Studies towards a catalytic antibody for an intramolecular Diels-Alder reaction

Hapten 6 was proposed as the transition-state analogue for both an intramolecular Diels-Alder reaction (13b to 46 ) and an ester hydrolysis (46 to 45). The key step in the synthesis of the hapten was the intramolecular Diels Alder reaction of (1E,7E)-1-diphenoxyphosphoryl-1,7,9-decatriene which resulted in a 2:1 mixture of isomeric cycloadducts. The stereochemistry of these isomers was determined by comparison of the 13C NMR spectra of the reduced cycloadducts to those of 1-methyl decahydronapthalenes. The cis-fused Diels-Alder adduct, which is formed via the exo transition state, was formed as the major isomer.*; Mice immunised with KLH6 led to the generation of high affinity antibodies to 6 as determined by a particle coated fluorescent immunoassay (PCFIA). Further binding studies showed that one monoclonal antibody, F325, had an equilibrium dissociation constant, KD, estimated to be 8.8 x 10-6 M using an indirect competition method with detection by an enzyme-linked immunosorbent assay (ELISA).; Kinetic studies of the hydrolysis reaction of 13b showed that F325 followed Michaelis-Menten kinetics with a Km of 265 +/- 32 muM and vmax of 0.13 +/- 0.007 muM/min in the presence of 1.0 muM F325. The activity of F325 was not affected by the general esterase inhibitor SBTI indicating that the hydrolysis reaction was being catalysed by the antibody and was not due to the presence of contaminating esterases. Studies on substrate specificity of F325 suggested that the conjugated diene moiety was important for the observed catalytic behaviour of the antibody. However, neither the cyclic p-nitrophenyl ester of the IMDA product nor the acid could be detected by an HPLC assay.; *Please refer to dissertation for diagram.