β-Lactamase-catalysed hydrolysis of cephalexin: evolution of the cephalosporoate intermediate

Abstract

Hydrolysis of cephalexin catalysed by P99 β-lactamase from Enterobacter cloacae has been studied at pD 6.4 and 8.0. The enzymatic hydrolysis product is the cephalosporoate intermediate, which in aqueous media undergoes tautomerization of the double bond in the dihydrothiazine ring from position 3–4 to 4–5, with the consequent uptake of a proton at C(3). This latter process is governed by steric constraints. NMR results show that the cephalosporoate compound bears the aromatic group, present in the side chain at C(7), folded over the dihydrothiazine ring. Under these conditions the proton is chiefly taken up by the α face of the dihydrothiazine ring. Subsequently, the six-membered ring can be subjected to several reactions according to the pH.