[Studies on development of disulfide bond forming reaction and the application to regioselective disulfide formation].
An efficient method for the disulfide bond formation in peptides by the silylchloride-sulfoxide system is described. Methyltrichlorosilane in trifluoroacetic acid, in the presence of diphenylsulfoxide, is found to cleave various S-protecting groups of cysteine to form cystine directly within 10 to 30 min. No side reactions were observed with nucleophilic amino acids such as Met, His, or Tyr, except for Trp, under the reaction conditions of the silylchloride-sulfoxide treatment. A chlorination of the indole moiety of unprotected Trp, rather than the sulfur-sulfur bond formation, is a dominant reaction when the peptide containing unprotected Trp is treated with the chlorosilane-sulfoxide. However, the disulfide bond can be formed efficiently with no modification at the indole ring by the treatment of the peptide having formyl-protected Trp residue with the silylchloride-sulfoxide system. The formyl group is removed by a brief treatment at basic pH without affecting the disulfide bond formed by the silylchloride-sulfoxide treatment. Total synthesis of human insulin, a two chain peptide containing three disulfide bonds, was achieved unambiguously by sequential and selective formation of disulfide bonds in the protein for the first time. The key reaction in the synthesis is regioselective formation of three disulfide bonds separately using the silyl chloride method described above. Prior to the insulin synthesis, it was confirmed by the syntheses of double-disulfide peptides: b-hANP, unnatural parallel dimer of a-hANP, and human endothelin-1 that no disulfide exchange occurred during the silyl chloride treatment. Using three orthogonal thiol protecting groups, Trt, Acm, and But, three disulfide bonds of human insulin were efficiently constructed by the successive reactions using thiolysis, iodine oxidation, and the sily1 chloride method. Each reaction for the stepwise disulfide formation proceeded within 15 to 60 min with no polymeric product and no solubility problem. The synthetic human insulin had the correct structure and was indistinguishable from natural human insulin.
Akaji
K
K
Kyoto Pharmaceutical University, Japan.
D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000138:chemical synthesis; D004220:Disulfides / Q000737:chemistry; D016232:Endothelins / Q000138:chemical synthesis; D006801:Humans; D007328:Insulin / Q000138:chemical synthesis; D008969:Molecular Sequence Data
D004220:Disulfides; D016232:Endothelins; D007328:Insulin; D009320:Atrial Natriuretic Factor