Efficient Synthesis Method And Characterization Of Conotoxin Containing Tyrosine Sulfate Modification

Protein tyrosine sulfate(Sulfotyrosine,sY)is a post-translational modification commonly found in organisms.Most sulfated proteins will participate in protein-protein interactions(PPI)to affect physiological functions such as hemostasis,leukocyte adhesion,etc.Some of the PPI process is involved in the recognition of sulfate on tyrosine,therefore,the relationship between sY modification and protein biological activity has attracted much attention.Currently,the widely adopted strategy is to protect the sulfate radical by neopentyl(nP)to prevent its hydrolysis in peptide synthesis,but most of the sulfated peptides currently synthesized have no cysteine(Cys)residues,which is related to the instability of the side chain thiol under nP removal conditions.However,Cys exists in most important physiological proteins/peptides,so it is necessary to develop a more general synthesis method of cysteine-containing sulfated modified peptides to promote the basic research of related peptides/protein drugs.Conotoxin is known as "the treasure trove of marine medicine",and its medicinal activity has become one of the hot spots in the field of international biomedicine research.The α4/7-conotoxin is generally composed of 15-22 amino acids and contains 4 Cys.It is considered to be a highly promising drug for neurological diseases because it can specifically block the neuronal acetylcholine receptor.Previously,some researchers found that some α4/7-conotoxins were modified by sY through mass spectrometry.However,due to the difficulty in obtaining uniform sulfated peptides,their activities and related research were not successfully carried out.In this thesis,we conducted a series of explorations on the nP removal method of sY,and finally got the general law of the removal condition.It is believed that nP can be removed in most aqueous solutions.We applied this method to the synthesis of 8 sulfated α4/7-conotoxins: linear peptides were prepared by the Fmoc-SPPS strategy,and the peptides were directionally folded in a step-by-step and selective method,while the nP group is removed at the same time as the folding step,realizing the one-pot method to efficiently prepare the target peptide containing sY.In addition,we have successfully avoided the problem of sulfate hydrolysis under acidic conditions,and finally prepared conotoxin containing sY modification by conventional HPLC.The animal serum experiment showed that conotoxin has good stability in serum and sY modification will not reduce the stability of conotoxin.Finally,in order to investigate the effect of sY modification on the interaction between conotoxin and acetylcholine binding protein,we also conducted a preliminary test on the recombinant expression of acetylcholine binding protein,and found that the protein is very easy to polymerize and hard to control during dialysis renaturation and protease cleavage.In summary,we have developed an efficient method for synthesizing cysteine-containing sulfated peptides to promote the synthesis of sulfated peptides/proteins and lay a solid foundation for the study of the pharmaceutical mechanism of sulfated modified peptides/proteins.