A Chemoenzymatic Platform for the Discovery of Thiopeptide Therapeutics

Thiopeptides are macrocyclic, bioactive natural products derived from ribosomally synthesized and post-translationally modified peptides. We have described a new chemoenzymatic route to access these scaffolds by exploiting a remarkable set of enzymes that catalyze an intramolecular formal [4 + 2] cycloaddition to form a trithiazolyl pyridine core, the hallmark structural component of these natural products. These pyridine synthases act late in thiopeptide biosynthesis to simultaneously affect macrocyclization and cleavage of an N-terminal leader peptide that is required for enzymatic recognition of the substrate. We have defined minimal recognition sites on both the N- and C-terminus of the linear substrates that are necessary and sufficient for enzymatic catalysis. Furthermore, we have showcased the extreme flexibility of these pyridine synthases by demonstrating they are capable of catalyzing both intra and inter-molecular cycloadditions. Linear substrates are readily synthesized via standard solid-phase peptide synthesis (SPPS). This results in a high throughput, modular and diversifiable route to thiopeptide analogs with atomic level control capable of generating variants inaccessible by other strategies. This enables a true exploration of these scaffold’s potential by allowing investigation of the main pharmacophore, the macrocycle, which has gone largely untouched due to the limitations of other methods.Doctor of Philosoph