Enantioselective hydrolysis of racemic esters using pig liver esterase

The utilization of enzymes is a recent achievement in organic synthesis. There are many good reasons for the organic chemist to consider this approach. Enzymatic reactions are very fast and specific. The same transformations carried out by classical means in several days or weeks, can be achieved in hours or days using enzymes as catalysts. Enzymes act in mild conditions with high yields, and do not require sophisticated equipment. As compared with conventional catalysts, enzymes have an excellent reaction, substrate and stereo chemical specifity. Many enzymes are now commercially available and their prices are not prohibitive. Moreover, the enzymes can be immobilized through simple techniques on inert supports. The immobilized enzymes can be easily recovered from the reaction medium and reused many times. Enantioselective synthesis is an important area in which enzymes found large applications. Hydrolases, as lipase and pig liver esterase (PLE), have been successfully used to obtain optically pure alcohols, acids and esters. In this paper we report a new method for the immobilization of PLE on modified inorganic substrates and some applications of the immobilized PLE in synthesis of enantiomeric mono esters of dicarboxylic acids. As inorganic support we used silica with particle size of 40–80 mesh and average pore diameter of 550 Å. The surface isoelectric point of this material is relatively low. The support was derivatized with p-phenylenediamine and functionalized by diazotization to reaction with protein. The enzyme was covalently bonded to the support by reaction between diazonium salt and phenolic group of tyrosine residues. The immobilization yield was higher than 50% and the preparation was stored for one year at 4°C without significant loss of activity. Kinetic studies on immobilized PLE emphasized that Michaelis constant K M was very close to that of free enzyme, that means that no conformational changes in active site occurred during immobilization. Dimethyl 2-methyl-2-phenylmalonate was hydrolysed using immobilized PLE with slightly higher enatiomeric excess than reported earlier. The immobilized PLE can be removed from the reaction mixture by simple filtration and reused to hydrolyse other quantities of substrate without significant loss of enantioselectivity and with moderate reduction in chemical yield. Attempts to hydrolyse diethyl (3,5-di- tert-butyl-4-hydroxybenzyl)-malonate and ethyl (3,5-di- tert-butyl-4-hydroxybenzyl)cyanoacetate lead