Synthesis And Application On The Stable Analogue Of Phosphoarginine

Protein phosphorylation is one of the most common and widely studied post-translational modifications(PTMs).The major efforts to date have focused on 0-phosphorylation.Compared to the O-phosphorylation on Ser,Thr and Tyr residues,our understanding about N-phosphorylation on Arg,His and Lys residues are relatively limited,both in prokaryotes and eukaryotes.The lack of efficient tools for studying on these PTMs likely relates to the fact that the P-N bond of N-phosphorylation(pArg,pHis,pLys)is highly unstable under the acidic and heat conditions.Our research focused on design and applicationon about the stable analogue of phosphoarginine.To provide effective and convenient technology and research strategies for protein arginine phosphorylation.This thesis include four topics.First of all,we designed and synthesised a stable pArg analogue,pAIE,in which the labile N-P bond was replaced with a non-hydrolyzable C-P bond.The analogue would be obtained conveniently on the gram-scale,with a total yield of 19.4%.Secondly,this analog was successfully used as a hapten to raise an immune response and the first mouse polyclonal antibody that specifically recognized pArg-containing peptides and proteins was produced using conjugated analog-KLH as the immunogen.The generated antibody showed excellent specificity towards pArg-containing peptides and proteins,and would be used for a variety of biological detection methods.Thirdly,we designed an amino acid form phosphoarginine analogue Fmoc-Aai(PO3Et2)-OH.Our synthesis of Fmoc-Aai(P03Et2)-OH had enabled us to demonstrate that the analogue was compatible with Fmoc-solid phase peptide synthesis(SPPS)condition.Standard SPPS yielded the diethyl phosphonate-protected peptide.Fourthly,Molecularly imprinted polymers(MIPs)for phosphoarginine(pArg)-containing peptides pAIE-MIPs were synthesized by precipitation method.Then,pAIE-MIPs was used for pArg enrichment from complex samples containing pArg peptide and tyrisin digestion of BSA,where it demonstrated a clear preference for pArg peptide over other peptides.Based on the above-mentioned research,we hope to deepen our understanding of arginine phosphorylation and provide the basis for the further investigation.