Basic Research Synthesis And Separation And Purification Of Four Polypeptides

Almost all cells in vivo are regulated by peptides, such as cell differentiation, neurotransmitters neurohonnonal regulation and immunoregulation. Peptide has a dual function of regulating the body’s physiological functions and providing the body nutrients, so the research and development of peptide drugs and health care products is of important significance and has great application prospects. Active peptides could be obtained by biological separation, bio technology and chemical synthesis. For chemical synthesis has the ability of changing the primary structure of the peptide easily, adding a special amino acid, modifying the peptide terminus and etc., several bioactive peptides were prepared by chemical synthesis in this paper, the preparation method, separation and purification, identification techniques and biological immune applications were also preliminarily studied, details are as follows:1. Synthesize, purify and characterize Fl-23(QVKLEESGGGLVQAGDSLRVSCA) and peptide AA12(CERVIGTGWVRC)Peptide Fl-23was synthesized by Boc protocol, the synthesis conditions especially the condensing agents and the resin types were optimized. The experimental results show that HCTU and HBTU have similar condensation efficiency and HBTU has higher resolution in HPLC performance, so choosing HBTU as condensing agent is conducive to the purification of peptides. Peptide AA12was synthesized by two synthesis protocols of Boc and Fmoc, since the Fmoc protocol avoids acid treatment during the synthesis process, the losses of the peptide from resin was reduced and the stability of Trp was increased. The experimental results show that the Fmoc method is conducive to the synthesis of peptide AA12and is also suitable for the synthesis of such peptides like AA12containing unstable amino acids for strong acid treatment.2. Study on the synthesis, characterization and photolysis efficiency of two photosensitive peptideThe unnatural Fmoc protected amino acid3-amino-3-(2-nitrophenyl) propionic acid was synthesized and was proved to be racemic enantiomers by using circular dichroism spectrum and polarimeter. The influence of different photosensitive positions and illumination time on the cleavage efficiency was studied through the synthesis of two photosensitive peptides. The experimental results show that:the cleavage rate could reach86.12%after2hour’s illumination, indicating that it is more prone to photolyze when the photosensitive amino acid was located in the fourth position of peptide KILGFVFTV. The peptide KILJFVFTV(J represents photosensitive amino acids) would be used to complex with MHC I to form the ligands which could be recognized by specific immune T cells. The photosensitive peptide act as a powerful tool for the analysis of biological immune without the requirement of renaturation and purification of complex.3. Study on the purification method of peptides using photosensitive responsivenessTo purify the peptides using the strong adsorption between the cysteine thiol group and the gold nanoparticles, a photosensitive cysteine derivative containing a mercapto group and a bromo group was synthesized. The bromo group could act as the "N-terminus" to connect with the-NH2of the peptide. After cleavage from resin, only the target peptide could be absorbed by gold nanoparticles, because the unreacted amino groups is capped by the addition of acetic anhydride and pyridine afte every step of reaction coupling amino acid. The complex of the peptide and the gold nanoparticles is purified from the impurities using high-speed centrifugation, then the complex was irradiated by ultraviolet to obtain the target peptide. This method is applicable to isolate and purify peptide of any primary sequence and has provided a new method and a new idea for the separation and purification of peptides.