Chemical Synthesis And Oxidative Folding Of Conotoxin LvMA

Conotoxins are small, structured peptide toxins secreted by carnivorous marine mollusks which lives in the tropical marine,and are used as prey and defensive weapons. Conotoxins are disulfide-rich peptides and generally consist of7-40amino acid residues.Many of these peptides display a high selectivity for particular membrane receptors such as ionic channels or G-protein coupled receptors. This property makes them very promising tools for the study of receptors and potential new drugs.So far conotoxins have been divided into superfamilies,,including A-,T-,O-,M-,P-,I-and S-superfamily. For there aren’t enough natural conotoxins in the earth and,further, these peptides are difficult to be separated, native conotoxins can not satisfied the followed researches about pharmacological activities and drug developments. As a result, We managed to synthesize these peptides by chemical methods,.In this study,a novel conotoxin LvMA native to Hainan was synthesized by three solid-phase synthesis strategies. Firstly, linear conotoxin LvMA was synthesized and cysteine thiols were protected with group Trt.Secondly,LvMAa was produced by Fmoc method and cysteine thiol of peptide LvMAa contained two pairs of different protecting groups.Thirdly,LvMa and LvMAb were synthesized with three pairs of cysteine thiol protecting groups. In this study,various parameters affecting four kinds of conotoxin LvMA and LvMAa oxidative folding were optimized, including different kinds of buffers, glutathione, temperature, additives and other factors. Furthermore, different protecting groups of linear conotoxins LvMA were removed by one-step two-step, three-step and one-pot protocol The results showed the linear conotoxin LvMA can be synthesized successfully by Fmoc solid-phase synthesis. When conotoxin LvMA was oxidized in NH4HCO3buffer, at20℃, the target product yield can reach40%, and the process of folding was relatively simple. For linear conotoxin LvMAa with protecting groups (-Trt/-Acm), folding condition was similar to previous description:in NH4HCO3buffer,20℃,and GSSG shoud be added, and ultimately oxidative folding efficiency of conpeptide was up to60%by two-step method. For conotoxin LvMa(-Trt/-Acm/-MeBzl), the final product of folding rate only about20%;Moreover, it was unsuccessful to synthesize nature conotoxin LvMA by one-pot method for LvMAb(Trt/-tBu/-MeBzl) Because many kinds of byproducts were produced, it was hard to identify native isomer. The experiment results showed that one-pot strategy was not suit for synthesizing native conotoxin LvMA.In this study, a novel conotoxin LvMA which is native to hainan was successfully synthesized,and we also systematically explored the conditions of reduced peptides oxidation. Furthermore,the reuslts of this study offered a excellent protocol to synthesize peptides resembled to native conotoxin LvMA.Lastly, we also provided a thinking to characterize pattern of disulfide bonds of peptides.