【作者】 林敏；

【导师】 屠春燕；

【作者基本信息】 南京工业大学 ， 生物化工， 2005， 硕士

【摘要】 本文以Fmoc策略固相合成胸腺五肽体系为研究对象,根据生产工艺特点,针对原料、氨基酸活化反应液及胸腺五肽粗品,分别建立了方便、有效的定性、定量分析方法。 首先建立了HPLC法分析固相合成原料—Fmoc保护氨基酸的方法,可以在一次进样的前提下对五种Fmoc保护氨基酸同时进行定性、定量分析。同时讨论了盐浓度、pH值对分离的影响。 固相合成多肽过程中,可溶性的杂质易除去,但连接在树脂上的副产物在合成过程中间无法从目的产物中分离出去,只能且必须在合成的终了阶段除去。因此,要获得高纯度的目标产物,必须保证脱保护和耦合反应充分有效且副反应少。目前,合成过程的中间监测方法主要集中在胺酰化及脱保护反应,而对于中间体一活化酯的反应进程没有监测方法。 有鉴于此,针对固相合成胸腺五肽过程中Fmoc-Tyr(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH羧基活化反应液建立了HPLC分离分析方法。同时采用LC/ESIMS液质联用技术,对体系中的中间体进行质谱定性分析,很好地解析了反应的整个过程,有助于建立N—保护氨基酸的活化反应动力学方程,改进合成工艺,减少氨基酸用量,回收未反应的保护氨基酸,从而最大限度地利用资源。 在采用液质联用技术对氨基酸活化反应液进行定性分析的过程中,我们发现五种活化反应液中均存在一对分子量相同组分,即这些中间体以成对的形式出现在反应液中。经分离纯化鉴定初步推测它们可能是同分异构体,在溶液中存在一个平衡关系。 最后用HPLC法分析了TP-5粗肽,该法对卫生部颁布的TP-5原料药检测方法进行了改进,适用性大大扩展,可适用于不同合成工艺的粗品、纯品分析;同时应用HPLC/MS/MS方法对TP-5粗肽进行成分解析,可确定出目标肽并对主要副产物可能的结构进行了推测。 综上所述,本文应用HPLC及HPLC/MS/MS技术对固相合成胸腺五肽体系进行过程分析,取得了良好的效果,为进一步优化合成工艺提供了有力的监测手更多还原

【Abstract】 We investigated the system of solid-phase pep tide synthesis (SPPS) of Fmoc chemistry for Thymopentin (TP-5). According to the process characteristics, we have established convenient and efficient methods for the qualitative and quantitative analysis of raw material, amino acid activation solution and crude TP-5.Firstly, we have established the HPLC method for the raw material of SPPS-Fmoc protected amino acid. With a single sampling, it enables simultaneous qualitative and quantitative analysis for the five Fmoc protected amino acid. We also discussed the influence of salt concentrations and pH on separation.In the progress of SPPS, the soluble impurities can be removed easily, but it is hard to remove the by-products attached ,to the resin from the desired products. The by-products could only and have to be removed at the end of the synthesis. Therefore, in order to obtain high purity final product, the deprotection and coupling reaction must be ensured to be complete and effective with few side reaction. Currently, the reaction progress monitoring focuses on amide acylation and deprotection reaction, while there is no method for the reaction progress of the intermediate-active esters.Under such background, we established HPLC methods for the carboxyl activation solution of Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH and Fmoc-Arg(Pbf)-OH. We have also used LC/ESIMS technique to analyze intermediates in the system. The qualitative analysis provides us a comprehensive understanding on the whole process. It helps to establish the kinetic equation for N-protected amino acid activation, to improve the synthesis technique, reduce amino acids consumption, recover protected amino acids which did not participate the reaction, therefore make full use of resources.When analyzing the amino acid activation solution with LC-MS technique, we found compound pairs with identical molecular weight in all five amino acid activation solution, that is, these intermediates exist in reaction solution in pairs. Byseparation and purification assay, we speculated that those pairs may be isomers, between which a chemical equilibrium existed in solutions.Finally, we established the HPLC method for crude TP-5. This method has improved the analysis method for TP-5 raw material drug issued by MOH, which makes it much more applicable for analysis of crude or purified products produced from various synthetic techniques. In addition, we have analyzed the chemical composition of crude TP-5 by HPLC/MS/MS method, through which the target peptide could be detected, and the structure of the main by-products has been speculated. ’.In conclusion, in this paper, we have analyzed the progress of SPPS for TP-5 by HPLC and HPLC/MS/MS technique, and obtained a sound effect, which provide a potential monitoring method for optimal synthetic technique.更多还原