Antibody catalysis of some organic and biochemical reactions: Dissertation

Antibodies are immunoglobulins that bind to stable ground-state molecules and recognise their respective antigens with high affinity and high specificity. Enzymes in turn are natural catalysts that bind and stabilise selectively the transition-state of the reaction and accelerate the rate of a (bio)chemical reaction by lowering the free energy of activation. Enzymes are also able to act as "entropy traps" in reducing the rotational and translational degrees of freedom that are prerequisites for the formation of the activated complex between the reactants. In addition, enzymes use general acid and base catalysis, nucleophiles and co-factors in enhancing the rates of reactions. It was nearly fifty years ago when Linus Pauling first proposed in his lecture entitled "Chemical Achievement and Hope for the Future" that antibodies binding the ground state molecules might act as enzyme-type catalysts for chemical reactions. Twenty-one years later, William Jencks suggested that it should be possible to obtain an antibody with enzymatic properties by raising it against the antigen that resembles the transition-state of the reaction. It is only eleven years ago that the first reports of the catalytic antibodies emerged from the laboratories of Richard Lerner and Peter Schultz. Indeed, the antibodies elicited against stable, natural or synthetic transition-state analogues of numerous reactions have been found to possess enzyme-like activities. These catalytic antibodies generally display the Michaelian type saturation kinetics, competitive inhibition by the transition-state analogue, selective binding to the transition-state and remarkable substrate specificities. In the present investigation, structurally different antigens (haptens) were used to study whether it was possible to obtain antibody catalysts for the acyl-transfer, Diels Alder and peptidyl-prolyl cis-trans isomerisation reactions. Acyl-transfer reactions, such as hydrolytic reactions are important transformations both in bio-chemistry and synthetic organic chemistry; the Diels Alder reaction is synthetically useful in constructing substituted cyclohexenes; and the peptidyl-prolyl cis-trans isomerisation reaction is a highly substantial biochemical reaction which plays a significant role in protein folding, transport and transmembrane signalling. Monoclonal antibodies were raised against two a-keto amide moiety containing antigens. They were anticipated to induce antibodies for hydrolytic acyl-transfer reactions, i.e. ester and amide hydrolyses. a-Keto amide substructures found in natural macrolides such as FK506, rapamycin and cyclotheonamide A are known to mimic the twisted amide bond that is one possible transition-state for the amide bond hydrolysis. During the study, a new, synthetically useful concurrent alkylative de-carbonylation and decarboxylation reaction of methoxy-substituted 3-phenyl-2-oxo-propanoic acids was discovered. It turned out to be a viable method for the preparation of isopropyl anisoles and veratroles, producing them in high yields. The elicitation of antibodies against the freely-rotating, lipophilic and highly aromatic ferrocene haptens as loose transition-state mimics was successful. Both endo and exo selective antibodies catalysing the Diels Alder reaction between 4-carboxy-benzyl trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide were found. High regio-, diastereo- and enantioselectivities and no product inhibition were observed. Moreover, the found Diels Alderases had effective molarities comparable to those of antibodies elicited against the constrained bicyclo[2.2.2]octene haptens. The dicarbonyl moiety in natural products FK506 and rapamycin and less complex pyruvylamides adopts an orthogonal conformation and possibly serves as a twisted-amide mimic. The a-keto Val-Pro-Phe hapten was anticipated to induce anti-body binding sites that were complementary to the twisted a-keto amide functionality and of hydrophobic character. Indeed, two antibodies were found to catalyse the cis to trans isomerisation of the fluorophoric tripeptides and the 4-nitroanilide substrates as characterised using both direct fluorescence quench and chymotrypsin-coupled assays, respectively. Both catalyst showed competitive inhibition by the antigen derivative, and the product inhibition, i.e. binding to the trans isomer, did not appear to be significant. In catalysis and binding the peptide substrates, factors other than simple hydrophobic interactions are possibly involved, such as transition-state stabilisation and ground-state destabilisation.