Microwave-promoted lipase-catalyzed reactions. Resolution of (±)-1-phenylethanol

Article abstract of DOI:10.1021/jo960309u

Microwave irradiation increased enzymatic affinity and selectivity of supported lipases in esterification and transesterification reactions carried out in dry media and at temperature near 100°C.

Full text of DOI:10.1021/jo960309u

7746
J . Org. Chem. 1996, 61, 7746-7749
Micr ow a ve-P r om oted Lip a se-Ca ta lyzed Rea ction s. Resolu tion of
(()-1-P h en yleth a n ol
J ose´-Ramon Carrillo-Munoz,1 Denis Bouvet, Eryka Guibe´-J ampel,* Andre´ Loupy,* and
Alain Petit
Laboratoire des Re´actions Se´lectives sur Supports, Institut de Chimie Mole´culaire d’Orsay,
URA CNRS 478, Universite´ Paris-Sud, Baˆtiment 410, 91405 Orsay Ce´dex, France
Received February 14, 1996X
Microwave irradiation increased enzymatic affinity and selectivity of supported lipases in
esterification and transesterification reactions carried out in dry media and at temperature near
100 °C.
In tr od u ction
librium shifting by evaporation of light polar molecules
which are strongly interacting with the electromagnetic
field. Electromagnetic field of high frequency (2450 MHz)
induces molecular rotation which is accompanied by
intermolecular friction of polar species and subsequent
dissipation of energy by heating in the core. Toward this
aim, microwave activation has to be coupled with solvent-
free methods to facilitate and to keep the procedure safe.7
The use of focused microwaves allows one to combine the
advantages of an homogenous field with very high
energetic yields8 (therefore without local heating effects)
and with close control of the reaction temperature.9
Results are then compared with those obtained under
classical heating strictly under the same conditions.
In recent years, chemoenzymatic methodology has
become a standard technique for the preparation of a
wide variety of enantiomerically pure molecules.2 Among
them, lipase-catalyzed acylations and transacylations
represent an important class of enzymatic transforma-
tions in organic chemistry. The use of biological catalysts
for technological purposes requires that enzymes be
stable and functional in nonphysiological environments.
Reactions in organic media, under pressure or at elevated
temperatures, are increasingly used.3a
Until now no general correlation of reaction enantio-
selectivity with physicochemical constants of solvents
could be established.4 Temperature has long been known
to be inversely correlated with enzyme enantiospecificity
(E). However, since 1992, numerous examples in the
opposite way have been accumulated.3 To date, temper-
ature dependent variations in E values are employed as
a practical means to achieve reaction stereoselectivity
even near 100 °C.3a
Resu lts a n d Discu ssion
To work under dry media conditions, enzymes were
immobilized on solid supports10 of adequate pH; thus only
weak interactions with microwaves would occur, avoiding
high-temperature enhancement. Four types of solid
supports were a priori considered: three of them are
mineral (Florisil, Celite 545, and Hyflo Super Cel )
HSC11) and the last one is organic (a polypropylene resin
) Accurel). Their behavior was tested under microwaves
for 30 min at a power level of 90 W (Figure 1 and Table
1).
The devising of enzymatic systems for use in organic
media is in constant progress. New thermostable en-
zymes now available through recombinant DNA technol-
ogy (Novozym SP 435) or isolation from thermophilic
microorganisms allow one to work at relatively high
temperatures.19
On the basis of experimental results, Florisil was
eliminated on the ground that it absorbs microwaves too
strongly. We subsequently found HSC and Accurel to
be the best supports for lipase-catalyzed transesterifica-
tion, and only results related to these two supports will
be presented in this report.
The lipases selected for our study were the Pseudomo-
nas cepacia lipase (LP) and Candida antarctica lipase
(SP 435).
One of the major limitations of enzymatic synthesis is
the reversible nature of the reactions which result in low
rates and low selectivities.2 A technique to displace the
equilibrium toward the desired direction, which has
industrial applications, is to continuously remove mol-
ecules such as water or ethanol formed in the process by
azeotropic distillation5 or evaporation under reduced
pressure.6
The purpose of this paper is to show it is possible to
take advantage of microwave exposure to induce equi-
(5) Gerlach, D.; Schreier, P. Biocatalysis 1989, 2, 257-263.
(6) (a) Adelhorst, K.; Bjo¨rkling, F; Godtfredsen, S. E.; Kirk, O.
Synthesis 1990, 112-115. (b) Bjorkling, F.; Godtfredsen, S. E.; Kirk,
O. J . Chem. Soc., Chem. Commun. 1989, 934-935. (c) Ohrner, N.;
Martinelle, M.; Mattson, A.; Norin, T.; Hult, K. Biotech. Lett. 1992,
14, 263-268. (d) Ohrner, N.; Martinelle, M.; Mattson, A.; Norin, T.;
Hult, K. Biocatalysis 1994, 9, 105-114.
X Abstract published in Advance ACS Abstracts, October 1, 1996.
(1) On leave from Department of Organic Chemistry, Universidad
de Castilla la Mancha, Ciudad Real, Spain.
(2) (a) Faber, K.; Riva, S. Synthesis 1992, 895-910. (b) Santaniello,
E.; Ferraboschi, P.; Grisenti, P.; Manzocchi, A. Chem. Rev. 1992, 92,
1071-1140. (c) Wong, C. H.; Whitesides, G. M. Enzymes in Synthesis
Organic Chemistry; Tetrahedron Organic Chemistry Series, Vol. 12;
Pergamon: New York, 1994. (d) Wu, S. H.; Guo, Z.-W.; Sih, C. J . J .
Am. Chem. Soc. 1990, 112, 1990-1995. (e) Chen, C. S.; Sih, C. J .
Angew. Chem., Int. Ed. Engl. 1989, 28, 695-707.
(3) (a) Adams, M. W. W.; Kelly, R. M. Biocatalysis at Extreme
Temperatures. ACS Symp. Ser. 1992, 498. (b) Phillips, R. S. TIBTECH
1996, 14, 13-16. (c) Parmar, V. S.; Prasad, A. K.; Singh, P. K.; Gupta,
S. Tetrahedron: Asymmetry 1992, 3, 1395-1398.
(4) Orrenius, C.; Norin, T.; Hult, K.; Carrea, G. Tetrahedron:
Asymmetry 1995, 6, 3023-3030.
(7) (a) Bram, G.; Loupy, A.; Villemin, D. Solid Supports and
Catalysts in Organic Synthesis; Ellis Horwood, Ed.; PTR Prentice
Hall: London, 1992; pp 302-326. (b) Loupy, A. Spectra Anal. 1993,
175, 33-38.
(8) Grillo, A. C. Spectroscopy 1988, 4, 16-20.
(9) J acquault, P. Eur. Pat. 1992, 549 495 (21-12-92).
(10) Guibe´-J ampel, E.; Rousseau, G. Tetrahedron Lett. 1987, 28,
3563-3564.
(11) Hyflo Super Cel composition according to Fluka informations:
silica 89.3%; Al2O3 4.2%; Na2O,K2O 3.5%; Fe2O3 1.4%. Silica structures
are a mixture of R-cristobalite and R-tridymite.
S0022-3263(96)00309-X CCC: $12.00 © 1996 American Chemical Society

Microwave-Promoted Lipase-Catalyzed Reactions
J . Org. Chem., Vol. 61, No. 22, 1996 7747
a
F igu r e 1. Thermal behavior of supports (1 g) under micro-
wave exposure (power ) 90 W)
b
Ta ble 1. Su p p or t Beh a vior a n d P r op er ty
final temperature (°C)
after MW exposure
Support
pH
30 W
90 W
Florisil
Celite 545
HSC
8.5-9
7.5
8.5-9
120
50
50
55
55
65-70
Accurel
a
MW ) microwave.
Specification of the enzyme purchased indicates that
(i) LP presents good stability in the dry state during at
least 10 h at 100 °C (Figure 2a)13 and (ii) Novozym 435
is thermostable with a maximum activity in the range
of 70-90 °C (Figure 2b).12
F igu r e 2. (a) Heat stability of LP (powder). (b) Activity of
Novozym versus temperature.
We first studied the stability of these enzymes under
our reaction conditions. P. cepacia lipase was therefore
dispersed on HSC (10-30% w/w) whereas C. antarctica
was impregnated on Accurel from pH 7 buffered solution
(commercial product Novozym SP 43512).
The supported enzymes were placed during 30 min
under microwave irradiation at the temperature range
of 70-100 °C and subsequently tested in the trans-
esterification reactions of racemic 1-phenylethanol. Within
experimental errors, their activities were found to be the
same as those of freshly prepared, nonheated, supported
enzymes.
The resolution of racemic 1-phenylethanol through
esterification or transesterification, a reaction quite often
described in the literature about enzymatic reaction,14
was then studied under microwave irradiation. Trans-
esterification was performed both with nonactivated
(ethyl valerate) and with activated esters (isopropenyl
acetate).
F igu r e 3. Thermal evolution of SP 435 catalyzed phenyl-
ethanol transesterification.
The substrates were solubilized in organic solvents and
then impregnated on the enzyme-loaded supports by
subsequent evaporation of these solutions (Scheme 1).
Reactions were then performed in dry media inside a
monomode microwave reactor with focused waves, since
the energy distribution is much more homogeneous and
consequently much more efficient than with domestic
ovens. The temperature was maintained at a fixed value
through control of incident power9 (Figure 3). For the
sake of comparison, and to check specific microwave
(purely nonthermal) effects, reactions were also per-
formed in a thermostatted oil bath at the same temper-
ature for the same time with as close as possible heating
profiles.
(12) Novozym 435, product sheet from Enzyme Process Division,
Novo-Nordisk, Bagsvaerd, Denmark.
(13) Lipase LP, a new lipolytic enzyme preparation, product sheet
from Amano Pharmaceutical Co. Ltd, Nagoya, J apan.
(14) (a) Laumen, K.; Breitgoff, D.; Schneider, M. P. J . Chem. Soc.,
Chem. Commun. 1988, 1459-1461. (b) Morgan, B.; Oehlschlager, A.
C.; Stokes, T. M. J . Org. Chem., 1992, 57, 3231-3236. (c) Kirchner,
G.; Scollar, M. P.; Klibanov, A. M. J . Am. Chem. Soc. 1985, 107, 7072-
7076. (d) Guibe´-J ampel, E.; Chalecki, Z.; Bassir, M.; Gelo-Pujic, M.
Tetrahedron 1996, 52, 4397-4402.

7748 J . Org. Chem., Vol. 61, No. 22, 1996
Carrillo-Munoz et al.
Ta ble 2. Acyla tion of (RS)-1-P h en yleth a n ol u n d er Micr ow a ve Ir r a d ia tion or Cla ssica l Hea tin g
run
no.
t
T
(°C)
conv
(%)
ee alcohol
S (%)
lipase
R
R′
C2H5
mode of activtn
(mn)
E
a
LP/HSC
C4H9
1
2
∆
180
180
77
77
46
50
32
47
3
8
MWb 90 W
CH3
H2CdCCH3
3
4
∆
15
15
85
85
38
47
50
79
16
42
MW 240 W
SP 435
C7H15
C2H5
5
6
∆
60
60
70
70
34
36
46
51
28
36
MW 60 W
7
8
9
∆
10
10
15
90
90
90
40
43
47
62
75
88
50
>100
>100
MW 90 W
MW 90 W
H
10
11
12
13
MW 30 W
∆
MW 60 W + 20 W
MW 300 W + 80 W
10
10
67
78
78
95
45
48
52
47
66
62
93
86
18
10
44
10 (5 + 5)
5 (1 + 4)
>100
a
b
∆ ) classical heating. MW ) microwave exposure.
Sch em e 1
Sch em e 2
Ta ble 3. Resolu tion of (RS)-1-P h en yleth yl Va ler a te (3)
We found that the supported enzymes could be reused
three more times in the reactions under study without
loss of activity, thus showing the good stability and the
possibility of recycling of such systems.
mode
t
T
(°C)
conv
(%)
ee alcohol
R (%)
of activtna
(mn)
MW 40W
MW 40-20W
∆
5
15
5
80
80
80
80
46
50
47
47
100
100
100
100
These results (average values of three experiments) in
terms of reaction time (t), temperature (T), substrate
conversion (conv), enantiomeric excess (ee), and enan-
tiomeric ratio of the reaction (E) calculated according to
the Sih equation15 are reported in Table 2.
15
a
MW ) microwave exposure. ∆ ) classical heating.
Con clu sion
In both cases (classical heating and microwave irradia-
tion) all reactions were very fast when compared, for
instance, to the results obtained under reduced pressure
in solution of acylating agent where 24 h were needed.6b
At optimal temperature (90 °C for water or 85 °C for
ethanol elimination), the initial rates and enantiomeric
ratios E are significantly enhanced under microwave
irradiation. When compared to ethyl ester (runs 1 and
2), the increase in selectivity is more pronounced for
isopropenyl acetate (runs 3 and 4) and octanoic acid (runs
11 and 12), a result which may be connected with the
enhanced polarities of these species and leading to their
more pronounced interactions with microwaves.16
We have taken advantage of the complementarity of
two recent and eco-friendly technologies, enzymatic
catalysis using immobilized enzymes in dry media and
microwave activation in solvent-free conditions, to en-
hance both reactivity and selectivity of enzymatic reac-
tions. The specificity of microwave effects as compared
to classical heating have also been evidenced, as already
described for several classical organic reactions.17 Such
specificity may result from an improvement of irrevers-
ibility of the reaction due to more expedited removal of
water or ethanol molecules and (or) to a decrease in
activation parameters ∆Hq and ∆Sq as demonstrated by
Lewis for the imidization of polyamic acid.18
Another advantage of microwaves in terms of reactivity
lies in the fact that the associated rate enhancements
may allow the reaction to go to completion, in cases where
it is barely attainable under classical heating. To il-
lustrate this point, we have studied the resolution of (()-
1-phenylethyl valerate (3) by transesterification with
butanol (Scheme 2, Table 3).
Exp er im en ta l Section
Gen er a l. All chemicals, including supports, were pur-
chased from Fluka and used without further purification. P.
cepacia lipase (LP) was obtained from Amano Pharmaceutical
Co. and Novozym 435 (SP 435) from Novo Nordisk Co.
(17) (a) Bram, G.; Loupy, A.; Majdoub, M.; Gutierrez, E.; Ruiz-
Hitzky, E. Tetrahedron 1990, 46, 5167-5176. (b) Barnier, J . P.; Loupy,
A.; Pigeon, P.; Ramdani, M.; J acquault, P. J . Chem. Soc., Perkin Trans.
1, 1993, 397-398. (c) Loupy, A.; Pigeon, P.; Ramdani, M.; J acquault,
P. Synth. Commun. 1994, 24, 159-165. (d) Bougrin, K.; Kella Bennani,
A.; Fkih Te´touani, S.; Soufiaoui, M. Tetrahedron Lett. 1994, 35, 8373-
8376. (e) Bougrin, K.; Soufiaoui, M.; Loupy, A.; J acquault, P. New J .
Chem. 1995, 19, 213-219. (f) Perez, E. R.; Marrero, A. L.; Perez, R.;
Autie, M. A. Tetrahedron Lett. 1995, 36, 1779-1782.
(18) Lewis, D. A.; Summers, J . D.; Ward, T. C.; McGrath, J . E. J .
Polym. Sci. Part A 1992, 30, 1647-1653.
(19) Adams, M. W. W.; Kelly, R. M. Chem. Eng. News, 18 Dec 1995,
32-42.
Quantitative yield could be obtained (50%) under
microwave exposure whereas it is limited to 47% (which
was constant even after prolongation of the reaction time)
by conventional heating, thus necessitating further sepa-
ration of enantiomers.
(15) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J . J . Am. Chem.
Soc. 1982, 104, 7294-7299.
(16) Gedye, R. N.; Smith, F. E.; Westaway, K. C. Can. J . Chem. 1988,
66, 17-26.

Microwave-Promoted Lipase-Catalyzed Reactions
J . Org. Chem., Vol. 61, No. 22, 1996 7749
Micr ow a ve Rea ctor . Reactions were performed in a
monomode microwave reactor (Synthewave 402 from Prolabo),
fitted with a stirring system and an IR temperature detector
which indicates the surface temperature.9 Reaction conditions
were controlled using the algorithm tout ou peu which allows
temperature control at the given value during the reaction
time by varying the power between an adequate value and 20
W (to operate under electromagnetic field all along the
reaction).
concentration under reduced pressure, the remaining alcohol
and the ester were purified by silica gel chromatography, using
a pentane/ethyl acetate gradient. Conversions and enantio-
meric excess were determinated by GC capillary chiral column
(Cydex B) with ethyl benzoate as an internal standard (GC
Carlo Erba Fractovap 2900: oven temperature 105 °C, gas
carrier pressure 1 kPa, retention times alcohol R ) 7.80 min,
alcohol S ) 8.33 min).
Ack n ow led gm en t. Financial support from Univer-
sidad de Castilla la Mancha for a postdoctoral position
to J .-R.C.M. is acknowledged as well as Novo-Nordisk
and Amano Pharmaceutical Co. for kind gifts of No-
vozym 435 and LP, respectively. We are also grateful
to Prolabo, France, for useful discussions and providing
microwave equipment.
Typ ica l P r oced u r e. Into a pyrex tube (12 cm3) were
introduced 1 g of SP 435, 2 mL of an ethereal solution of 1
(244 mg, 2 mmol), and 3 equiv (6 mmol) of octanoic acid (862
mg) or 1 g of LP/HSC (250 mg, 750 mg), 2 mL of an ethereal
solution of 1 (122 mg, 1 mmol), and 4 equiv (4 mmol) of
isopropenyl acetate (400 mg).
The mixture was heated as indicated in the tables. After
the solution was cooled to rt, the solid was transferred on a
fritted funnel and the product eluted with diethyl ether. After
J O960309U