A BIOMIMETIC STRATEGY IN THE SYNTHESIS AND FRAGMENTATION OF CYCLIC PROTEIN

This paper describes a simple biomimetic strategy to prepare small cyc lic proteins containing multiple disulfide bonds. Our strategy involve s intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqu eous solution. To illustrate our strategy, we synthesized the naturall y occurring circulin B and cyclopsychotride (CPT), both consisting of 31 amino acid residues tightly packed in a cystine-knot motif with thr ee disulfide bonds and an end-to-end cyclic form. The synthesis of the se small cyclic proteins can be achieved by orthogonal ligation of fre e peptide thioester via the thia zip reaction, which involves a series of reversible thiol-thiolactone. exchanges to arrive at an cr-amino t hiolactone, which then undergoes an irreversible, spontaneous ring con traction through an S,N-acyl migration to form the cyclic protein. A t wo-step disulfide formation strategy is employed for obtaining the des ired disulfide-paired products. Partial acid hydrolysis through intram olecular acyl transfer of X-Ser, X-Thr, Asp-X, and Glu-X sequences is used to obtain the assignment of the circulins disulfide bond connecti ves. Both synthetic circulin B and CPT are identical to the natural pr oducts and, thus, the total synthesis confirms the disulfide connectiv ity of circulin B and CPT contain a cystine-knot motif of 1-4, 2-5, an d 3-6. In general, our strategy, based on the convergence of chemical proteolysis and aminolysis of peptide bonds through acyl transfer, is biomimetic and provides a useful approach for the synthesis and charac terization of large end-to-end cyclic peptides and small proteins.