacia
BCL) was selected to evaluate the influence of various
reaction conditions. The conversion degree and the
enantioselectivity were shown to be dependent on the acyl
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p
on the ginger starch/PEO blend (7:3 m/m) preserved the
enzyme activity and was reused for 6 cycles.
1
2. Santos, A. A.; Costa, C. E.; Princival, J. L.; Comasseto, J. V.;
Tetrahedron: Asymmetry 2006, 17, 2252.
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1
3. Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan,
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011.
Submitted: February 9, 2011
Published online: May 12, 2011

J. Braz. Chem. Soc., Vol. 22, No. 8, S1-S4, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Supplementary Information
SI
Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases
Immobilized in Starch Films
Isabel Hoffmann, Vanessa D. Silva and Maria da G. Nascimento*
Departamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil
Characterization by optical microscopy of grains extracted
from ginger starch
Chemical synthesis of racemic (R,S)-1-phenylethyl acetate
(3)
O
O
8
3
1
4
2
7
5
6
(R,S)-3
The racemic (R,S)-1-phenylethyl acetate was prepared
by chemical acetylation of the precursor alcohol (R,S)-1
(4.9 mL, 4.1 mmol) employing acetic anhydride (19.4 mL,
2
0.5 mmol) in dichloromethane (30 mL) and acetic acid
Figure S1. Micrograph of ginger starch grains (Zingiber officinale),
magnification of 200 ×. No scale given.
1
as the catalyst, as described in the literature. A yellow oil
was obtained in 70% yield after purification by columm
chromatography on silica gel using a mixture of n-hexane
and ethyl acetate (9:1 v:v) as the eluent. This compound
was used as a standard in the chiral gas chromatography
1
analysis and was analyzed by H NMR, IR, chiral GC
and specific rotation. H NMR (CDCl , 400 MHz) d 7.23
1
3
(
m, 5H), 5.82 (q, 1H, J 6.8), 2.10 (s, 3H), 1.50 (d, 3H,
-
1
J 6.8 Hz); IR (KBr) nmax/cm : 3064-2872, 1734, 1242;
chiral GC t /min: 5.0 and 5.4; [a] 0.00 (2.56 × 10 CHCl ).
-2
R
D
3
*e-mail: graca@qmc.ufsc.br

S2
Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases Immobilized
J. Braz. Chem. Soc.
1
Figure S2. H NMR spectrum of (R,S)-3 (CDCl , 400 MHz).
3
Figure S3. IR spectrum of compound (R,S)-3 (film).

Vol. 22, No. 8, 2011
Hoffmann et al.
S3
Figure S4. Chromatogram of the racemic mixture of (R,S)-3:(S)-3 t 5.0 and (R)-3 t 5.4 min.
R
R
Enzymatic resolution of (R,S)-1
Figure S5. Chromatogram of (R,S)-1-phenylethanol resolution catalyzed by LBC immobilized in ginger starch film. (R)-3 t 5.4 min. (R)-1 t 5.8 min,
R
R
(
S)-1 t 6.1 min.
R

S4
Enantioselective Resolution of (R,S)-1-Phenylethanol Catalyzed by Lipases Immobilized
J. Braz. Chem. Soc.
Spectral data of (R)-1-phenylethyl acetate (3)
References
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. Fessender, R. J.; Fessenden, J. S.; Techniques and Experiments
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2
. Chênevert, R; Pelchat, N; Morin, P; Tetrahedron: Asymmetry
3
2
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1
4
2
7
5
6
(
R)-3
1
H NMR (CDCl , 400 MHz) d 7.23 (m, 5H), 5.82 (q,
3
1
H, J 6.8), 2.10 (s, 3H), 1.50 (d, 3H, J 6.8 Hz); IR (KBr)
-1
nmax/cm : 3064-2872, 1734, 1242; chiral GC t /min: 5.4;
R
2
[
a] +36 (0.03 CHCl ), [a] +43 (2.1 CHCl ).
D
3
D
3