Chemoenzymatic Synthesis and in Vitro Selection of De Novo Thiazole‐Containing Macrocyclic Peptides

Abstract

AbstractBackbone 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.