Research On Synthesis And Anti-inflammatory Activity Of Small Molecule Peptide Derivatives

Inflammatory reaction is a complex defense mechanism of the body when it is damaged by the outside factor.Moderate inflammation is beneficial,but excessive inflammation can cause a threat to people's physical and mental health.The men in the military personnel training exercise often suffer from military training injuries characterized by acute inflammation and pain,which seriously affects the combat effectiveness of the military.Treatment with anti-inflammatory drugs is one of the effective measures against the inflammatory response,but the drug can cause gastrointestinal damage side effects or damage to the cardiovascular system.Therefore,it has great significance to developefficient and safe new anti-inflammatory drugs.Small molecule peptide drugs have become a new target for research and development because of their small molecular weight,high safety,strong specificity and easy structure modification.On the other hand,they also have defects including being easily degraded by enzymes in the body,short half-life and low membrane permeability,which limits their further development and application.In this paper,anti-inflammatory linear peptides were synthesized and modified into cyclic peptides and staple peptide precursor peptide to improve their anti-inflammatory activity and obtain the law of structure-activity relationship,which laid the foundation for further development of new anti-inflammatory peptide drugs.This article has made the following work progress:Part ?: Synthesis of cyclic peptides.The anti-inflammatory linear peptide was modified to cyclic peptides by using a third-dimensional protecting group-photolabile protecting groups.Based on the previous study of early photolabile protecting group 2-(o-nitrophenyl)-ethyl(Npp-OH),four new photolabile protecting groups were designed and synthesized by introducing alkylsat the benzyl sites or changing primary alcohols into secondary alcohols.It found that 3-(o-nitrophenyl)butan-2-ol(Npb-OH)was stable to acid-base and had a fast photolysis rate,which was effectively against the side reaction of diketopiperazine.Furthermore,it was successfully used to modify the amino acid side chain of the linear peptide to synthesize two cyclic peptides,which had the advantage of removing the active groups of the peptide chain without affecting the other active groups in the peptide.Part ?: Synthesis of Linear Peptides and Staple Peptide Precursor Linear Peptides.Firstly,ten anti-inflammatory linear peptides were synthesized by standard solid-phase synthesis,and based on the staple peptide strategy,10 linear peptides were modified to prepare staple peptideprecursor linear peptides.The modification strategy: design and synthesis of a propylene alanine(hydrophobic amino acid)with two configurations of ?-disubstituted,and replacing the hydrophobic amino acid residue of the anti-inflammatory linear peptide at i~i+4 sites,nineteen new structural linear peptides were prepared for further synthesis of the staple peptide.The product was tested and purified by HPLC and molecular weight confirmed by Q-FT-ICR-MS.Part ?: Evaluation of anti-inflammatory activity of 31 synthetic cyclic peptides,linear peptides and peptide-precursor linear peptides.The effect of tested peptides on the viability of RAW264.7 cells were investigated at concentrations of 20,10,5,2.5,1.25 and 0.625 ?mol/L.The results demonstrated that three peptides of LP11-26,MP11-26-R,R,MP11-26-S,Sat a concentration of 0.5 ?mol/L obtained;three peptides of LP4-18,MP4-18-R,R,MP4-18-S,S in the concentration of 1 ?mol/L;the remaining 25 peptides at 5 ?mol/L concentration could maintain RAW264.7 cell viability at above 90%.Accordingly,three concentrations can be used as the respective administration concentrations corresponding peptide activity screening.The effect of 31 peptides on the release of TNF-? from RAW264.7 cells induced by LPS was detected by enzyme-linked immunosorbent assay(ELISA).Dexamethasone used as a positive control drug.The result displayed that compared with the model group,dexamethasone and 31 peptides showed inhibitory activity(P<0.05);the modified peptide precursor peptide had no significant difference in activity compared with its corresponding linear peptide,indicating that this structural modification strategy was successful and had no significant effect on the inhibition of TNF-? release in RAW264.7 cells by 10 linear peptides;the anti-inflammatory activity of cyclic peptide is higher than that of the corresponding linear peptide,indicating that the cyclic peptide modification strategy can improve anti-inflammatory activity.The results of furtherscreening demonstrated that the inhibitory activity of TNF-? in the high concentration group was higher than that in the low concentration group,suggesting that there might be dose dependence;MP5-8S,S,showed TNF-? inhibitory activity at 5 and 2.5 ?mol/L;2.5 ?mol/L LP4-18 inhibited TNF-? significantly higher than dexamethasone;1 ?mol/L LP11-26,MP11-26-R,R of TNF-? inhibitory activitywas similar to that of the dexamethasone group.Evaluation of anti-inflammatory activity in vivo showed that the swelling inhibition rate of 5mg/kg in dexamethasone group was 52.3%,and the swelling inhibition effect of dexamethasone group was good.The swelling inhibition rates of peptide LP4-18,MP5-8S,S,LP11-26 and MP11-26 R,R were 36.0%,24.0%,47.1%,29.7%,respectively.Compared with dexamethasone group,the inhibition rate of melittin LP11-26 was not statistically significant(p > 0.05),which indicated that the swelling inhibition rate of melittin group was similar to that of dexamethasone group.In conclusion,10 anti-inflammatory linear peptides were synthesized and used as prototype molecules.Through the cyclic peptide and the staple peptide strategy,the prototype peptide was modified.Two cyclic peptides were successfully synthesized,and 19 precursor linear peptide was synthesized which can be further synthesizedto staple peptide.Thus,a total of 31 peptides were prepared.The anti-inflammatory activity of peptides was evaluated by RAW264.7 cell assay.The results demonstrated that the cyclic peptide strategy can significantly improve anti-inflammatory activity,there was no significant effect on the anti-inflammatory activity of the original linear peptide by modifying the precursor linear through the modifiecation of staple peptide strategy.The above work lays a theoretical and experimental foundation for the further preparation of staple peptides and the study of anti-inflammatory active peptides with high stability and better drug resistance.