Muraymycins are a subclass of naturally occurring nucleoside antibiotics with promising antibacterial activity. They inhibit the bacterial enzyme translocase I (MraY), a clinically yet unexploited target mediating an essential intracellular step of bacterial peptidoglycan biosynthesis. Several structurally simplified muraymycin analogues have already been synthesized for structure–activity relationship (SAR) studies. We now report on novel derivatives with unprecedented variations in the nucleoside unit. For the synthesis of these new muraymycin analogues, we employed a bipartite approach facilitating the introduction of different nucleosyl amino acid motifs. This also included thymidine- and 5-fluorouridine-derived nucleoside core structures. Using an in vitro assay for MraY activity, it was found that the introduction of substituents in the 5-position of the pyrimidine nucleobase led to a significant loss of inhibitory activity towards MraY. The loss of nucleobase aromaticity (by reduction of the uracil C5-C6 double bond) resulted in a ca. tenfold decrease in inhibitory potency. In contrast, removal of the 2′-hydroxy group furnished retained activity, thus demonstrating that modifications of the ribose moiety might be well-tolerated. Overall, these new SAR insights will guide the future design of novel muraymycin analogues for their potential development towards antibacterial drug candidates.

To overcome bacterial resistances, the need for novel antimicrobial agents is urgent. The class of so‐called nucleoside antibiotics furnishes promising candidates for the development of new antibiotics, as these compounds block a clinically unexploited bacterial target: the integral membrane protein MraY, a key enzyme in cell wall (peptidoglycan) biosynthesis. Nucleoside antibiotics exhibit remarkable structural diversity besides their uridine‐derived core motifs. Some sub‐classes also show specific selectivities towards different Gram‐positive and Gram‐negative bacteria, which are poorly understood so far. Herein, the synthesis of a novel hybrid structure is reported, derived from the 5′‐defunctionalized uridine core moiety of muraymycins and the peptide chain of sansanmycin B, as a new scaffold for the development of antimicrobial agents. The reported muraymycin–sansanmycin hybrid scaffold showed nanomolar activity against the bacterial target enzyme MraY, but displayed no significant antibacterial activity against S. aureus, E. coli, and P. aeruginosa.

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram‐negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL, to increase the cellular uptake into Gram‐negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux‐deficient E. coli strain, thus providing a proof‐of‐concept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram‐negative pathogens.