Preparation method of cellulose-based cationic antibacterial peptide

Abstract

The invention discloses a preparation method of cellulose-based cationic antibacterial peptide, and relates to a preparation method of antibacterial peptide. The invention aims to solve the technical problems that the existing antibacterial peptide preparation is easy to degrade, poor in stability, low in probability of contact with pathogenic bacteria, low in immobilization technology efficiency and uncontrollable in structure. The invention provides a preparation method of cellulose-based cationic antibacterial peptide, which is characterized in that polylysine polypeptide with alkynyl which is synthesized in advance and azidation modified cellulose are subjected to covalent coupling by utilizing high efficiency and specificity of click chemical reaction to form a stable cellulose-polypeptide brush composite structure, and the cellulose-polypeptide brush composite structure is used for preparing the cellulose-based cationic antibacterial peptide. And finally, activating the antibacterial activity of the cations through a deprotection reaction. The antibacterial peptide CP-NH3 prepared by the method disclosed by the invention has extremely strong inhibition capability on spore germination of colletotrichum gloeosporioides, the germination rate can be reduced to 3.3% or below, and the antibacterial peptide CP-NH3 is obviously superior to a physical mixed antibacterial agent.

Description

Technical Field

[0001] This invention relates to a method for preparing an antimicrobial peptide. Background Technology

[0002] Currently, chemical control is the primary method for plant disease prevention and control, but its overuse leads to increasingly prominent problems such as environmental pollution and pathogen resistance. Cationic antimicrobial peptides have attracted widespread attention due to their low tendency to induce resistance. However, traditional free antimicrobial peptides suffer from drawbacks such as easy degradation by proteases, short retention time on plant surfaces, and low contact efficiency with pathogens, limiting their agricultural applications. To address these issues, existing technologies have proposed several strategies for immobilizing antimicrobial peptides. Patent CN114805821A discloses a method for grafting linear antimicrobial peptides onto cellulose via amidation, but its grafting efficiency and controllability need improvement, and it primarily focuses on the hemolytic problem in the medical field. Patents CN120441916A and CN105641734A employ physical blending and metal ion loading strategies, respectively, without addressing the chemical grafting of antimicrobial peptides.

[0003] Therefore, there is an urgent need in the field for a novel method that can efficiently, stably, and densely immobilize antimicrobial peptides on biomass carriers and significantly enhance their antifungal activity, especially against plant pathogenic fungi. This invention is based on this need. Summary of the Invention

[0004] The present invention aims to address the technical problems of current antimicrobial peptide preparations, such as easy degradation, poor stability, low probability of contact with pathogens, low efficiency of immobilization technology, and uncontrollable structure, and provides a method for preparing cellulose-based cationic antimicrobial peptides.

[0005] The preparation method of the cellulose-based cationic antimicrobial peptide of the present invention is carried out according to the following steps, such as... Figure 1 As shown:

[0006] I. Preparation of acetylene-containing polypeptides (PP):

[0007] ① Dissolve N(e)-Boc-L-lysine (P1) and α-pinene in THF, place in an ice-water bath and purge with nitrogen gas for 10-15 minutes in a sealed environment to obtain the first solution; dissolve triphosgene in THF and slowly add it dropwise to the first solution in the ice-water bath using a dropping funnel, purging with nitrogen gas throughout the dropping process. Remove the ice-water bath after dropping is complete, and then react in a nitrogen atmosphere at 40-60℃ for 1-10 hours. Then, remove the solvent by rotary evaporation and dissolve in ethyl acetate.

[0008] The molar ratio of N(e)-Boc-L-lysine, α-pinene, and triphosgene is (2~5):6:1;

[0009] ②After adding ice water, shake it first and then let it stand to separate into layers. The water will be on the bottom layer. Drain the water from the bottom layer and keep the top layer.

[0010] The volume ratio of the ice water to the ethyl acetate in ① is 1:(3~3.5);

[0011] ③ Repeat step ② twice;

[0012] ④ After adding saturated ice-salt water, shake it first and then let it stand to separate into layers. Drain the water from the bottom layer and keep the top layer.

[0013] The volume ratio of the saturated ice-salt water to the ethyl acetate in ① is 1:(3~3.5);

[0014] ⑤ Repeat step ④ twice, then add anhydrous MgSO4 to the retained upper layer for drying, then filter out the MgSO4, and remove ethyl acetate by rotary evaporation;

[0015] The mass ratio of the anhydrous MgSO4 to N(e)-Boc-L-lysine in ① is (1.6~1.7):1;

[0016] ⑥ The product obtained by rotary evaporation is dissolved in ethyl acetate, then placed in n-hexane to settle naturally, and the precipitate is collected by filtration;

[0017] The volume ratio of ethyl acetate to n-hexane is 1:(10~11);

[0018] ⑦ Repeat step ⑥ twice. After vacuum drying, a white powder product P2 is obtained. P2 is dissolved in DMF with propargylamine and reacted at room temperature for 24-25 hours. The product is then placed in a 7 kDa dialysis bag and dialyzed in pure water for 48-50 hours. The water is changed 6 times during the dialysis process. The product is then freeze-dried in a freeze dryer to obtain a white flocculent solid product PP.

[0019] The mass ratio of P2 to propargylamine is (76~77):1;

[0020] The mass ratio of P2 to the volume ratio of DMF is (20g~21g):1L;

[0021] The volume ratio of DMF to pure water is 1:(100~110);

[0022] II. Preparation of cellulose azide (N3-6-Cell):

[0023] ① Add cellulose to DMAc and stir at 100℃~130℃ for 1h~5h. Then cool the mixture to 50℃~80℃, add LiCl and stir at 50℃~80℃ for 2h~5h. Then cool to -40℃~-20℃ and keep warm for 2h~5h. Then heat to 50℃~80℃ and keep warm for 2h~5h to completely dissolve the cellulose and form a bright yellow viscous cellulose solution.

[0024] The mass ratio of cellulose to LiCl is 1:(4~10);

[0025] The mass ratio of cellulose to DMAc is 1 g:(45 mL~50 mL);

[0026] ② Triethylamine was added to the bright yellow viscous cellulose solution prepared in ① at room temperature and under a nitrogen atmosphere. The mixture was then stirred at 0℃ for 1-10 h to obtain a second solution. p-Toluenesulfonyl chloride solution was added to DMAc and then slowly added to the second solution using a constant pressure separatory funnel. The second solution was kept at 0℃ throughout the addition process. The mixture was then stirred at 20℃-60℃ under a nitrogen atmosphere for 20-30 h. The mixture was then slowly poured into cold water at 4℃-5℃ and mixed. The precipitate was filtered and retained. The product was washed successively with distilled water and anhydrous ethanol. The product was then vacuum dried at room temperature to obtain Tos-6-Cell.

[0027] The ratio of cellulose, triethylamine, and p-toluenesulfonyl chloride in the bright yellow viscous cellulose solution is 5g:(5mL~20mL):(8g~10g);

[0028] The mass ratio of p-toluenesulfonyl chloride to DMAc is 1 g:(2.4 mL~3 mL);

[0029] The volume ratio of DMAc to cold water at 4℃~5℃ is 1:(50~60);

[0030] ③ Dissolve Tos-6-Cell and NaN3 together in DMAc and react under nitrogen atmosphere at 50℃~80℃ for 20h~30h; then add to ice water and mix to form a precipitate. Filter to retain the precipitate, wash it with distilled water and anhydrous ethanol in sequence, and then dry it under vacuum to obtain N3-6-Cell.

[0031] The mass ratio of Tos-6-Cell to NaN3 is 1 (1~10);

[0032] The mass ratio of Tos-6-Cell to DMAc is 1 g:(3.5 mL~4 mL);

[0033] The volume ratio of DMAc to ice water is 1:(30~35);

[0034] III. Preparation of cellulose-based polypeptide brush CP-Boc: N3-6-Cell was dissolved in DMF, then PP prepared in step one was added, followed by cuprous bromide, pentamethyldiethylenetriamine and copper wire that had been soaked in hydrochloric acid for 5 min. The reaction was carried out under nitrogen protection at 50℃~70℃ for 20h~30h. After the reaction was completed, the product was placed in a 7kDa dialysis bag and dialyzed in pure water for 24h~30h. The water was changed 6 times during the dialysis process. Finally, the powdered solid product CP-Boc was obtained by freeze drying.

[0035] The mass ratio of N3-6-Cell, PP, cuprous bromide and pentamethyldiethylenetriamine is (5~20):(120~170):1:(5~15);

[0036] The volume ratio of DMF to pure water is 1:(200~210);

[0037] The mass ratio of N3-6-Cell to DMF is (4g~5g):1L;

[0038] IV. Preparation of cellulose-based polypeptide brush CP-NH3: Trifluoroacetic acid was slowly added dropwise to CP-Boc under stirring at room temperature. The mixture was stirred at room temperature for 2-2.5 hours. Then, the trifluoroacetic acid was removed by blowing air to obtain a viscous liquid. The viscous liquid was dissolved in methanol and then added to diethyl ether to form a precipitate of cellulose-based polypeptide brush CP-NH3 with the N-Boc protecting group removed. The solid was collected after centrifugation and dried under vacuum at 30-40°C for 24-25 hours to obtain a white flocculent solid product CP-NH3.

[0039] The mass ratio of CP-Boc to the volume ratio of trifluoroacetic acid is (20g~100g):1L;

[0040] The volume ratio of trifluoroacetic acid, methanol and diethyl ether is (1~10):1:(20~50).

[0041] This invention provides a method for preparing cellulose-based cationic antimicrobial peptides. The core of this method lies in utilizing the high efficiency and specificity of click chemistry to covalently couple a pre-synthesized alkyne-containing polylysine peptide with azidated cellulose to form a stable "cellulose-peptide brush" composite structure. Finally, the cationic antimicrobial activity is activated through a deprotection reaction.

[0042] The beneficial effects of this invention are as follows:

[0043] 1. This invention is the first to utilize "alkyne-azide" click chemistry to construct cellulose-based cationic polypeptide brushes. The reaction is highly efficient, the conditions are mild, and the product structure is well-defined, which has significant advantages over traditional methods such as amidation.

[0044] 2. The antimicrobial peptide CP-NH3 prepared in this invention has a strong inhibitory effect on the germination of anthrax spores, and the germination rate can be reduced to below 3.3%, which is significantly better than physical mixed antimicrobial agents;

[0045] 3. The high-density cationic polypeptide brush in this invention can efficiently destroy the cell membrane structure of fungi, causing their contents to leak out, thereby killing the fungi, and is less likely to produce drug resistance.

[0046] 4. This invention uses renewable biomass cellulose as a carrier, avoiding the use of harmful substances such as heavy metals, which meets the development needs of green agriculture. Attached Figure Description

[0047] Figure 1 This is a flowchart of the preparation method of the cellulose-based cationic antimicrobial peptide of the present invention;

[0048] Figure 2 This is the 1H NMR spectrum of the PP prepared in step one of Experiment 1;

[0049] Figure 3 The infrared spectra of Tos-6-Cell and N3-6-Cell prepared in step two of Experiment 1 are shown.

[0050] Figure 4 This is the 1H NMR spectrum of CP-NH3 prepared in Experiment 1;

[0051] Figure 5 This is a graph showing the spore germination rate of anthrax strains after treatment with different concentrations of materials in Experiment 2 (1);

[0052] Figure 6 This is a comparison diagram of the penetration test results in Experiment 2 (2);

[0053] Figure 7 The image shows a comparison of scanning electron microscopy images of anthrax spores after treatment in Experiment 2 (1). Detailed Implementation

[0054] Specific Implementation Method 1: This implementation method describes a method for preparing a cellulose-based cationic antimicrobial peptide, which is carried out according to the following steps:

[0055] I. Preparation of polypeptides with alkyne groups:

[0056] ① Dissolve N(e)-Boc-L-lysine and α-pinene in THF, place in an ice-water bath and purge with nitrogen for 10-15 minutes in a sealed environment to obtain the first solution; dissolve triphosgene in THF and slowly add it dropwise to the first solution in the ice-water bath using a dropping funnel, purging with nitrogen throughout the dropping process. Remove the ice-water bath after dropping is complete, and then react in a nitrogen atmosphere at 40-60℃ for 1-10 hours. Then, remove the solvent by rotary evaporation and dissolve in ethyl acetate.

[0057] The molar ratio of N(e)-Boc-L-lysine, α-pinene, and triphosgene is (2~5):6:1;

[0058] ② Add ice water, shake it first and then let it stand to separate into layers. The water will be on the bottom layer. Drain the water from the bottom layer and keep the top layer.

[0059] The volume ratio of the ice water to the ethyl acetate in ① is 1:(3~3.5);

[0060] ③ Repeat step ② twice;

[0061] ④ After adding saturated ice-salt water, shake it first and then let it stand to separate into layers. Drain the water from the bottom layer and keep the top layer.

[0062] The volume ratio of the saturated ice-salt water to the ethyl acetate in ① is 1:(3~3.5);

[0063] ⑤ Repeat step ④ twice, then add anhydrous MgSO4 to the retained upper layer for drying, then filter out the MgSO4, and remove ethyl acetate by rotary evaporation;

[0064] The mass ratio of the anhydrous MgSO4 to N(e)-Boc-L-lysine in ① is (1.6~1.7):1;

[0065] ⑥ The product obtained by rotary evaporation is dissolved in ethyl acetate, then placed in n-hexane to settle naturally, and the precipitate is collected by filtration;

[0066] The volume ratio of ethyl acetate to n-hexane is 1:(10~11);

[0067] ⑦ Repeat step ⑥ twice. After vacuum drying, a white powder product P2 is obtained. P2 is dissolved in DMF with propargylamine and reacted at room temperature for 24-25 hours. The product is then placed in a 7 kDa dialysis bag and dialyzed in pure water for 48-50 hours. The water is changed 6 times during the dialysis process. The product is then freeze-dried in a freeze dryer to obtain a white flocculent solid product PP.

[0068] The mass ratio of P2 to propargylamine is (76~77):1;

[0069] The mass ratio of P2 to the volume ratio of DMF is (20g~21g):1L;

[0070] The volume ratio of DMF to pure water is 1:(100~110);

[0071] II. Preparation of cellulose azide:

[0072] ① Add cellulose to DMAc and stir at 100℃~130℃ for 1h~5h. Then cool the mixture to 50℃~80℃, add LiCl and stir at 50℃~80℃ for 2h~5h. Then cool to -40℃~-20℃ and keep warm for 2h~5h. Then heat to 50℃~80℃ and keep warm for 2h~5h to completely dissolve the cellulose and form a bright yellow viscous cellulose solution.

[0073] The mass ratio of cellulose to LiCl is 1:(4~10);

[0074] The mass ratio of cellulose to DMAc is 1 g:(45 mL~50 mL);

[0075] ② Triethylamine was added to the bright yellow viscous cellulose solution prepared in ① at room temperature and under a nitrogen atmosphere. The mixture was then stirred at 0℃ for 1-10 h to obtain a second solution. p-Toluenesulfonyl chloride solution was added to DMAc and then slowly added to the second solution using a constant pressure separatory funnel. The second solution was kept at 0℃ throughout the addition process. The mixture was then stirred at 20℃-60℃ under a nitrogen atmosphere for 20-30 h. The mixture was then slowly poured into cold water at 4℃-5℃ and mixed. The precipitate was filtered and retained. The product was washed successively with distilled water and anhydrous ethanol. The product was then vacuum dried at room temperature to obtain Tos-6-Cell.

[0076] The ratio of cellulose, triethylamine, and p-toluenesulfonyl chloride in the bright yellow viscous cellulose solution is 5g:(5mL~20mL):(8g~10g);

[0077] The mass ratio of p-toluenesulfonyl chloride to DMAc is 1 g:(2.4 mL~3 mL);

[0078] The volume ratio of DMAc to cold water at 4℃~5℃ is 1:(50~60);

[0079] ③ Dissolve Tos-6-Cell and NaN3 together in DMAc and react under nitrogen atmosphere at 50℃~80℃ for 20h~30h; then add to ice water and mix to form a precipitate. Filter to retain the precipitate, wash it with distilled water and anhydrous ethanol in sequence, and then dry it under vacuum to obtain N3-6-Cell.

[0080] The mass ratio of Tos-6-Cell to NaN3 is 1 (1~10);

[0081] The mass ratio of Tos-6-Cell to DMAc is 1 g:(3.5 mL~4 mL);

[0082] The volume ratio of DMAc to ice water is 1:(30~35);

[0083] III. Preparation of cellulose-based polypeptide brush CP-Boc: N3-6-Cell was dissolved in DMF, then PP prepared in step one was added, followed by cuprous bromide, pentamethyldiethylenetriamine and copper wire that had been soaked in hydrochloric acid for 5 min. The reaction was carried out under nitrogen protection at 50℃~70℃ for 20h~30h. After the reaction was completed, the product was placed in a 7kDa dialysis bag and dialyzed in pure water for 24h~30h. The water was changed 6 times during the dialysis process. Finally, the powdered solid product CP-Boc was obtained by freeze drying.

[0084] The mass ratio of N3-6-Cell, PP, cuprous bromide and pentamethyldiethylenetriamine is (5~20):(120~170):1:(5~15);

[0085] The volume ratio of DMF to pure water is 1:(200~210);

[0086] The mass ratio of N3-6-Cell to DMF is (4g~5g):1L;

[0087] IV. Preparation of cellulose-based polypeptide brush CP-NH3: Trifluoroacetic acid was slowly added dropwise to CP-Boc under stirring at room temperature. The mixture was stirred at room temperature for 2-2.5 hours. Then, the trifluoroacetic acid was removed by blowing air to obtain a viscous liquid. The viscous liquid was dissolved in methanol and then added to diethyl ether to form a precipitate of cellulose-based polypeptide brush CP-NH3 with the N-Boc protecting group removed. The solid was collected after centrifugation and dried under vacuum at 30-40°C for 24-25 hours to obtain a white flocculent solid product CP-NH3.

[0088] The mass ratio of CP-Boc to the volume ratio of trifluoroacetic acid is (20g~100g):1L;

[0089] The volume ratio of trifluoroacetic acid, methanol and diethyl ether is (1~10):1:(20~50).

[0090] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that: in step one ①, nitrogen gas is introduced into an ice-water bath and in a sealed environment for 10 minutes. Everything else is the same as in Specific Implementation Method One.

[0091] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 1 or 2 in that the mass ratio of N(e)-Boc-L-lysine to THF in step 1① is 1g:(16mL~17mL). Everything else is the same as in Specific Implementation Method 1 or 2.

[0092] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the mass ratio of triphosgene to THF in step one ① is 1g:(3mL~3.5mL). Everything else is the same as in Specific Implementation Methods One to Three.

[0093] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method Four in that the rotary evaporation temperature in step one ① is 50°C and the time is 1 hour. Everything else is the same as in Specific Implementation Method Four.

[0094] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Five in that the rotary evaporation temperature in step one (⑥) is 50°C and the time is 1 hour. Everything else is the same as in Specific Implementation Method Five.

[0095] The invention was verified using the following experiments:

[0096] Experiment 1: This experiment demonstrates a method for preparing a cellulose-based cationic antimicrobial peptide, which was carried out according to the following steps:

[0097] I. Preparation of polypeptides with alkyne groups:

[0098] ① Dissolve 12.3g of N(e)-Boc-L-lysine and 20.5mL of α-pinene in 200mL of THF, place in an ice-water bath and purge with nitrogen for 10min in a sealed environment to obtain the first solution; dissolve 6.5g of triphosgene in 20mL of THF, and then slowly add it dropwise to the first solution in the ice-water bath using a dropping funnel, purging with nitrogen throughout the dropping process. Remove the ice-water bath after the dropping is complete, and then react at 50℃ under a nitrogen atmosphere for 2h. Then, remove the solvent by rotary evaporation (50℃, 1h), and then dissolve in 100mL of ethyl acetate.

[0099] ② Add 33.3 mL of ice water, shake and let stand to separate the layers. The water will be in the bottom layer. Drain the water from the bottom layer and keep the top layer.

[0100] ③ Repeat step ② twice;

[0101] ④ After adding 33.3 mL of saturated ice-salt water, shake it first and then let it stand to separate into layers. Drain the water from the bottom layer and keep the top layer.

[0102] ⑤ Repeat step ④ twice, then add 20g of anhydrous MgSO4 to the retained upper layer and dry it. Then filter out the MgSO4 and remove the ethyl acetate by rotary evaporation.

[0103] ⑥ The product obtained by rotary evaporation (50℃, 1h) was dissolved in 20mL of ethyl acetate, and then placed in 200mL of n-hexane to settle naturally. The precipitate was collected by filtration.

[0104] ⑦ Repeat step ⑥ twice. After vacuum drying, a white powder product P2 is obtained. Dissolve 200 mg of P2 and 2.62 mg of propargylamine in 10 mL of DMF and react at room temperature for 24 h. The product is then placed in a 7 kDa dialysis bag and dialyzed in 1 L of pure water for 48 hours. The water is changed 6 times during the dialysis process. The product is then freeze-dried in a freeze dryer to obtain a white flocculent solid product PP.

[0105] II. Preparation of cellulose azide:

[0106] ① Add 5g of cellulose to 225mL of DMAc and stir at 120℃ for 2h. Then cool the mixture to 60℃, add 25g of LiCl and stir at 60℃ for 2h. Then cool to -24℃ and keep warm for 2.5h. Then heat to 60℃ and keep warm for 3h to completely dissolve the cellulose and form a bright yellow viscous cellulose solution.

[0107] ② At room temperature and under a nitrogen atmosphere, 11.88 mL of triethylamine was added to 200 g of the bright yellow viscous cellulose solution prepared in ①. The mixture was then stirred at 0 °C for 0.5 h to obtain a second solution. 8.14 g of p-toluenesulfonyl chloride solution was added to 20 mL of DMAc and then slowly added to the second solution using a constant pressure separatory funnel (taken 2 h). The second solution was kept at 0 °C throughout the addition process. The mixture was then stirred at 25 °C under a nitrogen atmosphere for 24 h. The mixture was then slowly poured into 1 L of cold water at 4 °C and mixed. The mixture was filtered and the precipitate was retained. The precipitate was washed successively with 1 L of distilled water and 500 mL of anhydrous ethanol. The product was dried under vacuum at room temperature to obtain Tos-6-Cell.

[0108] ③ Dissolve 4g of Tos-6-Cell and 4.68g of NaN3 together in 15mL of DMAc and react at 70℃ under nitrogen atmosphere for 24h; then add to 500mL of ice water and mix to form a precipitate. Filter to retain the precipitate, wash with 1L of distilled water and 500mL of anhydrous ethanol in sequence, and then vacuum dry to obtain N3-6-Cell;

[0109] III. Preparation of cellulose-based polypeptide brush CP-Boc: 20 mg of N3-6-Cell was dissolved in 5 mL of DMF, then 310 mg of PP prepared in step one was added, followed by 2 mg of cuprous bromide, 10 mg of pentamethyldiethylenetriamine, and copper wire (0.5 cm long and 0.1 cm in diameter) that had been soaked in hydrochloric acid for 5 min. The reaction was carried out under nitrogen protection at 60 °C for 24 h. After the reaction was completed, the mixture was placed in a 7 kDa dialysis bag and dialyzed in 1 L of pure water for 24 h. The water was changed 6 times during the dialysis process. Finally, the powdered solid product CP-Boc was obtained by freeze drying.

[0110] IV. Preparation of cellulose-based polypeptide brush CP-NH3: Under room temperature and stirring conditions, 2 mL of trifluoroacetic acid was slowly added dropwise to 40 mg of CP-Boc. The mixture was stirred at room temperature for 2 h, and then the trifluoroacetic acid was removed by blowing air to obtain a viscous liquid. The viscous liquid was dissolved in 1 mL of methanol and then added to 30 mL of diethyl ether to form a precipitate of cellulose-based polypeptide brush CP-NH3 with the N-Boc protecting group removed. After centrifugation, the solid was collected and dried under vacuum at 30 °C for 24 h to obtain a white flocculent solid product CP-NH3.

[0111] Figure 2 The image shows the 1H NMR spectrum of the PP prepared in step one of Experiment 1, revealing its structural formula.

[0112] Figure 3 The images show the infrared spectra of Tos-6-Cell and N3-6-Cell prepared in step two of Experiment 1.

[0113] Figure 4 This is the 1H NMR spectrum of CP-NH3 prepared in Experiment 1, which shows its structural formula.

[0114] Experiment 2: This experiment demonstrates the application of the cellulose-based cationic antimicrobial peptide CP-NH3 prepared in Experiment 1 in the field of antifungal treatment.

[0115] (1) Anthrax spore germination experiment: Under a microscope, the spore germination rate of anthrax strains under different concentrations of material treatment was observed every 3 hours: Experimental group: 1 mL of material solution (solvent is water, solute is CP-NH3 prepared in Experiment 1, concentration see Figure 5 ) and 100 μL of anthrax spore suspension (CFU=5×10) 7 mL -1 Mix the ingredients and shake to mix at 20°C for 30 min. After incubation, take 10 μL of the mixture and spread it on GB medium (20 g / L glucose, 5 g / L peptone, 15 g / L agar), then air dry it naturally. Place this GB medium in an incubator at 25°C and observe the spore germination rate under a microscope after different incubation times (see...). Figure 5Anthrax spores were collected from the remaining spore suspension by centrifugation, fixed with 2.5% glutaraldehyde, subjected to gradient dehydration, and dried at the CO2 critical point before spore morphology was observed using scanning electron microscopy. The control group was treated with 1 mL of pure water. Figure 7 As shown.

[0116] like Figure 5 As shown, when the concentration of CP-NH3 reached 187.5 µg / mL, the germination rate of its spores gradually decreased with the increase of material concentration. Until the concentration was 1500 µg / mL, the germination rate of anthracnose spores after CP-NH3 treatment was 2.56%, which was less than 3%. This indicates that CP-NH3 has a significant inhibitory effect on the germination of anthracnose spores.

[0117] like Figure 7 As shown in the SEM images, the spores in the control groups (a, b, and c) have plump morphologies and smooth cell membrane surfaces. In contrast, the cell membranes of the experimental groups (d, e, and f) ruptured, and the cell structures collapsed. This may be because the nanopesticide CP-NH3 inhibits fungi by disrupting their cell membranes.

[0118] (2) Penetration test: In the minimum nutrient medium (MM medium: 6g / L) -1 Sodium nitrate (NaNO3), 0.52g / L -1 Potassium chloride (KCl), 0.52 g / L -1 Magnesium sulfate heptahydrate (MgSO4·7H2O), 0.25 g / L -1 Anthrax spore suspension (5×10⁻⁶) treated with CP-NH₃ was inoculated onto cicada wings with potassium dihydrogen phosphate (KH₂PO₄). 6 mL -1 The treatment method was the same as (1) for the penetration test: 2 μL of spore suspension was dropped onto the cicada wings, and the cicada wings were carefully removed after 2 days. The wings were then incubated at room temperature for another 3 days to determine the penetration ability of the fungus. Compared with the control group (treated with 1 mL of pure water), the growth of colonies in the experimental group was significantly inhibited. Obviously, the material had a good inhibitory effect on the growth of anthracnose spore colonies (see Figure 6 a and b are control groups, c and d are experimental groups. a and c are cicada wings taken out after 2 days, and b and d are cicada wings that have been incubated at room temperature for another 3 days. The white glowing parts are bacterial colonies.

[0119] Experiment 3: To demonstrate the superiority of click chemical grafting, a comparative example was set up:

[0120] Comparative Example 1: Unmodified cellulose was physically mixed with free polylysine P1 (with a molecular weight equivalent to PP).

[0121] Comparative Example 2: Polylysine was grafted onto cellulose using an amidation method (EDC / NHS catalysis).

[0122] The inhibition rate of each sample against anthracnose spore germination was tested using the method described in Experiment 1. The results are shown in Table 1 below:

[0123] Table 1

[0124]

[0125] Conclusion: The click chemical grafting product of the present invention exhibits significantly better antifungal activity than physically mixed samples and traditional amidation grafted samples, demonstrating the inventiveness and outstanding technical effect of the present invention.

Claims

1. A method for preparing a cellulose-based cationic antimicrobial peptide, characterized in that... The preparation method is carried out according to the following steps: I. Preparation of polypeptides with alkyne groups: ① Dissolve N(e)-Boc-L-lysine and α-pinene in THF, place in an ice-water bath and purge with nitrogen for 10-15 minutes in a sealed environment to obtain the first solution; dissolve triphosgene in THF and slowly add it dropwise to the first solution in the ice-water bath using a dropping funnel, purging with nitrogen throughout the dropping process. Remove the ice-water bath after dropping is complete, and then react in a nitrogen atmosphere at 40-60℃ for 1-10 hours. Then, remove the solvent by rotary evaporation and dissolve in ethyl acetate. The molar ratio of N(e)-Boc-L-lysine, α-pinene, and triphosgene is (2~5):6:1; ② Add ice water, shake it first and then let it stand to separate into layers. The water will be on the bottom layer. Drain the water from the bottom layer and keep the top layer. The volume ratio of the ice water to the ethyl acetate in ① is 1:(3~3.5); ③ Repeat step ② twice; ④ After adding saturated ice-salt water, shake it first and then let it stand to separate into layers. Drain the water from the bottom layer and keep the top layer. The volume ratio of the saturated ice-salt water to the ethyl acetate in ① is 1:(3~3.5); ⑤ Repeat step ④ twice, then add anhydrous MgSO4 to the retained upper layer for drying, then filter out the MgSO4, and remove ethyl acetate by rotary evaporation; The mass ratio of the anhydrous MgSO4 to N(e)-Boc-L-lysine in ① is (1.6~1.7):1; ⑥ The product obtained by rotary evaporation is dissolved in ethyl acetate, then placed in n-hexane to settle naturally, and the precipitate is collected by filtration; The volume ratio of ethyl acetate to n-hexane is 1:(10~11); ⑦ Repeat step ⑥ twice. After vacuum drying, a white powder product P2 is obtained. P2 is dissolved in DMF with propargylamine and reacted at room temperature for 24-25 hours. The product is then placed in a 7 kDa dialysis bag and dialyzed in pure water for 48-50 hours. The water is changed 6 times during the dialysis process. The product is then freeze-dried in a freeze dryer to obtain a white flocculent solid product PP. The mass ratio of P2 to propargylamine is (76~77):1; The mass ratio of P2 to the volume ratio of DMF is (20g~21g):1L; The volume ratio of DMF to pure water is 1:(100~110); II. Preparation of cellulose azide: ① Add cellulose to DMAc and stir at 100℃~130℃ for 1h~5h. Then cool the mixture to 50℃~80℃, add LiCl and stir at 50℃~80℃ for 2h~5h. Then cool to -40℃~-20℃ and keep warm for 2h~5h. Then heat to 50℃~80℃ and keep warm for 2h~5h to completely dissolve the cellulose and form a bright yellow viscous cellulose solution. The mass ratio of cellulose to LiCl is 1:(4~10); The mass ratio of cellulose to DMAc is 1 g:(45 mL~50 mL); ② Triethylamine was added to the bright yellow viscous cellulose solution prepared in ① at room temperature and under a nitrogen atmosphere. The mixture was then stirred at 0℃ for 1-10 h to obtain a second solution. p-Toluenesulfonyl chloride solution was added to DMAc and then slowly added to the second solution using a constant pressure separatory funnel. The second solution was kept at 0℃ throughout the addition process. The mixture was then stirred at 20℃-60℃ under a nitrogen atmosphere for 20-30 h. The mixture was then slowly poured into cold water at 4℃-5℃ and mixed. The precipitate was filtered and retained. The product was washed successively with distilled water and anhydrous ethanol. The product was then vacuum dried at room temperature to obtain Tos-6-Cell. The ratio of cellulose, triethylamine, and p-toluenesulfonyl chloride in the bright yellow viscous cellulose solution is 5g:(5mL~20mL):(8g~10g); The mass ratio of p-toluenesulfonyl chloride to DMAc is 1 g:(2.4 mL~3 mL); The volume ratio of DMAc to cold water at 4℃~5℃ is 1:(50~60); ③ Dissolve Tos-6-Cell and NaN3 together in DMAc and react under nitrogen atmosphere at 50℃~80℃ for 20h~30h; then add to ice water and mix to form a precipitate. Filter to retain the precipitate, wash it with distilled water and anhydrous ethanol in sequence, and then dry it under vacuum to obtain N3-6-Cell. The mass ratio of Tos-6-Cell to NaN3 is 1 (1~10); The mass ratio of Tos-6-Cell to DMAc is 1 g:(3.5 mL~4 mL); The volume ratio of DMAc to ice water is 1:(30~35); III. Preparation of cellulose-based polypeptide brush CP-Boc: N3-6-Cell was dissolved in DMF, then PP prepared in step one was added, followed by cuprous bromide, pentamethyldiethylenetriamine and copper wire that had been soaked in hydrochloric acid for 5 min. The reaction was carried out under nitrogen protection at 50℃~70℃ for 20h~30h. After the reaction was completed, the product was placed in a 7kDa dialysis bag and dialyzed in pure water for 24h~30h. The water was changed 6 times during the dialysis process. Finally, the powdered solid product CP-Boc was obtained by freeze drying. The mass ratio of N3-6-Cell, PP, cuprous bromide and pentamethyldiethylenetriamine is (5~20):(120~170):1:(5~15); The volume ratio of DMF to pure water is 1:(200~210); The mass ratio of N3-6-Cell to DMF is (4g~5g):1L; IV. Preparation of cellulose-based polypeptide brush CP-NH3: Trifluoroacetic acid was slowly added dropwise to CP-Boc under stirring at room temperature. The mixture was stirred at room temperature for 2-2.5 hours. Then, the trifluoroacetic acid was removed by blowing air to obtain a viscous liquid. The viscous liquid was dissolved in methanol and then added to diethyl ether to form a precipitate of cellulose-based polypeptide brush CP-NH3 with the N-Boc protecting group removed. The solid was collected after centrifugation and dried under vacuum at 30-40°C for 24-25 hours to obtain a white flocculent solid product CP-NH3. The mass ratio of CP-Boc to the volume ratio of trifluoroacetic acid is (20g~100g):1L; The volume ratio of trifluoroacetic acid, methanol and diethyl ether is (1~10):1:(20~50).

2. The method for preparing a cellulose-based cationic antimicrobial peptide according to claim 1, characterized in that... In step 1①, nitrogen gas is introduced into the ice-water bath and in a closed environment for 10 minutes.

3. The method for preparing a cellulose-based cationic antimicrobial peptide according to claim 1, characterized in that... The mass ratio of N(e)-Boc-L-lysine to THF in step 1① is 1g:(16mL~17mL).

4. The method for preparing a cellulose-based cationic antimicrobial peptide according to claim 1, characterized in that... The mass ratio of triphosgene to THF mentioned in step 1① is 1g:(3mL~3.5mL).

5. The method for preparing a cellulose-based cationic antimicrobial peptide according to claim 1, characterized in that... The rotary evaporation described in step 1① is carried out at a temperature of 50℃ for 1 hour.

6. The method for preparing a cellulose-based cationic antimicrobial peptide according to claim 1, characterized in that... The rotary evaporation described in step 1, ⑥, is carried out at a temperature of 50°C for 1 hour.

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