The Synthesis Of Salmon Calcitonin Analogs And Their Bioactivity Study

Calcitonins are peptide hormones with 32 amino acid residues, well-known for their hypocalcemic effect and inhibition of calcium absorption. Although there are some differences in individual amino acid from different species, calcitonins all contain: 1) disulfide bond at N-terminus(Cys1, Cys7); 2) prolinamide at C-terminus; 3) potential amphiliphilicα-helical structure from residue 8 to 22. In general, calcitonins originated from fish have been found to be more potent than mammalian ones. Salmon calcitonin (sCT) and synthetic version of eel calcitonin analog have been used for the treatment of hypocalcemia, postmenopausal osteoporosis and Paget's disease. Natural calcitonins have short serum half-lives (t1/2), for example, the t1/2 of sCT is just 14mins. PEGylation is a widely used technique to improve biological stability of polypeptides and proteins. The effects of PEGlyation include better stability, reduced immunogenicity and antigenicity,and decreased toxicity. A lot of PEG-modified proteins have been applied in clinic since 1990.Disulfide bond in sCT is not necessary for its activity, and linear sCT analogs without disulfide bond have showed comparable potencies. Based on these active linear sCT analogs and information about enzyme susceptive sites of sCT in literatures, novel sCT analogs were designed, synthesized and modified with PEG in a site specific way. The biological activities were also assayed in vivo.Structural modifications of sCT are involved: 1) deletion of residues 19-22 or more amino acid residues to obtain shorter sequences with bioactivity; 2) modifications and PEGylations at or adjacent to the enzyme susceptive sites; 3) biological evaluation of the resulting sCT analogs and derivatives.PEGylations could be coupled in case of amino, carboxyl and thiol groups of a peptide. For example, the N-terminalα-amino group in Cys1 andε-amine groups in Lys11 and Lys18 in sCT can react with amino-reactive PEG reagent at the same time, and get a mixture containing mono-, di- and tri-PEGylated products. Importantly, mono-PEGylation at one specific site is crucial to maintain the peptide's activity. An effective approach to realize the site-specific PEGylation is to use the highly reactive thiol group with mPEG-MAL.MBHA resin was used as the supporting scaffold, and the peptide analogs were coupled by HOBt/DCC reagents in the way of Fmoc- strategy. To increase the target peptide content in the crude product, deprotections was firstly performed to the side chain protected groups, followed by cleavage from the resin with HF.Twelve sCT analogs and elven corresponding PEGylated products were obtained, purified by RP-HPLC and determined by MALDI-TOF-MS.Results from biological assays indicated that two Cys substituted peptide analogs exhibited comparable potencies with sCT, and the D-Ala replacements at Cys1 and Gly30 even higher. Preliminary hypocalcemic potency data indicated that PEGylated product LK-YZ-101, administrated s.c. once a week, exhibited the same potency as sCT, followed by s.c. injection twice a week, which demonstrated that PEGylated products have longer action in vivo than sCT.