Synthesis Of Cyclic Peptides And Their Antitumor Activity Based On Photoinduced Single Electron Transfer Reaction

Cyclic peptides have become an attractive modality for drug development due to their excellent binding affinity,target selectivity and low toxicity.However,the bioactive difference caused by the structure of chemically synthesized and natural cyclic peptides has always troubled chemists.The drug design based on natural cyclic peptides and their analogs has become a new way for development of anti-tumor drugs.In order to explore the influence of the secondary structure of chemically synthesized cyclic peptides on its biological activity and the apoptosis pathway of tumor cells,this paper used the regioselective intramolecular photo-induced single electron transfer(SET)reaction to synthesize series of isoindole-Phakellistatin2 analogs and isoindole-Phakellistatin 6 analogs with special conformational characteristics,tried to study the in vitro activity and analyze the action by transcriptome sequencing.(1)The absolute configuration C-3 of Phakellistatin 2 analogs was determined by ECD spectroscopy combined with quantum chemistry calculations.Cyclic peptide 1was defined as‘DS'isomer and cyclic peptide 2 was‘LR'.In the secondary structure of cyclic peptide 1,we found a strong intramolecular hydrogen bond between the C=O of Phe³ and the N-H of Ile⁵(the distance is 1.92?),there is a classic?-turn structure in the DS isomer.Subsequently,the MTT colorimetric experiment was carried out on the Phakellistatin 2 analogs.The IC₅₀ of cyclic peptide 1 and 2 on Hep G-2 cells were 12.28±0.50 and 22.17±0.72?g/m L,respectively.The results showed that the compounds containing D-Pro residues presented stronger anti-tumor activity on Hep G-2 cells than that of compounds containing L-Pro residues,proving the importance of the secondary structure of cyclic peptides to biological activity.(2)The antitumor activity of the synthesized Phakellistatin 6 analogs was screened by MTT assay,and the results showed that compound 10 is the most toxic to Hep G-2liver cancer cells,with IC₅₀ of 7.79?g/m L.From subsequent flow cytometry experiments,it can be found that cyclic peptide 10 could lead tumor cells to apoptosis.We also analyzed the conformational characteristics of the synthesized Phakellistatin 6cyclic peptides.The spatial configuration characteristics were systematically studied using ECD and NMR combined with theoretical calculations.Cyclic peptide 10 with a unique?VIa turn and two cis-Xaa-Pro bonds exhibited the strongest inhibitory activity,implying this secondary structure might affect the interaction between the peptide and the target,andresulted in a significant biological difference.We also found that the anti-tumor activities of the prepared cyclic peptide analogs are mostly stronger than that of the anti-cancer drug paclitaxel,showing a promising applicable potential.The results of LDH release experiment and SEM electron microscopy confirmed that Phakellistatin cyclopeptide analogs have the ability to destroy tumor cell membranes.(3)Through the analysis of flow cytometry,we found a strong apoptotic signal to the liver cancer cell Hep G-2 caused by Phakellistatin cyclopeptides analogs.In order to further explore the apoptosis pathway,we performed transcriptome sequencing of RNA in the control group and the drug group.The RNA sequencing(RNA-seq)results were analyzed from differentially expressed genes,and the relative expression of candidate genes m RNA was verified by Real Time PCR.In this RNA-seq,a total of 20317 genes were identified in the cells,and a total of 2306 differentially expressed genes were selected according to the differential expression screening criteria,of which 2147 were up-regulated and 159 were down-regulated.The GO term and KEGG pathway enrichment analysis were performed on the differentially expressed genes,focusing on the differentially expressed genes related to the MAPK and Wnt signaling pathways.The genes related to MAPK pathway such as FGF21,HSPA1B,HSPA1A,JUN,DUSP1,HSPA6 and GADD45A were significantly up-regulated.The SESN2,GADD45,PMAIP1,GORAB,MDM4 and CDKN1A gene of p53 signaling pathway was significantly up-regulated.q RT-PCR verified the reliability of sequencing results.Investigations revealed that phakellistatin 6 induced marked apoptosis that was related to changes in m RNAs in the p53 signaling pathway and MAPK signaling pathway.(4)Two independent intramolecular electron donating and accepting systems are bridged by the disulfide bond of L-cystine,and the intramolecular photo-induced electron transfer cyclization reaction was used to bi-cyclized the Bisisoindolinone-cyclic peptide.This was a novel method for synthesizing bicyclic peptides.Through ECD and ¹³C-NMR spectroscopy,it was determined that the C-3chiral feature of bicyclic peptide 20 is the R configuration.