| Cyclophosphamide is an anti-tumor chemotherapy medicine, which is used widely in present tumor treatment due to its obvious effects on various tumors, but its toxicity and side effects on patient are remarkable. Therefore, studying on new anti-tumor medicine with high efficiency and low toxicity has become an important and significant topic in the field of biochemistry .MP-2（Myelopeptide-2） is one kind of small peptide which separated from the spinal cord peptides mixture by Russian scientists. It is composed by six amino acids, which can effectively suppress the growth of many kinds of transplant tumors. retro-MP-2 is composed by the same amino acids with reversed acylation constitution, which has similar anti-tumor activity to MP-2.However, the modified molecule could combine biological activity intrinsic for MP-2 with an increased stability to biodegradation due to the reversed acylation of peptide chain. Theoretically, this medicine has better pharmacodynamic action than MP-2, and it’s efficacy would last long. MP-2 and retro-MP-2 would probably become new clinical anti-tumor medicines be widely used.Hence, the study on synthesis and purification of them has become important topic. Fmoc solid phase peptide synthesis method avoids shortcomings （such as reaction time being long, process of operating complicated etc.） in traditional liquid phase synthesis, and be considered as an important direction of peptide synthesis development.In the paper, Fmoc-OSU, amino acids and wang resin were used to synthesize intermediary products that would be used in the solid phase synthesis, the purity of amino acids derivatives achieved above 90%, the load of Leu-wang resin achieved 0.40mmol ? g-1, which met the need of next step. The optimization of synthesis was carried out on PSSM-8 peptide auto-synthesizer, and the results were as follow: The dipping time of resin was 30min, the removing time of temporary protection group Fmoc was 7min per time and 14min totally, the pre-activating time of amino acid was 10min, and the coupling time was 40min. final yield of crude peptide achieved 82%. With the comparison of different activation reagents and different cleavage cocktail, it showed that HBTU/DIEA/HOBT catalysis system were the best activating reagents and the optimization of cleavage cocktail was TFA/DCM/EDT/H2O/Phenol = 70%: 20%: 2.5%: 5%: 2.5%, which has high productivity and less by-product.This paper applied RP-HPLC to separate and purify the crude peptides from synthesis. The conditions like mobile phase, pH values, column temperature, the flow rates etc. were optimized to find best solution for RP-HPLC analysis. Meanwhile, LC-MS was used to check the goal peak. The crude peptides were purified by preparative medium performance liquid chromatography （MPLC） and the solvent was removed by lyophilization. Finally, white fluffy easy reserved powder was produced. The purity of synthesized products reaches more than 95%.The antitumor activity of MP-2 was evaluated by experiments on S180 tumor bearing KM mice. The optimum dose of 0.25mg·kg-1 was determined. The restraint rate of tumor growth could reach 50.4%, slight lower than cyclophosphamide group. retro-MP-2 had very similar effect on S180, which meant the reversed acylation of peptide didn’t change the activity. Compared to cyclophosphamide, MP-2 and retro-MP-2 had little side effect, even could improve the growth of young mice.This paper optimized synthesis and purification conditions of MP-2 and retro-MP-2, and set foundation for further pharmacodynamic research. |
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