脊髓肽MP-2的Fmoc法固相合成、HPLC纯化及其活性检测  

Fmoc solid phase synthesis,HPLC purification and activity determination of myelopeptide-2

作　　者：王焕灵[1] 张兴群[1] 李湛军[2] 徐康森[2] 

机构地区：[1]东华大学,上海201620 [2]中国药品生物制品检定所,北京100050

出　　处：《药物分析杂志》2008年第12期2095-2099,共5页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:用固相法合成脊髓肽 MP-2并验证其生物活性。方法:采用 Fmoc 固相法对脊髓肽 MP-2进行仪器自动合成,通过正交实验对反应条件进行了优化,肽-树脂复合物酸裂解后获得小肽粗品。用高效液相色谱法对所得粗品纯化制备,并利用 LC-MS 色谱仪确定目标产物,最后对纯品的生物活性进行检测。结果:通过正交实验优化 PSSM-8多肽合成仪合成MP-2的反应条件,粗品得率达到82%,HPLC 峰归一化法检测纯度为62.9%,中压柱制备后纯品纯度达到96.7%。纯品对S180肿瘤细胞进行抑瘤试验,证明 MP-2 0.25 mg·kg^(-1)对 S180有明显抑制作用,抑瘤率50.4%,且毒性低,不影响动物的发育生长。结论:合成实验证明,Fmoc 固相合成法合成脊髓肽 MP-2,操作条件温和且粗品得率较高,质谱检定主峰为目标峰后中压柱进行制备,分离操作简单,可以进行小批量生产;活性试验证明 MP-2具有抑制肿瘤S180生长的效果,为进一步进行药效学验证提供了初探基础。Objectives：Fmoc solid phase peptide synthesis principle was used to synthesize MP- 2 and the anti - tuomr activity of MP -2 was checked. Methods：MP -2 was synthesized by PSSM -8 automatic peptide synthesizer, experimental factors were optimized by orthogonal experiment, peptide - resins were cleaved by trifluoroacetic acid （TFA） , crude peptides were assessed by LC - MS and purified by HPLC. The purified MP - 2 was farther characteried by conti - fumeor assay. Results： Reaction conditions of synthesis were optimized with a purity of more than 69.2% and a recovery yield of 82% ,which was checked by HPLC. The crude peptides were purified by pre- parative medium performance liquid chromatography （MPLC） and the purity of products reached more than 95%. The anti -tumor activity of MP -2 was evaluated on Km mice bearing S180 tumor, and the optimum dose of 0. 25 mg · kg-1 was determined, the inhibition of tumor growth could reach 50. 4%, a little lower than cyclophosphamide group. But MP -2 showed no side effect on mice＇s growth. Conclusions：With simple process, but high productivity and high purity, Fmoc principle was found to be a perfect method to synthesize MP -2, and the purity of products that purified by MPLC was high. Paper showed that MP -2 could suppress the growth of S180 efficiently, and set foundation for further pharmacodynamic research.

关 键 词：Fmoc固相合成法 脊髓肽MP一2 HPLC MPLC 抑瘤 肿瘤细胞S180 

分 类 号：R917[医药卫生—药物分析学]