Broad-spectrum low drug resistance amphiphilic antibacterial peptide and application thereof

Abstract

The application discloses a broad-spectrum low-drug-resistance amphiphilic antibacterial peptide and application thereof. The antibacterial peptide is obtained by modifying an alpha-helix antibacterial peptide template (X1X2Y1Y2)3 (X1 and X2 are hydrophilic amino acids, and Y1 and Y2 are hydrophobic amino acids), replacing the hydrophilic amino acid at the 10th position with a hydrophobic amino acid, and replacing the amino acid residues at the 8th and 12th positions with hydrophobic or hydrophilic amino acids, and the hydrophilic amino acids at other positions are defined as Arg, and the hydrophobic amino acids are Phe or Trp, so that the amphiphilic antibacterial peptide with better activity is obtained. The structure general formula is as follows: RRWFRRWXRWFX-NH2, and is recorded as H-X, wherein X=R, K, T, E, G, F, I, L and W. The antibacterial peptide has broad-spectrum antibacterial activity, the preferred antibacterial peptide has low hemolytic toxicity, has good antibacterial activity on clinically drug-resistant bacteria, and is not prone to inducing drug resistance of bacteria. The antibacterial peptide is simple in design, low in manufacturing cost, and has a good application prospect in preparation of clinical antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and relates to a class of broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptides. This invention also relates to the application of such antimicrobial peptides in the preparation of clinical antimicrobial drugs. Background Technology

[0002] In recent decades, antibiotic resistance caused by overuse has posed a significant threat to global public health (Nat. Rev. Drug Discov. 2020; 19:311-332). According to the latest survey by the U.S. Centers for Disease Control and Prevention, millions of people die each year from infections caused by drug-resistant bacteria, and this number is projected to reach tens of millions by 2050 (Adv. Mater. 2019; 31:1805092; Lancet Infect. Dis. 2013; 13:1057-1098). Therefore, there is an urgent need to develop new antibiotics with low resistance.

[0003] Antimicrobial peptides (AMPs) are widely found in animals, plants, and microorganisms, playing a crucial role in maintaining the homeostasis of the organism's internal environment and resisting the invasion of external pathogenic microorganisms (Nat. biotechnol. 2006; 24(12):1551-1557). Due to their specific hydrophilicity and generally positive charge, antimicrobial peptides can interact with lipids on bacterial cell membranes, causing membrane dissolution, leakage of bacterial contents, and ultimately, bacterial cell death. Furthermore, studies have shown that some antimicrobial peptides can also exert their antimicrobial effects by interfering with bacterial metabolic processes or targeting specific intracellular structures (ACS Appl. Mater. Interfaces. 2019; 12:2129-2144; Nat. Rev. Microbiol. 2015; 13:42-51; Chem. Rev. 2019; 119:6040-6085). The unique mechanisms of action of antimicrobial peptides make it difficult for bacteria to develop resistance, thus reducing the risk of antibiotic resistance, which is of great significance for solving the problem of antibiotic resistance.

[0004] Most antimicrobial peptides are amphiphilic and often possess secondary structures (Sci Adv. 2020; 6:eaaz4767). The amphiphilic α-helix is ​​the most common conformation. The positively charged hydrophilic side of the α-helix antimicrobial peptide can bind to the negatively charged bacterial membrane (Biochemistry. 1997; 36:1525-1531), while the hydrophobic portion can insert into the membrane, increasing its permeability (ProgLipid Res. 2012; 51:140-177), thereby causing the bacterial membrane to rupture and kill the bacteria. With the continuous development of de novo antimicrobial peptide design, there are currently two main templates for synthesizing α-helix antimicrobial peptides: one created by Wiradharma et al. in 2011 (X1X2Y1Y2). n Template, where X is a hydrophobic residue, Y is a cationic residue, n = 2-4 (Biomaterials. 2011; 32: 2204-2212); in addition, a heptanucleotide repeat sequence (abcdefg). n It is widely found in various natural proteins and antimicrobial peptides, such as Melittin, BMAP-27, and BMAP-28 (Biochemistry. 2009; 48:10905–10917; Biochim. Biophys. Acta (BBA) Biombr. 2009; 1788:2411-2420), (abcdefg) n The template provides a simple method for the synthesis of α-helical antimicrobial peptides, where positions a and d are hydrophobic amino acids, and the other positions are positively charged amino acids. However, considering that the α-helix structure rotates once every 3.6 amino acids, the amphiphilicity of the two α-helical antimicrobial peptide templates mentioned above is not perfect, with hydrophilic amino acids located on the hydrophobic side in both cases. This invention aims to address the α-helical antimicrobial peptide template (X1X2Y1Y2). n Further modifications will be made by designing antimicrobial peptides with different hydrophilic and hydrophobic properties through amino acid substitution, in order to obtain antimicrobial peptides with more perfect amphiphilicity, low toxicity, broad spectrum, and low drug resistance. Summary of the Invention

[0005] One of the objectives of this invention is to provide a class of broad-spectrum antimicrobial peptides with amphiphilic structures that are simple in design, easy to synthesize, low in preparation cost, have strong antibacterial activity, and exhibit low hemolytic activity and low drug resistance.

[0006] Another object of the present invention is to provide the application of the above-mentioned amphiphilic antimicrobial peptides in the preparation of clinical antimicrobial drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] I. Structural Design of a Class of Broad-Spectrum, Low-Drug-Resistance Amphiphilic Antimicrobial Peptides

[0009] This invention relates to a class of broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptides, obtained by modifying existing α-helical antimicrobial peptide templates (X1X2Y1Y2)3 (where X1 and X2 are hydrophilic amino acids, and Y1 and Y2 are hydrophobic amino acids). Specifically, based on the characteristics of the α-helical structure, calculated by rotating 3.6 amino acid residues once, the 10th amino acid in this template should be a hydrophobic amino acid, most of the 8th amino acid should be on the hydrophilic side, and all of the 12th amino acid should be on the hydrophilic side. Therefore, this invention replaces the 10th hydrophilic amino acid in commonly used α-helical antimicrobial peptide templates with hydrophobic amino acids, and simultaneously substitutes the 8th and 12th amino acid residues with either hydrophobic or hydrophilic amino acids. The hydrophilic amino acids at other positions are designated as Arg, and the hydrophobic amino acids as Trp or Phe. The resulting antimicrobial peptide is then C-terminated with amidation. The general structural formula of the newly designed broad-spectrum, low-drug-resistance antimicrobial peptide with near-perfect amphiphilicity is as follows:

[0010] RRWFRRWXRWFX-NH2, denoted as HX, where X = R, K, T, E, G, F, I,

[0011] L, W;

[0012] Specifically, the present invention provides a class of broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptides, the structural formula of which is as follows:

[0013] Arg-Arg-Trp-Phe-Arg-Arg-Trp-Arg-Arg-Trp-Phe-Arg, marked as HR;

[0014] Alternatively: Arg-Arg-Trp-Phe-Arg-Arg-Trp-Lys-Arg-Trp-Phe-Lys, marked as HK;

[0015] Alternatively: Arg-Arg-Trp-Phe-Arg-Arg-Trp–Thr-Arg-Trp-Phe-Thr, denoted as HT;

[0016] Alternatively: Arg-Arg-Trp-Phe-Arg-Arg–Trp-Glu-Arg-Trp-Phe-Glu, marked as HE;

[0017] Alternatively: Arg-Arg-Trp-Phe-Arg–Arg-Trp-Gly-Arg-Trp-Phe-Gly, marked as HG;

[0018] Alternatively: Arg-Arg-Trp-Phe-Arg-Arg–Trp-Phe-Arg–Trp-Phe-Phe, marked as HF;

[0019] Alternatively: Arg-Arg-Trp-Phe-Arg-Arg-Trp-Ile-Arg–Trp-Phe-Ile, marked as HI;

[0020] Alternatively: Arg-Arg-Trp-Phe-Arg-Arg-Trp-Leu-Arg–Trp-Phe Leu, marked as HL;

[0021] Alternatively: Arg-Arg-Trp-Phe-Arg-Arg-Trp-Trp-Arg-Trp-Phe-Trp, marked as HW.

[0022] The amino acid sequences of the HX series antimicrobial peptides are shown in SEQ ID No. 1 to SEQ ID No. 9, respectively.

[0023] As a preferred embodiment of the present invention, the antimicrobial peptide is HR, HK, HE, HT or HG; more preferably HR, HK or HT; and most preferably HR.

[0024] II. Application of a Class of Broad-Spectrum, Low-Drug-Resistance Amphiphilic Antimicrobial Peptides in the Preparation of Clinical Antimicrobial Drugs

[0025] 1. In vitro antibacterial test

[0026] The minimum inhibitory concentrations (MICs) of the above-mentioned antimicrobial peptides against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus, and Enterococcus faecalis were determined using the classic sequential two-fold microdilution method. Polymyxin was used as a positive control, and the results were replicated three times. The results are shown in Table 1.

[0027] Table 1. Minimum inhibitory concentrations of the antimicrobial peptides of this invention against standard strains.

[0028]

[0029] Table 1 shows that the relatively perfect amphiphilic antimicrobial peptides designed in this invention exhibit strong antimicrobial activity against common experimental strains, showing strong inhibitory effects against both Gram-positive and Gram-negative bacteria, demonstrating broad-spectrum antimicrobial activity. When the 8th and 12th positions of these antimicrobial peptides are hydrophilic amino acids, their antimicrobial activity is superior to peptides with hydrophobic amino acids at these positions. Peptides with positively charged hydrophilic amino acids at these positions exhibit superior antimicrobial activity compared to peptides with negatively charged hydrophilic amino acids and uncharged hydrophilic amino acids. HR, HK, and HT all showed strong antimicrobial activity.

[0030] 2. Hemolysis test

[0031] Fresh blood (1000g) from healthy mice was centrifuged at 4°C for 10 min. The supernatant plasma was discarded, and the lower blood cells were washed with physiological saline. A suspension containing 8% blood cells was prepared, and 100 μL / well was added to a 96-well plate. The antimicrobial peptide of this invention was dissolved in physiological saline and diluted twice to prepare peptide solutions of different concentrations. Equal volumes of these solutions were added to the 96-well plates containing the blood cell suspension and incubated at 37°C for 1 h. After centrifugation at 1200g for 15 min, 100 μL / well of the supernatant was transferred to a new 96-well plate, and the absorbance at 490 nm was measured using a microplate reader. The physiological saline group served as a negative control, and 1% Triton X-100 served as a positive control. The absorbance was determined according to the formula: Hemolysis rate (%) = [(OD...]]. 490nm peptides -OD 490nm negative control ) / (OD 490nm positive control -OD 490nm negative control )]×100%, calculate the hemolysis rate, and the result is as follows Figure 1 .

[0032] Figure 1 The results showed that when the 8th and 12th positions of the newly designed amphiphilic antimicrobial peptides of this invention were hydrophilic amino acids, the hemolysis rate of HR, HK, HE, HT, and HG was less than 10% at the highest detectable concentration (256 μM). When the 8th and 12th positions of the antimicrobial peptides were hydrophilic amino acids, the hemolytic toxicity of HF, HI, HL, and HW increased significantly, and hemolysis of 10% could be caused at relatively low concentrations (<10 μM). As preferred antimicrobial peptides of this invention, HR, HK, and HT exhibited low hemolytic toxicity and good safety.

[0033] 3. Experiments to induce bacterial resistance

[0034] The preferred antimicrobial peptide HR of this invention was selected, and its minimum inhibitory concentration (MIC) against *S. aureus* ATCC 25923 and *P. aeruginosa* ATCC 27853 was determined. Then, a 1 / 2×MIC group was inoculated into fresh MH medium and cultured to the logarithmic growth phase. The MIC of the antimicrobial peptide in the fresh bacterial culture was measured. This process was repeated 20 times, and the changes in MIC values ​​were observed to reflect the ability of the antimicrobial peptide to induce bacterial resistance. Polymyxin and gentamicin were used as control drugs. The results are as follows: Figure 2 .

[0035] Figure 2The results showed that after 20 consecutive passages, gentamicin easily induced resistance in S. aureus ATCC 25923 and P. aeruginosa ATCC 27853, while the newly designed amphiphilic antimicrobial peptide HR was less likely to induce bacterial resistance.

[0036] 4. MIC determination of clinically resistant bacteria

[0037] The minimum inhibitory concentration (MIC) of the preferred antimicrobial peptide HR of the present invention against clinically resistant bacteria was determined using the above-mentioned classic micro-dilution method. The antibiotics polymyxin, kanamycin, gentamicin, meropenem, vancomycin and methicillin were used as positive controls. The experiment was repeated three times in parallel, and the results are shown in Table 2.

[0038] Table 2. Minimum inhibitory concentrations of the preferred antimicrobial peptide HR of the present invention against clinically resistant strains.

[0039]

[0040] The results in Table 2 show that HR has strong antibacterial activity against both clinically resistant Gram-positive and Gram-negative bacteria.

[0041] The advantages of this invention compared to the prior art are as follows:

[0042] 1. The newly designed, more perfect amphiphilic antimicrobial peptide of this invention is a modification of the commonly used α-helical antimicrobial peptide template. It is simple to design, has low manufacturing cost, broad antimicrobial spectrum, and is easy to screen for clinical antimicrobial drugs.

[0043] 2. The amphiphilic antimicrobial peptide designed in this invention has broad-spectrum antimicrobial activity and low hemolytic toxicity.

[0044] 3. Compared with traditional antibiotics, the improved amphiphilic antimicrobial peptide of the present invention has a low incidence of drug resistance.

[0045] 4. The amphiphilic antimicrobial peptides of the present invention have good antimicrobial activity against clinically resistant bacteria.

[0046] Therefore, the relatively perfect amphiphilic antimicrobial peptide designed in this invention has good application prospects in the preparation of clinical antimicrobial drugs. Attached Figure Description

[0047] Figure 1 This is a graph showing the experimental results of the hemolytic toxicity of the antimicrobial peptides of this invention;

[0048] Figure 2 This is a diagram showing the experimental results of the antimicrobial peptide-induced drug resistance of this invention;

[0049] Figure 3-11 The mass spectrum of the antimicrobial peptide HX of this invention is shown, where X = R, K, T, E, G, F, I, L, W. Detailed Implementation

[0050] The synthesis method of the broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptide of the present invention will be described in detail below through specific embodiments.

[0051] Example 1: Synthesis of antimicrobial peptide HR

[0052] (1) Resin activation and pretreatment

[0053] Accurately weigh 0.444 g (0.2 mmol) of MBHA resin (substitution value 0.45 mmol / g) and place it in a synthesizer. After swelling in dichloromethane solution for 30 min and washing with DMF, test the resin using the ninhydrin colorimetric method. The resin is colorless and transparent, indicating that the resin is normal.

[0054] (2) Synthesis of HR-resin

[0055] The MBHA resin that passed the above test was treated with a DMF solution (v / v) containing 20% ​​piperidine to remove the Fmoc protecting group. The resin was tested using the ninhydrin colorimetric method; a blue-purple color indicated that the Fmoc protecting group had been removed. After washing with DMF, three times the excess (0.6 mmol) of Fmoc-Arg(Pbf)-OH, three times the excess (0.6 mmol) of HBTU and HOBT, and six times the excess (1.2 mmol) of DIEA were dissolved in redistilled DMF and added to the synthesizer. The condensation reaction was carried out for 1 hour. The resin was tested using the ninhydrin colorimetric method; a colorless and transparent color indicated that the condensation reaction was successful, yielding Fmoc-Arg(Pbf)-resin.

[0056] Following the above method, the following amino acids are sequentially condensed: Fmoc-Phe-OH, Fmoc-Trp(Pbf)OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc -Arg(Pbf)-OH and Fmoc-Arg(Pbf)-OH, with the same amounts of amino acids, HOBT, HBTU, and DIEA as above, yield Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-resin. The terminal Fmoc protecting group is removed with a DMF solution containing 20% ​​piperidine to obtain Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-resin.

[0057] (3) Peptide cleavage: The obtained Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-resin was washed successively with dichloromethane and methanol, thoroughly compressed and dried, and then cleaved with a cleavage reagent (trifluoroacetic acid:triisopropylsilane:water = 9.5:0.25:0.25) for 3 hours to obtain Arg-Arg-Trp-Phe-Arg-Arg-Trp-Arg-Trp-Phe-Arg-NH2. After extraction with ice-cold ether and water, it was freeze-dried to obtain crude peptide freeze-dried powder.

[0058] (4) Peptide purification

[0059] The crude peptide lyophilized powder obtained above was separated and purified by RP-HPLC. The eluent was collected, then freeze-dried, and identified by mass spectrometry as RRWFRRWRRWFR-NH2 with a molecular weight of 1962.11 Da. The mass spectrum is shown below. Figure 3 The RP-HPLC purification conditions were as follows: mobile phase A: 0.1% TFA / water; mobile phase B: 0.1% TFA / acetonitrile; linear gradient elution, and collection of the eluent with the main absorption peak.

[0060] Example 2: Synthesis of HK

[0061] (1) Resin activation and pretreatment

[0062] Same as Example 1.

[0063] (2) Synthesis of HK-resin

[0064] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Arg(Pbf)-Trp(Boc)-Phe-Lys(Boc)-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Lys(Boc)-Arg(Pbf)-Trp(Boc)-Phe-Lys(Boc)-resin was obtained.

[0065] (3) Peptide cleavage

[0066] Same as Example 1.

[0067] (4) Peptide purification

[0068] Same as in Example 1, mass spectrometry identification revealed RRWFRRWKRWFK-NH2 with a molecular weight of 1906.10 Da. The mass spectrum is shown below. Figure 4 .

[0069] Example 3: Synthesis of HT

[0070] (1) Resin activation and pretreatment

[0071] Same as Example 1.

[0072] (2) Synthesis of HT-resin

[0073] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Thr-Arg(Pbf)-Trp(Boc)-Phe-Thr-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Thr-Arg(Pbf)-Trp(Boc)-Phe-Thr-resin was obtained.

[0074] (3) Peptide cleavage

[0075] Same as Example 1.

[0076] (4) Peptide purification

[0077] Same as in Example 1, mass spectrometry analysis revealed RRWFRRWTRWFT-NH2 with a molecular weight of 1852.00 Da. The mass spectrum is shown below. Figure 5 .

[0078] Example 4: Synthesis of HE

[0079] (1) Resin activation and pretreatment

[0080] Same as Example 1.

[0081] (2) Synthesis of HE-resin

[0082] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Glu(OtBu)-Arg(Pbf)-Trp(Boc)-Phe-Glu(OtBu)-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Glu(Otbu)-Arg(Pbf)-Trp(Boc)-Phe-Glu(Otbu)-resin was obtained.

[0083] (3) Peptide cleavage

[0084] Same as Example 1.

[0085] (4) Peptide purification

[0086] Same as in Example 1, mass spectrometry analysis revealed RRWFRRWERWFE-NH2 with a molecular weight of 1907.99 Da. The mass spectrum is shown below. Figure 6 .

[0087] Example 5: Synthesis of HG

[0088] (1) Resin activation and pretreatment

[0089] Same as Example 1.

[0090] (2) Synthesis of HG-resin

[0091] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Gly-Arg(Pbf)-Trp(Boc)-Phe-Gly-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Gly-Arg(Pbf)-Trp(Boc)-Phe-Gly-resin was obtained.

[0092] (3) Peptide cleavage

[0093] Same as Example 1.

[0094] (4) Peptide purification

[0095] Same as in Example 1, mass spectrometry identification revealed RRWFRRWGRWFG-NH2 with a molecular weight of 1763.95 Da. The mass spectrum is shown below. Figure 7 .

[0096] Example 6: Synthesis of HF

[0097] (1) Resin activation and pretreatment

[0098] Same as Example 1.

[0099] (2) Synthesis of HF-resin

[0100] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Trp(Boc)-Phe-Phe-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Trp(Boc)-Phe-Phe-resin was obtained.

[0101] (3) Peptide cleavage

[0102] Same as Example 1.

[0103] (4) Peptide purification

[0104] Same as in Example 1, mass spectrometry analysis revealed RRWFRRWFRWFF-NH2 with a molecular weight of 1944.04 Da. The mass spectrum is shown below. Figure 8 .

[0105] Example 7: Synthesis of HI

[0106] (1) Resin activation and pretreatment

[0107] Same as Example 1.

[0108] (2) Synthesis of HI-resin

[0109] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Ile-Arg(Pbf)-Trp(Boc)-Phe-Ile-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Ile-Arg(Pbf)-Trp(Boc)-Phe-Ile-resin was obtained.

[0110] (3) Peptide cleavage

[0111] Same as Example 1.

[0112] (4) Peptide purification

[0113] Same as in Example 1, mass spectrometry analysis revealed RRWFRRWIRWFI-NH2 with a molecular weight of 1876.07 Da. The mass spectrum is shown below. Figure 9 .

[0114] Example 8: Synthesis of HL

[0115] (1) Resin activation and pretreatment

[0116] Same as Example 1.

[0117] (2) Synthesis of HL-resin

[0118] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Leu-Arg(Pbf)-Trp(Boc)-Phe-Leu-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Leu-Arg(Pbf)-Trp(Boc)-Phe-Leu-resin was obtained.

[0119] (3) Peptide cleavage

[0120] Same as Example 1.

[0121] (4) Peptide purification

[0122] Same as in Example 1, mass spectrometry analysis revealed RRWFRRWLRWFL-NH2 with a molecular weight of 1876.07 Da. The mass spectrum is shown below. Figure 10 .

[0123] Example 9: Synthesis of HW

[0124] (1) Resin activation and pretreatment

[0125] Same as Example 1.

[0126] (2) Synthesis of HW-resin

[0127] The amino acids were sequentially condensed using the same method as in Example 1 to obtain Fmoc-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Trp(Boc)-Arg(Pbf)-Trp(Boc)-Phe-Trp(Boc)-resin. After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Phe-Arg(Pbf)-Arg(Pbf)-Trp(Boc)-Trp(Boc)-Trp(Boc)-Arg(Pbf)-Trp(Boc)-Phe-Trp(Boc)-resin was obtained.

[0128] (3) Peptide cleavage

[0129] Same as Example 1.

[0130] (4) Peptide purification

[0131] Same as in Example 1, mass spectrometry identification revealed RRWFRRWWRWFW-NH2 with a molecular weight of 2022.07 Da. The mass spectrum is shown below. Figure 11 .

Claims

1. A class of broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptides, characterized in that, The antimicrobial peptide is modified from the α-helical antimicrobial peptide template (X1X2Y1Y2)3, and its general structural formula is as follows: RRWFRRWXRWFX-NH2, denoted as HX, where X = R, K, T, E, G, F, I, L, W, and the amino acid sequences are shown in SEQ ID No. 1 to SEQ ID No. 9, respectively.

2. The broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptide as described in claim 1, characterized in that, The antimicrobial peptide is HR, HK, HE, HT or HG.

3. The broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptide as described in claim 2, characterized in that, The antimicrobial peptide is HR, HK, or HT.

4. The broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptide as described in claim 3, characterized in that, The antimicrobial peptide is HR.

5. The application of the broad-spectrum, low-drug-resistance amphiphilic antimicrobial peptide as described in any one of claims 1-4 in the preparation of clinical antimicrobial drugs, characterized in that, The antimicrobial peptide is used to prepare drugs against Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and / or Klebsiella pneumoniae.

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