Studies On Thymopentin Peptide Library And Microwave Assisted Synthesis Of Vapreotide

Solid-phase peptide synthesis (SPPS) method is the key technique in modern protein chemistry. This thesis mainly discussed two SPPS method: combinatorial chemistry method, microwave-assisted synthesis. We used thymopentin and vapreotide as the model peptides.With the double immunoregulational effects, thymopentin can improve body's ability of immunity. It has a proposed use in clinical application. But it will be quickly degraded by proteinase and aminopeptidease in human's blood plasma. The half-life of thymopentin is 30s. One of the methods to increase peptides' stability is to add D-amino acids into peptide chain. In order to increase thymopentin's stability, we synthesized the D-amino acid substituted thymopentin peptide library using tea-bag method. And we also studied the pharmacody activity of the library to find peptides with higher immune activity and stability.We found that using tea-bag method, the peptide library could be synthesized in short time. The method was simple and convenient. It could cut the usage of reagents, reduce the cost and increase the synthesis efficiency. The yield of raw peptide was 54%. And after purified, the yield of peptide was about 36%. In the library, we found two analogs had the more obvious effects to stimulate the multiplication of T cell compared with thymopentin.Vapreotide is the analog of somatostain with an intramolecular disulfide bond. Compared with somatostain, it has a higher stability and longer half life time. And it also has a proposed market prospect. Microwave radiation can increase the reaction rate, shorten the reaction time and raise the yield of production if it is used in organic chemistry.In this paper, we added microwave assisted technique to the SPPS coupling reaction. The optimum coupling reaction conditions, including microwave radiating time, temperature and proportion of reactants, were found by orthogonal test. Results were analyzed by multiple nonlinear regression method and response surface optimization. We also discussed the process of deprotection, washing, cleavage andformation of disulfide bond that would influence the final yield of vapreotide.The result showed that the activating time should be 2~3h and the usage of amino acids should be 3 times of resin peptide load when using DCC-HOBt compound reagents. As to the valine, the optimal coupling conditions were 6 minutes of coupling reaction time, including 2 minute of ramping time and 4minutes of holding time, 60°C-maximum reaction temperature and 600w power. As to the other amino acids, all the conditions were the same as valine except that the ramping time was 45s and the power was 300w. Compared with the conventional reaction, the microwave-enhanced coupling reaction time was shortened about 12-36 times. The efficiency of coupling reaction was increased greatly and reached 90%. Only NMP, MeOH and DCM were alternate used to wash the resin for 15 times could the washing be complete. When deprotecting, we used 20% PiP/NMP 3 times and each time last for 4min. To get a higher yield and purity of vapreotide, we used reagent 1 as the cleavage reagent and DMSO/HiO/MeOH as oxidants. The time of overall process of vapreotide synthesis was shortened to 6 days which usually need 2~3 weeks. The efficiency of synthesis increased about 2~3 times. The yield of vapreotide also increased from 48% to 80%.