| Chemotherapy is one of the main methods of cancer treatment.Among them,peptide drugs have become a hot spot in the development of anti-tumor drugs in recent years due to their low toxicity,clear specificity and small molecular weight.Natural peptides are widely available,especially in the marine environment,which is one of the main sources of natural peptides because of their biodiversity.Samoamide A is a cyclic octapeptide extracted from the genus Coccinellium,and its structure is cyclo(-[Val-Leu-Pro-Pro-Phe-Ile-Pro-Phe]-),In vitro activity experiments show that it has good cytotoxicity,but the yield of natural Samoamide A is very low.No research has been found to solve this problem.Therefore,in order to increase its yield,further research is needed.This project uses Fmoc solid phase synthesis method,2-chlorotrityl chloride(CTC)resin as solid phase carrier,Fmoc protected phenylalanine,valine,valine,leucine,isoleucine The acid is used as a raw material to synthesize a linear peptide.After the linear peptide is cleaved from the resin,the cyclic peptide is cyclized by a liquid phase method to synthesize the cyclic peptide Samoamide A,and the reaction solvent,the condensing agent,the cleavage condition and the pH are synthesized.The conditions were optimized.The results showed that 60%DCM/DMF was used as the reaction solvent,TBTU was used as the condensing agent,and the solution of TFA/EDT/PhOH/H20/thioanisole was 80:2.5:7.5:5:5.When the pH of the cyclization system is 8.0,the synthesis purity and yield are the highest,and the total yield of Samoamide A can reach 54.5%.The structure of the synthesized compound Samoamide A was confirmed by high performance liquid chromatography-mass spectrometry(HRMS),nuclear magnetic resonance spectroscopy('H NMR)and nuclear magnetic resonance spectroscopy('3C NMR).The molecular weight was 910.40,and the theory The molecular weight is consistent,and the results of nuclear magnetic resonance spectroscopy indicate that Samoamide A is successfully synthesized,which provides technical support for its further development and utilization.On the basis of the previous one,in order to improve the anti-tumor activity of Samoamide A,it is necessary to modify Samoamide A.In this paper,the alanine scanning method was used to explore the inactive site of Samoamide A,and the alanine protected by Fmoc was substituted for Samoamide A.The 8 amino acids were synthesized in the same way as the previous section,and 8 new Samoamide A derivatives were obtained.The structure of the eight new Samoamide A derivatives was confirmed by HRMS,1H NMR and 13C NMR.The results showed that the molecular weight of the derivative was consistent with the theoretical molecular weight.The results of nuclear magnetic resonance and hydrogen spectrum analysis were also consistent with the theory.The chemically synthesized Samoamide A has good antitumor activity by cytotoxicity assay(MTT)and DPP4 enzyme inhibition assay.When the concentration of Samoamide A was 50?g/mL,the survival rate of 4T1 cells was 20.79%.When the concentration was 100 ?g/mL,the inhibition rate of DPP4 enzyme activity was 85.72%.Then the antitumor activity of 8 Samoamide A derivatives was analyzed.The results showed that Samoamide A-6 had the best antitumor effect,and its 4T1 cell survival rate was the lowest,only 25.35%,and the DPP4 enzyme activity inhibition rate was the highest,which was 82.44%.,the closest to Samoamide A.Therefore,the substitution of proline(Pro)at this site with alanine(Ala)did not significantly affect the antitumor activity of Samoamide A,demonstrating that this site is an inactive site of Samoamide A.In summary,this study has developed a new Samoamide A synthesis method,which has improved its yield,and found a non-active site of Samoamide A through alanine scanning and anti-tumor activity studies.The cyclic modification of Samoamide A provides a theoretical basis for subsequent modification and modification,and has important research significance and application value. |
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