Pseudomonas sp. lipase immobilized in polymers versus the use of free enzyme in the resolution of (R,S)-methyl mandelate

Article abstract of DOI:10.1016/S0040-4039(02)01057-2

In the present report, we describe the preparative-scale resolution of (R,S)-methyl mandelate by acylation with vinyl acetate catalyzed by free Pseudomonas sp. lipase or immobilized in poly(ethylene) oxide (PEO) and agar gel, in organic media. Under exper

Full text of DOI:10.1016/S0040-4039(02)01057-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 5225–5227
Pseudomonas sp. lipase immobilized in polymers versus the use
of free enzyme in the resolution of (R,S)-methyl mandelate
Neide Queiroz and Maria da Grac¸a Nascimento*
Departamento de Qu´ımica, Universidade Federal de Santa Catarina, Floriano´polis, SC 88040-900, Brazil
Received 9 November 2001; revised 29 November 2001; accepted 6 June 2002
Abstract—In the present report, we describe the preparative-scale resolution of (R,S)-methyl mandelate by acylation with vinyl
acetate catalyzed by free Pseudomonas sp. lipase or immobilized in poly(ethylene) oxide (PEO) and agar gel, in organic media.
Under experimental conditions the method was very effective using the enzyme immobilized in PEO. The degree of conversion was
near 50% and yielded both enantiomers with practically 100% of optical purity. © 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
organic media may be circumvented by immobilization
in a solid support. In general, immobilized enzymes are
more stable in relation to changes in pH and tempera-
ture when compared with soluble forms. Moreover,
they remain stable for months and the catalyst can be
used repetitively. Thus, they are easy to handle and can
be recuperated from the solution, which makes the
process practical and economically viable. Enzymes can
be immobilized in organic and/or inorganic materials to
be used for synthetic purposes. These materials include,
glass, silica, alumina, biopolymers, organogels and
chrysotille.5–8
Mandelic acid and its derivatives are useful chiral
reagents widely employed both for synthetic purposes
and for stereochemical investigations. In fact, the enan-
tiomers of mandelic acid can be used for the resolution
of alcohols, amines, etc., thus permitting the determina-
tion of enantiomeric excess by HPLC or NMR.1–3
Lipases are versatile catalysts, as they catalyze a
plethora of reactions such as esterification, amidation,
and transesterification of esters in addition to the natu-
ral reaction of fatty ester hydrolysis. Applications of
lipases include production of food additives, chiral
intermediates, and pharmaceutical products.4 Many of
the characteristics which make their use difficult in
In this work, Pseudomonas sp. lipase (PSL) was immo-
bilized in two polymers, poly(ethylene oxide) (PEO)
Scheme 1. Acylation reaction of R,S-methyl mandelate.
* Corresponding author. Tel.: +55-48-3319219 r. 212; fax: +55-48-3319711; e-mail: graca@qmc.ufsc.br
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01057-2

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N. Queiroz, M. G. Nascimento / Tetrahedron Letters 43 (2002) 5225–5227
and agar of which the latter is a complex mixture of
polysaccharides including agarose as the principal
gelling component. These systems were used in the
acylation reaction of R,S-methyl mandelate with vinyl
acetate in several organic solvents (Scheme 1). The
enzyme immobilization in PEO was performed by dis-
solving 500 mg of the polymer and 100 mg of PSL in 20
mL of water, with further solvent evaporation forming
a film, which was then cut into several regular sections.
The enzyme immobilization in agar was performed by
dissolving 0.4 g of agar into 9.0 mL of water at 100°C.
After the system reached ꢀ50°C, 100 mg PSL was
added and it was shaken until it became homogeneous.
Regular sections of agar gel containing enzyme were
obtained by passing it through a wire sieve at room
temperature. The reaction’s progress and enantiomeric
excess values were measured by gas chromatography
(GC) equipped with a chiral column (CP-chirasil-Dex
CB) using H2 as the carrier gas, with a detector, an
injector set at 275°C, and a column set to a tempera-
tures of 80–140°C (2°C/min). The enantiomeric ratio
(E) values were calculated from the degree of conver-
sion and the e.e. of the product, according to Sih,
Sharpless and Fajans’ equation.9
results showed the dependence of the enzymatic activity
according to the support employed. No product was
detected using PSL immobilized in agar gel. However,
immobilization of PSL in PEO increased its activity.
One explanation for this result was related to diffusion
process of the reagents and products from the support
to the reaction media. Using PEO, the diffusion was
faster since it formed the products in a shorter time
than when agar gel was used. All reactions were per-
formed under the same experimental conditions. Table
1 shows the effects of organic solvents on the enantiose-
lectivity of PSL free and immobilized in PEO in the
resolution of (R,S)-methyl mandelate with vinyl acetate
as the acylating agent. The results showed that PSL had
the same stereochemical preference for the S enan-
tiomer, regardless of the system used. It was verified
that the solvent had a profound effect on the % conver-
sion of the product, but had no effect on the enzyme
enantioselectivity. In all solvents, R-4 and S-3 were
obtained, confirmed by GC comparison with an
authentic sample. Kinetic resolution of 1 can be better
achieved by using PSL immobilized in PEO. S-3 was
obtained with e.e. >99% at 50% conversion with 96 h
reaction, while only 13% conversion was obtained with
the free form. Considering the nature of the organic
solvents and support, isopropyl ether as solvent and
PEO as support were the most appropriate for the
biocatalytic conversion of 1 (Fig. 1). The influence of
temperature was also evaluated in the acylation of 1
using PSL immobilized in PEO and isopropyl ether as
solvent. No macroscopic changes in the physical
integrity of the system PSL/PEO were observed after 72
h at 35°C. The variation of the conversion percentage
with temperature was evaluated by measuring the
2. Results and discussion
Many devices are commonly suggested to improve the
performance of the catalytic system. Thus, for compar-
ative purposes, PSL was immobilized in agar gel and in
PEO film. As reported by Wang et al., the lipase
immobilization can increase the reaction rate.10 Our
Table 1. Effects of solvents on the immobilized and free Pseudomonas sp.a lipase-catalyzed acylation of R,S-methyl mande-
late 1 with vinyl acetate
Solvents
t (h)
E.e.sb (%)
PSL/PEO
E.e.pc (%)
PSL/PEO
Conversion (%)
PSL/free PSL/PEO
Ed
PSL/PEO
PSL/free
PSL/free
PSL/free
Isopropyl
ether
24
2
33
99
99
1
25
200
274
48
72
96
24
48
72
96
3
9
15
0
1
6
7
0
1
3
47
69
99
17
39
56
72
20
33
47
61
5
99
99
99
0
99
99
99
99
99
99
99
99
99
99
99
99
99
99
99
3
8
13
0
1
6
7
0
1
3
32
41
50
17
28
36
42
15
25
32
38
5
205
216
230
0
200
211
214
0
200
205
205
0
315
413
1057
242
290
349
429
242
274
315
370
209
214
227
236
Hexane
0
99
99
0
99
99
99
0
0
99
99
tert-Butanol 24
48
72
96
24
48
72
96
3
0
0
2
3
0
0
2
Acetone
7
13
14
7
12
15
0
203
203
2
2
Reactions were conducted in each solvent (30 mL) containing R,S-methyl mandelate (0.0015 mol), lipase PS (100 mg) and vinyl acetate (0.015
mol) at 25°C.
a Amano 30, 30000 U/g.
b Enantiomeric excess of the recovered product 4.
c Enantiomeric excess of compound 3.
d Enantiomeric ratio, parameter describes the selectivity of enzyme between two enantiomers.

N. Queiroz, M. G. Nascimento / Tetrahedron Letters 43 (2002) 5225–5227
5227
Acknowledgements
This work was supported by the Federal University of
Santa Catarina (UFSC-Brazil) and the National
Research Council (CNPq-Brazil). We also thank
Amano Pharmaceutical Co. and Toyo Jozo Co. (Japan)
for their generous gifts of lipases and Dr. Valdir Soldi
for his gift of PEO.
References
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Figure 1. Immobilized Pseudomonas sp. lipase in PEO versus
free form in the resolution of (R,S)-methyl mandelate at
25°C.
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formed products at definite times (24, 48 and 72 h). In
the case of 48 h at 35°C, the (S)-(+)-3 was obtained
with 50% conversion, e.e. >99% and E >200, the yield
being 18% higher than at 25°C. However, 96 h at 25°C
was necessary to reach 50% conversion. This indicates
that the enzyme had higher catalytic activity at 35°C.
Therefore, it is clear that poly (ethylene oxide) when
used as a polymeric support to increase lipase reactiv-
ity, is far more efficient than the corresponding free
form and agar gel under the same conditions.
In conclusion, immobilization of PSL in PEO proves to
be an excellent methodology for the production of
(S)-(+)-3 on a laboratorial scale. In addition, the sup-
port increases the PSL reactivity, another further
advantage of this system is that it can be re-used three
times without losing its reactivity and enantioselectivity.