Acylation modified antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses an acylation modified antibacterial peptide as well as a preparation method and application thereof. Amino resin is used as a carrier of the fat acylation antibacterial peptide, a template peptide shown in SEQ ID NO: 1 is prepared through an Fmoc solid-phase synthesis method, and then the template peptide is coupled with palmitic acid, octanoic acid or dicaprylic acid. The lipid acylation antibacterial peptide not only can obviously inhibit the growth and reproduction of gram-negative bacteria and gram-positive bacteria, but also can efficiently inhibit and remove porphyromonas gingivalis biological membranes with the activity equivalent to that of polymyxin B. Meanwhile, the fat acylation antibacterial peptide has low hemolytic toxicity to human red blood cells and low cytotoxicity to mouse embryo fibroblasts, is high in serum stability and has remarkably improved anti-inflammatory activity; in an animal infection model, the compound shows a safe and effective peritonitis infection treatment effect, and is expected to replace traditional antibiotics to be used for treating diseases related to bacterial infection. The preparation method provided by the invention is simple and easy to implement, and the obtained product is high in yield and purity.

Description

Technical Field

[0001] This invention relates to an esterified antimicrobial peptide, its preparation method, and its application, belonging to the field of biomedical technology. Background Technology

[0002] Bacterial infection is the process by which pathogens invade the body, multiply within the host, and trigger an inflammatory response. Bacteria can not only damage tissues by releasing toxins, but can also cause purulent infections. Furthermore, excessive inflammatory responses caused by bacterial infections can lead to sepsis, septic shock, and even multiple organ failure. Currently, traditional clinical treatments still rely on antibiotics, but the overuse of antibiotics and the resulting development of bacterial resistance further increase treatment difficulty and mortality. In addition, bacterial communities form complex biofilms to resist external threats, providing bacteria with multiple protective mechanisms. Biofilms often lead to more severe chronic and refractory infections, such as surgical implant infections and periodontitis. Notably, biofilms promote the horizontal transfer of drug resistance genes, accelerating the emergence of drug-resistant strains, while their physical barriers make it difficult for traditional antibiotics to penetrate. Faced with the dilemma of traditional antibiotic ineffectiveness, antimicrobial peptides, due to their unique antibacterial mechanisms, have become a research hotspot, and they hold the promise of becoming the "next-generation weapon" to overcome the treatment bottlenecks of bacterial and biofilm infections and alleviate their social harm.

[0003] Antimicrobial peptides are a class of small molecule peptides composed of 5-50 amino acid residues produced by the innate immune system of organisms. They mainly exert their antibacterial effects by disrupting the integrity of bacterial cell membranes. Furthermore, antimicrobial peptides can rapidly penetrate the biomembrane matrix, disrupt bacterial cell membranes, and cleave their extracellular DNA networks, thereby killing bacteria in mature biomembranes and preventing the formation of new membranes. Buforin II, a 21-amino acid peptide derived from the stomach tissue of Asian toads, possesses broad-spectrum antimicrobial activity due to its unique "helix-hinge-helix" amphiphilic structure. Buforin II can penetrate bacterial cell membranes efficiently and non-lytically, leading to bacterial death by binding to bacterial nucleic acids and interfering with DNA / RNA function. Because it does not damage cell membranes, Buforin II effectively overcomes antibiotic resistance and exhibits better biocompatibility compared to other antimicrobial peptides, giving it greater potential for clinical translation. However, Buforin II still suffers from poor in vivo stability, susceptibility to protease degradation, and the need for further improvement in its antimicrobial activity, limiting its clinical translational application. To address the aforementioned issues, it is hoped that chemical modification techniques targeting antimicrobial peptides can be employed to enhance their stability and antimicrobial activity, such as stapling, glycosylation, and esterification. Summary of the Invention

[0004] The purpose of this invention is to provide an esterified antimicrobial peptide, its preparation method, and its application, in order to solve the problems of poor in vivo stability and the need to improve the antimicrobial activity of existing Buforin II antimicrobial peptides in clinical translation applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides an esterified antimicrobial peptide, wherein the esterified antimicrobial peptide is selected from any one of the following:

[0007] i) Using the sequence shown in SED ID NO:1 as a template peptide, palmitic acid is directly coupled to the N-terminus, denoted as BUFT-C16.

[0008] ii) Using the sequence shown in SED ID NO: 1 as a template peptide, the N-terminus is directly coupled with caprylic acid, denoted as BUFT-C8;

[0009] iii) Using the sequence shown in SED ID NO: 1 as a template peptide, the N-terminus is coupled with dioctanoic acid via a linker lysine (octanoic acid is coupled to each of the two amino groups of lysine), denoted as BUFT-2C8.

[0010] The chemical structural formulas of BUFT-C16, BUFT-C8, and BUFT-2C8 are shown below:

[0011]

[0012] Preferably, the esterified antimicrobial peptide is BUFT-C8 or BUFT-2C8.

[0013] More preferably, the lipid-acylated antimicrobial peptide is BUFT-2C8.

[0014] A second aspect of the present invention provides a product containing an esterified antimicrobial peptide as described in the first aspect of the present invention, the product comprising an antimicrobial drug or composition, a preservative, a bactericide, or a surfactant.

[0015] A third aspect of the invention provides the application of the esterified antimicrobial peptide as described in the first aspect of the invention, wherein the application is selected from any one of the following:

[0016] 1) Used in the preparation of antibacterial drugs or compositions;

[0017] 2) Used in the preparation of preservatives;

[0018] 3) Used in the preparation of bactericides;

[0019] 4) Used in the preparation of surfactants;

[0020] 5) Used for in vitro inhibition and removal of bacterial biofilms;

[0021] 6) Used in the preparation of products that inhibit and remove bacterial biofilms;

[0022] 7) Used to prepare products for the prevention and treatment of bacterial infectious diseases or conditions.

[0023] Preferably, the bacteria or fungi include Gram-positive bacteria and Gram-negative bacteria.

[0024] Preferably, the bacteria or fungi include Staphylococcus, Escherichia, Pseudomonas, Porphyromonas, and Acinetobacter.

[0025] Preferably, the bacteria or fungi include Staphylococcus aureus, Escherichia coli, Pseudomonas aeruinosa, Porphyromonas gingivalis, and Acinetobacter baumannii.

[0026] A fourth aspect of the present invention provides a method for preparing the esterified antimicrobial peptide described in the first aspect of the present invention, comprising the following steps:

[0027] Step 1): Remove the Fmoc protecting groups on the Rink Amide MBHA resin using a deprotecting agent;

[0028] Step 2): Use a condensation reagent to couple the carboxyl groups of the Fmoc-protected amino acids to the exposed amino groups on the resin.

[0029] Step 3): Use a deprotecting agent to remove the Fmoc protecting group from the amino acid;

[0030] Step 4): Repeat the coupling-deprotection operation described in steps 2) and 3) to synthesize the template peptide according to the amino acid sequence shown in SEQ ID NO: 1 (or couple all amino acids according to the chemical structure of the esterified antimicrobial peptide).

[0031] Step 5): Use a condensation reagent to couple the fatty acid (the product obtained in Step 4) to the exposed amino group at the N-terminus of the template peptide overnight to complete the esterification modification; the fatty acid is selected from palmitic acid (C16), octanoic acid (C8) or dioctanoic acid (2C8);

[0032] Step 6): Use a cleavage reagent to cut the peptide chain off the resin and precipitate it with ice-cold ether to obtain crude esterified antimicrobial peptide;

[0033] Step 7): The crude esterified antimicrobial peptide was separated and purified by high performance liquid chromatography to obtain the purified product.

[0034] Preferably, the deprotecting agent in step 1) is a mixed solution of piperidine and N,N-dimethylformamide (DMF) in a volume ratio of 1:4.

[0035] Preferably, the condensation reagent in step 2) is a mixed solution of ethyl 2-cyanoacetate (Oxyma), N,N-diisopropylcarbodiimide (DIC), and N,N-dimethylformamide (DMF), with a feeding ratio of resin:Fmoc protected amino acid:ethyl 2-cyanoacetate (Oxyma):N,N-diisopropylcarbodiimide (DIC):N,N-dimethylformamide (DMF) = 1 mol:4 mol:4 mol:4 mol:6 mL.

[0036] Preferably, the cutting reagent in step 6) is a mixed solution of triisopropylsilane (TIPS), water (H2O), and trifluoroacetic acid (TFA) in a volume ratio of 2.5:2.5:95, and the feeding ratio is cutting reagent: resin = 1 mL: 50 mg.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) Through extensive and in-depth research, the inventors discovered that using the truncated amino acid sequence RAGLQFPVGRVHRLLRK-NH2 at the N-terminus of the Buforin II antimicrobial peptide as a template peptide, esterification modification with different chain lengths and steric hindrances at its N-terminus, especially modification with palmitic acid, caprylic acid or dicaprylic acid, can significantly improve its antimicrobial activity. The esterified antimicrobial peptide can significantly inhibit the growth and reproduction of Gram-negative and Gram-positive bacteria. Among them, the antimicrobial peptide modified with dicaprylic acid (BUFT-2C8) has the best antimicrobial activity and can effectively inhibit and clear the biofilm of Porphyromonas gingivalis with activity comparable to polymyxin B. It has low hemolytic toxicity to human erythrocytes and low cytotoxicity to mouse embryonic fibroblasts, and high serum stability.

[0039] (2) The esterified antimicrobial peptide provided by the present invention has better anti-inflammatory activity. In a mouse peritonitis infection model, it showed anti-inflammatory activity comparable to polymyxin B and better safety than polymyxin B.

[0040] (3) The present invention provides a method for preparing lipid acylation modified antimicrobial peptides. The method is simple and easy to implement, and the product yield is high and the purity is high. Attached Figure Description

[0041] Figure 1 This is a chemical structure diagram of the esterified antimicrobial peptide described in this invention.

[0042] Figure 2 These are the high-performance liquid chromatograms and mass spectra of the esterified antimicrobial peptides described in this invention.

[0043] Figure 3 This is a summary of the minimum inhibitory concentration and minimum bactericidal concentration of the esterified antimicrobial peptides described in this invention.

[0044] Figure 4 This is a summary of the inhibition and clearance rates of the esterified antimicrobial peptides described in this invention against Porphyromonas gingivalis biofilm.

[0045] Figure 5 This is a hemolytic toxicity diagram of the esterified antimicrobial peptide described in this invention.

[0046] Figure 6 This is a cytotoxicity diagram of the esterified antimicrobial peptide described in this invention.

[0047] Figure 7 This is a serum stability graph of the esterified antimicrobial peptide described in this invention.

[0048] Figure 8 This is a diagram illustrating the anti-inflammatory activity of the esterified antimicrobial peptide described in this invention.

[0049] Figure 9 This is a survival rate curve of mice with peritonitis treated with the esterified antimicrobial peptide described in this invention.

[0050] Figure 10 This is a graph showing the bacterial survival rate in the blood and organs of mice treated with the esterified antimicrobial peptides of this invention. Detailed Implementation

[0051] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0052] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all strains, materials, reagents, etc., used in the following examples were obtained commercially available.

[0053] Example 1

[0054] This embodiment provides an acylated antimicrobial peptide and its preparation method, the chemical structural formula of which is as follows: Figure 1 As shown, the specific steps include the following:

[0055] (1) Take 1g of amino resin (sample loading amount is 0.34mmol / g) and add it to the polypeptide solid phase synthesis tube. Soak it in dichloromethane (DCM) for 20min until the resin swells. Then dry it for later use.

[0056] (2) Add 20% piperidine-N,N-dimethylformamide (DMF) solution to the solid phase synthesis tube until the resin is submerged. Shake the reaction at 25°C for 10 min × 2 to remove the 9-fluorenylmethoxycarbonyl (Fmoc) group on the resin. Wash the resin with dichloromethane (DCM) and N,N-dimethylformamide (DMF) three times each.

[0057] (3) The first Fmoc protected amino acid (1.36 mmol), ethyl 2-cyanoacetate (Oxyma) (193 mg, 1.36 mmol) and N,N-diisopropylcarbodiimide (DIC) (211 μL, 1.36 mmol) in the template peptide sequence were mixed in 6 mL of N,N-dimethylformamide (DMF) and added to a solid-phase synthesis tube. The reaction was carried out at 70 °C with shaking for 30 min. The resin was washed three times each with dichloromethane (DCM) and N,N-dimethylformamide (DMF).

[0058] (4) Repeat the coupling-deprotection operation in steps (2) and (3) until all amino acid linkage is complete.

[0059] (5) Fatty acid (1.36 mmol), ethyl 2-cyanoacetate (Oxyma) (193 mg, 1.36 mmol) and N,N-diisopropylcarbodiimide (DIC) (211 μL, 1.36 mmol) were mixed in 6 mL of N,N-dimethylformamide (DMF) and added to a solid-phase synthesis tube. The mixture was shaken and reacted overnight at 70 °C.

[0060] (6) Wash and dry the resin, add 20 mL of a mixed solution of triisopropylsilane (TIPS), water (H2O), and trifluoroacetic acid (TFA) with a volume ratio of 2.5:2.5:95, shake at 25°C for 4 h, filter, wash the resin with a small amount of TFA, and collect the filtrate. Dry the excess TFA with argon gas, add ice-cold ether to precipitate, centrifuge, discard the supernatant, and wash repeatedly with ice-cold ether and centrifuge 3 times. Dry with argon gas to obtain crude esterified antimicrobial peptide.

[0061] (7) The crude esterified antimicrobial peptide was purified by preparative high-performance liquid chromatography (RP-HPLC) using a SHIMADZU (LC-6A) system. A C18 reverse-phase column was used. Mobile phase A consisted of acetonitrile containing 0.1% trifluoroacetic acid (TFA), and mobile phase B consisted of water containing 0.1% TFA. The flow rate was 10 mL / min, and a linear gradient elution program was employed, starting with 10% buffer A and decreasing to 75% buffer A within 50 minutes. The crude solution was loaded onto the column and eluted for purification. The main peak was collected, and the acetonitrile was removed by rotary evaporation to obtain the concentrated target peptide solution. Finally, the purified peptide was obtained by lyophilization.

[0062] Example 2

[0063] This embodiment analyzes the purity and identifies the molecular weight of the esterified antimicrobial peptide prepared in Example 1:

[0064] Purity analysis of esterified antimicrobial peptides was performed using SHIMADZU (LC-20AD) RP-HPLC. A C18 reverse-phase column was used. Mobile phase A consisted of acetonitrile containing 0.1% trifluoroacetic acid (TFA), and mobile phase B consisted of water containing 0.1% TFA. The flow rate was 1 mL / min, and a linear gradient elution program was used, starting with 10% buffer A and reaching 90% buffer A within 20 minutes. Molecular weight identification of the esterified antimicrobial peptides was performed using SHIMADZU LCMS-8040 electrospray ionization mass spectrometry. Scan mode: positive ion; scan time: 2 min; molecular weight range: 400-2000; total flow rate: 0.2 mL / min; mobile phase A consisted of acetonitrile containing 0.1% formic acid, and mobile phase B consisted of water containing 0.1% formic acid. Analysis was performed while maintaining 80% mobile phase A for 2 minutes. Results are as follows: Figure 2 As shown, the purity of the prepared esterified antimicrobial peptide is greater than 96%, and the molecular weight is correct.

[0065] Example 3

[0066] This embodiment measures the antibacterial activity of the esterified antimicrobial peptide described in this invention:

[0067] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of esterified antimicrobial peptides were determined using the microbial dilution method, according to the Clinical and Laboratory Standards Institute (CLSI) methodology. 10 [units of measurement missing] were prepared in MH-II broth. 6CFU / mL bacterial suspension was used to prepare peptide solutions of different concentrations (0–256 μg / mL) in phosphate-buffered saline (PBS). Then, 100 μL of peptide solution was mixed with 100 μL of bacterial suspension in a 96-well plate and incubated at 37°C for 24 hours. The OD600 nm value of each well was measured using a microplate reader (SPECTROStar Nano, China). MIC was defined as the minimum peptide concentration required to completely inhibit bacterial growth. Additionally, 10 μL of AlamarBlue cell viability reagent (Thermo Fisher, UK) was added to each well, and the plates were incubated at 37°C for another 4 hours. MBC was defined as the peptide concentration at which AlamarBlue remained unchanged, indicating complete bacterial death. Results are as follows: Figure 3 As shown, the esterified antimicrobial peptides BUFT-C16, BUFT-C8, and BUFT-2C8 of this invention have significantly improved antibacterial ability compared with the template peptide BUFT, and can significantly inhibit the growth and reproduction of Gram-positive and Gram-negative bacteria, among which BUFT-2C8 has the best effect.

[0068] Example 4

[0069] This embodiment measures the anti-biofilm activity of the acylated antimicrobial peptide described in this invention:

[0070] *Porphyromonas gingivalis* strain 33277, purchased from the Shanghai Microbial Culture Collection Center, was cultured in brain and heart broth supplemented with heme chloride and vitamin K1. Ten... 6 CFU / mL bacterial suspension was used to prepare peptide solutions of different concentrations (0–256 μg / mL) in phosphate-buffered saline (PBS). Then, 100 μL of peptide solution was mixed with 100 μL of bacterial suspension in a 96-well plate and incubated at 37°C for 12 hours in an anaerobic incubator. Afterward, the medium was replaced with fresh medium and incubated for another 48 hours. The biofilm inhibition rate of the peptides was calculated using crystal violet staining and colony-forming unit (CFU) counting. Alternatively, 200 μL of bacterial suspension was inoculated into 96-well plates and incubated at 37°C for 48 hours to allow biofilm formation. Afterward, the medium was replaced with 200 μL of peptide solutions of different concentrations (0–256 μg / mL) and incubated for another 12 hours. The biofilm clearance rate of the peptides was calculated using crystal violet staining and CFU counting. Results are as follows: Figure 4 As shown, the esterified antimicrobial peptide BUFT-2C8 of this invention can significantly inhibit and clear Porphyromonas gingivalis biofilm compared with the template peptide BUFT, and its activity is comparable to that of polymyxin B (PMB).

[0071] Example 5

[0072] This embodiment tests the hemolytic toxicity of the acylated antimicrobial peptide described in this invention:

[0073] A peptide solution with a concentration of 256 μg / mL was prepared in phosphate-buffered saline (PBS) and then mixed with an equal volume (v / v) of 2% (v / v) human red blood cell suspension in a 96-well plate. The plates were co-incubated at 37°C for 1 hour, followed by centrifugation at 2500 rpm for 10 minutes, and the supernatant was collected. The absorbance at 540 nm was measured using a microplate reader (SPECTRO Star Nano, China), and the hemolysis rate was calculated using the following formula: Hemolysis rate (%) = (Abspeptide – Absblank) / (Abscontrol – Absblank), where Absblank and Abscontrol are the absorbance of the samples treated with PBS and 0.1% Triton X-100, respectively. Results are shown below. Figure 5 As shown, the esterified antimicrobial peptides BUFT-C8 and BUFT-2C8 described in this invention have comparable hemolytic toxicity to the template peptide BUFT, and both are less than 5%.

[0074] Example 6

[0075] This embodiment demonstrates the cytotoxicity of the acylated antimicrobial peptide described in this invention:

[0076] NIH-3T3 cells were used at a rate of 2 × 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated at 37°C for 24 hours. Cells were then treated with peptide solutions of varying concentrations (0–128 μg / mL) in fresh DMEM and incubated at 37°C for 24 hours, followed by the addition of 10 μL of CCK-8 and incubation for another hour. The absorbance of each well at 450 nm was measured using a microplate reader (SPECTRO Star Nano, China), and cell viability was calculated using the following formula: Cell viability (%) = (Abspeptide - Absblank) / (Abscontrol - Absblank) × 100%, where Abscontrol is the absorbance of PBS-treated cells and Absblank is the absorbance of cell-free DMEM medium. Results are shown below. Figure 6 As shown, the esterified antimicrobial peptide BUFT-2C8 of the present invention has lower cytotoxicity than the template peptide BUFT, with no statistically significant difference, and exhibits good biocompatibility.

[0077] Example 7

[0078] This embodiment demonstrates the serum stability of the acylated antimicrobial peptide described in this invention:

[0079] The serum stability of esterified antimicrobial peptides was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). A peptide solution with a concentration of 1 mg / mL was prepared in phosphate-buffered saline (PBS) and incubated with human serum at a final volume ratio of 4:1 at 37 °C. Samples were taken at various points after incubation, and 50 μL of the mixture was mixed with 50 μL of acetonitrile to terminate the enzymatic digestion. After centrifugation at 10,000 rpm for 10 min at 4 °C, the supernatant was collected and analyzed by RP-HPLC. The integrated peak area of ​​the intact peptide was recorded, and the percentage of remaining peptide was calculated based on the integrated area, as shown below: Remaining peptide (%) = Remaining peptide peak area / Intact peptide peak area × 100%. Results are as follows: Figure 7 As shown, the esterified antimicrobial peptide BUFT-2C8 of this invention has higher serum stability compared with the template peptide BUFT.

[0080] Example 8

[0081] This embodiment measures the anti-inflammatory activity of the acylated antimicrobial peptide described in this invention:

[0082] Animal experiments were approved by the Ethics Committee of Shanghai University (ECSHU). Adult female BALB / c model mice (6-8 weeks old, 18-20g) were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Jiangsu, China). For infected mice, after one week of acclimatization, 200 μL of the solution was administered intraperitoneally at a concentration of 5 × 10⁻⁶. 8 A peritonoseseptic model was established using a CFU / mL Escherichia coli suspension. Infected mice were then randomly divided into four groups (n=6 per group), receiving intraperitoneal injections of 0.9% sodium chloride saline, BUFT, BUFT-2C8, and PMB (10 mg / kg), respectively. Two days after treatment, all mice were euthanized, and blood samples were collected. After standing at room temperature for 4 hours, the samples were centrifuged at 2500 rpm for 10 minutes, and the supernatant was collected to obtain serum samples. The levels of IL-1β, IL-6, and TNF-α were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Lianke, China) according to the manufacturer's instructions. Results are as follows: Figure 8 As shown, the esterified antimicrobial peptide BUFT-2C8 of this invention has higher anti-inflammatory activity compared with the template peptide BUFT, and can downregulate inflammatory factors to the level of healthy mice, exhibiting anti-inflammatory activity comparable to PMB.

[0083] Example 9

[0084] This embodiment measures the survival rate of mice treated with the acylated antimicrobial peptide of the present invention for peritonitis:

[0085] By observing and recording the survival of peritonosebump mice in each group during the two-day treatment period in Example 8, survival rate curves were plotted within the treatment cycle to determine the treatment survival rate. The results are as follows: Figure 9 As shown, the esterified antimicrobial peptide BUFT-2C8 of this invention can significantly improve the survival rate of infected mice compared with the template peptide BUFT, and has a significant therapeutic effect.

[0086] Example 10

[0087] This embodiment measures the bacterial survival rate in the blood and organs of mice treated with peritonitis using the acylated antimicrobial peptide described in this invention:

[0088] Blood and major organ samples were collected from peritonitis mice that died or were euthanized two days after treatment in Example 8. Bacterial CFU were counted using sterile phosphate-buffered saline (PBS) homogenate, and the bacterial survival rate in the blood and major organs of each group of mice was calculated based on the counting results. The results are as follows: Figure 10 As shown, the esterified antimicrobial peptide BUFT-2C8 of the present invention, compared with the template peptide BUFT, can significantly reduce the bacterial content in the blood and major organs of infected mice, and the bacterial survival rate is comparable to that of the PMB group, basically returning to the level of healthy mice. This indicates that the esterified antimicrobial peptide BUFT-2C8 of the present invention can effectively treat peritonitis infection in mice.

[0089] The above embodiments demonstrate that the present invention successfully prepared an antimicrobial peptide based on the template peptide esterification modification of the truncated amino acid sequence RAGLQFPVGRVHRLLRK-NH2 (SEQ ID NO: 1) of the Buforin II antimicrobial peptide. The preparation method is simple, the product has high purity, and it has been proven that the esterified antimicrobial peptide of the present invention can significantly inhibit the growth and reproduction of Gram-negative and Gram-positive bacteria. Among them, BUFT-2C8 has the best effect, and BUFT-2C8 can effectively inhibit and clear the biofilm of Porphyromonas gingivalis with activity comparable to polymyxin B. It has low hemolytic toxicity to human erythrocytes and low cytotoxicity to mouse embryonic fibroblasts, high serum stability, and good anti-inflammatory activity. It can safely and effectively treat peritonitis infection in mice and has good application prospects.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A fatty acylated modified antibacterial peptide, characterized in that, The fatty acylated modified antibacterial peptide is selected from any one of the following: i) a template peptide with the sequence shown in SED ID NO: 1, directly coupled with palmitic acid at the N-terminus, denoted as BUFT-C16; ii) a template peptide with the sequence shown in SED ID NO: 1, directly coupled with octanoic acid at the N-terminus, denoted as BUFT-C8; iii) a template peptide with the sequence shown in SED ID NO: 1, coupled with dioctanoic acid at the N-terminus through a linker lysine, denoted as BUFT-2C8.

2. The fatty acylated antimicrobial peptide of claim 1, wherein, The fatty acylated modified antibacterial peptide is BUFT-C8 or BUFT-2C8.

3. The fatty acylated antimicrobial peptide of claim 2, wherein, The fatty acylated modified antibacterial peptide is BUFT-2C8.

4. A product containing the fatty acylated modified antibacterial peptide according to any one of claims 1-3, wherein the product comprises an antibacterial drug or composition, a preservative, a bactericide or a surfactant.

5. Use of the fatty acylated modified antibacterial peptide according to any one of claims 1-3, wherein the use is selected from any one of the following: 1) for preparing an antibacterial drug or composition; 2) for preparing a preservative; 3) for preparing a bactericide; 4) for preparing a surfactant; 5) for inhibiting and eliminating bacterial biofilm in vitro; 6) for preparing a product for inhibiting and eliminating bacterial biofilm; 7) for preparing a product for preventing and treating bacterial infectious diseases or conditions.

6. The use according to claim 5, wherein the compound is ###0002### The bacteria include gram-positive bacteria and gram-negative bacteria.

7. The use according to claim 5, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The bacteria include Staphylococcus, Escherichia, Pseudomonas, Porphyromonas, Acinetobacter bacteria.

8. The use according to claim 5, wherein the compound is ###0002### The bacteria include Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Porphyromonas gingivalis, Acinetobacter baumannii.

9. The method for preparing the fatty acylated modified antibacterial peptide according to any one of claims 1 to 3, characterized in that The method comprises the following steps: Step 1): removing the Fmoc protecting group on the Rink Amide MBHA resin using a deprotection reagent; Step 2): coupling the carboxyl group of the Fmoc-protected amino acid with the exposed amino group on the resin using a condensation reagent; Step 3): removing the Fmoc protecting group on the amino acid using a deprotection reagent; Step 4): repeating the coupling-deprotection operations described in steps 2) and 3) to synthesize the template peptide according to the amino acid sequence shown in SEQ ID NO: 1; Step 5): overnight coupling of the fatty acid with the exposed amino group at the N-terminus of the template peptide using a condensation reagent to complete the fatty acylation modification; the fatty acid is selected from palmitic acid (C16), octanoic acid (C8) or dioctanoic acid (2C8). Step 6): The peptide chain was cut from the resin using cleavage reagent, and precipitated using ice-ethanol to obtain crude fatty acylated antibacterial peptide; Step 7): The crude fatty acylated antibacterial peptide was separated and purified using high performance liquid chromatography to obtain the purified product.

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