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

Area of Science:

• Medicinal Chemistry
• Biochemistry
• Enzymology

Background:

• Metallo-β-lactamases (MβLs) are a significant class of enzymes responsible for antibiotic resistance.
• Development of specific substrates is crucial for studying MβL activity and for screening potential inhibitors.
• Existing fluorogenic substrates may have limitations in sensitivity or specificity.

Purpose of the Study:

• To synthesize and characterize a novel fluorogenic substrate for activity assays with metallo-β-lactamases.
• To evaluate the substrate's utility in kinetic studies of MβL enzymes.

Main Methods:

• Chemical synthesis of the fluorogenic substrate (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).
• Characterization of CA using fluorescence spectroscopy to determine quantum yield and fluorescence lifetimes.
• Steady-state kinetic assays to assess CA's substrate activity with specific MβLs.

Main Results:

• The synthesized compound CA demonstrated fluorescence properties, including a quantum yield of 0.0059 and two fluorescence lifetimes (3.63×10⁻¹⁰ s and 5.38×10⁻⁹ s).
• CA's fluorescence intensity was found to be concentration-dependent.
• Kinetic assays confirmed CA as a substrate for metallo-β-lactamases L1 and CcrA, with determined kinetic parameters (Km and kcat values).

Conclusions:

• The novel fluorogenic substrate CA is suitable for MβL activity assays.
• CA exhibits distinct kinetic properties with different MβL enzymes, enabling further characterization and inhibitor screening.
• This development offers a valuable tool for research into antibiotic resistance mechanisms mediated by MβLs.