Chemoenzymatic synthesis and in vitro selection of de novo thiazole‐containing macrocyclic peptides

Backbone thiazole moieties prevail in bioactive peptidic natural products and play important roles in their biological functions. However, the de novo discovery of artificial thiazole‐containing peptide ligands remains challenging. Here, we report an mRNA display‐based selection platform for thiazole‐containing macrocyclic peptides (ThzteMP), established through a dedicated posttranslational chemoenzymatic transformation. This method exploits the unique reactivity of ribosomally incorporated thioamides, enabling enzyme‐free spontaneous heterocyclization to form thiazoline, which is further oxidized using the substrate‐tolerant azoline dehydrogenase (GodE) to yield a thiazole moiety. By integrating this chemoenzymatic process with chloroacetyl‐mediated thioether macrocyclization and mRNA display, we have successfully discovered thiazole‐containing macrocyclic peptide ligands with high binding affinities against p‐21 activated kinase 4 (PAK4). This study establishes a robust system to expedite ligand discovery of pseudo‐natural peptides and to investigate the functional benefit of their backbone thiazoles.