Polypeptide Synthesis, Separation, Purification And Thiol Oxidation

Polypeptide is a type of bioactive substance that participates in all the cells' function. The synthesis of bioactive polypeptides and proteins with chemical methods has being greatly developed, since the method of Merrifield's solid phase peptide synthesis (SPPS) was set up. In this thesis, octreotide and other two peptides were synthesized by Fmoc - SPPS method, separated and purified, also various methods for the formation of disulfide bond within octreotide molecular were investigated.The thesis includes the following four parts:1. At first, the fundamental knowledge on polypeptide was introduced. Then the history and the principle of peptide synthesis, the coupling methods in SPPS, the strategies to form disulfide bond and the application of chromatography in separation and purification of polypeptide was also briefly reviewed. Total 94 references were cited.2. To synthesize the octreotide, Phe-Leu-Glu-Glu-Leu and C₁₅H₃₁-CO-Lys-Thr -Thr-Lys-Ser peptides, the strategies are as followings: Fmoc- group as protective group for α-NH₂; TBTU, DIEA and HOBt as coupling reagent; and ethanedithiol -phenol - thioanisole - H₂O - TFA ( 2.5:5:5:5:82.5 , V/V ) as cleaving solution. The structure of three crude peptides were identified to be correct by MALDI - TOF mass analysis.3. The separation and purification of octreotide, Phe-Leu-Glu-Glu-Leu and C₁₅H₃₁-CO-Lys-Thr-Thr-Lys-Ser peptides were carried out with reversed-phase liquid chromatography (RPLC). The chromatographic conditions were optimized, and the purified products were analyzed by RPLC and MALDI-TOF mass. The purity of purified octreotide was 97.8%.4. The disulfide formation methods of reduced octreotide were investigated. The four methods, air oxidation, DMSO/H₂O oxidation, potassium ferricyanide oxidation, Cu²⁺ oxidation and H₂O₂ oxidation, were used to promote the formation of disulfide bond in octreotide at different concentrations. On reaction rate and yield as evaluatingstandard for different methods, the potassium ferricyanide oxidation and H2O2 oxidation for octreotide were found to be superior to other methods in less reaction time and high oxidation yield.