Cyclic five-membered phosphinate esters as transition state analogues for obtaining phosphohydrolase antibodies

Abstract

Two novel cyclic five-membered phosphinate esters (6 and 7) were synthesized and used as transition state analogues (TSAs) for raising phosphohydrolase antibodies. The key step in the syntheses was a McCormack reaction between (2-chloroethyl)dichlorophosphite and 1,4-diphenyl-1,3-butadiene to form isomeric cyclic phosphinyl chlorides which upon further elaboration yielded the TSAs. X-ray crystal structures of two cyclic phosphinate TSA precursors (14a and 14b) show highly compressed CPC bond angles of 94°-97° indicating that the CPC bond angles in 6 and 7 are significantly distorted towards the ideal trigonal bipyramidal equatorial-apical transition state angle of 90° formed during phosphate ester hydrolysis. Antibodies were raised to 6 and 7 using standard hybridoma technology. One antibody, Jel 541, an IgM class antibody, was obtained that was capable of catalyzing the hydrolysis of phosphonate substrate 9 and, to a much lesser extent, phosphate substrate 8. This antibody bound TSA 6 approximately 100 times more tightly than TSA 7 as determined by solid phase radio immune assays. The activity of the antibody-catalyzed reaction was completely inhibited by the presence of TSA6. Although the relative instability and poor solubility of Jel 541 made it impossible to perform a detailed kinetic analysis of the antibody-catalyzed reactions, these results demonstrate that phosphohydrolase antibodies can indeed be obtained using cyclic five membered phosphinates as haptens.Key words: transition state analogues, phosphate ester hydrolysis, catalytic antibodies.