Synthetic Rifampicin‐Microcin J25 Conjugates Can Enter Both Gram‐Positive and Gram‐Negative Bacteria

ABSTRACT Rifampicin (RIF) is a widely used semi‐synthetic broad‐spectrum antibiotic that primarily targets Gram‐positive bacteria. Microcin J25 (MccJ25) is an antimicrobial lasso peptide with promising therapeutic potential that exhibits potent activity against Gram‐negative bacteria. To combine the distinct activity spectra and mechanisms of these two molecules, we designed and synthesized rifampicin‐MccJ25 conjugates. One end of the linker is connected to RIF through copper(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC), and the other through amide coupling to a MccJ25 variant engineered to carry a single lysine residue at a specific position. Even though the resulting conjugates were less active than the corresponding 1:1 mixture of rifampicin and MccJ25, they displayed broadened antibacterial activity and were able to suppress the growth of both Gram‐positive and Gram‐negative bacteria, and one of them has a moderate resistance suppression effect. Our data suggest that RIF‐MccJ25 conjugation is a promising strategy to build next‐generation chimeric antibiotics.