ENANTIOSELECTIVE HYDROLYSIS OF RACEMIC ESTERS USING PIG-LIVER ESTERASE

The utilization of enzymes is a recent achievement in organic synthesi s. There are many good reasons for the organic chemist to consider thi s approach. Enzymatic reactions are very fast and specific. The same t ransformations 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 sophistic ated equipment. As compared with conventional catalysts, enzymes have an excellent reaction, substrate and stereo chemical specificity. Many enzymes are now commercially available and their prices are not prohi bitive. Moreover, the enzymes can be immobilized through simple techni ques on inert supports. The immobilized enzymes can be easily recovere d from the reaction medium and reused many times. Enantioselective syn thesis is an important area in which enzymes found large applications. Hydrolases, as lipase and pig liver esterase (PLE), have been success fully used to obtain optically pure alcohols, acids and esters. In thi s paper we report a new method for the immobilization of PLE on modifi ed inorganic substrates and some applications of the immobilized PLE i n synthesis of enantiomeric mono esters of dicarboxylic acids. As inor ganic support we used silica with particle size of 40-80 mesh and aver age pore diameter of 550 Angstrom. The surface isoelectric point of th is material is relatively low. The support was derivatized with p-phen ylenediamine and functionalized by diazotization to reaction with prot ein. The enzyme was covalently bonded to the support by reaction betwe en diazonium salt and phenolic group of tyrosine residues. The immobil ization yield was higher than 50% and the preparation was stored for o ne year at 4 degrees C without significant loss of activity. Kinetic s tudies on immobilized PLE emphasized that Michaelis constant K-M was v ery close to that of free enzyme, that means that no conformational ch anges in active site occurred during immobilization. Dimethyl 2-methyl -2-phenylmalonate was hydrolysed using immobilized PLE with slightly h igher enatiomeric excess than reported earlier. The immobilized PLE ca n be removed from the reaction mixture by simple filtration and reused to hydrolyse other quantities of substrate without significant loss o f enantioselectivity and with moderate reduction in chemical yield. At tempts to hydrolyse diethyl (3,5-di-tert-butyl-4-hydroxybenzyl)-malona te and ethyl (3,5-di-tert-butyl-4-hydroxybenzyl)cyanoacetate lead to c omplex mixtures of products that must be further investigated.