Hyperbranched polylysine with a high content of ε-linear units, its preparation method and application

Abstract

The present invention belongs to the fields of polymer synthesis and antibacterial materials, and discloses a hyperbranched polylysine with a high content of ε-linear units, a preparation method thereof, and an application thereof. The preparation method comprises the following steps: (1) mixing lysine and a compound of formula I in a certain proportion and carrying out a polymerization reaction to obtain a polymer (II), wherein the molar proportion of lysine is 0-90%, and the molar proportion of the compound of formula I is 10-100%; (2) then removing the borane protecting group to obtain a hyperbranched polylysine (III) with a high content of ε-linear units; the present invention can achieve selective killing of fungi / bacteria through structure regulation, and in a specific structure, it can achieve only antifungal / killing of fungi, without killing effect on most bacteria, and can effectively protect the natural flora of the human body. The hyperbranched polylysine with a high ε-linear unit prepared by the present invention has high antifungal efficiency, low toxicity, and good biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the fields of polymer synthesis and antibacterial materials, and particularly relates to a hyperbranched polylysine with a high content of ε-linear units, a preparation method thereof, and an application thereof. Background Art

[0002] Since the lysine structure contains two amino groups: α-amino group and ε-amino group, starting from lysine, linear α-polylysine (α-PL) and ε-polylysine (ε-PL), as well as branched hyperbranched polylysine (HPL) with a branched structure can be synthesized respectively by using different polymerization processes. In the 1950s, Katchalski et al. found that α-PL can inhibit the growth of Gram-positive bacteria and Gram-negative bacteria, and copolymers containing lysine can inhibit the growth of Escherichia coli and Mycoplasma pyogenes. However, the antibacterial activity of α-PL is not very high and it has certain toxicity. ε-PL synthesized by fermentation method has excellent antibacterial effects against both Gram-positive bacteria and Gram-negative bacteria and low toxicity, but the inhibitory effect of ε-PL on fungi is poor, and its chemical synthesis process is not mature, the microbial fermentation production capacity is limited and the cost is high. In addition, existing antibacterial agents have almost no selectivity for fungi / bacteria.

[0003] Selective bactericidal has important applications in many fields. In the medical field, selective bactericidal drugs that only kill specific pathogens can, while effectively treating infections, minimize the damage to normal flora as much as possible, thereby reducing the risk of side effects and secondary infections. In addition, reducing the loss of beneficial bacteria can also slow down the development of drug resistance of pathogenic bacteria to antibiotics to a certain extent. In the food field, it is necessary to selectively kill harmful bacteria and spoilage-causing bacteria without killing probiotics such as lactic acid bacteria, while ensuring food safety and maintaining its quality and flavor. In the agricultural field, selectively killing harmful microorganisms can protect beneficial microorganisms, which is beneficial to ensuring the yield and quality of crops and maintaining the diversity of the ecosystem.

[0004] The paper published by Zuzana Kadlecova et al. (Macromolecular Bioscience, 2021, 12, 794-804) reported that hyperbranched polylysine (HBPL) was used as a multifunctional and biodegradable transfection agent for the production of recombinant proteins through transient gene expression and primary cell transfection. HBPL was synthesized by a thermal polycondensation reaction, using lysine hydrochloride as the raw material, first neutralizing it with one equivalent of KOH, and then carrying out the thermal polycondensation reaction, but the ratio of α-linear units and ε-linear units of HBPL was not regulated, and the antibacterial performance application of HBPL, especially the performance of selectively killing fungi, was not reported. Summary of the Invention

[0005] The object of the present invention is to provide a hyperbranched polylysine with a high content of ε-linear units, and its preparation method and application.

[0006] The technical solution of the present invention is as follows:

[0007] Using compounds such as 9-borabicyclo[3,3,1]nonane (9-BBN) to form a ring with the α-amino group and carboxyl group of lysine to obtain an intermediate (compound of formula I), then carrying out a polymerization reaction, and then obtaining a hyperbranched polylysine with a high content of ε-configuration through deprotection. The synthesis method of the intermediate has been published in Org. Lett. 2002, 4, 1249–1251. The polymerization reaction includes carrying out a melt polycondensation reaction on the intermediate, or carrying out a solution polycondensation reaction on the intermediate in solution; or copolymerizing the intermediate with lysine monomers in different ratios and carrying out a melt polycondensation or solution polycondensation reaction.

[0008] A preparation method of a hyperbranched polylysine with a high content of ε-linear units, comprising the following steps:

[0009] (1) Mix lysine and the compound of formula I in a certain ratio and carry out a polymerization reaction to obtain a polymer (II), wherein the molar proportion of lysine is 0-90%, and the molar proportion of the compound of formula I is 10-100%;

[0010] (2) Then, by removing the borane protecting group, a hyperbranched polylysine (III) with a high content of ε-linear units is obtained;

[0011]

[0012]

[0013] Among them, m, n, x, and y are the degrees of polymerization, 1≤m, n, x, y≤500, and the structure is an ε-linear unit, and the structure is an α-linear unit.

[0014] Preferably, the degree of polymerization of the hyperbranched polylysine is 1≤m, n, x, y≤200, 10≤m + n + x + y≤200, and the molecular weight distribution is 1.10-3.00; more preferably, the degree of polymerization of the hyperbranched polylysine is 10≤m, n, x, y≤60, 20≤m + n + x + y≤200, and the molecular weight distribution is 1.30-2.20.

[0015] Preferably, the molar proportion of the lysine is 0%-50%, and more preferably, the proportion of the lysine is 0%-20%.

[0016] Preferably, the polymerization reaction includes melt polycondensation reaction or solution polycondensation reaction. The solvent for the solution polymerization reaction is one or more of ethylene glycol, propylene glycol, ethylene glycol monomethyl ether, diethylene glycol monomethyl ether, anisole, dioxane, N,N-dimethylformamide, NMP, N-methylpyrrolidone, dimethyl sulfoxide, and sulfolane. In the melt polycondensation reaction, a catalyst may or may not be used during the polymerization process. The catalyst can be selected from one or more of metal salts; metal organic compounds or metal organic complexes; tertiary amines or quaternary amine salts; boric acid and its derivatives, phosphoric acid or phosphonic acid and its derivatives series; carboxylates.

[0017] Preferably, the temperature of the polymerization reaction in step 1) is 100°C - 220°C, and the time is 30 minutes - 48 hours; the reaction temperature in step 2) is controlled at 20 - 90°C, and the time is 30 minutes - 24 hours.

[0018] Preferably, the temperature of the polymerization reaction in step 1) is 140°C - 200°C, and the time is 1 hour - 24 hours; the compound of formula I is prepared by reacting lysine hydrochloride with 9-BBN.

[0019] Preferably, the removal of the borane protecting group is achieved by reacting the polymer (II) with a 25% - 38% hydrochloric acid solution.

[0020] Use of the hyperbranched polylysine with a high content of ε-linear units prepared by the above method in selectively killing fungi.

[0021] Preferably, the molar content of the ε-linear unit is more than 50% of the total molar number of the ε-linear unit and the α-linear unit.

[0022] Preferably, the molar content of the ε-linear unit is 55% - 95%, and more preferably, the molar content of the ε-linear unit is 70% - 90%.

[0023] Principle of the present invention: α-polylysine can only kill bacteria, and ε-polylysine has a certain bactericidal effect on fungi, but the effect is worse than that of hyperbranched polylysine. This may be because compared with ε-polylysine, hyperbranched polylysine has a higher charge density while containing ε-linear units. Therefore, by regulating the proportion of each structural unit of hyperbranched polylysine and increasing the proportion of ε-linear units, selective killing of fungi can be achieved.

[0024] 9-BBN can selectively protect the α-NH2 of lysine, thereby reducing the reactivity of α-NH2 during the polymerization process and increasing the reactivity of ε-NH2. Compared with the hyperbranched polylysine obtained by polymerizing lysine hydrochloride monomer, the hyperbranched polylysine obtained by polymerizing the 9-BBN-lysine intermediate (compound of formula I) has a higher content of ε-linear units. By copolymerizing the compound of formula I with lysine and adjusting the feeding ratio of the intermediate to the lysine monomer, the ratio of ε- / α-configurations in the hyperbranched polylysine can be regulated, thereby achieving the selective killing of bacteria / fungi.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) By adjusting the degree of branching of HPL and the ratio of α-linear units and ε-linear units, the antibacterial properties and toxicity of the present invention can be effectively regulated. In particular, the ratio of α-linear units and ε-linear units in HPL can be controllably regulated through a special reaction pathway, thereby regulating its antibacterial properties and toxicity.

[0027] (2) The present invention can achieve the selective killing of fungi / bacteria through structural regulation. In a specific structure, it can achieve only antifungal / killing of fungi and has no killing effect on most bacteria, effectively protecting the natural flora of the human body.

[0028] (3) The hyperbranched polylysine with high ε-linear units prepared by the present invention has high fungicidal efficiency, low toxicity, and good biocompatibility. Description of the Drawings

[0029] Figure 1 is the 1H NMR spectrum of the hyperbranched polylysine prepared in Example 1.

[0030] Figure 2 is the 13C NMR spectrum of the hyperbranched polylysine prepared in Example 1.

[0031] Figure 3 is the bar graph of the antibacterial properties of the hyperbranched polylysine in Example 2.

[0032] Figure 4 is the photo of the experimental results of the antibacterial properties in Example 2. The upper figure is the test result by the microbroth dilution method, and the lower figure is the test result by the plate dilution method. Escherichia coli (left), Staphylococcus aureus (middle), Candida albicans (right).

[0033] Figure 5 is the SEM images of Escherichia coli (upper), Staphylococcus aureus (middle), and Candida albicans (lower) after being treated with hyperbranched polylysine. Detailed Embodiments

[0034] The present invention will be further described in detail below in conjunction with specific embodiments. However, the implementation manners of the present invention are not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0035] Example 1

[0036] Synthesis of hyperbranched polylysine

[0037] (1) Melt polycondensation

[0038] 9-BBN-lysine (Compound of Formula I), lysine hydrochloride, and KOH were added to a 100 mL round-bottom flask. A water-separating device was connected. The temperature was raised to 180 °C under an anaerobic environment, and the mixture was stirred and reacted for 6 hours. Heating was stopped, and the product was dissolved in methanol and precipitated into ether. 2 g of the polymer intermediate was dissolved in 5 mL of methanol, 25 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 24 hours, then precipitated into tetrahydrofuran. The precipitation was repeated 3 times, and freeze-dried at -50 °C for 24 h to obtain hyperbranched polylysine.

[0039] (2) Solution polycondensation

[0040] The compound of Formula I, lysine hydrochloride, and KOH were dissolved in DMSO and added to a 100 mL round-bottom flask. A water-separating device was connected. The temperature was raised to 180 °C under an anaerobic environment, and the mixture was stirred and reacted for 6 hours. Heating was stopped, and the product was precipitated into ether. 2 g of the polymer intermediate was dissolved in 5 mL of methanol, 25 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 24 hours, then precipitated into tetrahydrofuran. The precipitation was repeated 3 times, and freeze-dried to obtain hyperbranched polylysine.

[0041] By adjusting the ratio of the compound of Formula I to lysine hydrochloride, hyperbranched polylysine with different ε-configuration contents can be obtained. The proton nuclear magnetic resonance spectrum of the obtained hyperbranched polylysine is as Figure 1 shown, and the carbon nuclear magnetic resonance spectrum is as Figure 2 shown. The specific feed ratio and ε-configuration content are shown in Table 1, where HPL-Bn is hyperbranched polylysine prepared by the melt polycondensation method, and S-HPL-Bn is hyperbranched polylysine prepared by the solution polymerization method.

[0042] Table 1

[0043]

[0044]

[0045] Comparative Example 1 91.32 g of lysine hydrochloride and 28.05 g of KOH were added to a 500 mL round-bottom flask. A water-separating device was connected, and the mixture was stirred and reacted at 180 °C for 4 h under a nitrogen atmosphere. Heating was stopped, and the reaction system was cooled to room temperature. The polymer was dissolved in methanol and precipitated into diethyl ether to obtain 75.2 g of hyperbranched polylysine. The ε-configuration content of the product of Comparative Example 1 was 30%.

[0046] As can be seen from the above comparison, by controlling the molar ratio of the compound of formula I to lysine hydrochloride, the structure of hyperbranched polylysine can be effectively regulated, and hyperbranched polylysine with a high content of ε-linear units can be obtained.

[0047] Example 2 Antibacterial Performance Test

[0048] Against bacteria: 36 mg of the hyperbranched polylysine of Example 1 and Comparative Example 1 was dissolved in 3 mL of sterile PBS to obtain a stock solution of 12 mg / mL for antibacterial performance testing. The testing method was as follows:

[0049] The antibacterial performance of hyperbranched polylysine was tested by the microbroth dilution method. A small amount of bacterial single colony was picked with an inoculation loop and placed in MH broth medium, and cultured overnight at 37 °C to revive the viability of the bacterial strain and grow to the mid-logarithmic growth phase. The concentration of the bacterial suspension was adjusted to ~10 6 CFU / mL with MH medium. 175 μL of the bacterial suspension was added to a 96-well plate, and then 25 μL of the gradient-diluted hyperbranched polylysine solution was added. The 96-well plate was placed in an incubator at 37 °C for 24 hours, and the OD 600 was detected with an enzyme-labeled instrument to determine the growth of bacteria, and the turbidity of the bacterial suspension in each well was observed visually for assistance. The minimum inhibitory concentration (MIC) was defined as the concentration of the antibacterial agent required to inhibit 90% of bacterial growth compared with the control group.

[0050] Against fungi: 36 mg of the hyperbranched polylysine of Example 1 and Comparative Example 1 was dissolved in 3 mL of sterile MOPS to obtain a stock solution of 12 mg / mL for antifungal performance testing. The testing method was as follows:

[0051] The antifungal performance of hyperbranched polylysine was tested by the microbroth dilution method. A small amount of fungal single colony was picked with an inoculation loop and placed in SDB medium, and cultured at 35 °C for 24 hours to revive the viability of the fungal strain and grow to the mid-logarithmic growth phase. The concentration of the fungal suspension was adjusted to ~10 3 CFU / mL with RPMI1640-MOPS medium. 175 μL of the fungal suspension was added to a 96-well plate, and then 25 μL of the gradient-diluted hyperbranched polylysine solution was added. The 96-well plate was placed in an incubator at 35 °C for 48 hours, and the OD 600Determine the growth of fungi and assist in visually observing the turbidity of the fungal solution in each well. The minimum inhibitory concentration (MIC) is defined as the concentration of the bacteriostatic agent required to inhibit 90% of fungal growth compared to the control group.

[0052] Table 2 Test results of antibacterial performance (MIC value, unit μg / mL)

[0053]

[0054] As can be seen from Table 2, compared with Comparative Example 1, the hyperbranched polylysine of the present invention can kill only fungi without killing bacteria, thus achieving the effect of selective sterilization.

[0055] Example 3 Blood compatibility test

[0056] Take 36 mg of the hyperbranched polylysine obtained in Example 1 and dissolve it in 3 mL of sterile PBS solution to obtain a stock solution of 12 mg / mL for in vitro hemolysis test. The test results are shown in Table 3, and the test method is as follows:

[0057] Take 1 mL of fresh rabbit blood, collect red blood cells after sufficient centrifugation, and dilute with sterile PBS to obtain a 2% (v / v) red blood cell suspension. Add 175 μL of the red blood cell suspension to a 1.5 mL sterile centrifuge tube, then add 25 μL of the gradient-diluted hyperbranched polylysine solution. Add 25 μL of 0.5% TX-100 solution to the positive control and 25 μL of sterile PBS to the negative control. Incubate at 37 °C for 1 hour, centrifuge, and pipette 150 μL of the supernatant into a 96-well plate, and detect OD with an enzyme-linked immunosorbent assay instrument 540 , HC 10 is defined as the polymer concentration that causes 10% hemolysis of red blood cells.

[0058] Table 3 Hemolysis test results (HC 10 value, unit μg / mL)

[0059] <![CDATA[HC 10 > HPL-B100 >1500 HPL-B95 >1500 HPL-B90 >1500 HPL-B80 >1500 HPL-B70 >1500 HPL-B60 >1500 HPL-B50 >1500 HPL-B40 >1500 HPL-B30 >1500 HPL-B20 >1500 HPL-B10 >1500

[0060] Example 4 Cytotoxicity test

[0061] Take 36 mg of the hyperbranched polylysine obtained in Example 1 and dissolve it in 3 mL of sterile PBS solution to obtain a stock solution of 12 mg / mL for cytotoxicity test. The test results are shown in Table 4, and the test method is as follows:

[0062] After digesting and eluting the well-grown mouse embryonic fibroblast NIH3T3 / L929 cells, a cell suspension of 40,000 cells / mL was prepared. 200 μL of the cell suspension was added to a 96-well plate and cultured in an incubator at 37 °C with 5% CO2 for 12 hours until the cells adhered. Then, 25 μL of the hyperbranched polylysine solution with gradient dilution was added, and the 96-well plate was cultured in an incubator at 37 °C with 5% CO2 for 24 hours. The cytotoxicity was tested by the MTT method. IC 50 is defined as the polymer concentration that inhibits 50% of cell growth.

[0063] Table 4 Cytotoxicity test results (IC 50 value, unit μg / mL)

[0064] <![CDATA[IC 50 (NIH3T3)]]> <![CDATA[IC 50 (L929)]]> HPL-B100 >1500 >1500 HPL-B95 >1500 >1500 HPL-B90 >1500 >1500 HPL-B80 >1500 >1500 HPL-B70 >1500 >1500 HPL-B60 >1500 >1500 HPL-B50 >1500 >1500 HPL-B40 >1500 >1500 HPL-B30 >1500 >1500 HPL-B20 >1500 >1500 HPL-B10 >1500 >1500

[0065] From the above examples and comparative examples, it can be seen that the hyperbranched polylysine with high ε-linear units prepared by the present invention can selectively and efficiently kill fungi without killing bacteria, and has low toxicity and good biocompatibility.

[0066] The above examples are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent substitution methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing hyperbranched polylysine with a high content of ε-linear units, characterized in that: The steps include: (1) mixing lysine and a compound of formula I in a certain ratio and performing a polymerization reaction to obtain a polymer (II), wherein the molar proportion of lysine is 0-90% and the molar proportion of the compound of formula I is 10-100%; (2) removing the borane protecting group to obtain a hyperbranched polylysine (III) with a high content of ε-linear units; Among them, m, n, x and y are the degree of polymerization, 1≤m, n, x, y≤500, the structure is an ε-linear unit, the structure is an α-linear unit.

2. The preparation method according to claim 1, characterized in that: The polymerization degree of the hyperbranched polylysine is 1≤m, n, x, y≤200, 10≤m+n+x+y≤200, and the molecular weight distribution is 1.10-3.

00.

3. The preparation method according to claim 2, characterized in that: The polymerization degree of the hyperbranched polylysine is 10≤m, n, x, y≤60, 20≤m+n+x+y≤200, and the molecular weight distribution is 1.30-2.

20.

4. The preparation method according to claim 3, characterized in that: The molar proportion of lysine is 0%-50%.

5. The preparation method according to claim 4, characterized in that: The lysine accounts for 0%-20%.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The polymerization reaction includes a melt polycondensation reaction or a solution polycondensation reaction. The solvent of the solution polymerization reaction is one or more of ethylene glycol, propylene glycol, ethylene glycol monomethyl ether, diethylene glycol monomethyl ether, anisole, dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, and sulfolane.

7. The preparation method according to claim 6, characterized in that: In step 1), the polymerization reaction temperature is 100° C.-220° C., and the reaction time is 30 minutes-48 hours; in step 2), the reaction temperature is controlled at 20-90° C., and the reaction time is 30 minutes-24 hours.

8. The preparation method according to claim 7, characterized in that: The polymerization reaction temperature in step 1) is 140°C-200°C, and the time is 1 hour-24 hours; the compound of formula I is prepared by reacting lysine hydrochloride with 9-BBN; and the removal of the borane protecting group is achieved by reacting the polymer (II) with concentrated hydrochloric acid.

9. Hyperbranched polylysine with a high content of ε-linear units obtained by the method according to any one of claims 1 to 8.

10. Use of the hyperbranched polylysine with a high content of ε-linear units according to claim 9 in selectively killing fungi.

11. The use according to claim 10, characterized in that: The molar content of the ε-linear unit accounts for more than 50% of the total molar number of the ε-linear unit and the α-linear unit.

12. The use according to claim 11, characterized in that: The molar content of the ε-linear unit is 55% to 95%.

13. The use according to claim 12, characterized in that: The molar content of the ε-linear units is 70% to 90%.

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