Study On The Synthetic Process Of Oreoch-2 And The Design,Synthesis And Biological Evaluation Of Its Analogues

In recent years,due to the abuse of antibiotics and the lack of new antibiotics,bacterial resistance has risen sharply,seriously endangering public health.It is estimated that 70 thousand patients die of drug-resistant bacteria infection every year,and drug-resistance has become one of the biggest public health problems in the world.Antimicrobial peptides(AMPs)are the first line of defense against a variety of pathogens(e.g.bacteria,fungi and viruses)and exist in almost all organisms.Compared with traditional antibiotics,AMPs are considered to be the most promising alternatives to traditional antibiotics because of their unique mechanism of action and targeting bacterial cell membrane.Natural AMP Oreoch-2 is an amphiphilic cationic peptide that contains 25 amino acid residues with a prominent α-helix structure isolated from Nile tilapia and its polypeptide sequence is FIHHIIGGLFSAGKAIHRLIRRRRR.Oreoch-2 has a variety of biological activities such as antibacterial,anticancer,immune regulation,and promotion of wound healing,which has broad commercial application prospects.This paper mainly includes two parts:The synthesis and technology of Oreoch-2;The design,synthesis and biological activity evaluation of Oreoch-2 analogues.Part one:As a widely studied polypeptide,the chemical synthesis of Oreoch-2 is based on solid-phase synthesizer,which has not been reported in patents or literatures up to now.However,the solid-phase synthesis method has high feed ratio,low product purity,difficult purification,and high production cost.Therefore,we used a new soluble hydrophobic support-assisted liquid phase synthesis method to synthesize Oreoch-2.This method combines the best performance of solid-phase and liquid-phase synthesis,and its advantages are that all reactions are homogeneous reactions,which can be monitored in real time by TLC method;All reaction operation methods are simple,and purification can be achieved only by simple crystallization,avoiding the interference of impurities in the next reaction.According to the structural characteristics of Oreoch-2,we divide the long peptide chain into four fragments,namely T-F1:Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-O-Tag;F2-Fmoc:Fmoc-Lys(Boc)-Ala-Ile-His(Trt)-Arg(Pbf)-Leu-Ile-OH;F3-Fmoc:Fmoc-Gly-Leu-Phe-Ser(tBu)-Ala-Gly-OH;F4-Boc:Boc-Phe-Ile-His(Trt)-His(Trt)-Ile-Ile-Gly-OH.Using 2,4-bis(docosyloxy)benzyl alcohol as a tag,the four fragments were synthesized separately by Fmoc chemical method.Then,F2-Fmoc,F3-Fmoc and F4-Boc were coupled to T-Fl in turn to obtain the full protective peptide with the tag support at the C-terminal.Finally,Oreoch-2 was obtained by cleaving protective groups and support on the polypeptide chain and then purified by preparative liquid chromatography.A total of 58 steps were taken.Next,we optimized the synthetic process of Oreoch-2,mainly investigated the effects of reaction conditions,feed ratio and catalyst types on the yield and product purity,and obtained the best synthetic process of Oreoch-2.(1)The effects of feed ratio,organic base and reaction temperature on the esterification reaction between C-terminal amino acid and support:For T-F1,the best reaction temperature was 40℃,the best organic base was DMAP,and the best feed ratio of amino acid and DIC were 1.5 eq and 3 eq,respectively;For F2-Fmoc,the best reaction temperature was room temperature,the best organic base was DMAP,and the best amino acid feed ratio was 1.2 eq.(2)The post-treatment method:The original crystallization solvent acetonitrile was adjusted to acetonitrile containing 20%water to reduce the amount of acetonitrile and the synthesis cost;In the post-treatment of each deprotection reaction of T-Fl,the pH of the reaction solution was adjusted from 7 to 6 to change the crystalline state the product and greatly improve the the yield;For intermediates with high viscosity in F3-Fmoc and F4-Boc:The product was not separated from diatomite,so that the mixture was directly used for the next step reaction,which not only improved the yield,but also saved the amount of THF and reduced the generation of "three wastes".(3)For F2-Fmoc and F4-Boc containing histidine:The amount of TFA was reduced from 1%to 0.8%and 0.7%,respectively,which weakened the acidic environment of the solution and effectively avoided the shedding of histidine side chain protective group triphenylmethyl.The yields were increased from 86.5%and 81.3%to 95.3%and 91.6%,and the purity of the product was increased from 78.45%and 67.35%to 98.25%and 97.51%,respectively(4)Coupling system between fragments:Using HATU and HOAt to couple F2-Fmoc,and DMT-MM to couple F3-Fmoc and F4-Boc.The total yield and purity of the crude Oreoch-2 were 47.25%and 43.46,respectively.We applied the optimized route to the total synthesis of Oreoch-2,and the crude product was purified by preparative liquid phase with a purity of 97.29%and a yield of 41.7%.A new soluble hydrophobic support assisted liquid-phase peptide synthesis method was used for the synthesis of Oreoch-2 for the first time,and a simple synthetic route was successfully established.The synthetic method has the advantages of simple operation,high purity and low cost.In addition,we also improved and optimized the method according to the technical problems encountered in peptide synthesis,such as high viscosity of intermediates and instability of amino acid side chain protection groups,which made the method more widely used,and laid a good foundation for the research of Oreoch-2 analogues.Part two:Aiming at the disadvantages of Oreoch-2,such as high hemolysis and long peptide chain,we designed and synthesized six Oreoch-2 analogues(ZN-1～ZN-6)by using bioinformatics prediction software and truncation strategy,and characterized their structures.On this basis,the antibacterial activity of Oreoch-2 analogues was evaluated systematically.The purpose of this study is to shorten the peptide chain,search for active fragments,improve antibacterial activity or reduce hemolysis,thereby obtaining new antimicrobial peptides with more potential.In vitro antibacterial activity study showed that ZN-5(22 peptide)and ZN-6(18 peptide)had the best activity against sensitive Gram-positive bacteria,which were equivalent to or even better than the parent peptide Oreoch-2.For example,ZN-5 exerted excellent antibacterial activity against S.aureus ATCC25923 and B.subtilis ATCC9372(MIC=1.5 μM),and good antibacterial activity against S.pyogenes PS(MIC=6.2 μM),which was twice as high as Oreoch-2.In addition,it exhibited equivalent antibacterial activity against B.pumilus ATCC63202 and S.epidermidis to Oreoch-2.ZN-6 had remarkable activity against S.aureus ATCC25923 and S.epidermidis,with MIC values of 1.8 μM,which was twice as high as Oreoch-2,and showed antibacterial activity equivalent to Oreoch-2 against B.subtilis ATCC9372(MIC=3.5 μM),B.pumilus ATCC63202(MIC=1.8μM)and S.pyogenes PS(MIC=14.2 μM).It is worth mentioning that the antibacterial activity of ZN-5 and ZN-6 against S.epidermidis is significantly higher than that of ciprofloxacin.In addition,ZN-4 showed the same antibacterial activity as Oreoch-2 against B.subtilis ATCC9372,B.pumilus ATCC63202,S.epidermidis and S.pyogenes PS,and especially the best activity against B.pumilus ATCC63202 with the MIC value of 1.5 μM.On the other hand,ZN-5 had the best activity against methicillin-resistant S.aureus ATCC43300 and penicillin-resistant S.aureus ATCC31007 with the MIC values of 3.1 μM and 6.2μM,respectively,which were equivalent to Oreoch-2.Surprisingly,ZN-5 and ZN-6 showed significantly activity against drug-resistant S.pyogenes PR,with MIC values of 12.3 μM and 28.4 μM,respectively,while Oreoch-2 had no activity.From the above results,it is not difficult to find that too many cationic amino acids and high hydrophobicity in the Oreoch-2 affect its antibacterial efficacy.In vitro bactericidal activity evaluation showed that the ZN-5 and ZN-6 were typical bactericidal agents,which were the same as the parent peptide Oreoch-2.In the study of bactericidal kinetics,ZN-5 showed concentration-and time-dependent manner,and all S.aureus ATCC43300 could be killed within 6 h at the concentration of 4 × MIC.The stability study indicated that ZN-5 and ZN-6 was slightly better than that of Oreoch-2 in acid,alkali,heat and physiological salts.In addition,the hemolytic activity of ZN-5 and ZN-6 on mice red blood cells was much lower than that of Oreoch-2.For example,at the concentration of 16 × MIC,the highest hemolysis rate of themwas only 4.84%.It also suggests that the high hemolysis is caused by high hydrophobicity to a certain extent.The inhibition of biofilm formation of bacteria showed that ZN-5 and ZN-6 had a significant inhibitory effect on the biofilm formation of S.aureus ATCC25923,and especially ZN-5 inhibited the biofilm formation by 46.0%at only 2 μg/mL.The damage of cell membrane integrity and the release of cell contents were directly observed by TEM in the treated S.aureus ATCC25923.These results indicate that ZN-5 and ZN-6 exert antibacterial effect by destroying bacterial cell membrane.In conclusion,a novel soluble hydrophobic support assisted liquid-phase peptide synthesis method was used for the synthesis of Oreoch-2 for the first time,and a simple synthetic route was successfully established.The method is simple,high purity and low cost.On this basis,six Oreoch-2 analogues were designed and synthesized by using the truncation strategy with Oreoch-2 as the lead compound,and their antibacterial activity,bactericidal activity,bactericidal kinetics,stability,hemolysis and inhibition of biofilm formation in vitro were systematically evaluated.The results showed that ZN-5 and ZN-6 are ideal new antimicrobial peptides,which fully conformed to the design idea of shortening peptide chain,improving antibacterial activity and reducing hemolysis,and could be used for systematic drug-likeness evaluation.