Sol–Gel Processes DOI: 10.1002/anie.200604615 Xerogel-Encapsulated W110A Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus PerformsAsymmetric Reduction of Hydrophobic Ketones in Organic Solvents** Musa M. Musa, Karla I. Ziegelmann-Fjeld, Claire Vieille, J. Gregory Zeikus, and Robert S. Phillips* The use of biocatalysts in organic synthesis has become an effective and sometimes preferable alternative to normal chemical methodologies for the production of optically active compounds. [1,2] The asymmetric reduction of ketones and the kinetic resolution (KR) of racemic alcohols are the most important reactions for producing optically active alcohols that then can be used to synthesize industrially important compounds like natural products. A practical technique to improve enzyme performance is enzyme immobilization. [3] Most enzyme-immobilization methods involve covalent attachment of the enzyme to an activated group on a solid or gel support, which may result in significant loss of activity. A simple and efficient noncovalent immobilization method is enzyme encapsulation in trans- parent porous silicate glasses prepared by the sol-gel method. [4] The resulting glasses allow the transport of small molecules, but not enzyme molecules, into and out of the glasses pores. [5] The sol-gel encapsulation of enzymes has a lot of advantages, such as ease of recycling, broad applicability, cost effectiveness, and safety. [3] Alcohol dehydrogenases (ADHs) are enzymes that cata- lyze the reversible reduction of aldehydes and ketones to the corresponding alcohols. [6] However, ADHs have not been widely used for synthetic purposes in organic chemistry laboratories in part because they require aqueous media, in which many ketone and alcohol substrates are poorly or not at all soluble; this leads to large reaction volumes and compli- cated product recovery. [2c,e] An obvious solution for this problem, using organic solvents, [7] was first demonstrated by Klibanov and co-workers. [8] Secondary ADH (EC 1.1.1.2) from Thermoanaerobacter ethanolicus (TeSADH), a nicotinamide adenine dinucleotide phosphate (NADP + )-dependent thermostable enzyme, [9,10] is a useful biocatalyst for synthetic applications because it tolerates organic solvents and it accepts ketones and alcohols as substrates with high activities. [11,12] TeSADH obeys Prelog)s rule, in which the coenzyme NADPH delivers its pro-R hydride from the Re face of ketone substrates. [13] Recently, we have reported a new mutant of TeSADH, in which trypto- phan-110 was replaced with alanine, W110A TeSADH. [14] Although this mutant is able to reduce phenyl-ring-containing ketones at concentrations of 35 mm to produce their corre- sponding S-configured alcohols in Tris-HCl buffer solution/2- propanol (70:30 v/v; Tris = tris(hydroxymethyl)aminome- thane), higher substrate concentrations are required for practical production of optically active alcohols. Herein, we report the use of encapsulated W110A TeSADH in sol-gel glasses to overcome the aforementioned limitation. In 2003, Gröger et al. reported a practical asym- metric enzymatic reduction of poorly water-soluble ketones by using an ADH-compatible biphasic reaction medium. [15] One problem associated with using mixed aqueous and organic solvents, water-miscible or -immiscible, for enzymatic reactions is the tendency of these solutions to form emulsions in the workup, which causes problems of product separation. If the water, necessary for enzyme activity, is entrapped with the enzyme within the sol gel, the workup procedure can be simplified by using water-immiscible organic solvents, and therefore emulsion formation can be avoided. Sol-gel-encapsulated W110A TeSADH was prepared as previously reported, [5,16] although with some modifications. The sol gel was kept in Tris-HCl buffer solution medium until it was used as a wet sol gel (hydrogel). The asymmetric reduction of 4-phenyl-2-butanone (1a) to (S)-4-phenyl-2- butanol ((S)-1b), a precursor for the synthesis of bufeniode and labetalol (antihypertensive agents), [17] was used as a model in the screening reactions in this study. The hydrogel- encapsulated W110A TeSADH was used to reduce 1a to (S)- 1b in several different solvent systems (Table 1). The reduction carried out in aqueous buffer solution gave almost the same yield as with the free enzyme. [14a] However, the same sol gel was reused three more times to give 56 %, 30%, and 10% conversion, respectively. It was necessary to add 2.0 mg of NADP + for every new reaction because NADP + molecules either escape from the pores of the sol- gel glasses or become inactivated during turnover. [18a] The asymmetric reduction of 1a was also carried out in Tris-HCl [*] M. M. Musa, Prof. R. S. Phillips Department of Chemistry University of Georgia 1001 Cedar St., Athens, GA 30602 (USA) and Department of Biochemistry and Molecular Biology University of Georgia B122 Life Sciences Building, Athens, GA 30602 (USA) Fax: (+ 1)706-542-9454 E-mail: rsphillips@chem.uga.edu K. I. Ziegelmann-Fjeld, Dr. C. Vieille, Prof. J. G. Zeikus Department of Biochemistry and Molecular Biology Michigan State University 410 Biochemistry Building, East Lansing, MI 48824 (USA) [**] We gratefully acknowledge financial support from the National Science Foundation (grant no. 0445511) to R.S.P., J.G.Z., and C.V. Angewandte Chemie 3091 Angew. Chem. Int. Ed. 2007, 46, 3091–3094 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim