A biomimetic strategy in the synthesis and fragmentation of cyclic protein.
This paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 31 amino acid residues tightly packed in a cystine-knot motif with three disulfide bonds and an end-to-end cyclic form. The synthesis of these small cyclic proteins can be achieved by orthogonal ligation of free peptide thioester via the thia zip reaction, which involves a series of reversible thiol-thiolactone exchanges to arrive at an alpha-amino thiolactone, which then undergoes an irreversible, spontaneous ring contraction through an S,N-acyl migration to form the cyclic protein. A two-step disulfide formation strategy is employed for obtaining the desired disulfide-paired products. Partial acid hydrolysis through intramolecular acyl transfer of X-Ser, X-Thr, Asp-X, and Glu-X sequences is used to obtain the assignment of the circulins disulfide bond connectives. Both synthetic circulin B and CPT are identical to the natural products and, thus, the total synthesis confirms the disulfide connectivity of circulin B and CPT contain a cystine-knot motif of 1-4, 2-5, and 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 characterization of large end-to-end cyclic peptides and small proteins.
Department of Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee 37232, USA. Tamjp@ctrvax.vanderbilt.edu
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D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000138:chemical synthesis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D047169:Cyclotides; D003545:Cysteine / Q000737:chemistry; D003553:Cystine; D004121:Dimethyl Sulfoxide; D004220:Disulfides / Q000138:chemical synthesis* / Q000737:chemistry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010070:Oxalates; D019815:Oxalic Acid; D010449:Peptide Mapping; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea
D019380:Anti-HIV Agents; D047169:Cyclotides; D004220:Disulfides; D010070:Oxalates; D010456:Peptides, Cyclic; C093020:cyclopsychotride A; C102385:circulin B; D003553:Cystine; D014508:Urea; D019815:Oxalic Acid; D003545:Cysteine; D004121:Dimethyl Sulfoxide