Ester Synthesis From Trimethylammonium Alcohols in Dry Organic Media Catalyzed by Immobilized Candida antarctica Lipase B Pedro Lozano, Mirta Daz,* Teresa de Diego, Jose ´ L. Iborra Departamento de Bioquı ´mica y Biologı ´a Molecular B e Inmunologı´a, Facultad de Quı´mica, Universidad de Murcia, P.O. Box 4021, E-30100 Murcia, Espan ˜ a; telephone: 34-968-36-7398; fax: 34-968-36-4148; e-mail: jliborra@um.es Received 16 May 2002; accepted 8 October 2002 DOI: 10.1002/bit.10580 Abstract: Twenty-one different organic solvents were as- sayed as possible reaction media for the synthesis of butyryl esters from trimethylammonium alcohols in dry conditions catalyzed by immobilized Candida antarctica lipase B. The reactions were carried out following a transesterification kinetic approach, using choline and L- carnitine as primary and secondary trimethylammonium alcohols, respectively, and vinyl butyrate as acyl donor. The synthetic activity of the enzyme was strictly depen- dent on the water content, the position of the hydroxyl group in the trimethylammonium molecule, and the Log P parameter of the assayed solvent. Anhydrous condi- tions and a high excess of vinyl butyrate over L-carnitine were necessary to synthesize butyryl-L-carnitine. The synthetic reaction rates of butyryl choline were practi- cally 100-fold those of butyryl-L-carnitine with all the as- sayed solvents. In both cases, the synthetic activity of the enzyme was dependent on the hydrophobicity of the sol- vent, with the optimal reaction media showing a Log P parameter of between −0.5 and 0.5. In all cases, 2-meth- yl-2-propanol and 2-methyl-2-butanol were shown to be the best solvents for both their high synthetic activity and negligible loss of enzyme activity after 6 days. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 352–358, 2003. Keywords: lipase; trimethylammonium ester; butyryl-L- carnitine; butyryl choline; ester synthesis INTRODUCTION The technological usefulness of enzymes can be greatly enhanced if they are used in organic media rather than in their natural aqueous reaction media. In this way, enzymes in nonconventional media may exhibit new catalytic prop- erties, such as synthetic activity (by hydrolytic enzymes), stereo- and regioselectivity for substrates, and enhanced sta- bility (Klibanov, 2001). Among enzymes, lipases have been used successfully as a catalyst for the synthesis of esters, both on a small scale and on an industrial scale (Balc ˜o et al., 1996; Jaeger and Reetz, 1998). Recently, research has been focusing on amphiphilic molecules, for example, fatty acid sugar esters (Arcos et al., 1998) and dihydroxyacetone fatty acid esters (Virto et al., 2000) for use as nonionic surfac- tants, esters of -hydroxyacids as humectants (Torres and Otero, 1999; Torres et al., 1999), and phospholipids (Virto et al., 1999; Virto and Adlercreutz, 2000) for their important food and pharmaceutical applications. Lipase-catalyzed synthesis of these compounds requires nearly anhydrous conditions and the use of nonpolar solvents as reaction me- dia to avoid the stripping of essential water molecules from the enzyme structure. However, polar acyl-acceptors exhibit limited solubility in common “enzyme-friendly” organic solvents (e.g., hexane). Several strategies have been used to overcome this problem, including the complexation of polar substrates (Castillo et al., 1994), the adsorption of polar substrates on inert silica-gel (Castillo et al., 1997), as well as the use of hydrophilic solvents and/or free-solvent systems containing partially soluble polar substrates (Ljunger et al., 1994). The use of polar solvents or free-solvent systems combined with an efficient removal of water from the media has proved to be an adequate strategy for enzymatic ester synthesis (Torres et al., 1999; Virto et al., 1999). Both direct esterification and transesterification approaches have been assayed in the lipase-catalyzed synthesis of amphiphilic es- ters. Among the numerous acyl-donors employed during transesterification, vinyl esters are the most popular, be- cause the vinyl alcohol formed during the process tautomer- izes to low-boiling-point acetaldehyde, thus shifting the equilibrium toward ester formation (Virto and Adlercreutz, 2000). Choline and L-carnitine [R-(-)-3-hydroxy-4-N,N,N-tri- methylaminobutyrate] are ubiquitous trimethylammonium alcohols, which play different roles in living cells. In addi- tion to the classical functions of these alcohols, many phar- macological applications have been described for their Correspondence to: J. L. Iborra *Permanent address: Departamento de Quı ´mica. Facultad de Ciencias Exactas, Universidad Nacional de Salta, Salta, Repu ´blica Argentina Contract grant sponsor: CICYT Contract grant number: BIO99-0492-C02-01 © 2003 Wiley Periodicals, Inc.