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

Abstract In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-β-lactamases, (6 R ,7 R )-8-oxo-7-(2-oxo-2 H -chromene-3-carboxamido)-3-((4-(2-oxo-2 H -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 K m and k cat values of 18 μM and 5 s −1 and 11 μM and 17 s −1 , respectively.