beta-secondary kinetic isotope effects in the clavaminate synthase-catalyzed oxidative cyclization of proclavaminic acid and in related azetidinone model reactions

Clavaminate synthase is an Fe(II)/alpha-ketoglutarate-dependent oxygenase t hat catalyzes three mechanistically distinct reactions in the course of cla vulanic acid biosynthesis. Clavulanic acid is of significant chemical impor tance as a potent: inhibitor/inactivator of beta-lactamase enzymes, a promi nent means of bacterial resistance to, for example, penicillin. Primary and alpha-secondary (T)(V/K) kinetic isotope effects have been determined in e arlier work for the clavaminate synthase-catalyzed oxidative cyclization of proclavaminic acid, one of the three reactions mediated by this enzyme. In this paper the beta-secondary deuterium kinetic isotope effect for this re action has been determined using remote H-3 and C-14 labels in an attempt t o distinguish between radical or cationic intermediates in the reaction as suggested by the magnitudes of the primary and secondary or-effects. The pr esence of the adjacent azetidinone nitrogen and the intervention of an azet inone intermediate, formally antiaromatic in the resonance form of the amid e, make interpretation of the low beta-secondary effect (1,056 +/- 0.002 fo r dideuteriation at C-3') problematic. To assist interpretation of this res ult, a 4-chloroazetidinone model system has been constructed dideuteriated at C-3 identically to proclavaminic acid and bearing remote radiolabels. Re action of this substrate at 25 degrees C under both radical and solvolysis conditions afforded beta-secondary kinetic isotope effect data for direct c omparison to the enzymic reaction. The measured effects are similarly small but strongly dependent on the polarity/acidity of the reaction medium. The se results are discussed in terms of the commitment to catalysis and the ex tent to which amide resonance may be favored in the transition state of the oxidative cyclization.