Direct Selection for Catalysis from Combinatorial Antibody Libraries Using a Boronic Acid Probe: Primary Amide Bond Hydrolysis

Abstract

This report describes a joint hybridoma and combinatorial antibody library approach to elicit catalysts for primary amide bond hydrolysis. By immunization with a trigonal boronic acid hapten 3a and construction of a Fab (antigen-binding fragment) library, a diastereoselective catalyst for hydrolysis of the tripeptide primary amide substrate 1a was selected. In contrast, no antibody catalyst was isolated by standard hybridoma methods of monoclonal antibody production following immunizations with hapten 3a. The active Fab, BL25, obeys Michaelis−Menten kinetic behavior (kcat/kuncat ca. 4 × 104, Km = 150 μM) and is competitively inhibited by a boronic acid hapten analog 3b (Ki = 9 μM). Kinetic and binding studies both point to Fab selection of a hydrated tetrahedral anionic form of the boronic acid hapten 3a which serves to mimic the putative transition state 4 for catalysis of water addition to the primary amide bond. Fab-BL25 exhibits exquisite substrate selectivity, as a methyl ester analog of 1a is not accepted as a substrate. This work emphasises the power of the direct selection strategy when linked to screening of antibody combinatorial libraries and discloses the utility of boronic acids as haptens in acyl transfer processes.