Lipase-catalyzed Irreversible Transesterification of Secondary Alcohols Using Isopropenyl Acetate

Article abstract of DOI:10.1007/s00706-003-0025-1

Asymmetric acetylation of a set of secondary alcohols with the innocuous acyl donor isopropenyl acetate catalyzed by a lipase from Pseudomonas cepacia immobilized on ceramic particles (PSL-C) in toluene as organic medium afforded the chiral alcohols and the corresponding acetates in high enantiomeric excess (up to 99%). An effective baseline separation of the enantiomers of both substrate and product was performed in one analysis without derivatization using gas chromatography on a new chiral stationary phase (CSP) Chirasil-β-Dex containing an undecamethylene spacer (C11-Chirasil-Dex).

Full text of DOI:10.1007/s00706-003-0025-1

Monatshefte f€ur Chemie 134, 1151–1157 (2003)
DOI 10.1007/s00706-003-0025-1
Lipase-catalyzed Irreversible
Transesterification of Secondary
Alcohols Using Isopropenyl Acetate
Ashraf Ghanem1;ꢀ and Volker Schurig2
1
Organic Chemistry Department, University of Geneva, CH-12 Geneva 4, Switzerland
2
€ €
Institute of Organic Chemistry, University of Tubingen, D-72076 Tubingen, Germany
Received November 27, 2002; accepted (revised) January 9, 2003
Published online June 2, 2003 # Springer-Verlag 2003
Summary. Asymmetric acetylation of a set of secondary alcohols with the innocuous acyl donor
isopropenyl acetate catalyzed by a lipase from Pseudomonas cepacia immobilized on ceramic particles
(PSL-C) in toluene as organic medium afforded the chiral alcohols and the corresponding acetates in
high enantiomeric excess (up to 99%). An effective baseline separation of the enantiomers of both
substrate and product was performed in one analysis without derivatization using gas chromatography
on a new chiral stationary phase (CSP) Chirasil-ꢀ-Dex containing an undecamethylene spacer
(C11-Chirasil-Dex).
Keywords. Enantiomeric resolution; Irreversible transesterification; Lipase; Secondary alcohols;
Gas chromatography.
Introduction
Enzymes and especially lipases are biological catalysts of extraordinary selectivity
and efficiency. Enantiomerically pure secondary alcohols are useful chiral auxil-
iaries in organic synthesis and they are preferably synthesized by enzymatic kinetic
resolution of the racemates. This biochemical transformation process has become a
standard reaction protocol in organic synthesis [1]. In the presence of a suitable
acyl donor and enzyme as well as solvent and at the optimum temperature one
enantiomer of the racemic mixture is selectively transferred to the corresponding
ester leaving the second unreacted enantiomer in optically pure form. The rever-
sible enzymatic process usually requires a long reaction time and a large excess of
the ester as acyl donor to achieve a reasonable degree of conversion [2]. In order to
render the process irreversible, the use of various activated esters like vinyl acetate
ꢀ
Dedicated to the Memory of Prof. E. Bayer 1927–2002
Corresponding author. E-mail: ashraf.ghanem@chiorg.unige.ch

1152
A. Ghanem and V. Schurig
[3], trifluoroethyl esters, chloroethyl esters [4], cyanomethyl esters [5], and acid
anhydrides [6, 7] have been reported. However, these methods have some draw-
backs, like the generation of toxic side products, thereby deactivating the enzyme
and inhibiting the formation of the required products [8], or the generation of water
which causes the undesired hydrolysis of the enantiomerically pure ester, leading
to a decrease in the conversion and in the enantiomeric excess. The generation of
acetaldehyde resulting from the tautomerization of vinylalcohol when using vinyl-
acetate as acyl donor can enter into reactions with many functional groups, parti-
cularly with the lysine side chains in enzymes leading to a deactivation of various
lipases, especially Geotrichum candidum and Candida rugosa. The latter is being
one of the most important lipases having a broad applicability to substrates in
organic synthesis [9]. In contrast to the transesterification with vinyl acetate, iso-
propenyl acetate is an innocuous acyl donor releasing acetone as a non-harmful
byproduct to enzymes.
The ways in which the efficiency and the practicability of the lipase-catalyzed
kinetic resolution of racemates are defined is depending on a large numbers of
factors [10, 11]. The development of accurate non-chiroptic methods for the deter-
mination of enantiomeric purity has been critical for the development of enantio-
selective synthesis. The modern and most sensitive methods used in the
determination of enantiomeric purity of the outcome of kinetic resolution reac-
tions, allowing a detection as little as 0.1% of one enantiomer in the presence of
another, are chiral GC and HPLC methods. For an efficient monitoring of the
reaction progress, enantioselective gas chromatography (GC) was the method of
choice for the simultaneous determination of the enantiomeric excesses of both
substrate and product in enantioselective reactions.
Although a large number of chiral stationary phases (CSPs) have been developed
[12, 13], the choice of an appropriate column is still difficult. Modified cyclodextrins
(CDs) have been widely used as chiral stationary phases for GC separation of racemic
chiral compounds. These CD derivatives are dissolved in polysiloxane phases and are
used for preparing efficient capillary columns [14, 15]. Chirasil-ꢀ-Dex, a polysilox-
ane-anchored permethylated ꢀ-cyclodextrin with 3, 5, and 8-spacer have been suc-
cessfully used as CSP in GC [16]. In this contribution, we report on the investigation
of Chirasil-ꢀ-Dex with a new 11-spacer (C11- Chirasil-Dex) [17] as CSP in GC for
the gas chromatographic enantiomers separation of a set of secondary alcohols and
their corresponding acetates. The utility of the titled CSP is demonstrated in the
analysis of the enantiomeric excesses of substrates and products resulting from the
lipase-catalyzed enantioselective transesterification of secondary alcohols using iso-
propenyl acetate as an innocuous acyl donor in toluene as organic solvent.
Results and Discussion
Chirasil-ꢀ-Dex with a new C11-spacer (C11-Chirasil-Dex, Fig. 1) has been synthe-
1
sized according to Ref. [16]. H, 13C NMR, and IR spectroscopy confirmed the
chemical link of the cyclodextrin moiety to the polysiloxane backbone, however,
the regioselective position of the C11 spacer (O6 vs. O2) is still elusive. The new
stationary phase was used in GC to separate a set of secondary alcohols and their
corresponding acetates. All secondary alcohols (Fig. 2) and their corresponding

Lipase-catalyzed Irreversible Transesterification
1153
Fig. 1. Structure of the permethylated ꢀ-cyclodextrin with a new C11-spacer (C11-Chirasil-Dex)
Fig. 2. A set of secondary alcohols tested in lipase-catalyzed resolution using isopropenyl acetate as
acyl donor in toluene; all have been base-line separated on Chirasil-ꢀ-Dex with a new C11- spacer
(C11-Chirasil-Dex)
acetates (racemic mixture or enantiomerically pure resulted from the enzymatic
reaction) tested in this investigation were baseline separated (for GC parameters
see Table 1).
The unusual elution order observed in the resolution of 3 and 5 using the new
CSP should be noted. The utility of the new CSP is demonstrated in the analysis of
the enantiomeric excesses of substrates and products resulting from the lipase-
catalyzed enantioselective transesterification of secondary alcohols using isopro-
penyl acetate as an innocuous acyl donor in toluene as an organic solvent (Scheme
1). The reaction was carried out at 40ꢁC at a molar ratio of isopropenyl acetate to
racemic alcohol of 2:1 to ensure the irreversibility of the reaction.
The results of the lipase-catalyzed transesterification of secondary alcohols
are summarized in Table 2. In the transesterification of (R,S)-secondary alcohols,
the (R)-alcohol was the faster reacting enantiomer yielding the (R)-acetate in

1154
A. Ghanem and V. Schurig
Table 1. Oven temperature (T), retention time (tR), resolution (Rs) and the separation factor (ꢁ) of the
simultaneous baseline separation of racemic secondary alcohols and their corresponding acetates
compound1
oven temperature=ꢁC2
tR(R)=min
tR(S)=min
Rs
5.83
ꢁ
1
1a
2
95
10.1
7.3
11.2
6.2
1.12
1.19
1.08
1.09
1.04
1.22
1.05
1.26
1.07
1.26
1.08
1.14
1.09
1.12
1.10
1.04
1.14
1.14
1.03
1.04
1.05
1.04
1.04
1.16
1.04
1.06
9.10
4.20
4.74
2.02
11.34
1.88
9.92
1.78
7.91
4.76
8.45
4.68
5.83
10.66
2.34
6.63
7.44
1.79
2.27
2.68
2.64
1.64
7.91
3.47
2.32
100
80
14.2
8.0
15.2
7.4
2a
3
27.1
30.2
4.6
26.1
24.9
4.8
3a
4
85
4a
5
3.6
4.4
50
5.4
5.8
5a
6
6.1
4.9
110
105
20.5
19.0
10.6
7.2
22.1
16.7
11.5
6.5
6a
7
7a
8
90 for 13 min, then increase
to 120 at the rate of 10=min
105
18.2
12.0
9.3
20.0
11.5
10.5
7.4
8a
9
9a
10
10a
11
11a
12
12a
13
13a
8.4
100
120
100
22.1
17.9
16.7
12.8
10.6
7.9
21.4
17.2
15.9
13.3
10.3
9.1
90 for 4 min, then increase
10.9
3.4
11.3
3.3
to 120 at the rate of 10=min
1
a is assigned to the acetate of the corresponding secondary alcohol; 2 the head pressure is 50 kPa,
the injector temperature was 200ꢁC and the FID temperature was 250ꢁC
Scheme 1
high ee and leaving (S)-alcohol as enantiomerically pure unreacted enantiomer.
The absolute configuration of the resulting alcohols and esters were determined
˚
by comparison with authentic samples. Molecular sieves (4 A) were added prior to
the enzymatic reaction to scavenge the liberated acetone (as a byproduct from the
enzymatic reaction), thereby shifting the equilibrium into the desired direction. In

Lipase-catalyzed Irreversible Transesterification
1155
Table 2. Lipase-catalyzed transesterification of secondary alcohols using isopropenyl acetate as acyl
donor in toluene as organic solvent
compound
time=h
ees=%
(S)-alcohol
eep=%
(R)-ester
conversion=%
E
1
2
4
4
>99
>99
>99
>99
94
78
92
93
5
56
52
51
94
55
53
56
73
67
84
77
49
65
93
287
3
4
>300
4
4
4
5
24
4
76
87
78
36
49
18
29
88
53
26
141
78
16
23
9
6
>99
>99
>99
>99
>99
99
7
4
8
15
24
4
9
10
11
12
13
4
10
42
29
15
15
85
>99
ees: enantiomeric excess of substrate (alcohol); eep: enantiomeric excess of product (ester); E:
enantiomeric ratio
Fig. 3. Lipase-catalyzed transesterification of 1 (4 h) and 3 (15 h) using isopropenyl acetate as acyl
donor in toluene
the case of alcohols 4, 10, and 11, the reaction was very fast. This was clear from
the fact that the faster reacting enantiomer reached its state of equilibrium more
rapidly and prolonged reaction time lead to some further conversion of the slower
reacting enantiomer and, thus, giving a less satisfactory enantiomeric excess of the
products (Table 2, compounds 4, 10 and 11).

1156
A. Ghanem and V. Schurig
Fig. 4. Lipase-catalyzed transesterification of 6 (4 h) and 12 (15 h) using isopropenyl acetate as acyl
donor in toluene
Conclusion
The lipase-catalysed enantioselective transesterification of a set of secondary alco-
hols was performed using the innocuous acyl donor isopropenyl acetate in toluene.
A baseline separation of both substrate and product was achieved using GC
equipped with a new CSP consisting of a polysiloxane-anchored permethylated
ꢀ-cyclodextrin with a new 11-spacer (C11-Chirasil-Dex).
Experimental
Chemicals and Instrumentation
All alcohols were commercially available and dried over molecular sieve prior to use. The racemic
esters were synthesized on an analytical scale to optimize a baseline separation of the enantiomers by
gas chromatography. Lipase from Pseudomonas cepacia immobilized on ceramic particles (PSL-C)
was a gift from Amano (Nagoya, Japan).
General Procedure for the Synthesis of Esters (Analytical Scale)
Acetic anhydride (30 mm3) is added to 20 mm3 of alcohol in 200 mm3 of pyridine. The mixture was
kept for 1 h at 100ꢁC and injected without further cleanup.
Lipase-catalyzed Irreversible Transesterification of Secondary Alcohols
˚
All reactants (alcohol, ester) were stored over activated molecular sieves (4A). Racemic alcohol
(0.5mmol) and 1 mmol of isopropenyl acetate were dissolved in 3 cm3 of toluene in a 5 cm3 reaction
vial. The reaction mixture was thermostated in an oil bath to 40ꢁC. Then, 100 mm3 of the reaction
mixture were withdrawn for GC analysis (t ¼ 0 of sample). Afterwards, 100 mg of lipase (PSL-C) were
3
˚
added followed by the addition of 100 mg of molecular sieves (4A). Samples (100 mm ) were taken
after several time intervals and centrifuged to separate the lipase. The organic layer was diluted by

Lipase-catalyzed Irreversible Transesterification
1157
100 mm3 of solvent and the reaction progress was monitored qualitatively by thin layer chromatogra-
phy. An aliquot of the supernatant was used for the GC analysis. When maximum conversion was
reached, the reaction was terminated by filtration, the enzyme was washed with solvent and then with
acetone, and the lipase powder was dried in air for further use.
GC Analysis Using a CSP
Gas-chromatographic analysis was performed on a Hewlett Packard instrument (Waldbronn, Germany)
equipped with a flame ionisation detector (FID). The chiral stationary phase C11-Chirasil-Dex was
coated on a non-deactivated 19 m ꢂ 0.25 mm fused silica capillary column (0.25 mm film thickness)
according to Ref. [18]. The analytical conditions were: injector temperature 200ꢁC; FID temperature
200ꢁC; oven temperature varying from 50 to 120ꢁC depending on the analyzed compound for the
simultaneous separation of enantiomers of both substrate and product. Hydrogen was used as carrier
gas (50 kPa column head pressure). The enantiomeric excess of both substrate and product as well as
conversion and enantiomeric ratio E were determined as described previously [10, 11].
Acknowledgements
This work was supported by Fonds der Chemischen Industrie and the Graduiertenkolleg Analytische
€
Chemie, University of Tubingen. Thanks are due to Prof. R. D. Schmid (University of Stuttgart,
Germany) and Prof. U. Bornscheuer (University of Greifswald, Germany) for their valuable advice.
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