Peracetylated β-cyclodextrin as additive in enzymatic reactions: Enhanced reaction rate and enantiomeric ratio in lipase-catalyzed transesterifications in organic solvents

Article abstract of DOI:10.1016/S0957-4166(01)00482-7

Peracetylated β-cyclodextrin has been employed as a macrocyclic additive to enhance the enantiomeric ratio E and reaction rate in Pseudomonas cepacia lipase (PSL)-catalyzed enantioselective transesterification of 1-(2-furyl)ethanol in organic solvents. The beneficial action of the cyclodextrin used as a regulator of lipase was tentatively interpreted as increasing the conformational flexibility of the enzyme and undergoing host-guest complexation with the product, thereby preventing product inhibition and leading to an enhancement of the enantiomeric ratio E and the reaction rate. The effect of the organic solvent on the present cyclodextrin-mediated enzymatic transesterification has been studied.

Full text of DOI:10.1016/S0957-4166(01)00482-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 2761–2766
Peracetylated b-cyclodextrin as additive in enzymatic reactions:
enhanced reaction rate and enantiomeric ratio in lipase-catalyzed
transesterifications in organic solvents
Ashraf Ghanem and Volker Schurig*
Institute of Organic Chemistry, University of Tu¨bingen, Auf der Morgenstelle 18, D-72076 Tu¨bingen, Germany
Received 16 October 2001; accepted 26 October 2001
Abstract—Peracetylated b-cyclodextrin has been employed as a macrocyclic additive to enhance the enantiomeric ratio E and
reaction rate in Pseudomonas cepacia lipase (PSL)-catalyzed enantioselective transesterification of 1-(2-furyl)ethanol in organic
solvents. The beneficial action of the cyclodextrin used as a regulator of lipase was tentatively interpreted as increasing the
conformational flexibility of the enzyme and undergoing host–guest complexation with the product, thereby preventing product
inhibition and leading to an enhancement of the enantiomeric ratio E and the reaction rate. The effect of the organic solvent on
the present cyclodextrin-mediated enzymatic transesterification has been studied. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
enzymes in organic solvents has also the drawback of
their decreased catalytic activities, which are generally
several orders of magnitude lower than in aqueous
solution. Several strategies have been explored to over-
come the lower activity of enzymes in organic solvents
to make them more appealing to organic chemists.
These include the methods of enzyme preparation,4
directed evolution,5 chemical modification of the
enzyme,6 control of the pH value,7 co-lyophilization
with lyoprotectants8 and salts,9 addition of water-mim-
icking compounds like formamide, glycol or DMF,10
immobilization techniques,11 imprinting with substrates
and substrate analogues,12 and cross-linking crystalliza-
tion.13 It has been reported that an additional improve-
ment in the rate and selectivity of the reaction in
non-aqueous media can be achieved by using macro-
cyclic organic additives, which might have a beneficial
influence on the microenvironment of the enzymes and
hence improve their catalytic activity and selectivity in
organic solvents.14
Enzymes, especially lipases, are now well established as
valuable catalysts in organic synthesis. These natural
catalysts have also been shown to be very useful tools
for access to chiral molecules under non-aqueous condi-
tions.1 Compared to aqueous conditions, the use of an
organic reaction medium can have some interesting
advantages, including enhanced thermal stability of the
undissolved enzyme, easy separation of the suspended
enzyme from the reaction medium for recycling,
increased solubility of the substrate, favorable equi-
librium shift to synthesis over hydrolysis in transesterifi-
cation reactions, the elimination of undesired
side-reactions caused by water and the generation of
novel selectivities of certain enzymes.2
Many non-aqueous enzymatic reactions are prone to
substrate or product inhibition, which deactivate the
enzyme at higher substrate or product concentration
leading to a decrease in the reaction rate and enan-
tiomeric ratio E. In fact, keeping the substrate concen-
tration at a low level through continuous addition can
circumvent substrate inhibition, however, product inhi-
bition is still difficult to control even by gradual
removal of the product by physical means.3 The use of
Among these supramolecular additives, crown ethers
and especially thiacrown ethers have been successfully
used to enhance the catalytic activity and the enan-
tiomeric ratio E of PSL in the formation and hydrolysis
of carboxylic esters.15–17 In fact, co-lyophilization of
various enzymes with crown ether leads to enhance-
ment of the catalytic activity of the enzyme and also
increases the enantiomeric ratio E in organic solvents.
It is conceivable that the crown ethers bind to the
* Corresponding author. Tel.: +49(0) 7071-2976257; fax: +49(0) 7071-
295538; e-mail: volker.schurig@uni-tuebingen.de
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00482-7

2762
A. Ghanem, V. Schurig / Tetrahedron: Asymmetry 12 (2001) 2761–2766
enzyme in the co-lyophilization process leading to
increased solubility of the enzymes in the organic phase,
thus accelerating the rate of transesterification.18 Also,
such binding can increase the flexibility of the enzymes
in organic solvents leading to enhancement of its activ-
ity and the enantiomeric ratio E.19 The enhancement of
the enzyme activity by crown ethers may also be
attributable to the capability of the crown ether to
facilitate the removal of water molecules from the
active site upon substrate binding.18
Herein, we report the utility of peracetylated b-
cyclodextrin employed as an additive to enhance the
reaction rate and enantiomeric ratio E in the Pseu-
domonas cepacia lipase (PSL)-catalyzed enantioselective
transesterification of 1-(2-furyl)ethanol in an organic
solvent.
2. Results and discussion
Two methods of enzyme preparation have been per-
formed. The first consisted of the lyophilization of P.
cepacia lipase (PSL), the present model enzyme, from
phosphate buffer pH 6.0 without the cyclodextrin addi-
tive. The second enzyme preparation was concerned
with the co-lyophilization of PSL with peracetylated
b-cyclodextrin using the same phosphate buffer. The
effect of the cyclodextrin on the reaction rate and E of
PSL-catalyzed transesterification in various organic sol-
vents was studied in the model kinetic resolution of
lipase-catalyzed transesterification of 1-(2-furyl)ethanol
using isopropenyl acetate as acyl donor (cf. Scheme 1).
Cyclodextrins (CDs), another class of compound with a
macrocyclic structure, have been successfully used to
improve enzymes activity and to increase the reaction
rate and E in enzyme-catalyzed reactions in organic
solvents.20,21 They are a family of chiral cyclic a-(1-4)-
linked
D-glucose oligomers with six, seven, or eight
glucose units, corresponding to a-, b-, and g-homo-
logues, possessing toroidal conformation in the solid
state and in solution. The internal hydrophobic cavity
and the external hydrophilic rim of chemically modified
cyclodextrins render them ideal for modelling enzyme-
substrate binding,22 drug delivery,23 catalysis,24 host–
guest association,25 chiral separation26 and molecular
recognition in self-assembled monolayers27 etc.
Compared to the commercially available PSL used as
purchased, a slight decrease in the catalytic activity was
observed when the enzyme was lyophilized from phos-
phate buffer pH 6. This finding is accounted for by
partial denaturation of PSL during lyophilization.
Interestingly, no such decrease in catalytic activity was
observed when the enzyme was co-lyophilized with
peracetylated b-cyclodextrin using the same phosphate
buffer. Since the magnitude of the enzyme activation in
organic solvents by macrocyclic compounds depends on
the ratio of the enzyme and the additive,18 three differ-
ent mixtures were investigated, i.e. 1:1; 1:2; 1:6 (enzyme
to cyclodextrin, w/w). When the peracetylated b-
cyclodextrin was employed at a 1:6 weight ratio of
enzyme to cyclodextrin, a significant activation of PSL
has been detected as compared to the data of the
lyophilized PSL from buffer alone (cf. Fig. 2). The
lyophilized lipase from buffer alone catalyzed kinetic
resolution of 1 was characterized by the following data:
time=24 h, e.e.s=99%, e.e.p=61%, conv.=60% and
E=28.
Previously, permethylated b-cyclodextrin has been used
to enhance the reaction rate and enantiomeric ratio, E
of the reactions of subtilisin Carlsberg suspended in an
organic solvent.21 In terms of bulkiness and hydrogen-
bonding ability, the introduction of an acetyl group to
free b-cyclodextrin may drastically alter its role as
regulator for enzymes in organic solvents. Therefore,
peracetylated b-cyclodextrin (cf. Fig. 1) is expected to
exhibit novel behavior in enhancing the reaction rate
and E of PSL-catalyzed transesterification reactions
under non-aqueous conditions when added prior to the
lyophilization of the enzyme in buffer.
The enhancement of the reaction rate and the E of the
PSL-catalyzed transesterification of 1 was in the order
1:6>1:2>1:1 (enzyme to peracetylated b-cyclodextrin,
w/w) (cf. Fig. 3).
Figure 1. Structure of peracetylated-b-CD (R=-COCH3).
O
O
Lipase/CD
+
+
+
O
O
O
O
OH
O
OH
O
2
(S)-1
1
(R)-3
Scheme 1. Co-lyophilized PSL with peracetylated-b-cyclodextrin catalyzed transesterification of ( )-1-(2-furyl)ethanol 1 using
isopropenyl acetate 2 in toluene.

A. Ghanem, V. Schurig / Tetrahedron: Asymmetry 12 (2001) 2761–2766
2763
smaller than that observed when lipase was co-
lyophilized with cyclodextrin in phosphate buffer using
the same weight ratio. Therefore, the beneficial effect of
the cyclodextrin additive is believed to arise mainly
from the direct interaction with the enzyme rather than
complexation with either substrate or product. Thus, in
the co-lyophilization step, the cyclodextrin may interact
with the enzyme in a specific but as yet unknown way
by changing its conformation and hence influencing its
catalytic behaviour as already inferred above.
Since the cyclodextrin is used in excess, host–guest
complexation in solution with the substrate and
product cannot be excluded. This might explain why
the reaction rate was higher when using the 1:6 as
compared to the 1:1 ratio (enzyme to cyclodextrin,
w/w). In fact, the ability of cyclodextrins to form
host–guest complexes may indeed prevent substrate as
well as product inhibition in enzymatic reactions.20 The
inclusion of product in the cavity of the cyclodextrin
may also shift the equilibrium in the desired direction.
This thermodynamic effect, however, would not explain
the enhancement of the reaction rate. The ability of
1-(2-furyl)ethanol 1 as well as its ester 3 to undergo
inclusion in the peracetylated b-cyclodextrin cavity was
inferred by (upfield/downfield) complexation-induced
shifts in 1H and 13C NMR spectroscopic studies.
Whether enantioselective binding of substrate 1 or
product 3 with cyclodextrin, rendering one enantiomer
more accessible to the enzyme, contributes to the
observed enhancement of enantiomeric ratio is still
open to question.
Figure 2. Co-lyophilized lipase with peracetylated b-cyclodex-
trin (1:6 weight ratio) catalyzed enantioselective transesterifi-
cation of 1(2-furyl)ethanol in toluene. The reaction was
terminated in 8 h with 56% conv., >99% e.e.s, 80% e.e.p,
E=52.
Indeed, when performing the esterification of 1 with
acetyl chloride with pyridine as the catalyst instead of
lipase, and peracetylated b-cyclodextrin as chiral addi-
tive, enantioselective esterification to give
3 was
Figure 3. Gas-chromatographic separation of the enantiomer
of both substrate 1 and product 3 on heptakis-(2,3-di-O-
methyl-6-O-tert-butyldimethylsilyl)-b-cyclodextrin of the co-
lyophilized PSL with peracetylated b-cyclodextrin-catalyzed
transesterification of 1 in toluene at t=8 h. (a) 1:1 enzyme/Ac
b-cyclodextrin weight ratio, e.e.s=13.7%, e.e.p=94.3%,
conv.=12.7%, E=39. (b) 1:2 enzyme/Ac b-cyclodextrin
weight ratio, e.e.s=14%, e.e.p=94%, conv.=13%, E=38.
(c) 1:6 enzyme/Ac b-cyclodextrin weight ratio, e.e.s=99.9%,
e.e.p=79.1%, conv.=56%, E=88.
observed. It was found that the inclusion of enan-
tiomers of 1 in the peracetylated b-cyclodextrin cavity
occurred in a preferential way allowing the (R)-1 enan-
tiomer to react faster with acetyl chloride to form the
enantiomerically biased ester. The following data were
measured: time=2 days, e.e.s=5.2% (S)-1, e.e.p=31%
(R)-3, conv.=15% and E=2.03. Also, this effect has
been observed when using permethylated b-cyclodextrin
as additive. The following data were measured: time=2
days, e.e.s=0.6% (S)-1, e.e.p=7.0% (R)-3, conv.=8.0%
and E=1.16. This supports the assumption that the
enhancement of E in the PSL and cyclodextrin co-
lyophilized catalytic transesterification of 1 in organic
solvent is based on a beneficial effect of the cyclodex-
trin on the enzyme rather then preferential enantioselec-
tive complexation of 1 with the cyclodextrin.
In a control experiment it was found that no reaction
took place when peracetylated b-cyclodextrin was
employed without any lipase. It is concluded from this
finding that peracetylated b-cyclodextrin does not cata-
lyze the enantioselective transesterification of 1 by itself
but regulates enzyme activity, probably by changing the
conformation of the enzymatic catalytic site or under-
goes host–guest complexation with substrate and/or
product. In an additional experiment, PSL was first
lyophilized from buffer alone, suspended in toluene and
only afterwards peracetylated b-cyclodextrin was added
to the reaction mixture (at the 1:6 weight ratio as
before). A slight enhancement in the reaction rate and
E was observed. However, this enhancement was much
It should be noted that simple esters like peracetylated
b-cyclodextrin cannot be used as an acylating agent for
1 in the presence of an activated ester like isopropenyl
acetate. In general, acylation with a normal ester as the
acyl donor for the transesterification of alcohols in the
presence of lipase are often slow and reversible with an
equilibrium constant of near to one. This was confi-
rmed by allowing 1 to react with peracetylated b-
cyclodextrin in the presence of lipase in toluene under

2764
A. Ghanem, V. Schurig / Tetrahedron: Asymmetry 12 (2001) 2761–2766
the same conditions as detailed above, no reaction was
observed and the recovered cyclodextrin was found to
be the peracetylated b-cyclodextrin. In an additional
experiment, perbutyrated b-cyclodextrin was synthe-
sized and used instead of peracetylated b-cyclodextrin
as additive and in co-lyophilized form with lipase to
catalyze the enantioselective transesterification of 1 in
toluene in the presence of isopropenyl acetate as the
acyl donor, surprisingly, reaction inhibition was
observed. We concluded that the acetyl group is
involved in the enhancement of the reaction rate as well
as the enantiomeric ratio of lipase-catalyzed transester-
ification of 1.
hydrophobicity in biocatalytic reactions33 and is defined
as the ratio of the concentration of a substance in two
immiscible phases (1-octanol and water) at equilibrium.
We tried to correlate the observed E with various
solvents in dependence on their log P. However, for the
present enzyme preparations (co-lyophilized lipase with
peracetylated b-cyclodextrin and lyophilized enzyme
only) no straightforward correlation between the
enzyme prepared, solvents and E was found (cf. Table
1). For example, as compared with lyophilized lipase
only, the enhancement of E using co-lyophilized lipase
with peracetylated b-cyclodextrin was 6.7 fold higher in
magnitude in isooctane (log P=4.5), while the enhance-
ment was 6.2 times higher in magnitude in tetra-
hydrofuran (log P=0.49). Also, in 1,4-dioxane
(log P=−1.1) the enhancement in enantiomeric ratio E
was 5.8 times higher in magnitude, while in toluene
(log P=2.5) the enhancement was only 1.8 times higher
in magnitude. With regard to the E value of the reac-
tion, isooctane was the best solvent employed in the
transesterification of 1 using the co-lyophilized lipase
with peracetylated b-cyclodextrin. However, with
regard to the reaction rate and the enantiomeric excess
of both substrate and product, toluene proved to be the
best solvent employed.
The solubility of lipase in toluene could also play an
important role in the enhancement of the reaction rate
and enantioselectivity, Reinhoudt et al.18 reported that
crown ethers could solubilize the lipase in organic
solvents and accelerate the rate of transesterification.
The solubility of lipase as well as co-lyophilized lipase
with peracetylated b-cyclodextrin and cyclodextrin
alone has been studied in toluene using light scattering
techniques. We found that only lipase dissolved in
toluene forms aggregates. Co-lyophilized lipase with
peracetylated b-cyclodextrin and cyclodextrin alone did
not form aggregates. We concluded from this observa-
tion that peracetylated b-cyclodextrin increases the sol-
ubility of the P. cepacia lipase in toluene when they are
mixed prior to lyophilization in buffer.
3. Experimental
3.1. Materials and methods
This increase in solubility of the lipase in toluene might
contribute to the enhancement of the reaction rate as
well as the enantiomeric ratio of co-lyophilized lipase-
catalyzed transesterification of 1 in toluene.
1
3.1.1. Instrumentation. H and 13C NMR spectra were
recorded at 250 and 400 MHz, respectively, on Bruker
spectrometers (Karlsruhe, Germany). Chemical shifts
(l) are given in ppm relative to TMS as internal
standard. Specific rotations were measured with a
Perkin-Elmer 241 polarimeter operating at the sodium
D line and at room temperature.
2.1. Effect of organic solvents on the PSL and
cyclodextrin co-lyophilized catalytic transesterification
of 1
The choice of the organic solvent for a lipase-catalyzed
reaction is known to be crucial in determining the E
values.30,31 It has been reported that solvents with log P
values more than two exhibit high E, whereas solvents
with log P of less than two show detrimental effects on
the E of the enzyme-catalyzed reactions in organic
solvents.32 P is employed as an index for the solvent
3.1.2. Chemicals and enzymes. All chemicals were pur-
chased from Fluka (Switzerland) and dried over molec-
ular sieves prior to use. Lipase from P. cepacia (PSL)
was a gift from Amano (Nagoya, Japan). b-Cyclodex-
trin was purchased from Wacker-Chemie GmbH (Bur-
ghausen/Germany). The peracetylated b-cyclodextrin
has been synthesized as described later.
Table 1. Enantiomeric ratio E enhancement of co-lyophilized lipase with peracetylated b-cyclodextrin-catalyzed transesterifi-
cation of 1 in different organic solvents
Solvent
Log Pa
Lyophilizedb lipase
Peracetylated b-cyclodextrin co-lyophilisatec
Enhancement
Isooctane
THF
1,4-Dioxane
n-Hexane
Acetonitrile
Toluene
4.5
0.49
−1.1
3.5
−0.33
2.5
Ed=46.2
E=93.6
E=27.9
E=11.3
E=99.7
E=28.9
E=\300
E=\300
E=161.6
E=40.1
E=\300
E=51.4
6.7
6.2
5.8
3.5
3.4
1.8
a P=partition coefficient; defined as the ratio of the concentration of a substance in two immiscible phases at equilibrium (1-octanol and water).
b PSL lyophilized from phosphate buffer alone, pH 6.
c Lyophilized from aqueous phosphate buffer containing peracetylated b-cyclodextrin at a 1:6 weight ratio of lipase to cyclodextrin.
d E=enantiomeric ratio.29

A. Ghanem, V. Schurig / Tetrahedron: Asymmetry 12 (2001) 2761–2766
2765
3.1.3. Synthesis and biochemical transformation reac-
tions. The racemic ester 3 was synthesized on an analyt-
ical scale to optimize a baseline separation of the
enantiomers using heptakis(2,3-di-O-methyl-6-O-tert-
butyldimethylsilyl)-b-cyclodextrin as a stationary phase
in GC. The separation factor h=1.32 and resolution
Rs=14.3 for the ester 3 and h=1.08 and Rs=2.5 for
the alcohol 1. Peak integration for the racemic sample
showed the unusual elution order (S)-3, <(R)-1, <(S)-1,
<(R)-3 which should be noted. The retention times were
14.7, 15.5, 16.8, and 20.2 min, respectively.
was used for the GC analysis. When maximum conver-
sion was reached the reaction was terminated by filtra-
tion. The enzyme was washed with solvent and then
with acetone. Substrate (S)-1 and product (R)-3 were
distilled and the recovered cyclodextrin was purified
and analyzed.
3.1.7. Enantioselective gas-chromatographic analysis.
Enantioselective analysis of 1-(2-furyl)ethanol 1 (sub-
strate) and acetoxy-1-(2-furyl)ethane 3 (product) was
performed simultaneously on a gas chromatograph
(Hewlett Packard 580, Waldbronn, Germany) equipped
with a flame ionization detector (FID). The chiral
stationary phase heptakis-(2,3-di-O-methyl-6-O-tert-
butyldimethylsilyl)-b-cyclodextrin, 20% (w/w) was dis-
solved in PS 86 (Gelest, ABCR GmbH & Co.,
Karlsruhe, Germany) and coated on a 25 m×0.25 mm
fused silica capillary column (0.25 mm film thickness)
according to literature.28 The analytical conditions
were: injector temperature, 200°C; FID temperature,
250°C; oven temperature 70°C for the simultaneous
separation of enantiomers of 1 and 3. Hydrogen was
used as the carrier gas (40 KPa column head pressure).
The retention time of (S)-3, (R)-1, (S)-1, (R)-3, were
14.7, 15.5, 16.8, 20.2 min, respectively. The substrate 1
and product 3 were identified by using a GC/MSD-sys-
tem HP 6890/5973 (Hewlett Packard, Waldbronn, Ger-
many) equipped with an HP 7683 autosampler. The
enantiomeric excess e.e. of both substrate (e.e.s) and
product (e.e.p) as well as conversion (conv.) and enan-
tiomeric ratio (E) were determined by the computer
program available on the internet http://www.
orgc.TUGraz.at/orgc/programs/selectiv/selectiv.htm,de-
veloped by Faber et al.29
3.1.4. Peracetylated b-cyclodextrin. b-Cyclodextrin (5.6
g) was reacted with acetic anhydride (113 mL) in pyri-
dine (225 mL). The reaction was stirred for 24 h at
room temperature. Water was added and the extraction
was performed using ethyl acetate. The organic layer
was washed with 1 M HCl followed by water, dried
with anhydrous sodium sulphate, and the excess solvent
was removed by rotary evaporator. The residue was
purified by column chromatography using CH2Cl2/ace-
tone (4:1, v/v). The yellowish product was crystallized
with ethanol/diethyl ether (1:1, v/v) to give white crys-
tals; yield 60%; mp 201; [h]2D4 125 (c 0.01, CHCl3); MS
(positive FAB, 0.1% in methanol+0.1% acetic acid)
m(nominal mass)/z 2017 [M+H]+; anal. calcd for
C84H112O56: C, 49.97; H, 5.55. Found: C, 49.80; H,
5.20%; 1H NMR (benzene-d6) l 5.26 (dd, 1H, H-3),
5.03 (d, 1H, H-1), 4.7 (t, 1H, H-2), 4.52 (d, 1H, H-6a),
4.22 (m, 1H, H-6b), 3.67 (t, 1H, H-4), 2.10 (s, 3H,
CH3); 13C NMR (benzene-d6) l 170.44 (CO), 170.31
(CO), 169.24 (CO), 97.48 (C-1), 77.53 (C-4), 71.23
(C-3), 70.69 (C-2), 70.33 (C-5), 63.11 (C-6), 20.62
(CH3), 20.54 (CH3), 20.47 (CH3).
3.1.5. Co-lyophilization of PSL with peracetylated b-
cyclodextrin. P. cepacia lipase (PSL, 100 mg) was dis-
solved (2 mg/1 mL) in 20 mM phosphate buffer (pH
6.0) and lyophilized for 48 h (control). The co-
lyophilization of lipase with peracetylated b-cyclodex-
trin was performed by the same method, except that the
cyclodextrin was added prior to lyophilization with
different ratios of lipase to cyclodextrin (1:1; 1:2; 1:6,
w/w). After lyophilization, the enzyme preparation was
dried under vacuum and stored at −18°C for further
use.
Acknowledgements
This work was supported by Fonds der Chemischen
Industrie. A.G. thanks Professor R. D. Schmid (Uni-
versity of Stuttgart) and Professor U. Bornscheuer
(University of Greifswald) for their advice and support
and the Graduate College Analytical Chemistry, Uni-
versity of Tu¨bingen, for a scholarship.
3.1.6. Lipase-catalyzed transesterification of 1-(2-
furyl)ethanol. All reactants (alcohol, ester) were stored
References
,
over activated
4
A
molecular sieves. ( )-1-(2-
1. Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in
Organic Synthesis; Wiley-VCH: Weinheim, Germany,
1999; ISBN 3-527-30104-6.
2. Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141.
3. Faber, K. Biotransformations in Organic Chemistry, 3rd
ed.; Springer Verlag: Heidelberg, Germany, 1997; ISBN
3-540-61688-8.
Furyl)ethanol (56 mg, 0.5 mmol) and isopropenyl ace-
tate (108.8 mg, 1 mmol) were dissolved in organic
solvent (3 mL) in a reaction vial (5 mL). The reaction
mixture was thermostated in an oil bath to 40°C. Then,
a 100 mL sample of the reaction mixture was withdrawn
for GC analysis (t=0 of sample). Afterward, 100 mg of
lyophilized lipase or lipase co-lyophilized with 100, 200
or 600 mg peracetylated b-cyclodextrin was added. 100
mL samples were taken after several time intervals. The
samples were centrifuged to separate lipase. The
organic layer was diluted by 100 mL toluene. The
reaction progress was monitored qualitatively by thin
layer chromatography. An aliquot of the supernatant
8
4. Almarsson, O.; Klibanov, A. M. Biotechnol. Bioeng.
1996, 49, 87–92.
5. Reetz, M. T.; Jaeger, K. E. Biocatalysis 1999, 200, 31–57.
6. Babonneau, M. T.; Jacquier, R.; Lazaro, R.; Viallefont,
P. Tetrahedron Lett. 1989, 30, 2787.
7. Yang, Z.; Zacherl, D.; Russell, A. J. J. Am. Chem. Soc.
1993, 115, 12251–12257.

2766
A. Ghanem, V. Schurig / Tetrahedron: Asymmetry 12 (2001) 2761–2766
8. Dabulis, K.; Klibanov, A. M. Biotechnol. Bioeng. 1993,
21. Griebenow, K.; Laureano, Y. D.; Santos, A. M.;
Clemente, I. M.; Rodriguez, L.; Vidal, M. W.; Barletta,
G. J. Am. Chem. Soc. 1999, 121, 8157–8163.
22. D’Souza, V. T.; Bender, M. L. Acc. Chem. Res. 1987,
20, 146–152.
23. Pfannemu¨ller, B.; Burchard, W. Macromol. Chem. 1969,
121, 1–17.
24. Ye, H.; Tong, W.; D’Souza, V. T. J. Am. Chem. Soc.
1992, 144, 5470–5472.
25. Andersson, T.; Sundahl, M.; Westman, G.; Wenner-
stroem, O. Tetrahedron Lett. 1994, 35, 7103–7106.
26. Schurig, V. J. Chromatogr. A 2001, 906, 275–299.
27. Rojas, M. T.; Koeniger, R.; Stoddart, J. F.; Kaifer, A.
E. J. Am. Chem. Soc. 1995, 117, 336–343.
28. Dietrich, A.; Maas, B.; Messer, W.; Bruche, G.; Karl,
V.; Kaunzinger, A.; Mosandl, A. High Resolut. Chro-
matogr. 1992, 15, 590–593.
29. Kroutil, W.; Kleewein, A.; Faber, K. Tetrahedron:
Asymmetry 1997, 8, 3251–3261.
30. Wescott, C. R.; Klibanov, A. M. J. Am. Chem. Soc.
1993, 115, 1629–1631.
41, 566–571.
9. Khmelnitsky, Y. L.; Welch, S. H.; Clark, D. E.;
Dordick, J. S. J. Am. Chem. Soc. 1994, 116, 2647–2648.
10. Kitaguchi, H.; Klibanov, A. M. J. Am. Chem. Soc.
1989, 111, 9272–9273.
11. Reslow, M.; Adlercreutz, P.; Mattiasson, B. Eur. J.
Biochem. 1988, 172, 573–578.
12. Rich, J. O.; Dordick, J. S. J. Am. Chem. Soc. 1997,
119, 3245–3252.
13. Lalonde, J. J.; Govardhan, C.; Khalaf, N.; Martinez, A.
G.; Visuri, K.; Margolin, A. L. J. Am. Chem. Soc.
1995, 117, 6845–6852.
14. Theil, T. Tetrahedron 2000, 56, 2905–2919.
15. Takagi, Y.; Teramoto, J.; Kihara, H.; Itoh, T.;
Tsukube, H. Tetrahedron Lett. 1997, 37, 4991–4992.
16. Takagi, Y.; Ino, R.; Kihara, H.; Itoh, T.; Tsukube, H.
Chem. Lett. 1997, 1247–1248.
17. Itoh, T.; Takagi, Y.; Murakami, T.; Hiyama, Y.;
Tsukube, H. J. Org. Chem. 1996, 61, 2158–2163.
18. Bross, J.; Sakodinskaya, I. K.; Engbersen, J.; Verboon,
W.; Reinhoudt, D. N. J. Chem. Soc., Chem. Commun.
1995, 255–256.
19. Bross, J.; Visser, A. J. W. G.; Engbersen, J. F. J.;
Verboom, W.; Hoek, A. V.; Reinhoudt, D. N. J. Am.
Chem. Soc. 1995, 117, 12657–12663.
31. Nakamura, K.; Kinoshita, M.; Ohno, A. Tetrahedron
1994, 50, 4681–4690.
32. Hansch, L. C.; Elkins, D. Chem. Rev. 1971, 71, 525–
616.
33. Terradas, F.; Tenston-Henry, M.; Fitzpatrick, P. A. J.
Am. Chem. Soc. 1993, 115, 390–396.
20. Harper, J. B.; Easton, C. J.; Lincoln, S. F. Curr. Org.
Chem. 2000, 4, 429–454.