Solid-Phase Synthesis and Biological Evaluation of Des-hydroxy Pseudouridimycin Analogs

Pseudouridimycin (PUM) is a C-nucleoside/peptide antibiotic that selectively inhibits bacterial RNA polymerase (RNAP) and exhibits potent activity against drug-resistant pathogens. However, PUM suffers from chemical instability due to self-immolative cleavage of its central hydroxamate bond. Here, we employed cryo-electron microscopy to determine structures of PUM (<b>1</b>) and a chemically stabilized des-hydroxy analog of PUM (<b>2a</b>) bound to an <i>Escherichia coli</i> RNAP transcription complex. Guided by the observed bound conformation, we developed an efficient solid-phase synthesis of 50 des-hydroxy PUM analogs modified at the Gln residue and Gdn-Gly tail. Several analogs retained low-micromolar RNAP-inhibitory activity, with a <i>para</i>-substituted phenyl amidine analog (<b>54</b>) emerging as the most potent inhibitor (IC₅₀ = 0.95 μM). These results establish a versatile synthetic platform and structural framework for optimizing stabilized PUM derivatives and provide a foundation for the development of RNAP-targeted therapeutics against resistant bacterial pathogens.