Study On The Synthetic Process Of Meucin-18 And The Design,Synthesis And Biological Evaluation Of Its Derived Peptides

In recent decades,the continuous development of antibiotic resistance has led to a serious threat to public health from multiple drug resistance bacteria infection.However,in the past three decades,the number of new antibiotics has been declining,and the number of antibiotic substitutes for the treatment of drug-resistant bacteria has been decreasing as well.Therefore,there is an urgent need to develop antibacterial agent substitutes with different mechanisms,which are not easy to induce drug resistance.Antimicrobial peptides(AMPs)and its derived peptides have a broad prospect in the development of new antimicrobial agents because of their unique membrane destruction mechanism that can reduce drug resistance.Meucin-18 is a natural antibacterial peptide extracted from the venom of Mesoboutus eupeus scorpion,with 18 amino acid residues and free carboxyl terminal.The sequence of Meucin-18 is FFGHLFKLATKIIPSLFQ,containing 2 lysines,so it has 2 net positive charges.Its secondary structure is α-helix and amphiphilic.Meucin18 has various biological activities such as antibacterial and antiviral activities,showing broad application prospects.At present,the research on Meucin-18 is few,and mainly focuses on its antiinfective activity,while the research on its synthesis is few.Meucin-18 is mainly obtained through solid phase synthesis.The solid phase synthesis method has become increasingly mature since its establishment,but it still has shortcomings,such as the inability to remove by-products in the peptide synthesis process,and excessive feeding,etc.Therefore,we considered applying the hydrophobic tag-assisted liquid-phase synthesis method to the synthesis of Meucin-18,to establish a new synthetic route to adapt to the future pilot scale of Meucin-18.For this method,we mainly conducted the following experimental studies:(1)We divided Meucin-18 into three segments for segmented synthesis,which were fragments F-Boc(Boc-Phe-Phe-Gly-His(Trt)-Leu-OH),G-Fmoc(Fmoc-PheLys(Boc)-Leu-Ala-Thr(t-Bu)-Lys(Boc)-OH)and Tag-fragment H(Ile-lle-Pro-Ser(tBu)-Leu-Phe-Gln(Trt)-O-Tag).(2)The effects of the feed ratio of hydrophobic tag to amino acid,temperature and organic base on the coupling of different amino acids with hydrophobic tag were investigated,and the optimal conditions for coupling Fmoc-Gln(Trt)-OH to hydrophobic tag were to use DCM as a solvent under heating at 40℃,and successively add Tag-OH,Fmoc-Gln(Trt)-OH,DIC,and DMAP to the solvent,according to the feed ratio of Tag-OH:Fmoc-Gln(Trt)-OH:DIC:DMAP=1:1.2:1.5:0.2.The optimal conditions for the coupling of Fmoc-Gln(Trt)-OH with the tag were suitable for FmocLys(Boc)-OH with the tag.(3)The effect of the amount of TFA on the last step of the hydrophobic tag removal reaction of fragment F-Boc(reducing the amount of by-products)was investigated.It was determined that when the amount of TFA was 0.7%,the yield and purity of fragment F-Boc were the highest,with 84.4%and 78.3%,respectively.(4)The effects of temperatures and solvents on the deprotection reaction of fragment G-Fmoc were investigated.It was determined that the most suitable reaction conditions were DCM as solvent,reaction temperature 25℃,and the addition of 10%TFE and 1%TFA in turn.The highest yield and purity of the product were obtained,with 74.2%and 88.9%,respectively.(5)The post-treatment of fragment G-Fmoc was investigated.the result showed that the filtration viscosity was reduced by adding diatomite,making the filtration easier to operate.This greatly shortened the filtration time,saved time and cost,and reduced the product loss caused by adhesion,which was helpful for improving the yield.(6)The effects of the coupling conditions of fragment to fragment on the yield and purity of Meucin-18 were investigated.Finally,it was determined that the optimal coupling conditions were Tag-fragment H:fragment G-Fmoc:HATU:HOAt:DIPEA:DIC=1:1.05:1.05:1.05:5:1,Tag-fragment H,G:fragment F-Boc:HATU:HOAt:DIPEA:DIC=1:1.05:1.05:1.05:5:1.The yield and purity of Meucin-18 crude peptide obtained at this time were the highest,70.9%and 82.7%,respectively.In addition to studying the synthesis of Meucin-18,we also explored its structure and activity.We designed several derived peptides of Meucin-18,with the design strategies such as shortening the peptide chain,changing the net positive charge quantity,and changing the peptide sequence,etc.Then we predicted the physical properties of the derived peptides using bioinformatics prediction software.Finally,four derived peptides(CH-1,CH-2,CH-3,CH-4)were determined for synthesis and their biological activities were evaluated.The study of antibacterial activity in vitro showed that CH-1 dispalyed excellent antibacterial activity against the tested strains,CH-2 and CH-3 did not exhibit significant antibacterial activity against the tested strains,and CH-4 only had good antibacterial activity against S.aureus ATCC25923 and B.subtilis ATCC9372.Specifically,CH-1 exhibited excellent antibacterial activity against six Gram-positive sensitive strains,seven Gram-positive drug-resistant strains,and two Gram-negative strains tested.Compared with Meucin-18,CH-1 showed excellent antibacterial activity against Gram-negative bacteria,with the MIC values against E.coli ATCC25922 and P.aeruginosa ATCC27853 of 3.8 μM and 7.5 μM,respectively,16 times more active than Meucin-18.For Gram-positive drug-resistant strains S.pyogenes EMA-R,E.faecalis ATCC51299 and E.faecium ATCC51559,their MIC values were 7.5 μM、15.0μM and 7.5 μM,respectively,4 times better than the antibacterial activity of Meucin18.CH-1 had excellent antibacterial activity against Gram-positive drug-resistant bacteria S.aureus ATCC31007 and S.aureus ATCC43300,with the MIC values of 1.9μM and 1.9 μM.CH-1 exerted strong antibacterial activity against S.aureus CI and S.epidermidis,with an MIC value of 3.8 μM.The antibacterial activity of CH-1 against Gram-positive sensitive bacteria S.pyogenes EMA-S and E.faecium ATCC19434 was 4 times stronger than that of Meucin-18,and the corresponding MIC value of CH-1 was 7.5 μM,showing good antibacterial activity.CH-1 possessed excellent antibacterial activity against B.pumilus CMCC63202,B.subtilis ATCC9372 and S.aureus ATCC25923,with an MIC value of 1.9 μM.In addition,an MIC value of CH-1 for E.faecalis ATCC29212 was 7.5 μM,displaying good antibacterial activity.The MBC/MIC value of CH-1 was 2,belonging to bactericidal agent.The time-kill kinetics study showed that at 1×MIC concentration,CH-1 could kill all S.aureus ATCC25923 within 9 h,and at 2×MIC concentration,CH-1 could kill all S.aureus ATCC25923 within 3 h,which indicated that the bactericidal effect of CH-1 increased with time or concentration.Moreover,CH-1 had high tolerance to strong acid,strong alkali,high temperature and common physiological saline.Besides,even at 4 × MIC,the hemolysis rate of CH-1 is less than 10%,while at 1 × MIC and 2 × MIC,CH-1 had no obvious hemolysis and its hemolysis rate was lower than that of Meucin-18.The inhibition of CH-1 to biofilm was enhanced,which promoted the enhancement of its antibacterial ability to a certain extent.TEM observation showed that CH-1 could cleave bacterial cell membrane,causing bacterial death,and played a bactericidal role.Compared to Meucin-18,CH-1 with replacing histidine at position 4 with arginine generally increased a net positive charge on the antibacterial peptide,thereby strengthening the electrostatic attraction of CH-1 to negatively charged bacterial membrane,which enhanced its inhibition of biofilms,and its destruction of cell membranes,leading to increased antibacterial activity.In addition,electrostatic interactions selectively bound to negatively charged bacterial cell membranes,which could increase the targeting of CH-1 to bacterial cell membranes and reduce its hemolytic activity.To sum up,we have explored a new synthetic method and route for Meucin-18.This method can monitor the reaction process in real time,reduce the use of reaction raw materials and costs,improve the yield and purity of product.In addition,our systematic studies on the biological activities of Meucin-18 derived peptides have showed that CH-1 is a promising antibacterial agent and deserves further study.