Intramolecular Cooperativity in the Reaction of Diacyl Phosphates with Serine β-Lactamases

Abstract

Asymmetric diaroyl phosphates (ArCOOPO(2)(-)OCOAr', where Ar = Ph, Ar' = 4-biphenyl, 2-benzothiophenyl and 2-benzofuranyl) have been prepared, evaluated as serine (classes A, C, and D) beta-lactamase inhibitors, and compared with respect to the latter with their symmetric parents, where Ar = Ar'. The asymmetric compounds, in general, were found to react with the beta-lactamases in two modes, corresponding to different orientations with respect to the active site, whereby either of the two aroyl groups may acylate the enzyme to form two different inert acyl-enzymes, E-COAr and E-COAr' . In all cases, the asymmetric compounds, in one orientation, react more rapidly with the enzymes tested than one symmetrical parent but not both. From comparisons of activation free energy differences, it was found that the changes in free energy on changing from one aryl group to another, in either the acyl group or the leaving group, were not additive, i.e., that the effect of changing one aroyl group to another depended on the leaving group and vice versa. Thus, intramolecular cooperativity between the aroyl groups must exist, arising either from direct interaction between them or from protein-mediated interaction or from a combination of both. Such cooperativity brings fresh opportunities and challenges to the search for novel beta-lactamase inhibitors.