| The pentapeptide Thymopentin (TP5) is the fragment of thymopoietin II(32-36) with basic sequence H-Arg-Lys-Asp-Val-Tyr-OH. It has the same biological activity as the thymopoietin II, which has the double-direction immunities to our bodies. Now TP5 has been applied as one effective medicine with good market prospects. According to above reasons, the studies on the solid phase synthesis of TP5 are not only for the good commercial values but also for establishing the solid peptide phase synthesis (SPPS) technical platform. Firstly, general methods for SPPS, current TP5 synthesis level and its disadvantages had been reviewed. Secondly, several following key steps involved in SPPS were particularly studied: (1) swelling of resin; (2) attaching the first amino acid to resin;(3) analyzing the content of racemisation and purify of the first amino acid attaching to resin; (4) selecting the coupling reagents in the elongation; (5) cleavaging peptide-resin. Eventually, there had been established one high-efficient and low-cost TP5 synthesis process, which indicated perfect feasibilities to the large-scale synthesis. Finally, the conclusions can be summarized as following: (1) Compared with different substitutions resins and swelling solvents, it came to a conclusion that either the enhanced substitution resins or increased polarity of solvents did decrease the resin swelling properties. (2) Attaching amino acid to the resin is the sticking point in the whole synthesis, because it has the most important effects on the yield and purification of ultimate product. By adding catalyst, selecting different solvents and changing kind of resins, the coupling yields had been increased from 30-40% to 100%. In Addition, the effects of catalyst and solvents had been dissertated in detail. Furthermore, the reaction mechanisms of carbodiimide (DCC and DIC) and 2-6-dichlorobenzyl chloride (DCB) methods were proposed. (3) D-Tyr and Tyr-Tyr are the byproducts in the first attaching reaction, which also have direct influences on the purifications and activities of final products. Through establishing a HPLC analytical method, the impacts of different conditions on both two byproducts had been quantified. (4) Three coupling reagents DCC, DIC, HBTU and PyBOP had been used in the elongation TP5 peptide chain process. It had been proved that DIC was the best one in both facets of yield and purification. (5) In the cleavaging TP5 from peptide-resin complexity, a high-yield and harmless condition has been build up by comparing the cleavage agents and their dosages, washing agents, time and temperature. That process was cleavaging the resin by TFA: TIS: H2O=95: 2.5: 2.5 under 24℃for 3.5h , and then washing the resin by 95% TFA/DCM to get the maximum yield of peptide. Based on above studies, the small-scale (0.2mmol) and large scale (2.2mmol) of TP5 were synthesized by our proposed optimum procedure. In the former test, the both yield and purity of TP5 reached 90%, while the cost was decreased by 60% comparing to the cost of original procedure. In the latter test, both yield and purity of TP5 reached to 86%, and the cost was further decreased by 6% comparing to the small-scale (0.2mmol). Consequently, the proposed optimum procedures had showed their feasibilities and benefits in both small and large scale TP5 synthesis process.
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