Chemical Synthesis And Oxidative Folding Of Conotoxin B179 And B8.1T

Conotoxins are small,disulfide-rich peptides (10-46aa) derived from the venom of cone snails.Compared to other natural peptide toxins,conotoxins possess the superior chemical and pharmacological features,such as small molecular,stable structure,high activity and selectivity. So far the conotoxins can be classified into several superfamilies,A-,T-,O-,M-,P-,I-and S-superfamily.Conotoxins can specifically target nicotinic acetylcholine receptors and other receptors,and conopeptides also selectively bind with submit of ion channals,including calcium, sodium or potassium channels. Due to their highly pharmacological potency and target selectivity,conotoxins are not only used as perfect drug design templates and probes for neurobiological research, but also are developded into drugs directly.Two novel conotoxins B179 and B8.1T native to Hainan were synthesized by solid-phase synthesis method.Various parameters affecting conotoxin B179 and B8.1T oxidative folding were optimized, which included different kinds of buffers, concentration and ratio of reduced and oxidized glutathione. and temperature. Besides,conotoxin B8.1T was oxidized by two-step method and one-pot method to determine the framework of disulfide bond.The results showed the linear conotoxin B179 and conotoxin B8.1T can be synthesized successfully by Fmoc solid-phase synthesis. The conotoxin B179’s oxidative products contained correctly folded peptide with one pair of disulfide bond mainly. There were other by-products also in the oxidative products, which might be the peptide mixture with the intermolecular disulfide bond. The folded by-products were increased with high concentration of reduced glutathione in the oxidative buffer. The conotoxin B179 oxidatvie folding reaction was speeded with higher temperature.For conotoxin B8.1T, glutathione、BSA and solvents can improve the efficiency of oxidatve folding of conotoxin B8.1T. Two-step oxidation method and One-pot method proved the framework of disulfide bond.As there aren’t adequate natural conotoxins on the earth, and they are difficult to be separated,native conotoxins can not meet the minimal requirements of the followed researches about pharmacological activities and drug developments.As a result,We managed to synthesize linear peptides by chemical methods,then oxidize them to obtain more native conntoxins.Two novel conotoxins which are native to hainan were successfully synthesized in this research.We also systematically explored the conditions of reduced peptides oxidation. In this study ,we established a good protocol to synthesize and oxidize peptides resembled to native conotoxins B179 and B8.1 T.Moreover, we also provided a particular thinking to locate fashion of disulfide bonds linking of peptides.