聚乙二醇化促胰岛素分泌肽类似物的结构确认

发布时间：2018-05-07 09:15

本文选题：促胰岛素分泌肽类似物 + PEG修饰　；参考：《重庆理工大学》2014年硕士论文

【摘要】：研究目的: 通过化学合成法合成促胰岛素分泌肽类似物(Byt-C)，Byt-C是以上市药物艾塞那肽（Byetta）的39个氨基酸序列为基础，在其C末端第39位后连接一个半胱氨酸（Cys）而成，采用长效修饰剂马来酰亚胺活化单甲氧基40kDa分子量的聚乙二醇（mPEG-MAL-40K）对Byt-C的半胱氨酸游离巯基进行单一位点的定点修饰，并且通过优化其修饰、纯化工艺，达到高修饰率，，得到高纯度目标产物。通过质谱分子量和质量肽图确认结构中半胱氨酸游离巯基的定点修饰以及修饰剂中马来酰亚胺为闭环结构的稳定产物。研究方法: 1)通过化学合成法获得Byt-C。①Byt-C纯度检测，包括SDS-PAGE，RP-HPLC方法的建立。②Byt-C结构确认，包括氨基酸全序测定，质谱分子量测定，质量肽图分析验证。 2) Byt-C的mPEG-MAL-40K修饰。包括修饰比例，修饰缓冲条件，修饰缓冲体系，修饰浓度，保护剂优化。 3) mPEG-MAL-Byt-C分离纯化。包括阳离子交换层析、反相层析、凝胶过滤层析。 4) mPEG-MAL-Byt-C非C末端修饰异构体研究。包括胰蛋白酶酶切质量肽图，修饰位点，氨基偶联分析。 5)马来酰亚胺为闭环结构的确证研究。包括水解开环，氢谱核磁共振分析。研究结果: 1. Byt-C多肽的质谱分子量与理论值完全一致，氨基酸测序完全正确，RP-HPLC纯度99%以上。 2. mPEG-MAL-Byt-C经修饰、纯化条件优化研究，可获得纯度高达98%的单一目标产物。 3. mPEG-MAL-Byt-C非C末端修饰异构体研究显示，证明了mPEG-MAL-Byt-C为修饰位点是单一的Byt-C多肽C末端Cys的游离巯基。 4. mPEG-MAL-Byt-C中马来酰亚胺为闭环结构的确认研究表明，目标产物为Byt-C多肽与聚乙二醇修饰剂mPEG-MAL-40k的闭环马来酰亚胺偶联而成的单一产物，且在修饰、纯化以及保存过程中均不会产生PEG的开环共轭物。
[Abstract]:Objectives of the study: The synthesis of insulin stimulating peptide analogue Byt-C by chemical synthesis is based on the 39 amino acid sequence of the listed drug Eisenapeptide Byettaand is connected to a cysteine CysA at the 39th position of its C-terminal. The polyethyleneglycol mPEG-MAL-40K, activated by Maleimide, was used to modify the cysteine free sulfhydryl group of Byt-C at a single site. By optimizing the modification and purification process, the modification rate was high. High purity target product was obtained. The fixed point modification of cysteine free sulfhydryl group in the structure and the stable product of Maleimide in the modifier were confirmed by mass spectrometry molecular weight and mass peptide map. Research methods: 1) the purity of Byt-C.1Byt-C was determined by chemical synthesis method, including the establishment of SDS-PAGEGE-RP-HPLC method, the confirmation of the structure of 2.2Byt-C, including the determination of total sequence of amino acids, the determination of molecular weight by mass spectrometry, and the verification of mass peptide analysis. 2) mPEG-MAL-40K modification of Byt-C. It includes modification ratio, modification buffer condition, modified buffer system, modification concentration, protection agent optimization. 3) mPEG-MAL-Byt-C separation and purification. It includes cation exchange chromatography, reverse phase chromatography and gel filtration chromatography. 4) study on non-C-terminal modified isomers of mPEG-MAL-Byt-C. It includes trypsin digested mass peptide map, modification site and amino coupling analysis. 5) confirmation of Maleimide as closed loop structure. Including hydrolysis ring opening, hydrogen NMR analysis. Results of the study: 1. The molecular weight of Byt-C polypeptide was completely consistent with the theoretical value, and the purity of RP-HPLC was over 99% by amino acid sequencing. 2. After modification of mPEG-MAL-Byt-C and optimization of purification conditions, a single target product with purity up to 98% was obtained. 3. The non-C-terminal modified isomers of mPEG-MAL-Byt-C showed that mPEG-MAL-Byt-C was the free sulfhydryl group of the C-terminal Cys of a single Byt-C polypeptide. 4. The confirmation of the closed loop structure of Maleimide in mPEG-MAL-Byt-C shows that the target product is a single product of Byt-C polypeptide coupled with the closed loop Maleimide of polyethylene glycol modifier mPEG-MAL-40k. No open loop conjugate of PEG is produced during purification and preservation.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914.5

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