Chemoenzymatic Synthesis and in Vitro Selection of <i>De Novo</i> Thiazole‐Containing Macrocyclic Peptides

Backbone thiazole (Thz) moieties prevail in bioactive peptidic natural products and play important roles in their biological functions. However, the de novo discovery of artificial Thz-containing peptide ligands remains challenging. Here, we report an mRNA display-based selection platform for Thz-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 (Thn), which is further oxidized using the substrate-tolerant azoline dehydrogenase (GodE) to yield a Thz moiety. By integrating this chemoenzymatic process with chloroacetyl-mediated thioether macrocyclization and mRNA display, we have successfully discovered Thz-containing macrocyclic peptide ligands with high binding affinities against p21-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 Thzs.