Synthesis of five enantiomerically pure haptens designed for in vitro evolution of antibodies with peptidase activity Original Research Article

A series of five haptens have been synthesized for use in in vitro selection experiments from combinatorial antibody libraries. Haptens were designed for the recruitment of serine and cysteine protease reaction mechanisms for the cleavage of Phe-Ala and Phe-Phe (l,l) dipeptide analogues. For the selection of transition state stabilization, PheP(O)Ala (7) and PheP(O)Phe (10) derivatives were synthesized using the Mitsunobu approach where PheP represents the phosphonic acid analogue of phenylalanine and (O)Phe and (O)Ala represent (l)-β-phenyllactic and (l)-lactic acid, respectively. Optically pure puptidyl diazomethyl ketones 16 and 22 were synthesized for selection of the catalytic ensemble of cysteine proteases. An optically pure dipeptidyl boronic acid 26 was synthesized for the selection of the catalytic ensemble of serine proteases. A strategy for the evolution of catalytic antibodies using these haptens was developed which includes mechanism-based selections. Since mechanism based selections result in covalent trapping of species from libraries, diol and disulfide containing haptenic linkers were developed for the oxidative or reductive release of selected catalysts.