Diglycine Enables Rapid Intrabacterial Hydrolysis for Activating Anbiotics against Gram‐negative Bacteria

Antimicrobial drug resistance demands novel approaches for improving the efficacy of antibiotics, especially against Gram‐negative bacteria. Herein, we report that conjugating a diglycine (GG) to an antibiotic prodrug drastically accelerates intrabacterial ester‐bond hydrolysis required for activating the antibiotic. Specifically, the attachment of GG to chloramphenicol succinate (CLsu) generates CLsuGG, which exhibits about an order of magnitude higher inhibitory efficacy than CLsu against Escherichia coli. Further studies reveal that CLsuGG undergoes rapid hydrolysis, catalyzed by intrabacterial esterases (e.g., BioH and YjfP), to generate chloramphenicol (CL) in E. coli. Importantly, the conjugate exhibits lower cytotoxicity to bone marrow stromal cells than CL. Structural analogues of CLsuGG indicate that the conjugation of GG to an antibiotic prodrug is an effective strategy for accelerating enzymatic prodrug hydrolysis and enhancing the antibacterial efficacy of antibiotics. A blessing in disguise: Conjugating a diglycine (GG) to an antibiotic prodrug (chloramphenicol succinate) increases the rate of prodrug activation by increasing the rate of ester‐bond hydrolysis catalyzed by bacterial esterases. Dipeptide conjugation to antibiotic prodrugs can increase antibiotic efficacy and reduce adverse drug effects.