Study On The Liquid Phase Synthesis Of The Bioactive Tripeptide Bursin

Bursin was a tripeptide first discovered in the bursa of fabricius which was the special primary lymphoid organ for humoral immunity of poultry, whose chemical composition was L-Lys-His-Gly-NH₂. Indicated from the research, BS can effectively promote the differentiation and themultiplication of the B lymphocyte precursor of poultry, it also has the obvious immunity activeness and physiology activeness to the mammal and the humanity lymphocyte; in addition. BS may promote the animal to increase weight heavily in the short time, the highly concentrated BS can also cause the hybrid tumor cell to perish weakly, therefore, it is one kind of highly effective immunity intensifier, hopefully, it would be equally applied to clinic like the thymosin and the interferon, to enhance the organism immunity and treat the immunodeficiency disease and cancer. In this thesis, the method of peptides synthesis and the bioactivity of bursin was summarized, the liquid phase synthesis method and technology of bursin was studied.The bioactive tripeptide bursin was synthesized from carboxy-terminal to amino-terminal by four steps in the liquid phase with dicyclohexylcarbodiimide(DCC) as the condensing agent, Boc-Lys-(Boc)-OH·DCHA, Fmoc-His-(Trt)-OH, Gly-NH₂·HCl as the raw materials which were all protected amino acides, TLC as the following and monitoring method. The whole yield of BS was 62.7%, and the purity was not less than 90%.By Optimization, the synthetic technolgy of BS in order was established as follows: In the synthetic reaction of Fmoc-His-(Trt)-Gly-NH₂, after the raw material Fmoc-His-(Trt)-OH, Gly-NH₂·HCl mixed in the 50%(V/V)tetrahydrofuran water solution, DCC was joined at 0℃, the mole ratio of reactant is: Fmoc-His-(Trt)-OH : DCC : Gly-NH₂·HCl=1 : 6 : 15, responded the most ideal yield 95%. The Fmoc-base was removed thoroughly by 50% (V/V) diethylamine dichloromethane solution more than 8 hours in room temperature, and the post-processing is simple. the synthetic reaction of Boc-Lys-(Boc)-His-(Trt)-Gly-NH₂ was carried on at 0℃, the mole ratio of reactant is :L-His-(Trt)-Gly-NH₂ : Boc-Lys-(Boc)-OH : DCC = 1 : 2 : 2, the yield can reach 90%. The removal of Boc-base and the Trt-base was carried on at the room temperature for 30 minutes with 50%(V/V)trifluoroacetic acid dichloromethane solution only by one step.The influence factors in the DCC condensation reaction was discussed with emphasis. Tetrahydrofuran was finally choosed as the basic solvent with the low boiling point and the water mixedable characters, and its effect is ideal. The carboxyl group was premixed with amino group 30 min to form the ionicbond before activation, thus prevent the occurrence of the side reaction, but the amino freeed or not dose not have obviously affects to the reaction. When the mole ratio of Fmoc-His-(Trt)-Gly-NH₂ reactant is: Fmoc-His-(Trt)-OH : DCC : Gly-NH₂·HCl=1 : 6 : 15, the use of Fmoc-His-(Trt)-OH is high, and there is less occurrence of side reaction. When the mole ratio of Boc-Lys-(Boc)-His-(Trt)-Gly-NH₂ reactant is: L-His- (Trt)-Gly-NH₂ : Boc-Lys-(Boc)-OH : DCC = 1 : 2 : 2, the yield may reach 90%. the optimum condensation condition is: 0℃, 6-8 h, standing over night. After pretest, the Fmoc-base was removed by 50% (V/V) diethylamine dichloromethane solution more than 8 h, the Boc-base and Trt-base was removed by 50% (V/V) trifluoroacetic acid dichloromethane solution 30 min at room temperature. The condensation reaction was post-processed by the acidpickling and alkalipickling to except the substrate, with the ethylacetate to precipitate and except DCU. The base-removed reaction was post-processed by precipitation, crystalization and extraction. The purification of the final product BS uses the flash column chromatography with silica gel to separation. The raw material Gly-NH₂·HCl was recycled through extraction of water and crystallization of the methyl alcohol, Boc-Lys-(Boc)-OH was recycled by alkaline washing, acidulation and extraction of ethylacetate.