Intermediate in beta-lactam hydrolysis catalyzed by a dinuclear zinc(II) complex: Relevance to the mechanism of metallo-beta-lactamase

Inactivation of beta -lactam antibiotics by metallo-beta -lactamase enzymes is a well-recognized pathway of antibiotic resistance in bacteria. As part of extensive mechanistic studies, the hydrolysis of a beta -lactam substra te nitrocefin (1) catalyzed by dinuclear zinc(TI) model complexes was inves tigated in nonaqueous solutions. The initial step involves monodentate coor dination of the nitrocefin carboxylate group to the dizinc center. The coor dinated substrate is then attacked intramolecularly by the bridging hydroxi de to give a novel intermediate (2') characterized by its prominent absorba nce maximum at 640 nm, which affords a blue color. The NMR and IR spectrosc opic data of 2' are consistent with it being zinc(II)-bound N-deprotonated hydrolyzed nitrocefin that forms from the tetrahedral intermediate upon C-N bond cleavage. Protonation of the leaving group is the rate-limiting step in DMSO solution and occurs after the C-N bond-breaking step. Addition of s trong acids results in rapid conversion of 2' into hydrolyzed nitrocefin (3 ). The latter can be converted back to the blue species (2') upon addition of base. The low pK(a) value for the amino group in hydrolyzed nitrocefin i s explained by its involvement in extended conjugation and by coordination to zinc(II). The blue intermediate (2') in the model system resembles well that in the enzymatic system, judging by its optical properties. The greate r stability of the intermediate in the model, however, allowed its characte rization by C-13 NMR and infrared, as well as electronic, spectroscopy.