Ribosomal Synthesis of Topologically Defined Thioisoindole‐Bridged Bicyclic Peptides

Macrocyclic peptides are promising scaffolds for drug discovery due to their structural rigidity and high target specificity. Here, we report a strategy for in vitro ribosomal translation of thioisoindole-bridged bicyclic peptides. Central to this approach is a newly developed flexizyme substrate, Ac-Ala(NtBA)^Sc-CME, which features a semicarbazone-masked 2-nicotinoyl benzaldehyde sidechain. We show that this amino acid can be efficiently charged onto tRNA with flexizyme and incorporated into ribosomal peptides using a customized flexible in vitro translation (FIT) system. The semicarbazone group can be post-translationally removed under mild conditions, triggering spontaneous intramolecular cyclization to cysteine and lysine sidechains in the same substrate to yield thioisoindole-bridged bicyclic (TiB) peptides. This strategy was leveraged to synthesize structurally diverse bicyclic peptides with varying sequences and ring sizes. The method maintains the integrity of mRNA and is therefore compatible with mRNA display, which opens the possibility of constructing topologically defined bicyclic peptide libraries for therapeutic peptide discovery.