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

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-β-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-β-lactamases (MβLs) L1 and CcrA, exhibiting Km and kcat values of 18 μM and 5 s−1 and 11 μM and 17 s−1, respectively.

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