Chang Rao

Acyl disulfide-mediated intramolecular acylation for orthogonal coupling between unprotected peptide segments. Mechanism and application

A highly efficient orthogonal coupling approach for peptide bond formation using unprotected peptide segments was described. The key element of this approach consisted of capturing an Npys modified N-Cys side-chain thiol of the amino segment with a Cα-thiocarboxylic acid of the acyl segment to form an acyl disulfide which undergoes rapid intramolecular acylation to generate an amide bond. A final product with a native Cys residue at the ligation site was obtained after a thiolytic reduction step.

Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid

Backbone-cyclic proteins are of great scientific and therapeutic interest owing to their higher stability over their linear counterparts. Modification of such cyclic proteins at a selected site would further enhance their versatility. Here we report a chemoenzymatic strategy to engineer site-selectively modified cyclic proteins by combining butelase-mediated macrocyclization with the genetic code expansion methodology. Using this strategy, we prepared a cyclic protein which was modified with biotin or a cell-penetrating peptide at a genetically incorporated noncanonical amino acid, making the cyclization-stabilized protein further amenable for site-specific immobilization and intracellular delivery. Our results point to a new avenue to engineering novel cyclic proteins with improved physicochemical and pharmacological properties for potential applications in biotechnology and medicine.