Synthesis of a molt-inhibiting hormone of the American crayfish, Procambarus clarkii, and determination of the location of its disulfide linkages

A molt-inhibiting hormone (Prc-MIH) of the American crayfish, Procambarus clarkii, a member of the type II CHH family, was chemically synthesized and the location of its three disulfide linkages was determined. Pre-MIM consists of 75 amino acid residues and was synthesized by a thioester method. Two peptide segments, Boc-[Cys(Acm)(7,24,27), Lys(Boc)(19)]-Prc-MIH(1-39)-SCH2CH2CO-Nle-NH2 and H-[Cys(Acm)(40,44,53), Lys(Boc)(42,51,67)]-Prc-MIH(40-75)-NH2, were prepared using peptides obtained via the Boc solid-phase method. Condensation of the building blocks in. the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, and N,N-diisopropylethylamine, followed by removal of the protecting groups, gave the reduced form of Prc-MIH(1-75)-NH2. This product was converted to the native form of Pre-MIM (synthetic Pre-MIM) in a buffer which contained cysteine and cystine, The synthetic Pre-MIM showed the same behavior by RP-HPLC and biological activity assays as the natural Pre-MIM, The disulfide bond between Cys7 and Cys44 was determined by isolation of a fragment from an enzymatic digest of the synthetic Prc-MIH by RP-HPLC, followed by mass analysis. The disulfide bonds between Cys24 and Cys40 and between Cys27 and Cys53 were determined by comparing the elution position of an enzymatic digest of the synthetic Pre-MIM with authentic chemically synthesized samples, which contained three types of possible disulfide linkages.