Intermediate in β-Lactam Hydrolysis Catalyzed by a Dinuclear Zinc(II) Complex: Relevance to the Mechanism of Metallo-β-lactamase

Inactivation of β-lactam antibiotics by metallo-β-lactamase enzymes is a well-recognized pathway
of antibiotic resistance in bacteria. As part of extensive mechanistic studies, the hydrolysis of a β-lactam
substrate nitrocefin (1) catalyzed by dinuclear zinc(II) model complexes was investigated in nonaqueous
solutions. The initial step involves monodentate coordination of the nitrocefin carboxylate group to the dizinc
center. The coordinated substrate is then attacked intramolecularly by the bridging hydroxide to give a novel
intermediate (2‘) characterized by its prominent absorbance maximum at 640 nm, which affords a blue color.
The NMR and IR spectroscopic 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
strong 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 pKa value for the amino group in hydrolyzed nitrocefin
is 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
greater stability of the intermediate in the model, however, allowed its characterization by 13C NMR and
infrared, as well as electronic, spectroscopy.