Solid Phase Synthesis And Immunity Study Of Myelopeptide 1

MP1 （Phe-Leu-Gly-Phe-Pro-Thr） was hexapeptides originally isolated from porcine bonemarrow cell culture. It had been shown that MP1 possesses good immunoregulatory activity. It could increase the number of antibody-forming cells （AFC） at the peak of immune response, and stimulate ConA-induced proliferation of spleen cells.Solid phase synthesis, resin cleavage, and impurity separating strategy of MP1 were studied in this thesis. The structure of MP1 was confined by LC-MS, RP-HPLC, and amino acid analysis. Immunity of MP1 was also studied by spleen lymphocyte transformation.MP1 was synthesized by Fmoc solid phase method. The protocol was optimized by studying the effects of coupling time, resin marinating time, excessive multiple and activation conditions of amino acid, concentration of HBTU/HOBt and DIEA, mass of resin, volume of solvent, and times of washing resin on the yield and purity of MP1 during synthesis. Coupling time was 50min, resin marinating time was 40 min, excessive moore multiple of amino acid was 2, activation of amino acid was carried out 2 times and through 7min every time, the concentration of HBTU/HOBt was 0.5mmol·L^-1, the concentration of DIEA was 1.5mmol·L^-1, 80mg resin was dissolved in 900μL solvent of DMF, and resin was washed 7 times after wiping off Fmoc, that was the preferable term. On the condition, the synthetical efficiency of MP1 could reach 90%.Resin and protecting function groups were cleaved by acids. By studying the effects of cleavage cocktails, cleavage time, and cleavage temperature on cleavage efficiency, the protocol was optimized. TFA-Water-EDT-TES （90:6:3:1） could corking finish cleavage in 3.5h under 20"C. Under this condition, the cleavage efficiency was more than 99%.The analysis of crude MP1 was carried through RP-HPLC. By studying the effects of grads, flow, concentration of TFA and ACN, and chromatogram column on RP-HPLC efficiency, the protocol was optimized. The separating of crude MP1 was carried on Waters-RP-HPLC with VP-ODS C₁₈（150×4.6mm, 5μm） as chromatogram column, 214nm as working wavelength, with 0.1%TFA/water as Eluent A and 0.1%TFA/70%ACN/water as Eluent B, 5min, 0→35%B and 35min, 35%B→55%B, as washing grads and with 0.7mL·min^-1 as velocity of flow.The structure of MP1 was confined by LC-MS. The spectrum offered charge （M+H）⁺ 681.4 and double charge （M+H）²⁺ 340.2. The result of molecular weight analysis was same to the theoretical value of MP1.Amino acid analysis was used to quantitate the content and the ratio of the amino acids, and the result was consisted with the theoretical value.Immunity of MP1 was also researched by spleen lymphocyte transformation. In vitro experiment results had determined that the stimulation of MP1 was observed in ConA-induced proliferation of spleen cells of Cy-treated mice. When the dosage of MP1 was 1.00×10^-3μg·mL^-1, the immune modulation of MP1 was best remarkableMP1 was successfully synthesized by Fmoc solid phase synthesis and it showed the same immunopotentiation.