Tryptophan-containing antibacterial peptide, synthesis method and application thereof

Abstract

This invention provides a self-assembly-induced tryptophan-containing antimicrobial peptide, its synthesis method, and its application, belonging to the field of antimicrobial agent preparation technology. First, an antimicrobial tryptophan-containing antimicrobial peptide with antimicrobial properties, namely a lysine-tryptophan-lysine tripeptide protected by Fmoc (Fmoc-KWK), is synthesized. Then, Fmoc-KWK is self-assembled to obtain the Fmoc-KWK assembly with better antimicrobial properties. The Fmoc-KWK assembly is in the form of nanofibers, which promotes the binding of the peptide nanostructure to the bacterial cell membrane through the chiral arrangement of tryptophan residues induced by self-assembly, thereby achieving the purpose of killing bacteria. The Fmoc-KWK and Fmoc-KWK assembly of this invention have well-defined structures and controllable sequences, and do not contain antigenic epitopes that may trigger specific recognition, thus avoiding adaptive immune responses. They are more advantageous than long-chain peptides when used as building blocks for antimicrobial materials.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agent preparation technology, specifically relating to a tryptophan-containing antibacterial peptide, its synthesis method, and its application. Background Technology

[0002] Currently, widespread antibiotic resistance in pathogenic bacteria has become a global threat. Many antibiotics in clinical development still belong to existing compound families, and their availability may decrease due to the rapid development of resistance in clinical strains. Therefore, there is an urgent need to develop antimicrobial agents with novel mechanisms of action.

[0003] Unlike traditional antibiotics, which primarily exert their effects through specific receptor-protein interactions, antimicrobial peptides (AMPs) typically exert their antimicrobial activity through non-receptor-mediated membrane lysis of pathogens, thus they do not induce bacterial resistance. Most antimicrobial peptides possess both cationic and hydrophobic amino acid residues, making them amphiphilic in polar organisms and enabling them to disrupt bacterial membranes. The antimicrobial mechanism of AMPs is as follows: Electrostatic interactions exist between the bacterial membrane and AMPs. Because bacterial membranes are composed of anionic lipids such as phosphatidylglycerol and cardiolipin, and the amphiphilic ionic lipid phosphatidylethanolamine, once the polar side of an AMP rich in charged residues binds to the anionic bacterial membrane, the hydrophobic region of the peptide chain further penetrates into the hydrophobic region of the phospholipid bilayer, thereby inducing membrane permeability and killing the bacteria. Furthermore, unlike the negatively charged bacterial cell membranes, the surface of normal mammalian cells is mainly composed of neutrally charged phospholipids, such as sphingolipids or phosphatidylcholine. Therefore, AMPs selectively kill bacteria without harming normal cells. AMPs have attracted much attention as potential alternatives to traditional antibiotics due to their high specificity, lack of drug resistance tendency, and good biocompatibility, as they possess broad-spectrum antibacterial activity.

[0004] In recent years, domestic and international scholars have mostly focused on the design of antimicrobial peptides on natural, long-chain polypeptides, or on coupling them with other polymers and metal nanoparticles with antimicrobial capabilities. For example, HnMc peptide complex micelles are self-assembled from chimeric antimicrobial lipopeptides (DSPE-PEG-HnMc) and amphiphilic biodegradable polymers. They have high specificity and can easily detect a wide range of bacterial infections, making them a good targeted antimicrobial agent for drug-resistant bacterial infections. The shell-core structure composite material AgPW@PDA@Nisin can disrupt the cell membrane of Staphylococcus aureus, causing nucleotide leakage, altering its permeability, destroying cell integrity, and killing bacteria. TAT (YGRKKRRQRRR) is a peptide derived from HIV glycoprotein. TAT is used as an antimicrobial peptide against drug-resistant bacteria, as well as a transporter for other polypeptides, proteins, nanoparticles, or anticancer drugs. It is very effective in inhibiting Staphylococcus aureus in vivo. However, the applications of AMPs, including those mentioned above, mainly focus on natural complex long-chain polypeptides and their derivatives, most of which contain 20-50 amino acid residues, making synthesis difficult and production costs high. Therefore, there is a need to provide a method for synthesizing short peptide AMPs with well-defined structures and controllable sequences. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a self-assembly-induced tryptophan-containing antimicrobial peptide, its synthesis method, and its applications. First, an antimicrobial tryptophan-containing antimicrobial peptide, namely a lysine-tryptophan-lysine tripeptide protected by Fmoc (Fmoc-KWK), is synthesized. Then, Fmoc-KWK is self-assembled to obtain an Fmoc-KWK assembly with improved antimicrobial properties. The Fmoc-KWK assembly is in the form of nanofibers, which promotes the binding of the peptide nanostructure to the bacterial cell membrane through the chiral arrangement of tryptophan residues induced by self-assembly, thereby achieving the purpose of killing bacteria. The Fmoc-KWK and Fmoc-KWK assemblies of this invention have well-defined structures and controllable sequences, and do not contain antigenic epitopes that may trigger specific recognition, thus avoiding adaptive immune responses. They are more advantageous than long-chain peptides when used as building blocks for antimicrobial materials.

[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means.

[0007] This invention first provides a tryptophan-containing antimicrobial peptide, denoted as Fmoc-KWK, with the following structural formula:

[0008]

[0009] This invention also provides a method for synthesizing the above-mentioned tryptophan-containing antimicrobial peptide, specifically comprising the following steps:

[0010] (1) Add activated resin to the polypeptide solid phase synthesis column, and then add Fmoc deprotecting agent to remove Fmoc protecting groups from the activated resin. Repeat the Fmoc protecting group removal reaction several times to obtain resin A.

[0011] (2) Fmoc-protected lysine (Fmoc-Lys(Boc)-OH), condensing agent benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIPEA) were dissolved in dimethylformamide (DMF) to obtain amino acid solution A;

[0012] Amino acid solution A was added to a polypeptide solid-phase synthesis column containing resin A. Then, the polypeptide solid-phase synthesis column containing resin A was placed in a polypeptide synthesizer for condensation reaction. After the reaction was completed, the column was washed and dried to obtain resin B.

[0013] (3) Fmoc-protected tryptophan (Fmoc-Trp-OH(Boc)-OH), condensing agent HBTU, HOBt and DIPEA are dissolved in DMF to obtain amino acid solution B;

[0014] Repeat step (1) to remove the Fmoc protecting group in resin B, then add amino acid solution B to the polypeptide solid-phase synthesis column containing resin B, then put the polypeptide solid-phase synthesis column containing resin B into a polypeptide synthesizer for condensation reaction, wash and dry after the reaction to obtain resin C.

[0015] (4) Fmoc-protected lysine (Fmoc-Lys(Boc)-OH), condensing agent HBTU, HOBt and DIPEA are dissolved in DMF to obtain amino acid solution C;

[0016] Repeat step (1) to remove the Fmoc protecting group in resin C, then add amino acid solution C to the peptide solid-phase synthesis column containing resin C, then put the peptide solid-phase synthesis column containing resin C into a peptide synthesizer for condensation reaction, wash and dry after the reaction to obtain resin D.

[0017] (5) Add a deprotection reagent to the polypeptide solid-phase synthesis column containing resin D, and then place the polypeptide solid-phase synthesis column into a polypeptide synthesizer for deprotection reaction. After the reaction is completed, rinse and dry. Repeat the above steps several times to obtain resin E.

[0018] (6) Add lysis buffer to the polypeptide solid-phase synthesis column containing resin E, and then place the polypeptide solid-phase synthesis column into a polypeptide synthesizer for lysis reaction. After the reaction is completed, filter and collect the filtrate.

[0019] Next, the filtrate was bubbled with nitrogen. After bubbling, pre-cooled diethyl ether was added, the precipitate was collected by centrifugation, washed, and dried to obtain the crude polypeptide product.

[0020] The crude peptide product was separated and purified by high performance liquid chromatography, and then freeze-dried to obtain an antimicrobial peptide containing tryptophan, denoted as Fmoc-KWK.

[0021] Preferably, in step (1), the resin comprises Rink-Amide-AM resin;

[0022] The Fmoc deprotection agent was prepared by mixing piperidine and DMF in a volume ratio of 1:4.

[0023] The ratio of the activated resin to the Fmoc deprotecting agent is 5 mL: 100 mg;

[0024] The Fmoc protecting group removal reaction was carried out at room temperature for 15–20 min, and the Fmoc protecting group removal reaction was repeated 2–3 times.

[0025] Preferably, in step (2), the ratio of Fmoc-Lys(Boc)-OHHBTU, HOBt, DIPEA and DMF is 92mg:74mg:30mg:50mg:3.5mL;

[0026] The ratio of the amount of amino acid solution A to the activated resin in step (1) is 5 mL: 100 mg;

[0027] The condensation reaction was carried out at room temperature for 4–8 hours, followed by alternating washing with DMF and dichloromethane (DCM) after the reaction was completed.

[0028] Preferably, in step (3), the ratio of Fmoc-Trp-OH(Boc)-OH, HOBt, DIPEA and DMF is 92mg:74mg:30mg:50mg:3.5mL;

[0029] The ratio of the amount of amino acid solution B to the activated resin in step (1) is 5 mL: 100 mg;

[0030] The condensation reaction was carried out at room temperature for 4–8 hours, followed by alternating washing with DMF and DCM after the reaction was completed.

[0031] Preferably, in step (4), the ratio of Fmoc-Lys(Boc)-OH, HOBt, DIPEA and DMF is 92mg:74mg:30mg:50mg:3.5mL;

[0032] The ratio of the amount of amino acid solution B to the activated resin in step (1) is 5 mL: 100 mg;

[0033] The condensation reaction was carried out at room temperature for 4–8 hours, followed by alternating washing with DMF and DCM after the reaction was completed.

[0034] Preferably, in step (5), the deprotecting agent is prepared from trifluoroacetic acid (TFA) and DCM in a volume ratio of 10:90.

[0035] The ratio of the deprotection reagent to the activated resin in step (1) is 5 mL: 100 mg;

[0036] Repeating the above steps several times constitutes repeating 2 to 3 times.

[0037] Preferably, in step (6), the pyrolysis solution is prepared from TFA, triethylsilane (TES) and H2O in a volume ratio of 94:5:1;

[0038] The pyrolysis reaction was carried out at room temperature for 1.5–2 hours.

[0039] The nitrogen bubbling time is 10-20 minutes;

[0040] The ratio of the pre-cooled ether to the activated resin in step (1) is 100 mL: 100 mg.

[0041] The present invention also provides a tryptophan-containing antimicrobial peptide assembly, which is formed by the self-assembly of the above-mentioned tryptophan-containing antimicrobial peptide; the tryptophan-containing antimicrobial peptide assembly has a diameter of 7-10 nm, is in the form of nanofibers, and is denoted as Fmoc-KWK assembly.

[0042] The present invention also provides a method for synthesizing Fmoc-KWK assemblies, wherein the pH value of the Fmoc-KWK prepared above is adjusted to 7-7.5, and incubated at room temperature for 24 hours to obtain the Fmoc-KWK assemblies.

[0043] The present invention also provides the application of the above-mentioned tryptophan-containing antimicrobial peptide Fmoc-KWK and Fmoc-KWK assembly in killing bacteria.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] In the Fmoc-KWK of this invention, the lysine residues carry a positive charge, allowing them to target the bacterial cell membrane. Tryptophan, a nonpolar amino acid, can insert into the bacterial cell membrane, leading to cell lysis and death. Furthermore, the Fmoc-protected lysine-tryptophan-lysine tripeptide exhibits pH-induced self-assembly, which can be used to obtain the Fmoc-KWK assembly. Compared to Fmoc-KWK, the Fmoc-KWK assembly exhibits stronger antibacterial activity. Both Fmoc-KWK and the Fmoc-KWK assembly have the potential to serve as antibiotic alternatives.

[0046] The antibacterial mechanism of the Fmoc-KWK and Fmoc-KWK assemblies described in this invention is as follows: charged lysine residues (K) target the bacterial cell membrane through electrostatic interactions, accumulating on the bacterial cell membrane surface. Once a certain threshold is reached, the polypeptide begins to insert into the cell membrane, forming a stable barrel-shaped pore, leading to cell death. Compared to Fmoc-KWK, the ordered arrangement of tryptophan residues in the Fmoc-KWK assembly is more conducive to insertion into the cell membrane, thus exhibiting stronger antibacterial activity and killing bacteria at a lower dose and faster rate. Because the Fmoc-KWK and Fmoc-KWK assemblies kill bacteria by targeting the bacterial cell membrane, bacteria cannot evolve resistance to Fmoc-KWK and Fmoc-KWK assemblies.

[0047] Furthermore, the Fmoc-KWK and Fmoc-KWK assemblies described in this invention are short peptides composed of three amino acids, which are low-cost, easy to prepare, and have effects comparable to long-chain antimicrobial peptides. The Fmoc-KWK and Fmoc-KWK assemblies have well-defined structures and controllable sequences, and do not contain antigenic epitopes that could potentially trigger specific recognition, thus avoiding adaptive immune responses. Therefore, they are more advantageous than long-chain peptides when used as building blocks for antimicrobial materials. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the synthesis pathway of Fmoc-KWK.

[0049] Figure 2 This is the electrospray ionization mass spectrum (ESI-MS) of Fmoc-KWK.

[0050] Figure 3 This is the high-performance liquid chromatogram of Fmoc-KWK.

[0051] Figure 4 This is a TEM image of Fmoc-KWK at pH 2.7.

[0052] Figure 5 This is a TEM image of the Fmoc-KWK assembly at a scale of 100 nm.

[0053] Figure 6 This is a TEM image of the Fmoc-KWK assembly at a scale bar of 1 μm.

[0054] Figure 7 This is a TEM image of the Fmoc-KWK assembly at a scale of 200 nm.

[0055] Figure 8 The MICs of Fmoc-KWK assembly (a) and Fmoc-KWK (b) against Enterococcus faecalis ATCC 29212 are shown in the figure. The rightmost part of the figure is the control group of normally growing bacteria.

[0056] Figure 9 The MICs of Fmoc-KWK assembly (a) and Fmoc-KWK (b) against Bacillus subtilis ATCC 6633 are shown in the figure. The rightmost part of the figure is the control group of normally growing bacteria.

[0057] Figure 10 The MICs of Fmoc-KWK assembly (a) and Fmoc-KWK (b) against Escherichia coli ATCC 25922 are shown in the figure. The rightmost part of the figure is the control group of normally growing bacteria.

[0058] Figure 11 The MICs of Fmoc-KWK assembly (a) and Fmoc-KWK (b) against Escherichia coli ATCC 8739 are shown in the figure. The rightmost part of the figure is the control group of normally growing bacteria.

[0059] Figure 12 This is the UV absorption spectrum of the TX-100 positive control group.

[0060] Figure 13 The hemolysis experiment of Fmoc-KWK assembly and Fmoc-KWK is shown in the inset, which is a blood sample after centrifugation.

[0061] Figure 14 The antibacterial mechanism of Fmoc-KWK assembly and Fmoc-KWK. Detailed Implementation

[0062] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The source, trade name, and components of the reagents used are indicated when they first appear, and subsequent use of the same reagents are from the same source as initially indicated unless otherwise specified; the reagents and materials involved are all obtained for commercial use unless otherwise specified.

[0063] Example 1:

[0064] The schematic diagram of the synthetic pathway of the Fmoc-KWK tripeptide sequence described in this embodiment is shown below. Figure 1 As shown, the following content details the synthesis process of the Fmoc-KWK tripeptide sequence.

[0065] (1) Resin activation:

[0066] Clean the peptide solid-phase synthesis column, then weigh 100 mg of Rink-Amide-AM resin (loading capacity 0.35 mg / mL, hereinafter referred to as resin) into the peptide solid-phase synthesis column, add DMF to the peptide solid-phase synthesis column to rinse the resin three times to remove impurities on the resin surface, and then use a vacuum pump to dry the liquid in the peptide solid-phase synthesis column.

[0067] Add 5 mL of DCM to the peptide solid-phase synthesis column and soak the resin at 25°C for 1-2 hours. Then, rinse the column three times with DCM and use the liquid from the column to obtain activated resin. The activation process mainly involves swelling the resin beads to fully expose their carried groups, making it easier for them to undergo condensation reactions with amino acids.

[0068] (2) Removal of Fmoc protecting groups from the resin:

[0069] 5 mL of Fmoc deprotecting reagent, prepared by mixing piperidine and DMF in a volume ratio of 1:4, was added to the peptide solid-phase synthesis column. The column was then placed in a peptide synthesizer (Intelli-Mixer™ RM-2, Shanghai Weibai Technology) and the deprotection reaction was carried out at 25°C for 20 min. After the reaction, the resin was rinsed three times alternately with DMF and DCM, and the liquid in the peptide solid-phase synthesis column was dried. To ensure complete removal of the Fmoc groups, the deprotection reaction was repeated three times to obtain resin A.

[0070] In this step, the resin is rinsed three times alternately with DMF and DCM. The first rinse with alternating DMF and DCM removes the reaction solvent and allows the resin to swell again. The second rinse removes any residual DMF trapped within the resin. The third rinse completely removes any remaining reaction solution. The final rinse with DCM ensures the resin remains swollen, facilitating the next reaction step. Simultaneously, DCM's high volatility allows for easy drying, keeping the resin dry and preventing a decrease in the concentration of the subsequent reaction solution.

[0071] (3) Amino acid condensation of the polypeptide sequence:

[0072] Weigh out Fmoc-protected lysine Fmoc-Lys(Boc)-OH (92 mg, 3 eq) and condensing agents HBTU (74 mg, 3 eq), HOBt (30 mg, 3 eq), and DIPEA (50 mg, 6 eq) and add them to a 5 mL glass bottle. Then add 3.5 mL of pure DMF and dissolve under sonication to obtain amino acid solution A.

[0073] Then, amino acid solution A was added to the polypeptide solid-phase synthesis column containing resin A. The polypeptide solid-phase synthesis column containing resin A was then placed in a polypeptide synthesizer and a condensation reaction was carried out at 25°C for 4 hours. After the reaction was completed, the resin was washed three times with DMF and DCM alternately and then dried to obtain resin B.

[0074] To ensure the condensation reaction is complete, the presence of free amino groups on the resin can be qualitatively detected using the ninhydrin assay (Kaiser assay), thus determining the extent of the reaction. The Kaiser assay procedure is as follows: Take a small amount of cleaned resin into a small glass tube using a capillary tube, then add 2 drops of reagent 1 and 2 drops of reagent 2 in sequence, mix well, and heat in a 100°C oil bath for 3 minutes. After heating, remove the small glass tube and observe the color of the resin. Colorless resin indicates that the condensation reaction is complete.

[0075] Reagent 1: 5g ninhydrin dissolved in 100mL ethanol; Reagent 2: 80g phenol dissolved in 20mL ethanol.

[0076] (4) Fmoc-protected condensation of tryptophan:

[0077] Repeat the step of removing the Fmoc protecting group on the resin described in step (2) to remove the Fmoc protecting group in resin C in step (3). Then weigh the Fmoc-protected tryptophan Fmoc-Trp-OH (92 mg, 3 eq) and condensing agents HBTU (74 mg, 3 eq), HOBt (30 mg, 3 eq), and DIPEA (50 mg, 6 eq) and add them to a 5 mL glass bottle. Then add 3.5 mL of pure DMF and dissolve it under ultrasonic conditions to obtain amino acid solution B.

[0078] Then, amino acid solution B was added to the polypeptide solid-phase synthesis column containing resin B. The polypeptide solid-phase synthesis column containing resin B was then placed in a polypeptide synthesizer and a condensation reaction was carried out at 25°C for 4 hours. After the reaction, the resin was washed three times with DMF and DCM alternately and then dried to obtain resin C.

[0079] (5) Condensation of Fmoc-protected lysine:

[0080] Repeat the step of removing the Fmoc protecting group on the resin described in step (2) to remove the Fmoc protecting group in resin C in step (3). Then weigh the Fmoc-protected lysine Fmoc-Lys(Boc)-OH (92 mg, 3 eq) and condensing agents HBTU (74 mg, 3 eq), HOBt (30 mg, 3 eq), and DIPEA (50 mg, 6 eq) and add them to a 5 mL glass bottle. Then add 3.5 mL of pure DMF and dissolve it under ultrasonic conditions to obtain amino acid solution C.

[0081] Then, amino acid solution C was added to the polypeptide solid-phase synthesis column containing resin C, and the polypeptide solid-phase synthesis column containing resin C was placed in a polypeptide synthesizer and a condensation reaction was carried out at 25°C for 4 hours. After the reaction, the resin was washed three times alternately with post-reaction DMF and DCM and then dried to obtain resin D.

[0082] (6) Removal of the Boc protecting group from the lysine side chain:

[0083] Add 5 mL of a deprotecting reagent prepared from TFA and DCM at a volume ratio of 10:90 to a peptide solid-phase synthesis column containing resin D. Then, place the peptide solid-phase synthesis column in a peptide synthesizer and carry out the deprotection reaction at 25°C for 20 min. After the reaction, wash the resin three times with DCM and dry the liquid in the peptide solid-phase synthesis column. To ensure complete removal of the Boc protecting group on the lysine side chain, repeat the deprotection reaction three times to obtain resin E.

[0084] (7) Cleavage of peptide chains on resin:

[0085] Add 5 mL of a lysis buffer prepared from TFA, TES, and H2O in a volume ratio of 94:5:1 to a peptide solid-phase synthesis column containing resin E. Then, place the column in a peptide synthesizer and carry out the lysis reaction at 25°C for 1.5–2 h. The reaction time should not be too long to avoid degradation of the peptide chains by strong acid. After the reaction, the peptide chains will detach from the resin and be stored in the lysis buffer. Next, filter to remove the resin, collect the lysis buffer in an Erlenmeyer flask, and bubble with pure nitrogen for 10 min to remove excess TFA.

[0086] (8) Polypeptide precipitation:

[0087] 100 mL of pre-cooled diethyl ether was added to an Erlenmeyer flask containing lysis buffer. Since the peptide chain is insoluble in diethyl ether, it promotes precipitation. The solution was then transferred to a 50 mL centrifuge tube and centrifuged at 9000 rpm for 5 min. After centrifugation, the supernatant was removed to obtain the peptide precipitate, completing one washing step. This washing was repeated three times to remove most impurities from the peptide precipitate. The peptide precipitate was then dried in a vacuum desiccator to obtain a powdered crude peptide product.

[0088] (9) Purification of crude peptides:

[0089] The crude powdered peptide product was separated and purified by high performance liquid chromatography (HPLC) (1 mL / min) at ambient temperature (25 °C). A linear gradient elution was performed using CH3CN / H2O containing 0.1% TFA, with the timeline set to 0–20 min. After elution, the product was freeze-dried to obtain the powdered peptide product, which was stored at -8 °C for later use. The monomeric form of the tryptophan-containing antimicrobial peptide was obtained and designated as Fmoc-KWK.

[0090] Figure 2 The image shows the electrospray ionization mass spectra (ESI-MS) spectra of Fmoc-KWK. From the image, we can see the calculated molecular weight [M+H] of Fmoc-KWK. + The value is 683.82. The actual measured value [M+H] can be seen from the graph. + The value was 683.54, indicating that Fmoc-KWK was successfully synthesized.

[0091] Figure 3 The figure shows the high performance liquid chromatogram of Fmoc-KWK. As can be seen from the figure, the purity of the synthesized Fmoc-KWK is 99.33%.

[0092] Figure 4 The image shows a TEM image of Fmoc-KWK at pH 2.7. As can be seen from the image, Fmoc-KWK did not self-assemble under these conditions, because the TEM only showed some random aggregates and did not reveal an organized structure.

[0093] Example 2:

[0094] In this embodiment, the inhibitory effects of the unassembled Fmoc-KWK prepared in Example 1 and the Fmoc-KWK assembly obtained by self-assembling Fmoc-KWK on the growth of Escherichia coli (E. coli, ATCC 8739), E. coli (E. coli, ATCC 25922), Bacillus subtilis (B. subtilis, ATCC 6633), and Enterococcus faecalis (E. faecalis, ATCC 29212) were investigated by microdilution with broth.

[0095] (1) Sample preparation:

[0096] Since tryptophan-containing antimicrobial peptides in monomeric form may polymerize into oligomers such as dimers and trimers during storage and are no longer in monomeric form, they need to be treated with TFE before use to ensure that the Fmoc-KWK used is in monomeric form. Therefore, TFE treatment is required for the Fmoc-KWK obtained in Example 1. The treatment steps are as follows: Dissolve 100 mg of Fmoc-KWK prepared in Example 1 in 10 mL of TFE, remove the solvent by rotary evaporation, and then freeze-dry to obtain Fmoc-KWK that is completely in monomeric form.

[0097] Solutions of non-self-assembled Fmoc-KWK and Fmoc-KWK assembled products were prepared separately for later use.

[0098] Fmoc-KWK solution: Weigh 6.8 mg of Fmoc-KWK into a 3 mL vial at room temperature (25℃), add 2 mL of HPLC-grade water, and sonicate to mix thoroughly. The pH of the solution is 2.7 at this point. Incubate naturally at room temperature for 24 h to obtain 2 mL of Fmoc-KWK solution with a concentration of 5 mM.

[0099] Fmoc-KWK assembly solution: Weigh 6.8 mg of pure Fmoc-KWK monomer into a 3 mL vial at room temperature (25℃), add 2 mL of HPLC-grade water, and sonicate to mix thoroughly, obtaining 2 mL of 5 mM Fmoc-KWK aqueous solution. Adjust the pH of the Fmoc-KWK aqueous solution to 7.4 with 1 M NaOH / HCl solution, and incubate naturally at room temperature for 24 h to obtain a hydrogel-like Fmoc-KWK assembly.

[0100] Figures 5-7 The images show TEM images of the self-assembled Fmoc-KWK assembly at pH 7.4 at different scales. The images show that the monomeric Fmoc-KWK self-assembles into continuous nanofibers with diameters of 7–10 nm and lengths of tens of micrometers. Furthermore, the Fmoc-KWK nanofibers exist as well-defined monofilaments, which may be due to the charged surfaces of the nanofibers, thus enabling them to maintain a uniformly dispersed morphology.

[0101] (2) Examination of antibacterial ability:

[0102] Add 2 mL of Fmoc-KWK assembly to 18 mL of LB broth, then dilute the Fmoc-KWK assembly to a concentration of 0.5 mM; add 2 mL of Fmoc-KWK solution to 9 mL of LB broth, and dilute its concentration to 0.1 mg / mL, approximately 1.464 mM, for later use.

[0103] Add 100 μL of LB liquid medium (containing 10⁻⁶ bacteria) to wells 2 through 12 of a sterile 96-well plate.6 CFU / mL), take respectively

[0104] 200 μL of diluted Fmoc-KWK solution and Fmoc-KWK assembly were added to the first well. Then, 100 μL of the solution from the first well was added to the second well and mixed. 100 μL of the mixed sample solution was then added to the third well and mixed, and so on until the eleventh well. The sample solution was diluted twofold, and finally, 100 μL of the mixed sample solution from the eleventh well was discarded. The twelfth well contained 100 μL of LB liquid medium (containing 10... 6 (CFU / mL) was used as a control for normally growing bacteria.

[0105] After incubating the 96-well plates at 37°C for 24 hours, the MIC (minimum inhibitory concentration) was measured. The MIC is the minimum peptide concentration required to prevent turbidity in the bacterial culture after incubation. All MIC measurements are the average of three independent experiments. The results are shown in Table 1 and... Figures 8-11 As shown:

[0106] Table 1. Inhibitory effect of Fmoc-KWK assembly on the growth of different bacteria

[0107]

[0108] Combining Table 1 and Figure 8-11 It can be seen that the Fmoc-KWK assembly exhibits significant inhibitory effects on the growth of both common pathogenic Gram-negative and Gram-positive bacteria, demonstrating broad-spectrum antibacterial activity and stronger antibacterial capacity compared to existing antibiotics with limited use. This is in contrast to peptide analogs (KW) that also contain lysine and tryptophan. n It can be seen that the Fmoc-KWK assembly has comparable, or even better, antibacterial activity than peptides with longer chains, such as octapeptide (KW)4 and decapeptide (KW)5. However, the relationship between the chain length of the antimicrobial peptide and the hemolysis rate must also be considered; generally, the longer the chain, the higher the hemolysis rate. (KW)5, in particular, causes significant hemolysis, which does not meet medical requirements.

[0109] The MIC of Fmoc-KWK is 5 to 10 times larger than that of the Fmoc-KWK assembly, indicating that the spatial configuration of the Fmoc-KWK assembly, which evolves with self-assembly, namely the ordered arrangement of tryptophan residues, makes it much easier for the Fmoc-KWK assembly to insert into the bacterial cell membrane, leading to bacterial lysis and death.

[0110] The antibacterial mechanism of the Fmoc-KWK and Fmoc-KWK assembly described in this invention is as follows: Figure 14As shown in the figure, charged lysine residues (K) target the bacterial cell membrane through electrostatic interactions, accumulating on the surface. Once a certain threshold is reached, the polypeptide begins to insert into the cell membrane, forming a stable barrel-shaped pore, leading to cell death. Compared to Fmoc-KWK, the ordered arrangement of tryptophan residues in the Fmoc-KWK assembly is more conducive to insertion into the cell membrane, thus exhibiting stronger antibacterial activity and killing bacteria at a lower dose and faster rate.

[0111] Example 3:

[0112] In this embodiment, the human erythrocyte (hRBC) hemolysis assay was used to conduct a preliminary toxicity assessment of Fmoc-KWK assemblies and unassembled Fmoc-KWK solutions. PBS solution was used as the negative control (zero hemolysis), and 0.3 wt% Triton-100 (TX-100) aqueous solution was used as the positive control (100% hemolysis). The specific assessment steps are as follows:

[0113] (1) Preparation of red blood cell suspension:

[0114] Centrifuge 2 mL of anticoagulated whole blood containing EDTA in a centrifuge tube at 1000 rpm for 10 min. Then, add 0.2 mL of the lower red blood cell pellet to a 1.5 mL centrifuge tube, add 1 mL of physiological saline, gently invert to mix, and centrifuge again at 1000 rpm for 10 min to remove the supernatant. Finally, add 0.25 mL of physiological saline to the centrifuge tube to dilute the red blood cells and obtain a red blood cell suspension for later use.

[0115] (2) Determination of the wavelength of maximum absorbance:

[0116] The absorption spectrum of the supernatant of the positive control group in the wavelength range of 400–600 nm was measured using a UV spectrometer. The absorption spectrum is shown below. Figure 12 As shown.

[0117] Depend on Figure 12 It can be seen that the absorbance is directly proportional to the amount of hemoglobin released by hRBCs, and the wavelength at which the supernatant of the positive control group reaches its maximum absorbance value is determined to be 414 nm. Therefore, the wavelength of 414 nm is selected as the measurement wavelength.

[0118] (3) Sample determination:

[0119] The Fmoc-KWK assembly solution and the unassembled Fmoc-KWK solution obtained in Example 2 were diluted to 100 μM with deionized water and set aside. 1 mL of the Fmoc-KWK assembly solution and the Fmoc-KWK solution were added to 20 μL of erythrocyte suspension, and then incubated at 37°C with gentle stirring for 60 min. After incubation, the mixture was centrifuged at 3500 rpm (approximately 1000 g) for 5 min. The supernatant was collected and transferred to a 96-well plate to obtain test sample 1 containing the Fmoc-KWK assembly solution and test solution 2 containing the unassembled Fmoc-KWK monomer solution.

[0120] Then, the absorbance values ​​of the supernatant of sample 1, sample 2, negative control, and positive control were measured at a wavelength of 414 nm, with three replicates for each sample and control group. The hemolysis rate was calculated according to the following formula.

[0121]

[0122] In the formula A s A represents the absorbance value of the experimental sample at a wavelength of 414 nm. nc A represents the absorbance value of the negative control group at a wavelength of 414 nm. pc The absorbance value of the positive control group at a wavelength of 414 nm is shown.

[0123] Test results are as follows Figure 13 As shown in the figure (the inset shows a blood sample after centrifugation), it can be seen from the figure that after treatment with 100 μM Fmoc-KWK assembly solution (approximately 5 times the MIC of Fmoc-KWK) and Fmoc-KWK solution, the blood cell integrity rate is >95% and >90%, respectively, indicating that Fmoc-KWK has significant biocompatibility. In particular, the hemolysis rate of the self-assembled Fmoc-KWK assembly is <5%, which meets the requirements for medical materials.

[0124] In summary, the Fmoc-KWK and Fmoc-KWK assemblies described in this invention are short peptides composed of three amino acids, which are low-cost, easy to prepare, and have effects comparable to long-chain antimicrobial peptides. The Fmoc-KWK and Fmoc-KWK assemblies have well-defined structures and controllable sequences, and do not contain antigenic epitopes that could potentially trigger specific recognition, thus avoiding adaptive immune responses. Therefore, they are more advantageous than long-chain peptides when used as building blocks for antimicrobial materials.

[0125] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of tryptophan-containing antimicrobial peptides, or tryptophan-containing antimicrobial peptide self-assemblies, in the preparation of antibacterial agents; The bacteria in question is Enterococcus faecalis; The structural formula of the tryptophan-containing antimicrobial peptide is: 。 2. Use according to claim 1, characterized in that, The tryptophan-containing antimicrobial peptide self-assembly is formed by the self-assembly of tryptophan-containing antimicrobial peptides; the tryptophan-containing antimicrobial peptide self-assembly has a diameter of 7-10 nm and is in the form of nanofibers. The method for synthesizing the tryptophan-containing antimicrobial peptide self-assembly is as follows: the pH value of the tryptophan-containing antimicrobial peptide is adjusted to 7-7.5, and incubated at room temperature for 20-24 hours to obtain the tryptophan-containing antimicrobial peptide self-assembly.

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