Chemical Protein Synthesis Via Ligation Of Peptide Hydrazides

After the completion of the Human Genome Project, the structure-activityrelationships of protein have become the leading edge of biological research.Particularly, scientists pay more attention to post-translational modifications (PTMs)such as methylation, acylation, phosphorylation and glycosylation. The accessibility ofpurified proteins with multiple PTMs would be of significant value for its systematicstudy. However, the traditional recombinant DNA-based techniques can onlyincorporate the20genetically encoded amino acids into the target protein. Recently, the“misacylated” transfer RNAs technology developed by Schultz et al has been widelyused to study biological processes. However, this method is geneally limited to the lowefficiency incorporation of a single type of unnatural amino acid at a time. Proteinchemical synthesis can produce molecules with atomic precision, and it is a powerfultool to prepare proteins with predesigned modifications. Native chemical ligation (NCL)developed by Kent et al is the most widely used tool for protein chemical synthesis, andit has been applied in the synthesis of hundreds of proteins. NCL involves achemoselective amide formation reaction between a C-terminal peptide thioester and anN-terminal Cys peptide. Peptide thioester is sensitive to piperidine, thus it can not bedirectly synthesized by Fmoc SPPS. The use of toxic HF in Boc SPPS often causessafety problems, and peptide thioesters with acid-sensitive modification such asglycosidic and phosphate residues are hardly accessible by using Boc SPPS. Untill now,there are many methods that provide reliable access to peptide thioester by using FmocSPPS. However, the current methods are still too complex. In this paper, we developeda new protein synthesis technology named ligation of peptide hydrazides. Trifolitoxinand CssII were prepared efficiencily by the ligation of peptide hydrazides. Besides, wecompleted the semisynthesis of LC3with expressed protein hydrazide. Furthermore,we finished the fully convergent chemical synthesis of ribosomal protein S25employing2-(tert-butyldisulfanyl) ethyloxycarbonyl protection group.