An antibacterial heptapeptide, and a preparation method and application thereof

Abstract

The application discloses an antibacterial heptapeptide with an amino acid sequence of RFIRWKL, which has the advantages of strong bacteriostatic capacity, broad-spectrum bactericidal activity, safety performance, low-cost synthesis and application, etc., can efficiently inhibit escherichia coli and staphylococcus aureus, and has a minimum inhibitory concentration (MIC) of 75 muM; meanwhile, the antibacterial peptide still maintains low cytotoxicity in a high-concentration bacterial environment. Moreover, the antibacterial peptide provided by the application has a length of only 7 amino acids, and is easy to synthesize and prepare.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide synthesis technology, specifically to an antimicrobial heptapeptide, its preparation method, and its application. Background Technology

[0002] In the field of public health, bacterial infections remain a significant threat. The elderly, hospitalized patients, and those with weakened immune systems are at significantly higher risk of developing serious complications after exposure to pathogens. Traditionally, various antibiotics have been effective in controlling most bacterial infections. However, the widespread use of antibiotics has led to the continuous emergence of drug-resistant variants in pathogens, giving rise to a variety of multidrug-resistant strains, including methicillin-resistant Staphylococcus aureus (MRSA) and ESBL-producing (extended-spectrum β-lactamase)-resistant Escherichia coli. This has resulted in a reduction in available clinical drugs and a significant decrease in efficacy.

[0003] To address the infection challenges posed by drug-resistant bacteria, researchers worldwide are continuously exploring candidate molecules with novel mechanisms of action. Antimicrobial peptides are a class of naturally occurring bioactive polypeptides widely distributed in animals, plants, and microorganisms. Due to their broad-spectrum antibacterial activity, rapid bactericidal action, and low likelihood of inducing drug resistance, they have become a research hotspot. Their antibacterial mechanism primarily relies on accumulation on the bacterial membrane surface through electrostatic attraction, disrupting membrane structural integrity through membrane pores, and ultimately inducing pathogen death.

[0004] Although antimicrobial peptides show broad application prospects, the structural characteristics and physicochemical properties of most natural antimicrobial peptides limit their industrialization. Specifically, their peptide chain length is typically 20-50 amino acids, resulting in persistently high costs for solid-phase synthesis. Furthermore, most suffer from poor stability, short in vivo half-life, and potential cytotoxicity. For example: Patent CN101974082A discloses that the naturally matured antimicrobial peptide hepcidin from large yellow croaker exhibits clear antimicrobial activity, and its molecule contains four pairs of disulfide bonds, resulting in a complex structure. The patent's background technology indicates that the four pairs of disulfide bonds make chemical synthesis difficult and expensive, hindering industrial-scale production and restricting subsequent research into its commercialization.

[0005] The patent with publication number CN115043924A uses the green sea turtle-derived antimicrobial peptide Cm-CATH2 as a template for truncation modification. The parent peptide contains 33 amino acid residues, and its relatively long sequence leads to high synthesis costs. Although the truncated form NCM4 retains activity, it is a linear L-type peptide with limited enzyme stability, and further optimization of drug properties is needed by combining it with D-type amino acid replacement.

[0006] In summary, most existing antimicrobial peptide design strategies fail to simultaneously meet the requirements of economy, safety, and antibacterial efficacy. Achieving a balanced optimization of these three aspects remains a key challenge in this field. Therefore, developing novel peptides with simplified sequences, optimized structures, and simultaneously high antibacterial activity and low cytotoxicity has become the primary research objective. Ideal short-chain antimicrobial peptides should simultaneously possess characteristics such as high structural stability, low hemolytic toxicity, and a broad antibacterial spectrum. The development of such products has significant practical implications and application needs. Summary of the Invention

[0007] To address the above problems, the present invention provides an antibacterial heptapeptide.

[0008] The technical solution adopted in this invention is: an antibacterial heptapeptide with the amino acid sequence RFIRWKL.

[0009] This application also provides a method for preparing the above-mentioned antimicrobial heptapeptide, the preparation steps of which are as follows: S1: Add N,N-dimethylformamide and morpholine to the resin for a first deprotection. Then filter out the N,N-dimethylformamide and morpholine. Add the same amount of N,N-dimethylformamide and morpholine to the deprotected resin for a second deprotection. Then filter out the N,N-dimethylformamide and morpholine. Subsequently, wash the deprotected resin with N,N-dimethylformamide and dichloromethane, respectively. After each wash, filter out the washing solvent. The amount of N,N-dimethylformamide and dichloromethane added for each deprotection should only be enough to fill the peptide solid-phase synthesis tube.

[0010] S2: Fmoc-Leu-OH, 1-hydroxybenzotriazole, and N,N-dimethylformamide were added sequentially to the washed resin and sonicated until clear. Then, N,N'-diisopropylcarbodiimide was added at 0°C for a first reaction. After the reaction was completed, N,N-dimethylformamide was added to the good mixture for a condensation reaction to obtain Fmoc-Leu-Resin. The second addition of N,N-dimethylformamide was only needed to fill the peptide solid-phase synthesis tube. The sonication conditions were 40 kHz and 70% power.

[0011] S3: Wash Fmoc-Leu-Resin in S2 with N,N-dimethylformamide and dichloromethane; then add pyridine and acetic anhydride for end-capping treatment. After end-capping, wash alternately with N,N-dimethylformamide and dichloromethane. The pyridine and acetic anhydride added should only be enough to fill the peptide solid-phase synthesis tube.

[0012] S4: After alternating washing in S3, add N,N-dimethylformamide and morpholine to Fmoc-Leu-Resin for one deprotection. Then filter out N,N-dimethylformamide and morpholine. Add the same amount of N,N-dimethylformamide and morpholine to the deprotected resin for a second deprotection. Then filter out N,N-dimethylformamide and morpholine. The amount of N,N-dimethylformamide and dichloromethane added for each deprotection should be enough to fill the peptide solid-phase synthesis tube.

[0013] S5: Weigh out the deprotected Fmoc-Leu-Resin, 1-hydroxybenzotriazole, and N,N-dimethylformamide from S4, dissolve them, sonicate until clear, and then add N,N'-diisopropylcarbodiimide at 0℃ for a first reaction; after the reaction is complete, add N,N-dimethylformamide to the good mixture for a condensation reaction to obtain Fmoc-Lys-Leu-Resin; the second addition of N,N-dimethylformamide is only needed to fill the peptide solid-phase synthesis tube; the sonication conditions are 40kHz and 70% power.

[0014] S6: Repeat steps S4-S5, sequentially linking Fmoc-Trp-OH, Fmoc-Arg-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Arg-OH to the resin support from the C-terminus to the N-terminus until the peptide chain synthesis is complete; S7: After completing the last amino acid-Arg linking, the terminal Fmoc group is removed, followed by two methanol shrinkages. Then, the mixture is transferred to a flask, and trifluoroacetic acid, triisopropylsilane, and water are added for room temperature pyrolysis. The amount of trifluoroacetic acid, triisopropylsilane, and water added should be enough to fill the peptide solid-phase synthesis tube.

[0015] S8: After lysis, the filtrate is collected and rotary evaporated until trifluoroacetic acid is completely evaporated. Then, ice-crystal ether is added and recrystallized by sonication. After sonication, the mixture is allowed to stand, the supernatant is removed, and ice-crystal ether is added again for secondary recrystallization by sonication. The supernatant is removed, the precipitate is collected, and vacuum is applied to obtain crude peptide. Ice-crystal ether is used as a solvent and an excess is sufficient. The sonication conditions are 40 kHz and 70% power.

[0016] S9: Purify and freeze-dry the crude peptide in S8 to obtain the antibacterial heptapeptide product. The purification steps are as follows: 1) Dissolve the crude peptide in a 50% acetonitrile-water solution. Short-term sonication (about 2 minutes) can be used to help dissolve the peptide, and the mass-volume ratio of crude peptide to 50% acetonitrile-water solution is 40-100:1.

[0017] 2) Filter the dissolved crude peptides using a 0.45 μm filter membrane to obtain the filtered sample; 3) Analytical purity: Take 3 μL of the filtered sample into the analytical HPLC system, use water / acetonitrile as the mobile phase, and perform gradient elution for 30 min. The initial ratio is 95% water / 5% acetonitrile, and the final ratio is 5% water / 95% acetonitrile. Equilibrate for 5 min before injection to obtain the sample after gradient elution. 4) Preparation and purification: The sample after gradient elution was used for preparative HPLC. The equilibration time was 10 min. The initial gradient was 95% water / 5% acetonitrile, and the final gradient was 25% water / 75% acetonitrile. The gradient time was 40 min. The target peak was collected. 5) Purity and molecular weight identification: The collected target peaks were subjected to HPLC purity detection and mass spectrometry (MS) analysis.

[0018] Freeze-drying: The purified peptide solution is freeze-dried to obtain the finished antimicrobial peptide product.

[0019] Storage method: The obtained polypeptide is a white powder. It should be sealed in packaging and stored at -20℃ for a long time.

[0020] Preferably, resin pretreatment is performed before S1, specifically as follows: resin is placed in a reaction tube, N,N-dimethylformamide (DMF): dichloromethane (DCM) = 1:9 is added, the amount of DMF is 3 mL, the amount of DCM is 27 mL, and the mixture is shaken thoroughly on a shaker for 30 min to achieve resin swelling.

[0021] Preferably, after step S5, the obtained Fmoc-Lys-Leu-Resin is subjected to a cleaning step, specifically: after condensation, the synthesis tube is cleaned with DMF and methanol in sequence to remove residual activator.

[0022] Preferably, the mass ratio of N,N-dimethylformamide and morpholine in S1 is 1:1, and the time for both the first and second deprotection is 25-30 min; the washing is performed 6 times with N,N-dimethylformamide and 1 time with dichloromethane.

[0023] Preferably, the molar ratio of the starting resin, Fmoc-Leu-OH in S2, 1-hydroxybenzotriazole, the first addition of N,N-dimethylformamide and N,N'-diisopropylcarbodiimide is 1:3:3:5:10, the first reaction time is 9-11 min, and the condensation reaction time is 1.5-2.5 h.

[0024] Preferably, in S3, N,N-dimethylformamide is washed 3 times, dichloromethane is washed once, the mass ratio of pyridine to acetic anhydride is 1:1, the reaction time is 2h, and N,N-dimethylformamide and dichloromethane are washed alternately 5 times.

[0025] Preferably, the mass ratio of N,N-dimethylformamide to morpholine in S4 is 1:1, and the time for both the first and second deprotection is 25-30 min.

[0026] Preferably, the molar ratio of the starting resin, Fmoc-Leu-Resin after secondary deprotection in S5, 1-hydroxybenzotriazole, the first addition of N,N-dimethylformamide and N,N'-diisopropylcarbodiimide is 1:3:3:5:10, the first reaction time is 9-11 min, and the condensation reaction time is 1.5-2.5 h.

[0027] Preferably, the mass ratio of trifluoroacetic acid (TFA), triisopropylsilane and water in S7 is 95:2.5:2.5, and the pyrolysis time is 2 hours.

[0028] This application also provides the use of the above-mentioned antimicrobial heptapeptide in the preparation of products that inhibit bacterial growth.

[0029] The beneficial effects of this invention are: The antimicrobial peptide provided by this invention possesses advantages such as potent antibacterial activity, broad-spectrum bactericidal activity, safety, and low-cost synthesis and application. It can effectively inhibit *Escherichia coli* and *Staphylococcus aureus*, with a minimum inhibitory concentration (MIC) of 75 μM. Furthermore, this antimicrobial peptide maintains low cytotoxicity even in high-concentration bacterial environments. Moreover, the antimicrobial peptide provided by this invention is only 7 amino acids in length, making it easy to synthesize and prepare. It has outstanding practical value and broad application prospects. Attached Figure Description

[0030] Figure 1 The HPLC results of the antimicrobial heptapeptides in Examples 1-3 are shown in the figure.

[0031] Figure 2 The mass spectrometry results are for the antimicrobial heptapeptides in Examples 1-3.

[0032] Figure 3 This is a diagram showing the biocompatibility results of the antimicrobial heptapeptide in Example 1. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and a preferred embodiment.

[0034] The resin used in the following examples is rink amide resin.

[0035] Example 1 1. Solid-phase synthesis of antimicrobial peptides 1.1 Resin Pretreatment Place 1g of rink resin in a reaction tube, add N,N-dimethylformamide (DMF): dichloromethane (DCM) = 1:9, with 3mL of DMF and 27mL of DCM, and shake thoroughly on a shaker for 30min to allow the resin to swell.

[0036] 1.2 Deprotection After filtering out the DMF:DCM mixed solution, add N,N-dimethylformamide (DMF):morpholine (Morph) in a 1:1 ratio, with 15 mL of both DMF and Morph. Perform the first deprotection for 30 min, followed by the second deprotection for 30 min. After deprotection, wash 6 times with DMF and once with DCM.

[0037] 1.3 Grafting the first amino acid After removing DCM by filtration, 3 molar equivalents of Fmoc-Leu-OH, 3 molar equivalents of 1-hydroxybenzotriazole (HOBT), and 5 molar equivalents of dimethylformamide (DMF) were added sequentially to the resin to dissolve them. The solution was then sonicated until clear. 1.5 mL of 10 molar equivalents of N,N'-diisopropylcarbodiimide (DIC) was added at 0°C, and the reaction was allowed to proceed for 10 min. After the reaction was complete, the resulting mixture was added to a peptide solid-phase synthesis tube, which was then filled with DMF and allowed to react for 2 h.

[0038] 1.4 End capping After the first amino acid was added, the resin was washed three times with DMF and once with DCM. Then, pyridine (Py):acetic anhydride (Ac2O) in a 1:1 ratio was added, with 15 mL of both Py and Ac2O. The reaction was allowed to proceed for 2 hours. After the reaction was completed, the resin was washed five times alternately with DMF and DCM.

[0039] 1.5Fmoc group removal Add DMF:Morph=1:1 to remove protection, 30 minutes at a time, for two cycles.

[0040] 1.6 Subsequent amino acid condensation Weigh out 3 molar equivalents of amino acids, 3 molar equivalents of HOBT, and 5 molar equivalents of DMF, dissolve them, sonicate until clear, add 1.5 mL of 10 molar equivalents of DIC at 0℃, and react for 10 min. After the reaction is complete, add the good mixture to a peptide solid-phase synthesis tube, fill with DMF, and react for 2 h.

[0041] 1.7 Repeat the cleaning steps After condensation, the reaction tube was cleaned sequentially with DMF and methanol to remove residual activator.

[0042] 1.8 Sequence Stepwise Assembly Repeat steps 1.5-1.7, sequentially linking Fmoc-Trp-OH, Fmoc-Arg-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Arg-OH to the resin support from the C-terminus to the N-terminus until peptide chain synthesis is complete.

[0043] 1.9 Terminal deprotection and pyrolysis After linking the last amino acid -Arg, terminal Fmoc removal was performed, followed by two condensations with methanol. The resin was then transferred to a flask, and a mixture of trifluoroacetic acid (TFA):triisopropylsilane (TIPS):water = 95:2.5:2.5 was added, with 19 mL of TFA and 0.5 mL of both TIPS and water, for lysis.

[0044] 1.10 recrystallization After lysis, the filtrate is collected and rotary evaporated until only a small amount of liquid remains. An appropriate amount of ice-crystal ether is added for recrystallization. Ice-crystal ether is added during sonication. After sonication, the mixture is allowed to stand, the supernatant is removed, and ice-crystal ether is added again for a second recrystallization. The supernatant is removed, the precipitate is collected, and vacuum is applied to obtain the crude peptide.

[0045] 1.11 HPLC Purification Procedure 1) Dissolve 200 mg of crude peptide in 2-5 mL of 50% acetonitrile-water solution and sonicate briefly (2 min) to aid dissolution; 2) The solution obtained by filtration using a 0.45 μm filter membrane; 3) Analytical purity: Inject 3 μL of the sample into the analytical HPLC system, using water / acetonitrile as the mobile phase, and perform gradient elution for 30 min. The initial ratio is 95% water / 5% acetonitrile, and the final ratio is 5% water / 95% acetonitrile. Equilibrate for 5 min before injection. 4) Preparation and purification: The sample was subjected to preparative HPLC with an equilibration time of 10 min, an initial gradient of 95% water / 5% acetonitrile, an ending gradient of 25% water / 75% acetonitrile, and a gradient time of 40 min. The target peak was then collected. 5) Purity and molecular weight identification: The collected samples were subjected to HPLC purity detection and mass spectrometry (MS) analysis.

[0046] 1.12 Freeze-drying treatment The purified peptide solution is freeze-dried to obtain the finished antimicrobial peptide product.

[0047] 1.13 Saving Method The obtained polypeptide is a white powder, which is sealed in packaging and stored at -20°C for a long time.

[0048] The HPLC analysis results of the synthesized antimicrobial heptapeptide in this embodiment are as follows: Figure 1 As shown, the mass spectrometry results are as follows: Figure 2 As shown. (From the appendix) Figure 1 As shown in Figure 2, this embodiment synthesized an antimicrobial heptapeptide with the amino acid sequence RFIRWKL.

[0049] Example 2 1. Solid-phase synthesis of antimicrobial peptides 1.1 Resin Pretreatment Place 0.9g of rink resin in a reaction tube, add N,N-dimethylformamide (DMF): dichloromethane (DCM) in a ratio of 0.9:8.9, with 3mL of DMF and 29.7mL of DCM, and shake thoroughly on a shaker for 25 minutes to allow the resin to swell.

[0050] 1.2 Deprotection After filtering out the DMF:DCM mixed solution, N,N-dimethylformamide (DMF):morpholine (Morph) = 0.9:1 was added, with 15 mL of DMF and 16.7 mL of Morph. The first deprotection was performed for 28 min, followed by a second deprotection for 28 min. After deprotection, the solution was washed 6 times with DMF and 1 time with DCM.

[0051] 1.3 Grafting the first amino acid After removing DCM by filtration, 3 molar equivalents of Fmoc-Leu-OH, 3 molar equivalents of 1-hydroxybenzotriazole (HOBT), and 5 molar equivalents of dimethylformamide (DMF) were added sequentially to the resin to dissolve them. The solution was then sonicated until clear. 1.4 mL of 10 molar equivalents of N,N'-diisopropylcarbodiimide (DIC) was added at 0°C, and the reaction was allowed to proceed for 9 min. After the reaction was complete, the resulting mixture was added to a peptide solid-phase synthesis tube, topped with DMF, and reacted for 1.5 h.

[0052] 1.4 End capping After the first amino acid was added, the resin was washed three times with DMF and once with DCM. Then, pyridine (Py):acetic anhydride (Ac2O) = 0.9:1 was added, with 15 mL of Py and 16.7 mL of Ac2O. The reaction was allowed to proceed for 2 hours. After the reaction was completed, the resin was washed five times with DMF and DCM alternately.

[0053] 1.5Fmoc group removal Add DMF: Morph = 0.9:1 to deprotect the product, 25 minutes at a time, for two cycles.

[0054] 1.6 Subsequent amino acid condensation Weigh out 3 molar equivalents of amino acids, 3 molar equivalents of HOBT, and 5 molar equivalents of DMF, dissolve them, sonicate until clear, add 1.4 mL of 10 molar equivalents of DIC at 0℃, and react for 9 min. After the reaction is complete, add the good mixture to a peptide solid-phase synthesis tube, fill with DMF, and react for 1.5 h.

[0055] 1.7 Repeat the cleaning steps After condensation, the reaction tube was cleaned sequentially with DMF and methanol to remove residual activator.

[0056] 1.8 Sequence Stepwise Assembly Repeat steps 1.5-1.7, sequentially linking Fmoc-Trp-OH, Fmoc-Arg-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Arg-OH to the resin support from the C-terminus to the N-terminus until peptide chain synthesis is complete.

[0057] 1.9 Terminal deprotection and pyrolysis After linking the last amino acid, terminal Fmoc removal was performed, followed by two condensations with methanol. The resin was then transferred to a flask, and a mixture of trifluoroacetic acid (TFA):triisopropylsilane (TIPS):water = 95:2.5:2.5 was added, with 19 mL of TFA and 0.5 mL of both TIPS and water, for lysis.

[0058] 1.10 recrystallization After lysis, the filtrate is collected and rotary evaporated until only a small amount of liquid remains. An appropriate amount of ice-crystal ether is added for recrystallization. Ice-crystal ether is added during sonication. After sonication, the mixture is allowed to stand, the supernatant is removed, and ice-crystal ether is added again for a second recrystallization. The supernatant is removed, the precipitate is collected, and vacuum is applied to obtain the crude peptide.

[0059] 1.11 HPLC Purification Procedure 1) Dissolve 150 mg of crude peptide in 2-5 mL of 50% acetonitrile-water solution and sonicate briefly (2 min) to aid dissolution; 2) The solution obtained by filtration using a 0.45 μm filter membrane; 3) Analytical purity: Inject 3 μL of the sample into the analytical HPLC system, using water / acetonitrile as the mobile phase, and perform gradient elution for 30 min. The initial ratio is 95% water / 5% acetonitrile, and the final ratio is 5% water / 95% acetonitrile. Equilibrate for 5 min before injection. 4) Preparation and purification: The sample was subjected to preparative HPLC with an equilibration time of 10 min, an initial gradient of 95% water / 5% acetonitrile, an ending gradient of 25% water / 75% acetonitrile, and a gradient time of 40 min. The target peak was then collected. 5) Purity and molecular weight identification: The collected samples were subjected to HPLC purity detection and mass spectrometry (MS) analysis.

[0060] 1.12 Freeze-drying treatment The purified peptide solution is freeze-dried to obtain the finished antimicrobial peptide product.

[0061] 1.13 Saving Method The obtained polypeptide is a white powder, which is sealed in packaging and stored at -20°C for a long time.

[0062] The amino acid sequence of the antimicrobial heptapeptide synthesized in this embodiment is RFIRWKL, and the HPLC analysis results are as follows: Figure 1 As shown, the mass spectrometry results are as follows: Figure 2 As shown (since the synthesized substance is unique, the mass spectrum is the same as in Example 1).

[0063] Example 3 1. Solid-phase synthesis of antimicrobial peptides 1.1 Resin Pretreatment Place 1.1g of rink resin in a reaction tube, add N,N-dimethylformamide (DMF): dichloromethane (DCM) in a ratio of 1.1:9.1, with 3mL of DMF and 24.8mL of DCM, and shake thoroughly on a shaker for 35 minutes to allow the resin to swell.

[0064] 1.2 Deprotection After filtering out the DMF:DCM mixed solution, add N,N-dimethylformamide (DMF):morpholine (Morph) in a ratio of 1:1.1, with 15 mL of DMF and 16.5 mL of Morph. Perform the first deprotection for 26 min, followed by the second deprotection for 26 min. After deprotection, wash 6 times with DMF and once with DCM.

[0065] 1.3 Grafting the first amino acid After removing DCM by filtration, 3 molar equivalents of Fmoc-Leu-OH, 3 molar equivalents of 1-hydroxybenzotriazole (HOBT), and 5 molar equivalents of dimethylformamide (DMF) were added sequentially to the resin to dissolve them. The solution was then sonicated until clear. 1.7 mL of 10 molar equivalents of N,N'-diisopropylcarbodiimide (DIC) was added at 0°C, and the reaction was allowed to proceed for 11 min. After the reaction was complete, the resulting mixture was added to a peptide solid-phase synthesis tube, which was then filled with DMF and allowed to react for 2.5 h.

[0066] 1.4 End capping After the first amino acid was added, the resin was washed three times with DMF and once with DCM. Then, pyridine (Py):acetic anhydride (Ac2O) was added in a ratio of 1:0.9, with 15 mL of Py and 13.5 mL of Ac2O. The reaction was allowed to proceed for 2 hours. After the reaction was completed, the resin was washed five times with alternating amounts of DMF and DCM.

[0067] 1.5Fmoc group removal Add DMF: Morph=1:0.9 to deprotect the product, 27 minutes at a time, for two cycles.

[0068] 1.6 Subsequent amino acid condensation Weigh out 3 molar equivalents of amino acids, 3 molar equivalents of HOBT, and 5 molar equivalents of DMF, dissolve them, sonicate until clear, add 1.7 mL of 10 molar equivalents of DIC at 0℃, and react for 11 min. After the reaction is complete, add the good mixture to a peptide solid-phase synthesis tube, fill with DMF, and react for 2.5 h.

[0069] 1.7 Repeat the cleaning steps After condensation, the reaction tube was cleaned sequentially with DMF and methanol to remove residual activator.

[0070] 1.8 Sequence Stepwise Assembly Repeat steps 1.5-1.7, sequentially linking Fmoc-Trp-OH, Fmoc-Arg-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Arg-OH to the resin support from the C-terminus to the N-terminus until peptide chain synthesis is complete.

[0071] 1.9 Terminal deprotection and pyrolysis After linking the last amino acid, terminal Fmoc removal was performed, followed by two condensations with methanol. The resin was then transferred to a flask, and a mixture of trifluoroacetic acid (TFA):triisopropylsilane (TIPS):water = 95:2.5:2.5 was added. The volume of TFA was 19 mL, and the volumes of TIPS and water were 0.5 mL each. The mixture was then lysed for 2 h.

[0072] 1.10 recrystallization After lysis, the filtrate is collected and rotary evaporated until only a small amount of liquid remains. An appropriate amount of ice-crystal ether is added for recrystallization. Ice-crystal ether is added during sonication. After sonication, the mixture is allowed to stand, the supernatant is removed, and ice-crystal ether is added again for a second recrystallization. The supernatant is removed, the precipitate is collected, and vacuum is applied to obtain the crude peptide.

[0073] 1.11 HPLC Purification Procedure 1) Dissolve 250 mg of crude peptide in 2-5 mL of 50% acetonitrile-water solution and sonicate briefly (2 min) to aid dissolution; 2) The solution obtained by filtration using a 0.45 μm filter membrane; 3) Analytical purity: Inject 3 μL of the sample into the analytical HPLC system, using water / acetonitrile as the mobile phase, and perform gradient elution for 30 min. The initial ratio is 95% water / 5% acetonitrile, and the final ratio is 5% water / 95% acetonitrile. Equilibrate for 5 min before injection. 4) Preparation and purification: The sample was subjected to preparative HPLC with an equilibration time of 10 min, an initial gradient of 95% water / 5% acetonitrile, an ending gradient of 25% water / 75% acetonitrile, and a gradient time of 40 min. The target peak was then collected. 5) Purity and molecular weight identification: The collected samples were subjected to HPLC purity detection and mass spectrometry (MS) analysis.

[0074] 1.12 Freeze-drying treatment The purified peptide solution is freeze-dried to obtain the finished antimicrobial peptide product.

[0075] 1.13 Saving Method The obtained polypeptide is a white powder, which is sealed in packaging and stored at -20°C for a long time.

[0076] The amino acid sequence of the antimicrobial heptapeptide synthesized in this embodiment is RFIRWKL, and the HPLC analysis results are as follows: Figure 1 As shown, the mass spectrometry results are as follows: Figure 2 As shown. (Since the synthesized substance is unique, the mass spectrum is the same as in Example 1.) Test Example 1: Detection of antimicrobial activity of antimicrobial peptides.

[0077] The antimicrobial performance of the antimicrobial heptapeptide prepared in Example 1 was evaluated using a 96-well plate assay. All strains used were purchased from the American Type Culture Collection (ATCC) standard strain library.

[0078] The experimental steps are as follows: 1. Preparation of inoculum Escherichia coli and Staphylococcus aureus were inoculated onto sterile LB solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd., catalog number: HB0128) and incubated overnight at 37°C for 12 hours. Escherichia coli and Staphylococcus aureus were then inoculated onto LB liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd., catalog number: HB0128) and incubated at 37°C with shaking at 170 rpm for 18 hours.

[0079] 2. Preparation and inoculation of antimicrobial peptide gradients (1) The antimicrobial peptide was prepared into a stock solution with a concentration of 1000 μM using PBS (phosphate buffer), and then diluted to prepare 6 working solutions with concentrations of 300, 250, 200, 150, 100 and 50 μM. Each working solution was repeated 6 times, for a total of 36 working solutions, each 100 μL.

[0080] (2) Dilute the Escherichia coli liquid culture medium to 1×10⁻⁶. 3 CFU / mL, divided into 36 portions, each 100μL; (3) Dilute Staphylococcus aureus liquid culture medium to 1×10 3 CFU / mL, divided into 36 portions, each 100μL.

[0081] Mix the 36 working solutions in (1) with the 36 diluted E. coli culture solutions in (2) to obtain 6 groups of antimicrobial peptide concentrations (μM) of 25, 50, 75, 100, 125 and 150, for a total of 36 mixed solutions (E. coli).

[0082] The 36 working solutions from (1) were mixed with the 36 diluted Staphylococcus aureus culture solutions from (2) to obtain 6 groups of antimicrobial peptide concentrations (μM) of 25, 50, 75, 100, 125 and 150, for a total of 36 mixed solutions (Staphylococcus aureus). 72 mixed solutions were sealed with sealing film, placed in sealed bags, and incubated at 37℃ and 120rpm for 18h.

[0083] 3. Compare settings Add 100 μL of LB liquid culture medium to wells A1-A12 as a blank control, for a total of 12 aliquots, 100 μL per aliquot.

[0084] After sealing, the plate was sealed with sealing film, placed in a sealed bag, and incubated at 37°C and 120 rpm for 18 hours.

[0085] 4. OD value detection and MIC determination After cultivation, the OD600 values ​​of 72 mixed solutions and 12 blank control groups were measured using an ELISA reader. The concentration of the antimicrobial peptide corresponding to the lowest OD600 value is the minimum inhibitory concentration (MIC) of the strain. Table 1 shows the minimum inhibitory concentration (MIC, unit: μM) of the antimicrobial heptapeptide described in this invention against the target strain.

[0086] Table 1. Calculation results of antimicrobial peptide MIC

[0087] The smaller the MIC value, the stronger the inhibitory effect of the antimicrobial peptide on the strain. As shown in Table 1, the antimicrobial peptide described in this invention has antibacterial effects against both Gram-positive and Gram-negative bacteria, exhibiting good broad-spectrum antimicrobial properties compared to short peptides with the same number of amino acids.

[0088] Test Example 2: Biocompatibility test of antimicrobial peptides.

[0089] To evaluate whether the antimicrobial peptide in Example 1 has a strong killing effect on normal cells at an effective bactericidal concentration.

[0090] 1. Cell plating A total of 84 wells (A1-G12) of a 96-well plate were seeded with normal mammalian cells (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences, HFF-1) at a cell density of 5 × 10⁻⁶. 3 Cells per well were added to cell culture medium and incubated at 37 ℃ in a 5% CO2 incubator for 24 hours. The cell culture medium consisted of 90% DMEM (high glucose medium) + 10% FBS (fetal bovine serum) + 1% PS (penicillin-streptomycin mixture). 2. Drug administration The yellow, nutrient-free culture medium in each well was aspirated, and fresh cell culture medium was added. The 84 wells of cultured animal cells were divided into an experimental group (72 wells) and a control group (12 wells). Different concentrations of antimicrobial peptides were added to each well of the experimental group (100, 50, 25, 12.5, 6.2, and 3.1 μM), for a total of 6 groups, with 12 wells in each group. The control group (12 wells) was filled with culture medium but without antimicrobial peptides. The cells were cultured for another 24 hours. 3. Colorimetric detection After 24 hours of culture, a total of 84 wells were prepared (experimental group + blank group). 10 μL of MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) solution was added to each well and incubated in the dark for 4 hours. The yellow, nutrient-free culture medium and MTT in each well were removed, and 100 μL of LDMSO (dimethyl sulfoxide) solution was added to each well. The incubation was carried out for 15 minutes, and the absorbance was measured at 490 nm using a microplate reader. 4. Cell viability calculation and results

[0091] Figure 3 The diagram shows the biocompatibility results of the antimicrobial heptapeptide at various concentrations in Example 1. At a concentration of 50 μM, the cell viability was above 90%. This result indicates that the antimicrobial peptide of the present invention has relatively good biocompatibility.

[0092] In summary, the antimicrobial peptide of this invention exhibits significant in vitro antibacterial activity based on its unique amino acid composition and sequence. Of particular note is that its cytotoxicity remains low even at high concentrations. Therefore, this antimicrobial heptapeptide is suitable for preparing drugs to treat infectious diseases such as bacterial pneumonia and sepsis.

[0093] The broad-spectrum antibacterial activity of the antimicrobial heptapeptide of this invention stems from its mechanism of action in disrupting bacterial cell membranes. The antimicrobial heptapeptide molecule carries a positive charge and can adsorb onto the negatively charged bacterial cell membrane through electrostatic interactions. When the concentration of the antimicrobial heptapeptide adsorbed on the membrane surface accumulates to a critical threshold, its inherent amphiphilic structure causes the polypeptide molecule to insert into the phospholipid bilayer of the cell membrane, where it self-assembles to form transmembrane channels, ultimately leading to membrane disintegration and bacterial death. This mode of action enables it to effectively kill a variety of pathogens.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also within the protection scope of the present invention.

Claims

1. An antimicrobial heptapeptide, characterized in that, The amino acid sequence of the antimicrobial heptapeptide is RFIRWKL.

2. The antimicrobial heptapeptide according to claim 1, characterized in that, The preparation steps are as follows: S1: Add N,N-dimethylformamide and morpholine to the resin for a first deprotection, then filter out N,N-dimethylformamide and morpholine. Add the same amount of N,N-dimethylformamide and morpholine to the deprotected resin for a second deprotection, then filter out N,N-dimethylformamide and morpholine. Subsequently, wash the deprotected resin with N,N-dimethylformamide and dichloromethane respectively, and filter out the washing solvent after each wash. S2: Fmoc-Leu-OH, 1-hydroxybenzotriazole, and N,N-dimethylformamide were added sequentially to the washed resin and sonicated until clear. Then, N,N'-diisopropylcarbodiimide was added at 0°C for a single reaction. After the reaction was completed, N,N-dimethylformamide was added to the good mixture for a condensation reaction to obtain Fmoc-Leu-Resin. S3: Wash Fmoc-Leu-Resin in S2 with N,N-dimethylformamide and dichloromethane; then add pyridine and acetic anhydride for end-capping treatment, and after end-capping, wash alternately with N,N-dimethylformamide and dichloromethane. S4: Add N,N-dimethylformamide and morpholine to Fmoc-Leu-Resin after alternating washing in S3 for one deprotection, then filter out N,N-dimethylformamide and morpholine. Add the same amount of N,N-dimethylformamide and morpholine to the deprotected resin for a second deprotection, then filter out N,N-dimethylformamide and morpholine. S5: Weigh out the deprotected Fmoc-Leu-Resin, 1-hydroxybenzotriazole, and N,N-dimethylformamide from S4, dissolve them, sonicate until clear, and then add N,N'-diisopropylcarbodiimide at 0℃ for a first reaction; after the reaction is complete, add N,N-dimethylformamide to the good mixture for a condensation reaction to obtain Fmoc-Lys-Leu-Resin; S6: Repeat steps S4-S5, sequentially linking Fmoc-Trp-OH, Fmoc-Arg-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Arg-OH to the resin support from the C-terminus to the N-terminus until the peptide chain synthesis is complete; S7: After completing the last amino acid-Arg linking, the terminal Fmoc group is removed, then the mixture is condensed twice with methanol, and then transferred to a flask. Trifluoroacetic acid, triisopropylsilane and water are added, and the mixture is pyrolyzed at room temperature. S8: After lysis, collect the filtrate and rotary evaporate until trifluoroacetic acid is completely evaporated. Then add ice-crystal ether and recrystallize by sonication. After sonication, let stand, remove the supernatant, add ice-crystal ether again and recrystallize by sonication for a second time. Remove the supernatant, collect the precipitate, and vacuum to obtain crude peptide. S9: Purify and freeze-dry the crude peptide in S8 to obtain the antibacterial heptapeptide product.

3. The antimicrobial heptapeptide according to claim 2, characterized in that, The mass ratio of N,N-dimethylformamide to morpholine in S1 is: 0.9-1.1:1, the time for the first and second deprotection is 25-30 min; wash 6 times with N,N-dimethylformamide and wash once with dichloromethane.

4. The antimicrobial heptapeptide according to claim 2, characterized in that, The molar ratio of the starting resin, Fmoc-Leu-OH in S2, 1-hydroxybenzotriazole, the first addition of N,N-dimethylformamide and N,N'-diisopropylcarbodiimide was 1:3:3:5:

10. The reaction time was 9-11 min and the condensation reaction time was 1.5-2.5 h.

5. The antimicrobial heptapeptide according to claim 2, characterized in that, In S3, N,N-dimethylformamide was washed 3 times, dichloromethane was washed once, the mass ratio of pyridine to acetic anhydride was 0.9-1.1:1, the reaction time was 2 hours, and N,N-dimethylformamide and dichloromethane were washed alternately 5 times.

6. The antimicrobial heptapeptide according to claim 2, characterized in that, The mass ratio of N,N-dimethylformamide to morpholine in S4 is: The ratio is 0.9-1.1:1, and the time for both the first and second deprotection is 25-30 minutes.

7. The antimicrobial heptapeptide according to claim 2, characterized in that, The molar ratio of the starting resin, Fmoc-Leu-Resin after secondary deprotection in S5, 1-hydroxybenzotriazole, and the first addition of N,N-dimethylformamide and N,N'-diisopropylcarbodiimide was 1:3:3:5:

10. The reaction time was 9-11 min, and the condensation reaction time was 1.5-2.5 h.

8. The antimicrobial heptapeptide according to claim 2, characterized in that, The mass ratio of trifluoroacetic acid (TFA), triisopropylsilane, and water in S7 is 95:2.5:2.5, and the pyrolysis time is 2 hours.

9. The use of an antimicrobial heptapeptide as described in claims 1-8 in the preparation of a product that inhibits bacterial growth.

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