A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-ß-lactamases.

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-ß-lactamases, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and characterized. CA exhibited a fluorescence quantum yield (φ) of 0.0059, two fluorescence lifetimes of 3.63×10(-10) and 5.38×10(-9)s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic assays revealed that CA is a substrate for metallo-ß-lactamases (MßLs) L1 and CcrA, exhibiting Km and kcat values of 18µM and 5s(-1) and 11µM and 17s(-1), respectively.