Approach And Application Toward Chemical Synthesis And Modification Of Protein

As one of the most important components in body,proteins with various structure sustain the life of cells by carrying out all aspects of its biological functions.With the completion of the Human Genome Project,the relationship between protein structure and function has become the forefront of biological research.Especially,the post-translational modifications(phosphorylation,methylation,acetylation,ubiquitination and glycosylation)process,play an important role in regulation of the structure and function of cell,and are relate with many diseases.Therefore,post-translational modification process of proteins has attracted extensive attention of researchers.The preparation of proteins with high purity is crucial for the study of relationship between protein structure and function.The traditional method using biological expression is incapable of satisfying the need of current research.However,the method of chemical protein synthesis is theoretically able to overcome the limitations of biological expression methods.Besides,protein molecule with bioothogonally labelling or modification based on chemical methods to position the protein in cells,makes it possible to explain the interaction between protein and other components in cells or the pathway of protein biological metabolismin in detail.And these modified or labelled protein molecules are difficult to be obtained by biological expression.Researchers can obtain high purity natural proteins or specific site modification proteins by chemical synthesis or modification methods regardless of the site of modifications,the type of modifications and the number of modifications.These target molecules obtained by chemical synthesis or modification with defined structure help researchers to more clearly understand the relationship between protein structure and function in cells at the molecular level.Therefore,the chemical method of protein synthesis modification of proteins has been paid more and more attention by researchers.In recent years,researchers have developed a variety of chemical strategies for protein synthesis and modification,but there are still some challenges to be addressed,such as peptide fragments ligation and N-glycosylation.In this dissertation,it was mainly aimed at the synthesis and modification of peptide fragments of protein chemistry.Thus,photocatalytic peptide desulfurization,peptide N-glycosylation based on the aminolysis of 4-nitrophenyl thioester and bioothogonal click reaction were investigated.It may be summarized as follows:First,a brief overview of protein chemical synthesis and selective modification was made,and the approach and application of peptide fragment ligation and protein bioothogonal modification were summarized.Second,replacing free radical initiator and TCEP with TPPTS and ruthenium photocatalyst,using cysteine and glutataione as model substrates,the visible light induced desulfurization of peptide with thiol residue was achieved.The optimized reaction conditions were screened and applied to eleven cysteine-containing long peptides,to examine the scope of the photocatalytic desulfurization.It was confirmed that the photocatalytic desulfurization method was tolerance with the residues of natural amino acid,functional groups with sulfur,carbonhydrates,etc.Combining this desulfurization method with the native chemical ligation,we successfully made the merge of linear peptide fragments and synthesized the natural cyclic peptide.Besides,based on the aminolysis of simple active ester,using glycosylamine and Fmoc-protected glycine esters as substrates,the 4-nitrophenol thioester was demonstrated to be the final optimized active ester for the N-glycosylation process.Through aminolysis of peptide incorporating with 4-nitrophenylthiol ester and glycosylamine,N-glycosylation product between glycosylamine and peptide was obtained,forming the amide bond,expanding the substrate scope of this N-glycosylation reaction.Moreover,the N-glycosylation process based on the aminolysis of 4-nitrophenylthiol ester peptide is also applicable to the merge of oligosaccharides and peptide thiolesters.This method is combined with the ligation/desulfurization protocol to achieve the ligation of the glycopeptide fragments.Last,heterothiometallic copper clusters with various structure were firstly employed to catalyze the azide and alkyne cycloaddition reaction,and the catalytic activity of W(Mo)/S/Cu copper clusters with different structure was tested.[NH4]2[WS4Cu4I4]copper cluster was found to be the optimal catalyst and used to test the substrate scope of this catalytic reaction.In addition,[NH4]2[WS4Cu4I4]had been successfully applied to catalysis the modification of biocompatible molecules or materials,such as saccharides and polyethylene glycol,and it is possible to provide a new route for modification and ligation in protein chemistry.