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Fig. 6. Kinetic resolution of rac-6 catalyzed by lipases.
Fig. 7. Kinetic resolution of rac-9 catalyzed by lipases.

2.2. Sol-Gel Process

1. n-Butyltrimethoxysilane (ABCR, Germany).

2. Isobutyltrimethoxysilane (Lancaster, UK).

3. Tween-80 [poly-oxyethylene (20) sorbitan monooleate] (Fluka).

4. Sodium fluoride (Fluka).

6. Methyl-P-cyclodextrin (Aldrich).

7. Tetramethoxysilane (Fluka).

8. Polyvinyl alcohol (PVA; molecular weight [mol wt] = 15000) (Merck, Germany).

2.3. Lipases

The lipases from Candida antarctica (CaLB; Chirazyme L-2®), C. rugosa (CrL; Chirazyme L-3®), and Mucor miehei (MmL; ChirazymeL-9®) are accessible from Roche; Aspergillus niger (AnL; Amano AS), Burkholderia cepacia (BcL; Amano PS), and Pseudomonas fluorescens (PfL; Amano AK "20") from Amano Pharmaceutical Co.; C. rugosa type VII (CrL type VII) from Sigma; Penicillium roqueforti (PrL) from Fluka; Thermomyces lanuginosa (TIL; Novozym SP) from Novo Nordisk.

3. Methods

3.1. Sol-Gel Entrapment of Lipases General Procedure

1. A commercial lipase powder (lyophilizate), such as AnL (150 mg), BcL (150 mg), CaLB (125 mg), CrL (150 mg), CrL type VII (60 mg), MmL (150 mg), PfL (150 mg), PpL (150 mg), PrL (150 mg), or TIL (70 mg) is placed in a 50-mL Falcon tube (Corning) with 390 |L, 0.1 M Trid/HCl-buffer, pH 7.5, and the mixture is vigorously shaken with a vortex mixer. In the case of an additive or an additional porous solid support, these materials are included as needed (26). In a typical procedure, 0.5 mmol of 18-crown-6 as an additive is added. In the case of "double immobilization," the additive is used as above in addition to 50 mg of Celite.

2. Then 100 |L of aqueous 4% (v/v) PVA, 50 |L of 1 Maqueous sodium fluoride, and 100 |L isopropanol are added and the mixture is homogenized using a vortex mixer.

3. Then the alkylsilane (Fig. 2, 4d or 4e) (2.5 mmol) and TMOS (Fig. 2, 5) (0.5 mmol; 74 |L; 76 mg) are added. The mixture is agitated once more for 10 to 15 s. Gelation is usually observed within seconds or minutes while the reaction vessel is gently shaken.

4. Following drying overnight in the opened Falcon tube, 10 to 15 mL isopropanol is added in the order to facilitate removal of the white solid material (filtration).

5. The gel is washed with 10 mL of distilled water, 10 mL isopropanol, 10 mL and n-pentane. During this process a spatula is used to crush the gel.

6. The lipase immobilizate is placed in an open 2-mL plastic vessel and dried at room temperature (26).

3.2. Determination of Protein Content

In order to determine the protein content of the commercial lipases as well as the degree of loading, the BCA Protein Assay Kit (Sigma) can be used. Accordingly, solutions of the commercial lipase or the wash-solutions following the solgel process are incubated for 30 min and measured at 37°C using a UV/ Vis-spectrometer at 562 nm according to the Technical Bulletin (Sigma). Distilled water is used as a reference and bovine serum abumin (BSA) as standards. The degree of immobilization ranges between 0.3 and 0.9 (26).

3.3. Determination of Lipase Activity

1. Depending on the activity, between 1 and 50 mg of the sol-gel lipase-immobilizate is placed in a Falcon tube together with solutions of lauric acid (100 mg; 0.5 mmol) solution and n-octanol (158 |L; 130 mg; 1.0 mmol) in nondried (H2O-saturated) isooctane.

2. The mixture is then shaken at 180 rounds per minute (rpm) at 30°C. At defined intervals (usually after 15, 30, 45, and 60 min) 300 |L samples are taken and centrifuged (13,000 rpm) before the gas chromatographic determination of lauric acid and lauric acid n-octyl ester is carried out using 150 |L of the supernatant.

3. By applying a linear regression of the measured values the initial reaction rate in |imol/min and therefore the activity relative to 1 g of immobilizate can be determined. By considering the degree of loading, the specific activity is calculated.

4. The relative activity is determined by dividing the specific activity of the immobilizate by the specific activity of the commercial lipase powder (26).

3.4. Typical Kinetic Resolution on a Preparative Scale

1. Similar to the above protocol a mixture of rac-1-(2-naphthyl)-ethanol (10 g; 58 mmol), vinyl acetate (8.1 mL; 7.5 g; 87 mmol), and 250 mg of a sol-gel CaLB-immobilizate (prepared in the presence of 18-crown-6 as additive) in 300 mL toluene is shaken at 35°C for 48 h.

2. Following filtration gas chromotography analysis shows a conversion of 50.0%, the enantio meric excess of no-reacted (S)-1-(2-naphthyl)ethanol and product ((^-acetate) each being >99.9%.

3. The immobilizate is removed by filtration, washed with toluene and pentane, and can be reused, showing no significant loss in activity or enantioselectivity (26).


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