Selection of Solvents for Bioorganic Synthesis MATS RESLOW, PATRICK ADLERCREUTZ, AND BO MATTIASSON Department of Biotechnology Chemical Center University of Lund S-221 00 Lund, Sweden The use of biocatalysts in organic media has attracted much interest in recent years. The fact that enzymes and cells can be active in organic media has opened a totally new sector for enzyme technologists to exploit. Some interesting reactions catalyzed by enzymes in organic solvents are: (i) resolution of alcohols and acids by lipase-catalyzed esterification,’.’ (ii) protease-catalyzed peptide synthesis,’ and (iii) regioselective lipase-catalyzed interesterification of fats and oils.’ Enzymes can be used in reaction systems with very low water contents. A low water content favors mass transfer of hydrophobic substrates to the biocatalyst phase and the thermodynamic equilibrium in reversed hydrolytic reactions. The solvent chosen is important for the enzyme activity. Different parameters have been used to charac- terize the solvent. Log P (the partition coefficient of a given compound in the octanol-water two-phase system) has been useful in predicting the activity of whole cell catalysts in different solvents.’ The aim of this work was to study the influence of solvent and water content on the kinetics and stability of an enzyme-catalyzed reaction and to investigate if it was possible to rationalize the choice of organic solvent. MATERIALS AND METHODS a-Chymotrypsin (92 mg) from bovine pancreas (Sigma Chemical) was dissolved in 4.3 mL of 50 mM sodium phosphate buffer, pH 7.8, and mixed with 2.0 g porous glass (CPG-10; mesh size: 200-400; surface area: 7.4 m’/g; mean pore diameter: 2147 A; Sigma Chemical). The mixture was dried under reduced pressure for 3 h. The water content was determined after drying by analyzing a part of the preparation gravimetri- cally. This preparation had an enzyme content of 4.29% (w/w) and a water content of 0.6% (w/w). The activity of the enzyme was measured by following the esterification of Ac-Phe with ethanol. We added 1.9 mL of water-saturated solvent and different amounts of water to 100 mg chymotrypsin-glass in 10-mL stoppered glass bottles. The bottles were shaken on a reciprocal shaker (150 rpm) at 20 “C. After 20 min, the reaction was started by adding ethanol containing Ac-Phe so that final concentrations of 10 mM Ac-Phe and 1 .O M ethanol were obtained. We took 20-pL samples at various intervals to determine the content of Ac-Phe-OEt by HPLC (Nucleosil C,,-column eluted with H,O/MeCN/HAc, 55:40:5). 250