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A. Ghanem, V. Schurig / Tetrahedron: Asymmetry 14 (2003) 2547–2555
3. Experimental
reaction vial. The reaction mixture was thermostated in
an oil bath to 40°C. 100 ml of the reaction mixture was
then withdrawn for GC analysis (t=0 of sample).
Afterward, 100 mg of lipase (PSL-C) colyophilized with
either 100, 200 or 600 mg peracetylated b-cyclodextrin
and entrapped in sol–gel was added followed by the
3.1. Chemicals and instrumentation
All alcohols were commercially available and dried over
molecular sieves prior to use. The racemic esters were
synthesized on an analytical scale to optimize a baseline
separation of the enantiomers by gas chromatography.
Scanning electron micrographs (SEM) and energy dis-
persive X-ray analysis (EDX) were performed on a
DSM 962 scanning electron microscope (SEM) (Zeiss,
LEO) equipped with a DX-4 X-ray detection system by
EDAX. This consisted of an energy dispersive Si(Li)-
detector with an active area of 10 mm2 and the EDX
software package. Micrographs were recorded detecting
secondary electrons generated by a probe current of 168
pA, whereas a 623 pA probe current was applied for
carrying out elemental analysis by EDX.
,
addition of 100 mg molecular sieve (4 A). Samples (100
ml) were taken after several time intervals and cen-
trifuged to separate lipase. The organic layer was
diluted by solvent (100 ml). The reaction progress was
monitored qualitatively by thin layer chromatography.
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. The lipase
powder was then dried in air for further use.
3.5. Enantioselective gas-chromatographic analysis
3.2. General procedure for the synthesis of esters (ana-
lytical scale)
Enantioselective gas-chromatographic analysis was per-
formed on a Hewlett Packard instrument (Waldbronn,
Germany) equipped with a flame ionisation detector
(FID). The chiral stationary C11-Chirasil-Dex was
coated on a non-deactivated 19 m×0.25 mm fused silica
capillary column (0.25 mm film thickness) according to
Ref. 22. The analytical conditions were: injector tem-
perature, 200°C; FID temperature, 200°C; oven temper-
ature varying from 50 to 120°C depending on the
analysed compound for the simultaneous separation of
enantiomers of both substrate and product. Hydrogen
was used as the carrier gas (50 kPa column head
pressure). The enantiomeric excess of both the substrate
and product as well as conversion and enantioselectiv-
ity E was determined as described previously.24,25
Acetic anhydride (30 ml) was added to an alcohol (20
ml) in pyridine (200 ml). This mixture was then kept at
100°C for 1 h and injected without further clean-up.
3.3. Co-lyophilized lipase with cyclodextrin-containing
gel
The Pseudomonas cepacia lipase (PSL) was dissolved (2
mg/1 ml) in a 20 mM phosphate buffer (pH 6) and
lyophilized for 48 h (control). The co-lyophilization of
the lipase with peracetylated b-cyclodextrin was per-
formed by the same method, except that the CD was
added prior to the lyophilization in the weight ratio 1:1;
1:2; 1:6 of lipase to cyclodextrin. The co-lyophilized
lipase with peracetylated b-cyclodextrin was then added
into a flask and stirred in 4 ml of phosphate buffer (pH
6) for 5 min to generate a homogeneous lipase/CD
suspension. To this mixture 400 ml of 1 M NaF solu-
tion, 800 ml gelatin (4% w/w) and 628.2 ml (5 mmol)
MTMS were added. The two-phase mixtures were vig-
orously shaken by hand until the evolution of heat
began (1–3 min). The resulting milky emulsion cleared
up to form a white solid material. This solid was then
cooled down in an ice bath for 10 min and left in a
closed vessel for 24 h at rt. The material was dried in an
oven at 40°C for 3 days under atmospheric pressure.
The resulting solid was grounded in a mortar and
shaken with 16 ml of buffer (pH 6) for 2 h. The product
was collected on a glass frit (D4), washed with buffer
(20 ml) and n-pentane(20 ml). The resulting gel was
then dried at 40°C for 24 h, grounded and kept for
further use.
Acknowledgements
This work was supported by Fonds der Chemischen
Industrie and the Graduiertenkolleg Analytische
Chemie, University of Tu¨bingen. Thanks are due to
Professor R. D. Schmid (University of Stuttgart, Ger-
many) and Professor U. Bornscheuer (University of
Greifswald, Germany) for their valuable advice.
Thanks to Professor E. Plies, Dr. S. Steinbrecher and
D. Adam, (Institut fu¨r Angewandte Physik) and E.
Nadler (Institut fu¨r Physikalische Chemie) for the SEM
and EDX parts.
References
1. Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in
Organic Synthesis; Wiley-VCH: Weinheim, 1999.
2. Ghanem, A.; Schurig, V. Chirality 2001, 13, 118–123.
3. Ghanem, A.; Schurig, V. Tetrahedron: Asymmetry 2003,
14, 57–62.
3.4. Lipase-catalyzed irreversible transesterification of
secondary alcohols
All reactants (alcohol, ester) were stored over activated
molecular sieves 4 A.
4. Ghanem, A.; Schurig, V. Tetrahedron: Asymmetry 2001,
12, 2761–2766.
5. Ghanem, A. Org. Biomol. Chem. 2003, 1, 1282–1291.
6. Ghanem, A. Ph.D. Thesis, University of Tu¨bingen, Ger-
many, 2002.
,
Racemic alcohol (0.5 mmol) and isopropenyl acetate (1
mmol) were dissolved in toluene (3 ml) in a 5 ml