Synthesis And Antibacterial Activity Of Palm Point Turbulent Frog Antimicrobial Peptides

Brown points Amolops antimicrobial peptides, Hebei Normal University in 2006, Professor Liu Jingze specialty in Southwest China from the point of brown frog skin secretion of fluid in the turbulent separation and extraction of a class of biologically active peptides.It has obtained the national invention patent protection.In order to overcome the isolation of bioactive peptides cost constraints.In this paper, we have synthesized by Fmoc solid brown points Amolops 2 anti-microbial peptide,and its preliminary analysis of antibacterial activity.Objective:Brown points Amolops of antimicrobial peptide synthesis, synthetic products were analyzed on the identification and evaluation of antibacterial effect.Methods:1 Physical and Chemical Properties of antimicrobial peptide1.1 Peptide1Single chain polypeptide, molecular weight:1706.96Da, isoelectric:8.75. sequence:Phe-Leu-Pro-Pro-Ser-Pro-Trp-Lys-Glu-Thr-Phe-Arg-Thr-Thr Simplified as: FLPPSPWKGTFRTT1.2 Peptide2Single chain polypeptide, molecular weight:1335.7Da, isoelectric:8.75. sequence:Leu-Leu-Pro-Ile-Val-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Leu-NH2 Simplified as: LLPIVGKLLSGLL-NH22 Synthesis and purification of peptideFmoc-solid phase synthesis: Wang Resin as solid phase, HBTU,DIEA as shrinkage mixture, condensation reaction with protected amino acid according to sequence, after cleavaged from resin and purified by RP-HPLC, high purity peptide were got.3 Synthesis optimization3.1 Synthesis instrument chosenManual synthesis and CS136XT peptide synthesis, compare purity of different operation methods, according to yield of every gram Wang Resin,scale,reaction time,synthetic cost to judge which operation method is the best. 3.2 Wang Resin swelledWhen comparing 5-35min 65% DCM / DMF case of Wang resin swelling, and different swelling time resin Fmoc-Thr (oBt)-OH coupling efficiency, optimization of the activation time of Wang resin.3.3 Amino resin closedComparison is not, after acetylation and acetylation of amino acid resin purity of peptides generated, closed on the acquisition certificate amino resin product of the need for synthesis of high purity.3.4 Condensation agent chosenDifferent condensation agent DCC and HBTU were used in peptides, synthesis, from coupling rate and HPLC can judge which agent is good for reaction.3.5 Condensation reaction time chosenKziser method test whether the reaction is completed or not in different time ,from 0.5h to 3h,0.5h once, choose the best time for condensation reaction.3.6 Reaction solvent chosenCompare reaction solvent, DMF, DCM and 65%DCM/DMF(v/v),from the coupling rate to rate to choose the best reaction solvent.3.7 Shrinkage mixture ratio chosen AA:HBTU :DIEA=1 :0.9 :2 and AA: HBTU :DIEA=1 :1 :1, compare of amino acids connection rate and yield.3.8 Removal methods of protecting groups chosenIn the experiments that use of amino acids are protecting groups, Fmoc-solid phase synthesis used is piperidine mixed solution with DMF, compares the different concentrations of piperidine solution.3.9 Cracking reagent chosenFmoc-solid phase synthesis targets polypeptide, cracking peptides from resin, using four different way: A: TFA / phenol (v/v=95/5); B: TFA/ H₂O (v/v=95/5); C: TFA /H₂O/ EDT (v/v/v=95/2.5/2.5); D: TFA/phenol/H₂O/thioanisole/EDT (v/v/v/v=82.5/5/5/2.5); Select the best way of cracking.4 Large scale synthesis peptides Optimize technological conditions, 65%DCM/DMF (v/v) as reaction solvent, HBTU as condensation agent, reaction time 2h, CS136XT peptide synthesizer synthesis, Fmoc-solid phase synthesis peptide.5 Purification optimization5.1 Organic phase chosenRP-HPLC, compared two organic phase, acetonitrile and methanol, compare resolution and cost to chose the best one. From HPLC to get peak time, and figure out the best linear gradient (mobile phase B linear gradient).5.2 Chromatographic column chosen Respectively with C8 and C18 isolation and purification of peptides analysis spectrums of the separation efficiency, chose the best separation, purification of chromatographic column.6 Purification of synthesis productsRP-HPLC, C18 Semi preparative column, mobile phase A is composed of 0.1%TFA and water, mobile phase B is composed of 0.1%TFA and acetonitrile. Linear gradient elution, flow rate 3ml/min, detection wavelength 214nm.7 Analysis and identification of synthetic products and modifications Kr100-5-C18 analytical column, from area normalization method to get syntheticproducts and modifications, purity, analyze and identify it.Use MALDI-TOF-MS to confirm products.8 Analysis synthetic products with traditional antibiotics of antibacterial experimentBy using escherichia coli and golden staphylococcus bacteria for experimental determination. Respectively detection antibacterial peptides, gentamycin sulfate, penicillin and streptomycin and antimicrobial peptides and use a combination of three respectively bacteriostatic effect.Results:1 Peptide synthesis condition optimization1.1Peptide synthesis instrument chosenManual synthesis and CS136XT peptide synthesizer separately synthesis peptide1 ,same amino acid and resin weight, yield of crude peptides are 0.14g and 0.26g, purity are 57.39% and 67.94%. 1.2 Wang Resin swelledIn 65% DCM/DMF (v/v), swelled more than 30 minutes, and Fmoc-Thr (oBt)-OH coupling efficient.1.3 Coupling rate of different condensation agents Chose HBTU to synthesis FLPPSPWKGTFRTT, coupling rate and purity show HBTU is better than DCC.1.4 Coupling rate of different reaction solventSolid phase synthesis FLPPSPWKGTFRTT, 65% DCM/DMF (v/v) as reaction solvent, coupling rate is higher than DMF and DCM.1.5 Coupling rate of different timeKziser negative reaction test show, different amino acids have been sufficient reaction after 2h, during synthesis FLPPSPWKGTFRTT.1.6 Coupling rate of different shrinkage mixture proportion AA: HBTU : DIEA=1 :0.9 :2, in the conditions of condensation reaction time is short, and connection rate is high.1.7 Not reaction amino acetylatedAfter the amino acid condensation reaction completely, making the amino which is not reacted acetylation, which increases the purity of coarse peptide, easy purification.1.8 Concentration and time of pip/DMF 20%pip/DMF, 20min, efficiency can achieve 99%.1.9 Cutting results TFA/phenol/H₂O/thioanisole/EDT (v/v/v/v=82.5/5/5/2.5), after cutting the resin wasoriginally 20%. With ether processing filtrate without white substance.2 Peptides purification optimization2.1 Purification effect of different organic system Chose C18 analytical column purify FLPPSPWKGTFRTT, RP-HPLC analysis, methanol as organic phase, resolution is lower; acetonitrile as organic phase, peptides peak retention is between 13min to19min,far from impurity, resolution is higher than methanol.2.2 Purification effect of different chromatographic columnAcetonitrile as organic phase, RP-HPLC, C18 is batter than C8 on separation effect.3 Synthetic anti-microbial activity of the antimicrobial peptideSynthetic antimicrobial peptides against Gram-positive bacteria and Gram-negative bacteria have some bactericidal effect.MBC: 200μg/ml and 100μg/m.Synthetic peptide with penicillin, streptomycin antibiotic after using the enhanced role of the United,Which in combination with the effect of penicillin,on the MBC values of S. aureus to penicillin / synthetic peptide 0.625/25μg/ml,Synthetic peptide significantly increased the bactericidal effect of antibiotics classic.Conclusions:1. After process optimization synthesis, synthesis methods of FLPPSPWKGTFRTT: Fmoc-soild synthesis method, CS136XT peptide synthesizer, Wang resin as solid phase, reaction solvent: 65% DCM/DMF (v/v), condensation agent: HBTU, shrinkage mixture proportion: AA: HBTU : DIEA=1 :0.9 :2, reaction time: 2h, 20%pip/DMF, TFA/phenol/H₂O/thioanisole/EDT (v/v/v/v=82.5/5/5/2.5).Purification condition: RP-HPLC, C18 Semi preparative column, mobile phase A is composed of 0.1%TFA and water, mobile phase B is composed of 0.1%TFA and acetonitrile. Linear gradient elution, flow rate 3ml/min, detection wavelength 214nm. Study proved Fmoc-soild synthesis method is mild, products have high purity, goodrepetitiveness, MS results is correspond to anticipation, quality can be controlled.2. Peptide acting on Staphylococcus aureus and Escherichia coli inhibition was weaker than the natural brown points Amolops antimicrobial peptide.Synthetic antimicrobial peptides and natural products may have occurred between the change in spatial structure.Synthetic peptides Amolops brown points still need to analyze the structure-activity relationship based on the design of further structural reform.3. Use of antimicrobial peptides with the traditional classical mechanism of inhibitory effect of different antibiotics.Synthesis of antimicrobial peptides and antibiotics, combined with the traditional classical classic of antibiotics can increase the intensity.Help to improve the pathogenic bacteria resistant to traditional antibiotics.