Structure-Guided Semisynthesis of Blasticidin S–Amicetin Chimeras as Selective Ribosome Inhibitors

Peptidyl nucleosides are broad-acting inhibitors, but their dense functionality and complex reactivity have historically limited the modification of these scaffolds. Guided by structural overlays and molecular modeling, we designed blasticidin S-amicetin chimeras to exploit a bacterial-specific pocket of the ribosomal PTC while reducing eukaryotic ribosome engagement. To test this hypothesis, we developed a semisynthetic route enabling sequential C6' derivatization and C4 amine coupling on the blasticidin S scaffold, facilitated by counterion exchange to prevent side reactions. This approach furnished four C6' classes (acid, methyl ester, primary amide, phenethyl amide), each diversified at C4 with <i>para</i>-aminobenzoate motifs, delivering densely functionalized chimeras in as few as four steps and up to 38% yield. Across the series, antibacterial potency was retained while mammalian cytotoxicity dropped sharply, with selectivity indices approaching >50 and cytotoxicity values >256 μg/mL for the phenethyl amide series. Comparison of resolved ribosome structures supplemented by modeling rationalizes the observed selectivity gains as engagement of a termination-compatible bacterial pocket that is disfavored during eukaryotic elongation. These results demonstrate how structure-guided semisynthesis can transform a challenging natural product into selective translation inhibitors and establish a practical framework for diversifying chemically complex scaffolds.