Quality Monitoring Of Solid-phase Synthesis System For Thymopentin

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.