Guanidyl chitosan derivative capable of destroying bacterial membrane as well as preparation method and application of guanidyl chitosan derivative

Abstract

The invention discloses a guanidyl chitosan derivative capable of destroying a bacterial membrane as well as a preparation method and application of the guanidyl chitosan derivative, and belongs to the field of antibacterial materials. The derivative is prepared through four steps of reaction: reacting N, N-di-Boc-S-methylisothiourea with ethanolamine to prepare OBG; then, the OBG and lipoic acid are esterified under the catalysis of DCC / DMAP to obtain LBG; the preparation method comprises the following steps: reacting chitosan with N-acetylcysteine under the action of EDCI / HOBT to prepare CSSH; finally, LBG is subjected to trifluoroacetic acid deprotection to generate LGa, LGa and CSSH react in a hydrochloric acid-triethanolamine buffer solution and DMF mixed solution, and the guanidyl chitosan derivative is obtained. The derivative can adsorb negatively charged bacteria through electrostatic interaction, tear and destroy a bacterial membrane structure, effectively kill methicillin-resistant staphylococcus aureus and pseudomonas aeruginosa, is suitable for the field of biological sterilization, and provides support for development of novel antibacterial materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial materials, and particularly relates to a guanidine-based chitosan derivative for destroying bacterial membranes, and a preparation method and application thereof. BACKGROUND

[0002] With the extensive use of antibiotics, the problem of bacterial drug resistance is becoming increasingly serious, and has become one of the major challenges faced by global public health. Developing new antibacterial materials, especially antibacterial materials effective against drug-resistant bacteria, has become a current research hotspot. Cationic polymers have shown great potential in the field of antibacterial materials due to their unique structure and properties, such as broad-spectrum antibacterial properties and difficulty in developing drug resistance. However, their antibacterial activity still needs to be improved. Therefore, obtaining cationic polymers with high sterilization effect and further studying their antibacterial mechanism are the purposes of the present application. SUMMARY

[0003] An object of embodiments of the present application is to solve at least the above problems and / or deficiencies, and to provide at least the advantages described later.

[0004] The purpose of the present application is to provide a guanidine-based chitosan derivative that can tear bacterial membranes and a preparation method thereof. The material has a guanidine group on its surface, can combine with bacteria with negative potential through electrostatic action, tear and damage the bacterial membrane, destroy the homeostasis in the bacteria, has strong sterilization effect, can be applied in the field of biological sterilization, and the preparation method adopted has the advantages of simple operation, mild reaction conditions and high yield.

[0005] To solve the technical problems of the present application, the technical scheme proposed is: A guanidine-based chitosan derivative for destroying bacterial membranes has the following structural formula: Formula I Preferably, in the preparation method of the guanidine-based chitosan derivative for destroying bacterial membranes, the following steps are included: 1) N,N-di-Boc-S-methyl isothiourea, ethanolamine, N,N-diisopropylethylamine and dichloromethane are reacted at room temperature, and after the reaction is completed, purification is performed to obtain white solid 2-(2,3-bis(tert-butoxycarbonyl) guanidino) ethanol OBG; 2) lipoic acid, 2-(2,3-bis(tert-butoxycarbonyl) guanidino) ethanol, 4-dimethylamino pyridine and dichloromethane are stirred and uniformly mixed in an ice bath, then [N,N-dicyclohexyl carbodiimide (DCC) is dissolved in dichloromethane and added dropwise, the ice bath is stirred for 1 hour, and then room temperature reaction is performed to obtain yellow oily product LBG, which has the following structure: 3) stirring chitosan and 1-hydroxybenzotriazole in deionized water, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-acetyl cysteine to react, to obtain CSSH, the structure of which is shown below: 4) dissolving LBG in dichloromethane, adding trifluoroacetic acid to deprotect to obtain LGa, then dissolving LGa and CSSH in a mixed solution of hydrochloric acid-triethanolamine buffer and N,N-dimethylformamide, adding 2-iodoacetamide to terminate the reaction after a period of time, to obtain the guanidyl chitosan derivative for destroying bacterial membranes shown in formula I; Preferably, in the preparation method of the guanidyl chitosan derivative for destroying bacterial membranes, the mass ratio of chitosan, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-acetyl cysteine in step 3) is 1:0.7:4:1.7, the pH is adjusted to 5.0 before the reaction, and the reaction is completed after stirring at room temperature for 3 h.

[0006] Preferably, in the preparation method of the guanidyl chitosan derivative for destroying bacterial membranes, the mixing ratio of N,N-di-Boc-S-methyl isothiourea, ethanolamine, N,N-diisopropylethylamine and dichloromethane in step 1) is 5.8 mmol: 11 mmol: 17 mmol: 10 mL.

[0007] Preferably, in the preparation method of the guanidyl chitosan derivative for destroying bacterial membranes, after the reaction in step 1) is completed, methyl mercaptan byproduct is removed by bubbling nitrogen for 1 h, and the crude product is purified by silica gel column chromatography, with the eluent being n-hexane and ethyl acetate in a volume ratio of 1:3, to obtain white solid 2-(2,3-bis(tert-butoxycarbonyl)guanidyl)ethanol.

[0008] Preferably, in the preparation method of the guanidyl chitosan derivative for destroying bacterial membranes, the mixing ratio of thioctic acid, 2-(2,3-bis(tert-butoxycarbonyl)guanidyl)ethanol, 4-dimethylaminopyridine and dichloromethane in step 2) is 7.3 mmol: 4.9 mmol: 7.4 mmol: 15 mL, The use ratio of N,N-dicyclohexyl carbodiimide dissolved in dichloromethane is 7.3 mmol: 10 mL; After stirring in an ice bath for 1 h, the reaction continues at room temperature for 24 h, after the reaction is completed, the insoluble solid is filtered, and the organic solvent is removed to obtain the crude product, which is purified by silica gel column chromatography, with the eluent being n-hexane and ethyl acetate in a volume ratio of 1:5, to obtain yellow oily product LBG.

[0009] Preferably, in the preparation method of the guanidyl chitosan derivative for destroying bacterial membrane, in step 4), 1.0 g of LBG is dissolved in 10 mL of dichloromethane, 10 mL of trifluoroacetic acid is added, and the reaction is carried out for 24 h to obtain LGa: 0.59 g of LGa and CSSH are dissolved in a mixed solution of pH 7.0, 0.03 mol / L hydrochloric acid-triethanolamine buffer and N,N-dimethylformamide, 2-iodoacetamide is added after stirring at 30 ℃ for 30 min to terminate the reaction.

[0010] The guanidyl chitosan derivative for destroying bacterial membrane is applied to the preparation of a bactericidal material.

[0011] Preferably, in the application, the bactericidal pH of the guanidyl chitosan derivative is 7.0, and the guanidyl chitosan derivative is used for killing methicillin-resistant Staphylococcus aureus MRSA and Pseudomonas aeruginosa P. aeruginosa .

[0012] The advantages and effects of the present application compared with the prior art are as follows: The bactericidal material containing guanidyl obtained by the present application can adsorb negatively charged bacterial microorganisms, and the cytoplasmic contents flow out by tearing the bacterial membrane, so as to achieve the effect of killing bacteria, and is a scarce and ideal bactericidal material. The preparation method adopted has simple reaction operation, mild conditions and high yield.

[0013] Other advantages, objects and features of the embodiments of the present application will be embodied partly through the following description, and will be understood by those skilled in the art through research and practice of the embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0014] The present application will be further described below in combination with the drawings.

[0015] Figure 1 The nuclear magnetic hydrogen spectrum of the compound OBG involved in the present application.

[0016] Figure 2 The nuclear magnetic carbon spectrum of the compound OBG involved in the present application.

[0017] Figure 3 The high-resolution mass spectrum of the compound OBG involved in the present application.

[0018] Figure 4 The nuclear magnetic hydrogen spectrum of the compound LBG involved in the present application.

[0019] Figure 5 The nuclear magnetic carbon spectrum of the compound LBG involved in the present application.

[0020] Figure 6High resolution mass spectrum of the compound LBG involved in the present application.

[0021] Figure 7 NMR hydrogen spectrum of the compound CSSH involved in the present application.

[0022] Figure 8 NMR hydrogen spectrum of the compound CSSGa involved in the present application.

[0023] Figure 9 GPC of the compound CSSGa involved in the present application.

[0024] Figure 10 Zeta potential of the chitosan and chitosan derivatives involved in the present application.

[0025] Figure 11 Bar chart of the killing of (A) MRSA and (B) MSSA by the chitosan and chitosan derivatives involved in the present application. P. aeruginosa

[0026] Figure 12 Bar chart of the killing of (A) MRSA and (B) MSSA by the chitosan and chitosan derivatives involved in the present application. P. aeruginosa CLSM images of live / dead staining before and after treatment with CSSGa.

[0027] Figure 13 Bar chart of the killing of (A) MRSA and (B) MSSA by the chitosan and chitosan derivatives involved in the present application. P. aeruginosa SEM and TEM images before and after treatment with CSSGa.

[0028] Figure 14 Bar chart of the killing of (A) MRSA and (B) MSSA by the chitosan and chitosan derivatives involved in the present application. P. aeruginosa Potential before and after treatment with CSSGa.

[0029] Figure 15 Bar chart of the killing of (A) MRSA and (B) MSSA by the chitosan and chitosan derivatives involved in the present application. P. aeruginosa Absorbance analysis chart of the contents released by the bacteria after treatment with different concentrations of CSSGa.

[0030] Figure 16 Bar chart of the killing of (A) MRSA and (B) MSSA by the chitosan and chitosan derivatives involved in the present application. P. aeruginosa Fluorescence intensity analysis chart after treatment with different concentrations of CSSGa and propidium iodide (PI, only staining damaged membrane).

[0031] Figure 17 Bar chart of the killing of (A) MRSA and (B) MSSA by the chitosan and chitosan derivatives involved in the present application. P. aeruginosa Fluorescence intensity analysis chart of the active oxygen in the bacteria after treatment with different concentrations of CSSGa. DETAILED DESCRIPTION

[0032] ​To further illustrate the technical means adopted by the present application and its effects, the present application will be further described below in conjunction with examples. The specific embodiments described herein are merely intended to explain the present application, but not to limit the present application.

[0033] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0034] A guanidine chitosan derivative bactericidal material and a preparation method thereof, the structural formula of which is: Formula I To solve the technical problems of the present application, the technical scheme proposed is: a preparation method of a guanidine chitosan derivative bactericidal material, comprising the following steps: 1) N,N'-di-Boc-S-methyl isothiourea, ethanolamine, N,N-diisopropylethylamine (DIPEA) and dichloromethane (DCM) are sequentially added to a round-bottom flask, and stirred at room temperature for 12 h. After the reaction is completed, the methyl mercaptan by-product is removed by bubbling under nitrogen for 1 h. The crude product is purified by silica gel chromatography column method to obtain white solid 2-(2,3-bis(tert-butoxycarbonyl) guanidino) ethanol (OBG); the reaction route is as follows: 2) lipoic acid, OBG, 4-dimethylaminopyridine (DMAP) and dichloromethane are sequentially added to a round-bottom flask, and stirred and mixed under ice cooling. Then, N,N-dicyclohexyl carbodiimide (DCC) is dissolved in dichloromethane, and added dropwise into the round-bottom flask, and stirred under ice cooling for 1 h. The reaction is carried out at room temperature for 24 h. After the reaction is completed, the insoluble solid is filtered, and the crude product is purified by silica gel chromatography column method to obtain yellow oily product (-2-(2,3-bis(tert-butoxycarbonyl) guanidino) ethyl-5-(1,2-dithiolane-3-yl) pentanoate (LBG); the reaction route is as follows: E ​3) CS and HOBT were added into deionized water in turn, stirred until the solution was clear, then EDCI and NAC were added, the pH was adjusted to 5.0, and stirred at room temperature for 3 h. After the reaction was completed, the crude product was purified by dialysis and freeze-drying to obtain N-((2R)-1-((((2S,3R,5S,6R)-5-(((2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2-(((2R,3S,5R,6R)-5-amino-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)oxy)-3-mercapto-1-oxopropan-2-yl)acetamide (CSSH); the reaction scheme is as follows: 4) LBG was dissolved in dichloromethane, then trifluoroacetic acid was added, and deprotection reaction was carried out for 24 h to obtain LGa. Subsequently, LGa and CSSH were dissolved in triethanolamine buffer and N,N-dimethylformamide with pH = 7.0, mixed, stirred at 30 °C for 30 min, 2-iodoacetamide was added to terminate the reaction, and the crude product was finally purified by dialysis to obtain (15R)-1-amino-15-(((2S,3R,5S,6R)-5-(((2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2-(((2R,3S,5R,6R)-5-amino-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)oxy)carbonyl)-11-ethyl-6,17-dioxo-5-oxa-12,13-dithia-2,16-diazonadecan-1-ium (CSSGa); the reaction scheme is as follows: Example 1 Synthesis of CSSGa 1) N,N'-di-Boc-S-methyl isothiourea (1.7 g, 5.8 mmol), ethanolamine (0.69 mL, 11 mmol), N,N-diisopropylethylamine (3.0 mL, 17 mmol) and dichloromethane (10 mL) were added into a round-bottom flask in turn, and stirred at room temperature for 12 h. After the reaction was completed, the methyl mercaptan by-product was removed by bubbling nitrogen for 1 h. The crude product was purified by silica gel column chromatography, and the eluent was n-hexane and ethyl acetate (v / v, 1:3) to obtain 1.31 g of white solid OBG with a yield of 74.5 %. 1H NMR (400 MHz, CDCl3) δ 11.44 (s, 1H), 8.68 (t, 1H), 3.76 (t, 2H), 3.54-3.59 (m, 2H), 1.49 (d, 18H). 13 C NMR (100 MHz, CDCl3) δ 162.75, 157.39, 153.07, 83.50, 79.59,63.02, 44.36, 28.03. HRMS (m / z): 326.1696 [M+Na], theoretical value: 326.1692 [M+Na]. The H NMR spectrum of OBG is shown in Figure 1 , NMR carbon spectrum see Figure 2 , mass spectrometry see Figure 3 The compound was successfully synthesized through NMR and mass spectrometry characterization.

[0035] 2) Lipoic acid (1.5 g, 7.3 mmol), OBG (1.5 g, 4.9 mmol), 4-dimethylaminopyridine (0.91 g, 7.4 mmol), and dichloromethane (15 mL) were added sequentially to a round-bottom flask and mixed under an ice-water bath. N,N'-dicyclohexylcarbodiimide (1.5 g, 7.3 mmol) was then dissolved in dichloromethane (10 mL) and added dropwise to the round-bottom flask. The mixture was stirred on ice for 1 h, and the reaction was continued at room temperature for 24 h. After the reaction, the insoluble solid was filtered, and the crude product was purified by silica gel column chromatography using n-hexane and ethyl acetate (v / v, 1:5) as eluent to obtain 1.73 g of LBG as a yellow oil in a 71.9% yield. 1 H NMR (400 MHz, CDCl3) δ 11.44 (s, 1H), 8.51 (s, 1H), 4.17 (t, 2H), 3.61-3.67 (m, 2H), 3.48-3.55 (m, 1H), 3.02-3.15 (m, 2H), 2.37-2.44 (m, 1H),2.28 (t, 2H),1.81-1.89 (m, 1H), 1.59-1.66 (m, 4H),1.43 (d, 18H) 1.18 (s, 2H). 13C NMR (100 MHz, CDC13) δ 173.26, 171.12, 163.38, 156.31, 153.15, 83.28, 79.49, 62.71, 56.27, 40.21, 39.69, 38.50, 34.61, 33.95, 28.76, 28.08, 24.72, 24.61, 22.65, 21.06, 14.21. HRMS (m / z): 514.2023 [M+Na], Calcd: 514.2021 [M+Na]. The H NMR of LBG is shown in Figure 4 , the C NMR is shown in Figure 5 , and the mass spectrum is shown in Figure 6 The successful synthesis of the compound is indicated by the characterization of NMR and mass spectrum.

[0036] 3) 1.0 g of chitosan (degree of deacetylation > 85%) and 1-hydroxybenzotriazole (HOBT, 0.70 g, 5.1 mmol) were added into 92 mL of deionized water in turn, and stirred until clear. Then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 4.0 g, 0.02 mol) was dissolved in 8.0 mL of water and added into the reaction solution, and finally N-acetylcysteine (NAC, 1.7 g, 0.01 mol) was added. The pH was adjusted to 5.0 with 1.0 M HCl, and the reaction was stirred at room temperature for 3 h. The crude product was purified by dialysis (molecular weight cut-off 7000 Da, dialysis for 24 h). Specifically, on the first day, 5.0 mM HCl and 2.0 μM EDTA solution were used, on the second day, 5.0 mM HCl, 2.0 μM EDTA and 1.0% NaCl solution were used, and on the third day, 1.0 mM HCl solution was used. Finally, 0.87 g of CSSH was obtained by freeze-drying. 1 H NMR (400 MHz, DO2) δ 2.80 (s, 2H), 2.60 (s, 2H). The H NMR of CSSH is shown in Figure 7 .

[0037] 4) 1.0 g LBG was dissolved in 10 mL dichloromethane, and then 10 mL trifluoroacetic acid was added, and the reaction was carried out for 24 h to obtain LGa, which was directly used in the next step. 0.59 g LGa and 0.065 g CSSH obtained in the above reaction were dissolved in 3.5 mL hydrochloric acid-triethanolamine buffer (0.03 mol / L) and N,N-dimethylformamide (v / v, 1:2.5) with pH=7.0, and the reaction solution was mixed thoroughly and stirred at 30 °C for 30 min, and then 0.175 g 2-iodoacetamide was added to terminate the reaction, and the crude product was purified by dialysis to obtain 0.196 g CSSGa. 1 H NMR (400 MHz, DO2) δ 4.14 (m, 2H), 3.65 (m, 1H),3.40 (t, 2H), 2.34 (m, 2H), 2.28 (t, 2H), 1.54 (s, 6H), 1.36 (m,3H). The nuclear magnetic resonance spectrum of CSSGa is shown in Figure 8 The results of gel permeation chromatography (GPC) test are shown in Figure 9 The average molecular weight of CSSGa is 4536 g / mol, and the dispersion degree is 1.12.

[0038] Example 2 Potential test of CS, CSSH and CSSGa: The solutions of CS, CSSH and CSSGa were prepared at a concentration of 1.0 mg / mL. The concentration was diluted to 20 μg / mL with pure water, and the potential of chitosan and its derivatives was tested by Malvern Zetasizer Nano ZSE 3700 nanoparticle size potential instrument. The test results are shown in Figure 10 CSSGa has positive charge and has the potential to bind to bacteria.

[0039] Example 3 Bactericidal effect test of CS, CSSH and CSSGa on MRSA and P. aeruginosa : 1) Cultivation of bacteria: After the clean bench was sterilized for 20-30 min, the surface was wiped clean with 75% alcohol. 10 mL of LB liquid medium was taken into a 50 mL sterile centrifuge tube, and 10 μL of MRSA (ATCC43300) and P. aeruginosa (ATCC27853) strains were added, and the culture was carried out at a temperature of 37 °C, 180 rpm / min shaker for 8-10 h.

[0040] 2) Bactericidal rate test of MRSA and P. aeruginosa : Take 2.0 mL of bacterial solution and centrifuge at 7200 rpm / min for 2.5 min, discard the supernatant, pipette PBS evenly, and adjust the OD value using a UV spectrophotometer. 600 =1.0, washed 2-3 times with PBS; took 200 μL OD 600 = 1.0 bacterial solution and different concentrations of CS, CSSH and CSSGa were added to 1.0 mL system. The concentrations of CS, CSSH and CSSGa were 0 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL and 25 μg / mL respectively. The system was incubated for 24 h, diluted and 100 μL was taken to plate. The system was incubated in a 37 °C constant temperature incubator for 12 h and then taken out to count and calculate the bacterial survival rate. The results are shown in Figure 11 A and 11B, CSSGa for MRSA and P. aeruginosa It has a strong killing effect.

[0041] Example 4 MRSA and P. aeruginosa Live-dead staining test after CSSGa treatment 1) Bacterial culture: Same as in Case 3; 2) Antibacterial experiment: Same as in Case 3, except that the concentration of CSSGa was 25 μg / mL; 3) Live-dead staining: After the antibacterial experiment, add 40 μL of SYTO-9 / PI mixed stain solution and stain at room temperature in the dark for 30 minutes. Then add 1 mL of PBS and gently pipette the bacterial suspension repeatedly at 12,000 rpm / min for 3 minutes. Repeat this three times. Finally, add 10 μL of PBS to resuspend the bacteria. Take 5 μL of the bacterial solution and drop it on a glass slide. Cover with a coverslip and image with a confocal laser scanning microscope (CLSM). Figure 12 , MRSA and P. aeruginosa After being treated with different materials, CS showed green fluorescence, indicating that the bacterial survival rate was the highest, but in CSSGa, all showed red fluorescence, indicating that CSSGa killed all the bacteria, showing that CSSGa has a good antibacterial effect.

[0042] Example 5 MRSA and P. aeruginosa SEM and TEM imaging experiments 1) Bacterial culture: Same as in Case 3; 2) Antibacterial experiment: Same as in Case 3, except that the concentration of CSSGa was 25 μg / mL; 3) Imaging: After the antibacterial experiment, the sample was dropped on a silicon wafer, naturally ventilated and dried, fixed with 4% glutaraldehyde at 4°C overnight, the supernatant was discarded, and the sample was dehydrated with 20%, 40%, 60%, 80%, and 100% ethanol gradient, followed by freeze-drying. The silicon wafer containing bacteria was placed on an aluminum plate, sprayed with gold, and observed under a scanning electron microscope (SEM) for morphological changes. In addition, the sample was observed under a biological transmission electron microscope (TEM) for morphological changes. The results are shown in Figure 13 , the MRSA and P. of the Control group were in good condition, and the bacterial membrane was intact, but after the CSSGa treatment, the morphology of the MRSA and aeruginosa changed significantly, showing concave and shrunk, cell membrane rupture, cytoplasm leakage, and bacterial death. P. aeruginosa

[0043] Example 6 Potential test of MRSA and P. aeruginosa P. aeruginosa 1) Bacterial culture: same as Example 3; 2) Antibacterial experiment: same as Example 3, the concentration of CSSGa was 25 μg / mL; 3) Potential test: after the antibacterial experiment, the bacterial precipitate was washed with pure water for 2-3 times, and finally pure water was added to mix the bacterial precipitate. The potential of MRSA and P. aeruginosa planktonic bacteria and their materials was characterized by a particle size potential instrument. The results are shown in Figure 14 A and 14B, the MRSA planktonic colony potential showed negative electricity, CSSGa was positively charged, and after the material treatment, the whole showed positive electricity, indicating that the positively charged material could adsorb negatively charged bacteria to achieve the sterilization effect; P. aeruginosa showed the same experimental results as MRSA.

[0044] Example 7 Protein leakage test of MRSA and P. aeruginosa P. aeruginosa 1) Bacterial culture: same as Example 3; 2) Antibacterial experiment: same as Example 3, the concentration of CSSGa was 0 μg / mL, 5 μg / mL, 25 μg / mL, and 125 μg / mL; 3) Potential test: after the antibacterial experiment, the bacterial precipitate was blown and mixed with a pipette, centrifuged at 12000 rpm / min for 3 min, the supernatant was taken and incubated with the prepared BCA working solution at 37°C, the color change was observed, and the absorbance was measured at 562 nm to detect the bacterial protein leakage. The results are shown in Figure 15A and 15B, after MRSA was treated with different concentrations of CSSGa, the absorbance at 562 nm increased with the increase of concentration, indicating that more proteins in the cytoplasm were effluxed. P. aeruginosa The results were the same as those for MRSA.

[0045] Example 8 MRSA and P. aeruginosa Bacterial membrane integrity assay 1) Bacterial culture: Same as in Case 3; 2) Antibacterial experiment: Same as in Example 3, except that the concentrations of CSSGa were 0 μg / mL, 5 μg / mL, 25 μg / mL, and 125 μg / mL; 3) Bacterial membrane integrity determination: After the antibacterial experiment, PI staining solution was added to the bacterial pellet and stained in the dark for 30 minutes. The pellet was then centrifuged at 12,000 rpm / min for 3 minutes and washed 2-3 times with PBS. Finally, 500 μL of PBS was added to resuspend the bacterial pellet. The PI fluorescence intensity was measured to detect bacterial membrane integrity. PI only stains dead bacteria. Figure 16 A and 16B, MRSA and P. aeruginosa After being treated with different concentrations of CSSGa, the bacterial precipitates were stained with PI. The fluorescence intensity of PI increased with the increase of CSSGa concentration, indicating that CSSGa can destroy the bacterial membrane.

[0046] Example 9 MRSA and P. aeruginosa Bacterial membrane integrity assay 1) Bacterial culture: Same as in Case 3; 2) Antibacterial experiment: Same as in Example 3, except that the concentrations of CSSGa were 0 μg / mL, 5 μg / mL, 25 μg / mL, and 125 μg / mL; 3) Bacterial membrane integrity determination: After the antibacterial experiment, add DCFH-DA stain to the bacterial pellet and stain in the dark for 30 minutes. Then centrifuge at 12,000 rpm for 3 minutes and wash 2-3 times with PBS. Finally, add 500 μL of PBS to resuspend the bacterial pellet and measure the intensity using a fluorescence spectrophotometer. Figure 17 A and 17B, MRSA and P. aeruginosa After treatment with CSSGa and DCFH-DA, the accumulation of ROS increased with the increase of CSSGa concentration, leading to bacterial membrane damage and destruction of bacterial homeostasis.

[0047] The application discloses a guanidyl chitosan derivative capable of tearing bacterial membrane and a preparation method thereof. Specifically, the guanidyl chitosan derivative with high-efficiency antibacterial property is prepared by using chitosan as raw material through esterification reaction and addition reaction, and the antibacterial mechanism is explored. The guanidyl chitosan derivative prepared by the method has the advantages of simple operation, mild reaction condition, high yield and good antibacterial property, and can kill methicillin-resistant Staphylococcus aureus (MRSA) and pseudomonas aeruginosa (P. P. aeruginosa ). The guanidyl chitosan derivative can adsorb bacteria, destroy bacterial biofilm, tear bacterial membrane, and make cytoplasm contents flow out, so that the bactericidal effect is achieved. The research provides theoretical basis and technical support for development of a new antibacterial material.

[0048] Although embodiments of the application have been disclosed in connection with the above specification and drawings, it should be understood that they are not intended to limit the application to the specific form set forth, but on the contrary, it is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the application as defined by the appended claims and their equivalents.

Claims

1. A guanidino chitosan derivative for destroying bacterial membranes, characterized in that: Its structural formula is as follows: Formula I.

2. The method for preparing a guanidino chitosan derivative for destroying bacterial membranes as claimed in claim 1, wherein: The steps include: 1) N,N-di-Boc-S-methylisothiourea, ethanolamine, N,N-diisopropylethylamine, and dichloromethane are reacted at room temperature. After completion of the reaction, purification is performed to obtain a white solid 2-(2,3-bis(tert-butyloxycarbonyl)guanidino)ethanol OBG; 2) Thioctic acid, 2-(2,3-bis(tert-butyloxycarbonyl)guanidino)ethanol, 4-dimethylaminopyridine, and dichloromethane were stirred in an ice bath. [N,N-dicyclohexylcarbodiimide dissolved in dichloromethane was then added dropwise. The mixture was stirred in an ice bath for 1 hour and then reacted at room temperature to obtain a yellow oily product, LBG, with the structure shown below. 3) Chitosan and 1-hydroxybenzotriazole were added to deionized water and stirred, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-acetylcysteine ​​were added to react to obtain CSSH, the structure of which is shown below: 4) LBG is dissolved in dichloromethane and trifluoroacetic acid is added for deprotection reaction to obtain LGa. LGa and CSSH are then dissolved in a mixed solution of hydrochloric acid-triethanolamine buffer and N,N-dimethylformamide. After the reaction, 2-iodoacetamide is added to terminate the reaction to obtain the bacterial membrane-destroying guanidine chitosan derivative shown in Formula I. 。 3. The preparation method according to claim 2, wherein In step 3), the mass ratio of chitosan, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-acetylcysteine ​​is 1:0.7:4:1.

7. The pH is adjusted to 5.0 before the reaction, and the reaction is completed by stirring at room temperature for 3 h.

4. The preparation method according to claim 2, wherein In step 1), the mixing ratio of N,N-di-Boc-S-methylisothiourea, ethanolamine, N,N-diisopropylethylamine, and dichloromethane is: 5.8 mmol: 11 mmol: 17 mmol: 10 mL.

5. The preparation method according to claim 2, wherein After the reaction in step 1) was completed, nitrogen was bubbled for 1 h to remove the methyl mercaptan by-product, and the crude product was purified by silica gel column chromatography using n-hexane and ethyl acetate in a volume ratio of 1:3 as the eluent to obtain 2-(2,3-bis(tert-butyloxycarbonyl)guanidino)ethanol as a white solid.

6. The preparation method according to claim 2, wherein In step 2), the mixing ratio of lipoic acid, 2-(2,3-bis(tert-butyloxycarbonyl)guanidino)ethanol, 4-dimethylaminopyridine and dichloromethane is 7.3 mmol:4.9 mmol:7.4 mmol:15 mL. The mixing ratio of N,N-dicyclohexylcarbodiimide and dichloromethane is: 7.3 mmol: 10 mL; The mixture was stirred in an ice bath for 1 hour and then continued to react at room temperature for 24 hours. After the reaction, the insoluble solid was filtered and the organic solvent was removed to obtain a crude product. The crude product was purified by silica gel column chromatography using n-hexane and ethyl acetate in a volume ratio of 1:5 as the eluent to obtain a yellow oily product LBG.

7. The preparation method according to claim 2, wherein In step 4), 1.0 g of LBG was dissolved in 10 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added and reacted for 24 h to obtain LGa: 0.59 g of LGa and CSSH were dissolved in a mixture of hydrochloric acid-triethanolamine buffer (pH 7.0, 0.03 mol / L) and N,N-dimethylformamide. The mixture was stirred at 30 °C for 30 min and then 2-iodoacetamide was added to terminate the reaction.

8. Use of the guanidino chitosan derivative for destroying bacterial membranes as claimed in claim 1 in the preparation of bactericidal materials.

9. The use according to claim 8, characterized in that The bactericidal pH of the guanidino chitosan derivative is 7.0, and the guanidino chitosan derivative is used to kill methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa P. aeruginosa .

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