Beyond Making Thiazolines and Oxazolines: Expanding the Enzymatic Repertoire to incorporate other 5- and 6-membered heterocyclic rings in peptides

his project aims at developing a novel, efficient and eco-friendly enzymatic approach to incorporate a range of heterocyclic rings in cyclic peptides at a commercially viable cost. These modifications will: 1) improve activity, receptor binding affinity and selectivity by stabilizing the peptide secondary structure; 2) enhance the stability against metabolic and digestive enzymes thus improve oral bioavailability and 3) improve cellular permeability by decreasing the number of hydrogen bond donors (HBDs). We previously reported the use of enzymes to incorporate thiazolines and oxazolines in cyclic peptides by dehydrative cyclization of cysteine, serine and threonine. These enzymes were derived from the biosynthetic pathways of the ribosomally synthesized and posttranslationally modified peptides (RiPPs) in which a precursor peptide is ribosomally-synthesised and tailored by a set of processing enzymes to give the modified cyclic peptide. The precursor peptide contains a sequence recognised by the heterocyclase called "leader", typically followed by a protease cleavage signal and the sequence to be processed to a final product (the core peptide). The distant separation of the recognition and processing sites makes these enzymes highly tolerant to variations in substrate sequence and appealing for biotechnological applications. We have studied the structure and mechanism of some of these enzymes and engineered an enzyme which is fused with its substrate recognition sequence so it can process stand-alone core sequences. We aim to apply our expertise to add new enzymes to our toolbox and to explore the chemical flexibility of the nucleophilic side chain to enable the incorporation of other 5 and 6 membered heterocycles in cyclic peptides.