A method for preparing chitosan quaternary phosphonium salt
By reacting small molecule quaternary phosphonium salt with chitosan to form chitosan quaternary phosphonium salt, the problem of poor solubility of chitosan is solved, its antibacterial activity and ability to inhibit biological membranes are improved, and its effectiveness on bacterial biological membranes is achieved Clear.
Patent Information
- Application Number
- CN202310346103.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-03
- Publication Date
- 2025-01-17
- Estimated Expiration
- 2043-04-03
AI Technical Summary
The poor solubility of chitosan limits its application in antibacterial and inhibiting biofilm formation, and mature biofilm increases tolerance to antibiotics and disinfectants, resulting in bacterial infections and medical device degeneration problems.
A preparation method is adopted to react substance A and substance B in an organic solvent to form small molecule quaternary phosphonium salt, and react with chitosan in aqueous acetic acid solution, and graft the small molecule to the shell through the action of EDC and NHS. Chitosan quaternary salts are formed on the glycan molecular chains, improving their water solubility and antibacterial activity.
The prepared chitosan quaternary salt has high antibacterial activity, can effectively inhibit the formation of biological films and have a certain removal effect on mature bacterial biological films, improving the solubility and application performance of chitosan.
Smart Images
Figure CN116284497B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for preparing chitosan quaternary phosphonium salt, and belongs to the field of preparation and application of chitosan antibacterial agents. Background Art
[0002] Bacteria attach to the surface of materials to form biofilms. Various chronic infections and degradation of various medical devices caused by biofilms are also increasing, and mature biofilms have increased tolerance to antibiotics and disinfectants.
[0003] Chitosan has been shown to have antibacterial properties against both Gram-negative and Gram-positive bacteria, and can destroy the integrity of biofilms and promote the decomposition of biofilms, but the poor solubility of chitosan limits its application.
[0004] In view of this, the inventor of this case conducted in-depth research on the above-mentioned issues, which led to the emergence of this case. Summary of the invention
[0005] The object of the present invention is to provide a method for preparing chitosan quaternary phosphonium salt, which is simple to operate and easy to obtain materials, and the formed chitosan has the functions of antibacterial and inhibiting biofilm formation and removing biofilm.
[0006] In order to achieve the above object, the present invention adopts such technical solution:
[0007] A method for preparing chitosan quaternary phosphonium salt comprises the following steps:
[0008] Step 1, dissolving substance A in an organic solvent, wherein the mass percentage concentration of substance A is 0.5-20%, adding substance B, reacting at 50-140° C. for 4-48 hours, and after the reaction, filtering, washing and drying to obtain a small molecule quaternary phosphonium salt,
[0009] Wherein, substance A is p-chloromethylbenzoic acid, p-bromomethylbenzoic acid, chloroacetic acid, chloropropionic acid or bromoacetic acid; substance B is triphenylphosphine, tricyclohexylphosphine or trimethylphosphine;
[0010] Step 2, dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution with a mass percentage concentration of 1%-5%;
[0011] Step 3, dissolving a small molecule quaternary phosphonium salt in water or a mixed solution of water / ethanol, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) until the small molecule quaternary phosphonium salt is completely dissolved to obtain a small molecule quaternary phosphonium salt solution;
[0012] Step 4, adding the small molecule quaternary phosphonium salt solution to the chitosan solution for reaction, the reaction temperature is 20-50° C., and the reaction time is 12-48 hours;
[0013] Step 5: After the reaction is completed, the product is transferred to an ultrafiltration device, water is added to ultrafilter to remove the small molecule quaternary phosphonium salt that does not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary phosphonium salt.
[0014] As a preferred embodiment of the present invention, the organic solvent includes one or more of ethyl acetate, tetrahydrofuran, acetone, petroleum ether and acetonitrile.
[0015] As a preferred embodiment of the present invention, the molar ratio of the p-chloromethylbenzoic acid to the triphenylphosphine is 1:1-2.
[0016] As a preferred embodiment of the present invention, in step 2, the mass percentage concentration of the acetic acid aqueous solution is 1% to 5%.
[0017] As a preferred embodiment of the present invention, in step 3, the amount of small molecule quaternary phosphonium salt added is 1-3 times the molar amount of chitosan.
[0018] As a preferred embodiment of the present invention, in step 3, the mass percentage concentration of the small molecule quaternary phosphonium salt is 1%-10%.
[0019] As a preferred embodiment of the present invention, in step 3, the molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the N-hydroxysuccinimide is 1:1.
[0020] As a preferred embodiment of the present invention, the molar ratio of the small molecule quaternary phosphonium salt to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-3.
[0021] The invention has the following beneficial effects: firstly, a carboxyl-terminated small molecule is synthesized, and then, under the action of EDC and NHS, the small molecule is grafted onto the chitosan molecular chain, and the chitosan quaternary phosphonium salt is obtained by ultrafiltration and concentration. The chitosan quaternary phosphonium salt antibacterial agent synthesized by the preparation method of the invention has good water solubility, high inhibitory and antibacterial activity, can inhibit the formation of biofilm, and has a certain removal effect on mature bacterial biofilm. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 is N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride in Example 1 1 H NMR.
[0023] Figure 2 is N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride in Example 1 13 C NMR.
[0024] Figure 3 is N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride in Example 1 31 P NMR.
[0025] Figure 4 is the chitosan quaternary phosphonium salt in Example 1 1 H NMR.
[0026] Figure 5 is N-(4-carboxybenzyl)-N,NN-tricyclohexylphosphine chloride in Example 2 1 H NMR.
[0027] Figure 6 is N-(4-carboxybenzyl)-N,NN-tricyclohexylphosphine chloride in Example 2 13 C NMR.
[0028] Figure 7 is N-(4-carboxybenzyl)-N,NN-tricyclohexylphosphine chloride in Example 2 31 P NMR.
[0029] Figure 8 is the chitosan quaternary phosphonium salt in Example 2 1 H NMR.
[0030] Fig. 9 is N-(4-carboxybenzyl)-N,NN-trimethylphosphine chloride in Example 3 1 H NMR.
[0031] Fig.10 is N-(4-carboxybenzyl)-N,NN-trimethylphosphine chloride in Example 3 13 C NMR.
[0032] Fig.11 is N-(4-carboxybenzyl)-N,NN-trimethylphosphine chloride in Example 3 31 P NMR.
[0033] Fig.12 is the chitosan quaternary phosphonium salt in Example 3 1 H NMR. DETAILED DESCRIPTION
[0034] Reference Figures 1 to 12 In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments. The embodiments are only used to explain the content of the present invention and are not intended to limit the protection scope of the present invention.
[0035] Example 1
[0036] Step 1, weigh 1.70 kg of p-chloromethylbenzoic acid and dissolve it in 20 kg of ethyl acetate, and put it into a reactor, add 2.6 kg of triphenylphosphine under stirring, raise the temperature to 85 ° C, reflux and stir for 8 hours, a large amount of white solid precipitates during the reaction, after the reaction is completed, filter, wash the filter cake with ethyl acetate several times, and dry to obtain N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride.
[0037] Step 2: Weigh 1.61 kg of chitosan and dissolve it in 100 kg of 2% by volume acetic acid water to obtain a chitosan acetic acid solution, wherein the mass percentage concentration of chitosan is 1.6%; wherein chitosan can be directly purchased from the market.
[0038] Step 3, weigh 4.32kg of N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride and dissolve it in 50kg of 50% ethanol water by volume, add 1.92kg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.15kg of N-hydroxysuccinimide, and obtain a small molecule quaternary phosphonium salt solution after complete dissolution, wherein the mass percentage concentration of N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride in the solution is 7.7%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.
[0039] Step 4, slowly dropwise adding the small molecule quaternary phosphonium salt solution to the chitosan acetic acid aqueous solution in the reaction kettle, reacting at room temperature for 24 hours after the addition is completed. In this embodiment, the molar ratio of N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride to the chitosan added in step 2 can be 1:1;
[0040] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added to ultrafilter to remove N-(4-carboxybenzyl)-N,NN-triphenylphosphine chloride and carboxyl activator (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, the same below) that do not participate in the reaction, and the chitosan quaternary phosphonium salt is obtained by concentration and drying.
[0041] Example 2
[0042] Step 1, weigh 1.70 kg of p-chloromethylbenzoic acid and dissolve it in 20 kg of ethyl acetate, and put it into a reactor, add 3.1 kg of tricyclohexylphosphine under stirring, raise the temperature to 85 ° C, reflux and stir for 8 hours, a large amount of white solid precipitates during the reaction, after the reaction is completed, filter, wash the filter cake with ethyl acetate for several times, and dry to obtain N-(4-carboxybenzyl)-N,NN-tricyclohexylphosphine chloride.
[0043] Step 2: Weigh 1.61 kg of chitosan and dissolve it in 50 kg of 2% by volume acetic acid water to obtain a chitosan acetic acid solution. At this time, the mass percentage concentration of chitosan is 3.2%.
[0044] Step 3, weigh 4.50 kg of N-(4-carboxybenzyl)-N,NN-tricyclohexylphosphine chloride and dissolve it in 100 kg of water, add 2.30 kg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.3 kg of N-hydroxysuccinimide, and obtain a small molecule quaternary phosphonium salt solution after complete dissolution, wherein the mass percentage concentration of N-(4-carboxybenzyl)-N,NN-tricyclohexylphosphine chloride in the solution is 4.5%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.
[0045] Step 4, slowly dropwise adding the small molecule quaternary phosphonium salt solution to the chitosan acetic acid aqueous solution in the reaction kettle, reacting at 30° C. for 24 hours after the addition is completed. In this embodiment, the molar ratio of N-(carboxymethyl)-N,N-dimethyldodecylphosphonium chloride to the chitosan added in step 2 is 1.2:1;
[0046] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added for ultrafiltration to remove N-(4-carboxybenzyl)-N,NN-tricyclohexylphosphine chloride and carboxyl activator that do not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary phosphonium salt.
[0047] Example 3
[0048] Step 1, weigh 1.70 kg of p-chloromethylbenzoic acid and dissolve it in 20 kg of ethyl acetate, and put it into a reactor, add 0.84 kg of trimethylphosphine under stirring, raise the temperature to 85 ° C, reflux and stir for 10 hours, a large amount of white solid precipitates during the reaction, after the reaction is completed, filter, wash the filter cake with ethyl acetate several times, and dry to obtain N-(4-carboxybenzyl)-N,NN-trimethylphosphine chloride.
[0049] Step 2: Weigh 1.61 kg and dissolve it in 100 kg of 2% by volume acetic acid water to obtain chitosan acetic acid solution. At this time, the mass percentage concentration of chitosan is 1.6%.
[0050] Step 3, weigh 2.46 kg of N-(4-carboxybenzyl)-N,NN-trimethylphosphine chloride and dissolve it in 50 kg of 50% ethanol water by volume, add 1.92 kg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.15 kg of N-hydroxysuccinimide, and obtain a small molecule quaternary phosphonium salt solution after complete dissolution, wherein the mass percentage concentration of N-(4-carboxybenzyl)-N,NN-trimethylphosphine chloride in the solution is 2.46%, and the molar percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.
[0051] Step 4: slowly drop the small molecule quaternary phosphonium salt solution into the chitosan acetic acid aqueous solution in the reaction kettle, and react at room temperature for 24 hours after the addition is completed. In this embodiment, the molar ratio of N-(4-carboxybenzyl)-N,NN-trimethylphosphine chloride to the chitosan added in step 2 is 1:1.
[0052] Step 5: After the reaction at room temperature for 24 hours, the product is transferred to an ultrafiltration device, and water is added for ultrafiltration to remove N-(4-carboxybenzyl)-N,NN-trimethylphosphonium chloride and carboxyl activator that do not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary phosphonium salt.
[0053] 2. Antibacterial performance test
[0054] The antibacterial properties of chitosan quaternary phosphonium salts in the examples were tested by plate counting method.
[0055] The inhibition rate was calculated by formula 1.
[0056]
[0057] Table 1 Antibacterial effect of chitosan quaternary phosphonium salts at different concentrations on Staphylococcus aureus (S. aureus)
[0058] Group 2.5mg / mL 1.0mg / mL 0.5mg / mL Example 1 100％ 100％ 100％ Example 2 100％ 100％ 100％ Example 3 100％ 100％ 100％
[0059] Table 2 Antibacterial effect of different concentrations of chitosan quaternary phosphonium salt on Escherichia coli (E.coli)
[0060] Group 2.5mg / mL 1.0mg / mL 0.5mg / mL Example 1 83.76％ 62.37％ 58.51％ Example 2 81.67％ 61.78％ 50.28％ Example 3 81.13％ 61.17％ 41.97％
[0061] The minimum inhibitory concentration (MIC) and minimum biofilm inhibition concentration (MBC) of the chitosan quaternary phosphonium salt in the examples were determined by turbidimetry, and the minimum bactericidal concentration (MBC) and minimum biofilm killing concentration (MBBC) of the chitosan quaternary phosphonium salt in the examples were determined by plate count method.
[0062] Add 100 μL of diluted bacterial solution and 100 μL of chitosan solution diluted by half to a 96-well plate, and then make a control group with only chitosan quaternary phosphonium salt solution without bacterial solution and a control group with only bacterial solution without chitosan quaternary phosphonium salt solution, respectively. Make three parallel groups for each concentration of chitosan quaternary phosphonium salt, and observe the results after culturing in a 37°C incubator for 18 hours. Take the lowest concentration of chitosan quaternary phosphonium salt corresponding to the clarified wells as MIC, take 100 μL of the mixture of bacterial solution and sample solution in the clarified wells for plating, and record the lowest concentration corresponding to less than 5 colonies per dish as MBC.
[0063] Add diluted bacterial solution to a 96-well plate and place it in a 37°C incubator for 36-48 hours to allow the bacterial solution to form a biofilm on the well plate, then discard the floating bacteria, rinse with sterile saline or PBS, dry and fix, add 100 μL of diluted chitosan quaternary phosphonium salt sample solution and 100 μL of culture medium to the 96-well plate, use sterile water as a control group, place it in a 37°C incubator for 18 hours and observe the results, take the lowest chitosan quaternary phosphonium salt concentration corresponding to the clarified wells as MBIC, take 100 μL of the mixture of the bacterial solution and the sample solution in the clarified wells for plating, and record the lowest chitosan quaternary phosphonium salt concentration corresponding to less than 5 colonies per dish as MBBC.
[0064] Table 3 Changes in drug resistance of Staphylococcus aureus before and after BF formation
[0065]
[0066] Table 4 Changes in drug resistance of Escherichia coli before and after BF formation
[0067]
[0068] The crystal violet staining method can quickly and easily measure the intervention effect of drugs on biofilms. The OD value under the action of different drugs can reflect the inhibitory effect of chitosan quaternary phosphonium salt on biofilm formation.
[0069] 100 μL of diluted bacterial solution and 100 μL of chitosan quaternary phosphonium salt solution diluted to a certain concentration (4MIC, 2MIC, MIC) were added to a 96-well plate, with water as the control group, and the final concentration of chitosan quaternary phosphonium salt was 2MIC, MIC and 1 / 2MIC. Three groups of drugs were made in parallel for each concentration, and cultured in a 37°C incubator for 36-48 hours, then the floating bacteria and chitosan quaternary phosphonium salt solution were discarded, rinsed with sterile saline, dried and fixed, 0.1% crystal violet solution was added for staining for 15 minutes, the crystal violet solution was discarded and rinsed with sterile saline for 3 times, and dried with sterile air. 95% ethanol solution was added, and the absorbance was measured at 595nm with an enzyme marker after 5 minutes, and the biofilm formation inhibition rate was calculated by formula 2.
[0070]
[0071] Table 5 Inhibitory effect of chitosan quaternary phosphonium salt on biofilm formation of Staphylococcus aureus
[0072] Group 1 / 2MIC MIC 2MIC Example 1 62.22 86.16 94.25 Example 2 60.54 80.09 93.13 Example 3 53.84 61.47 84.79
[0073] Table 6 Inhibitory effect of chitosan quaternary phosphonium salt on Escherichia coli biofilm formation
[0074] Group 1 / 2MIC MIC 2MIC Example 1 56.62 64.24 78.33 Example 2 46.71 69.19 76.51 Example 3 33.95 50.06 62.42
[0075] Add 200 μL of diluted bacterial solution to a 96-well plate, then place it in a 37°C constant temperature incubator for 36-48 hours, discard the bacterial solution, rinse with sterile saline, dry and fix, then add 200 μL of chitosan quaternary phosphonium salt solution, and use sterile water as a control group. Keep the temperature in a 37°C incubator for 1 hour, discard the chitosan solution, rinse with sterile saline, stain with crystal violet, dry, add 95% ethanol solution, and measure the absorbance at 595 nm with an enzyme reader after 5 minutes. Calculate the clearance rate of the biofilm using Formula 3.
[0076]
[0077] Table 7 Effect of chitosan quaternary phosphonium salt on the removal of Staphylococcus aureus biofilm
[0078] Group 2.5mg / mL 5.0mg / mL Example 1 69.95 91.45 Example 2 62.70 84.03 Example 3 60.40 82.87
[0079] Table 8 Effect of chitosan quaternary phosphonium salt on the removal of Escherichia coli biofilm
[0080] Group 2.5mg / mL 5.0mg / mL Example 1 48.85 68.54 Example 2 47.16 68.32 Example 3 46.40 63.01
[0081] The above results show that the chitosan quaternary phosphonium salt prepared by this method has an inhibitory effect on Staphylococcus aureus and Escherichia coli. It can also inhibit the biofilm formation of Staphylococcus aureus and Escherichia coli, and has a certain clearing effect on mature biofilm.
[0082] The above embodiments and drawings do not limit the method of the present invention. Any appropriate changes or modifications made thereto by a person of ordinary skill in the art should be deemed to be within the patent scope of the present invention.
[0083] The product form of the present invention is not limited to the embodiments of this case. Any appropriate changes or modifications made by anyone with similar ideas should be deemed to be within the patent scope of the present invention.
Claims
1. A method for preparing a chitosan quaternary phosphonium salt, characterized in that: The steps include: Step 1, dissolving substance A in an organic solvent, wherein the mass percentage concentration of substance A is 0.5-20%, adding substance B, reacting at 50-140° C. for 4-48 hours, and after the reaction, filtering, washing and drying to obtain a small molecule quaternary phosphonium salt, Wherein, substance A is p-chloromethylbenzoic acid, p-bromomethylbenzoic acid, chloroacetic acid, chloropropionic acid or bromoacetic acid; substance B is triphenylphosphine, tricyclohexylphosphine or trimethylphosphine; Step 2, dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution with a mass percentage concentration of 1%-5%; Step 3, dissolving a small molecule quaternary phosphonium salt in water or a mixed solution of water / ethanol, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide until the small molecule quaternary phosphonium salt is completely dissolved to obtain a small molecule quaternary phosphonium salt solution; Step 4, adding the small molecule quaternary phosphonium salt solution to the chitosan solution for reaction, the reaction temperature is 20-50° C., and the reaction time is 12-48 hours; Step 5: After the reaction is completed, the product is transferred to an ultrafiltration device, water is added to ultrafilter to remove the small molecule quaternary phosphonium salt that does not participate in the reaction, and the product is concentrated and dried to obtain chitosan quaternary phosphonium salt.
2. The method for preparing a chitosan quaternary phosphonium salt according to claim 1, characterized in that: The organic solvent includes one or more of ethyl acetate, tetrahydrofuran, acetone, petroleum ether and acetonitrile.
3. A method for preparing a chitosan quaternary phosphonium salt as claimed in claim 2, characterized in that: The molar ratio of the p-chloromethylbenzoic acid to the triphenylphosphine is 1:1-2.
4. A method for preparing a chitosan quaternary phosphonium salt as claimed in claim 3, characterized in that: In step 2, the mass percentage concentration of the acetic acid aqueous solution is 1% to 5%.
5. A method for preparing a chitosan quaternary phosphonium salt as claimed in claim 4, characterized in that: In step 3, the amount of small molecule quaternary phosphonium salt added is 1-3 times the molar amount of chitosan.
6. The method for preparing a chitosan quaternary phosphonium salt according to claim 5, characterized in that: In step 3, the mass percentage concentration of the small molecule quaternary phosphonium salt is 1%-10%.
7. A method for preparing a chitosan quaternary phosphonium salt as claimed in claim 6, characterized in that: In step 3, the molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the N-hydroxysuccinimide is 1:
1.
8. The method for preparing a chitosan quaternary phosphonium salt according to claim 7, characterized in that: The molar ratio of the small molecule quaternary phosphonium salt to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-3.