Mechanistic studies of catalytic antibodies

Two haptens based on the strategy of transition state stabilization and entropic restriction were used to generate antibodies to catalyze the polymerization of beta-hydroxybutyrate monomers. Several of the antibodies specific for one of the haptens catalyzed the transesterification reaction between monomers, but lacked sufficient selectivity to generate polymeric material through iterative transesterification. Implications for improved design of antibody-based polymerization catalysts are presented.; Mechanistic studies of an antibody catalyzed oxy-Cope rearrangement are presented as well. Traditional kinetic and thermodynamic tools of mechanistic analysis were combined with transferred nuclear Overhauser effect (tr-nOe) studies of bound substrate conformation to provide a complete mechanistic scheme for the antibody catalyzed rearrangement. The tr-nOe studies indicate that the antibody combining site preferentially binds the substrate in a cyclic conformation. The substrate is thus preorganized for rearrangement by the combining site, as intended in the original hapten design. Appendix 1 describes further mechanistic studies of the antibody catalyzed oxy-Cope rearrangement using site directed mutagenesis.; Similar tr-nOe studies were applied to an antibody catalyzed transesterification reaction. The established tr-nOe techniques were extended to the study of bimolecular complexes, allowing determination of the three-dimensional structure of the Michaelis complex of the transesterification substrates bound in the antibody combining site. The structure indicates that a very high level of pre-organization can be achieved by antibodies raised against phosphonate-based transition state analogs, providing a structural basis for the general effectiveness of such haptens. Preliminary tr-nOe data on several different substrates are also presented, providing insight into the selectivity of the antibody catalyzed reaction.