论文部分内容阅读
目的:探索普兰林肽的固相合成、氧化条件及纯化方法。方法:采用Fmoc固相多肽合成法,以Rink Amide-AM树脂做载体,HBTU/HOBt/DIEA做缩合剂,逐步缩合得到全保护线性普兰林肽树脂,以TFA/苯甲硫醚/苯酚/H2O/EDT/TIS配比的裂解液脱除保护基团,分别采用空气,二甲基亚砜,双氧水氧化两个半胱氨酸的巯基形成一对二硫键,半制备反相高效液相色谱法纯化。结果:合成含37个氨基酸以及一对二硫键的普兰林肽经RP-HPLC和MALDI-TOF-MS确证,粗品纯度在50.0%以上,粗品经半制备型反相高效液相色谱纯化,所得精肽的纯度大于95.0%,总收率为30.5%。结论:该方法简单,合成的产品成本低,纯度高,可为工业化生产提供借鉴。
Objective: To explore the solid-phase synthesis, oxidation conditions and purification methods of pramlintide. Methods: The total protective linear pramlintide resin was obtained by Fmoc solid-phase peptide synthesis using Rink Amide-AM resin as carrier and HBTU / HOBt / DIEA as condensing agent. TFA / thiophenol / / EDT / TIS ratio of the lysate removal of protective groups, respectively, using air, dimethyl sulfoxide, hydrogen peroxide oxidation of two cysteine thiol to form a pair of disulfide bond, semi-preparative reversed-phase high performance liquid chromatography Purification. Results: Pramlintide with 37 amino acids and one pair of disulfide bonds was confirmed by RP-HPLC and MALDI-TOF-MS. The purity of the crude product was over 50.0%. The crude product was purified by semi-preparative reversed-phase high performance liquid chromatography The purity of the refined peptide is greater than 95.0% with a total yield of 30.5%. Conclusion: The method is simple, low cost and high purity of synthesized products, which can provide reference for industrialized production.