ANTIBODY-CATALYZED DECARBOXYLATIVE OXIDATION OF VANILLYLMANDELIC ACID

The most important industrial process for the synthesis of vanillin is performed in two steps involving an electrophilic aromatic substituti on of glyoxylic acid on guaiacol followed by an oxidative decarboxylat ion of the intermediary alpha-hydroxy acids formed, thereby producing not only vanillin, but also byproducts which have to be eliminated. In the present study, we took advantage of the high specificity of catal ytic antibodies to improve the synthesis of vanillin. Among ii monoclo nal antibodies elicited against the quaternary ammonium hapten H3, ant ibody H3-12 was found to catalyze the oxidative decarboxylation of van illylmandelic acid (VMA), the precursor of vanillin, in the presence o f sodium metaperiodate. The kinetic data of the antibody-catalyzed rea ction are consistent with an ordered binding mechanism. At pH 9.0, H3- 12 catalyzed the transformation of VMA into vanillin with a k(cat) of 2.70 min(-1), a Michaelis-Menten constant K-a for the binary complex o f 260 mu M, and a K-b for the ternary complex of 2100 mu M. The cataly zed reaction was fully inhibited by a hapten analogue with a K-i, of 1 0 mu M. The fine specificity of anti-H3 monoclonal antibodies was dete rmined using H3-related compounds with a competitive enzyme immunoassa y. Controls demonstrating that catalytic activity is actually related to antibody binding, and mechanistic studies, are also presented.