CN107011473A - Leucine methacrylate homopolymer, preparation method and application in antibacterial - Google Patents

Abstract

The invention discloses a leucine methacrylate homopolymer, a preparation method and application thereof in antibiosis, wherein the leucine methacrylate homopolymer is prepared by homopolymerizing monomer D-type or L-type leucine methacrylate, dithiobenzoic acid-4-cyanovaleric acid is taken as a chain transfer agent, and azobisisobutyronitrile is taken as an initiator to perform reversible addition-fragmentation chain transfer polymerization; the polymer of the invention has excellent antibacterial performance, and can obviously destroy the structure of a bacterial membrane to cause the death of bacteria; hemolysis experiment and in vitro smooth muscle cell compatibility experiment show that the biocompatibility is kept remarkably.

Description

Leucine methacrylate homopolymer, preparation method and application in antibacterial

technical field

The invention belongs to the field of biomedical polymer materials, and relates to a method for designing and synthesizing a novel imitation antimicrobial peptide polymer, in particular to the preparation of a cationic chiral amino acid methacrylate polymer and its antibacterial application.

Background technique

At present, the emergence of antibiotic-resistant bacteria has posed a great threat to public health. Bacterial adhesion, proliferation, and biofilm formation can lead to infection in patients and lead to a series of complications and even endanger the lives of patients. Although some progress has been made in the research of alternative new antibacterial polymers, there are still great challenges in their biomedical and clinical applications. Alternative new antibacterial polymers should not only have efficient antibacterial properties, but also not affect the normal physiological functions of body tissues. Simply put, they can selectively kill bacteria without toxicity to normal mammalian cells. Therefore, antibacterial polymers with high antibacterial activity and selectivity are still to be studied.

Contents of the invention

The purpose of the present invention is to prepare a cationic polymethacrylate polymer based on chiral amino acids, and to study its application in antibacterial aspects, so as to overcome the weak antibacterial activity and poor biocompatibility of existing antibacterial polymers Shortcomings.

The present invention adopts following technical scheme:

L-leucine methacrylate homopolymer, that is, P(L-Leu-HEMA), is formed by the homopolymerization of monomer D-leucine methacrylate. The molecular formula and structure are as follows:

n is the degree of polymerization, the number average molecular weight of the polymer is 10-23kDa, and the molecular weight distribution coefficient is 1.10-1.25.

The preparation method of the above-mentioned homopolymer is formed by homopolymerizing the monomer L-leucine methacrylate, using dithiobenzoic acid-4-cyanovaleric acid as the chain transfer agent, and azobisisobutyronitrile as Initiator for RAFT polymerization, the molar ratio of monomer L-leucine methacrylate, chain transfer agent and initiator is (45-60):1:0.2, preferably (50-60):1:0.2.

In the above technical scheme, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N,N-dimethylformamide provides the reaction Ambiance and environment.

In the above technical scheme, the monomer L-leucine methacrylate is prepared according to the following steps: L-leucine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydration condensation agent, reacted in an ice-water bath with dichloromethane as a solvent for 30-60 minutes, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein L-leucine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), L-leucine is L-leucine protected by tert-butoxycarbonyl.

In the above technical scheme, the prepared homopolymer is deprotected under the action of trifluoroacetic acid, preferably the homopolymer is dissolved in dichloromethane, and the deprotection reaction is carried out at 20-25 degrees Celsius under the action of trifluoroacetic acid After 2-4 hours, it can be dissolved and dialyzed after purification, then freeze-dried.

Due to the use of trifluoroacetic acid, there are trifluoroacetic acid groups in the polymer after the deprotection reaction. Since the isoelectric point of Leu is pH 6.01, and the isoelectric point of Lys is pH 9.60, when the pH is adjusted to 7.2, the -NH of leu ₃ ⁺ is deprotonated to generate -NH ₂ , as shown in the following chemical formula:

D-leucine methacrylate homopolymer, that is, P(D-Leu-HEMA), is formed by the homopolymerization of monomer D-leucine methacrylate. The molecular formula shows the structure as follows, n is the degree of polymerization, The number average molecular weight of the polymer is 10-23kDa, and the molecular weight distribution coefficient is 1.10-1.25.

The preparation method of the above-mentioned homopolymer is formed by homopolymerizing the monomer D-leucine methacrylate, using dithiobenzoic acid-4-cyanovaleric acid as the chain transfer agent, and azobisisobutyronitrile as The initiator is used for RAFT polymerization, and the molar ratio of monomer D-leucine methacrylate, chain transfer agent and initiator is (45-60):1:0.2, preferably (50-60):1:0.2.

In the above technical scheme, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N,N-dimethylformamide provides the reaction Ambiance and environment.

In the above technical scheme, the monomer D-leucine methacrylate is prepared according to the following steps: D-leucine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydrating condensation agent, reacted in an ice-water bath with dichloromethane as a solvent for 30-60 minutes, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein D-leucine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), D-leucine is D-leucine protected by tert-butoxycarbonyl.

In the above technical scheme, the prepared homopolymer is deprotected under the action of trifluoroacetic acid, preferably the homopolymer is dissolved in dichloromethane, and the deprotection reaction is carried out at 20-25 degrees Celsius under the action of trifluoroacetic acid After 2-4 hours, it can be dissolved and dialyzed after purification, then freeze-dried.

Due to the use of trifluoroacetic acid, there are trifluoroacetic acid groups in the polymer after the deprotection reaction. Since the isoelectric point of Leu is pH 6.01, and the isoelectric point of Lys is pH 9.60, when the pH is adjusted to 7.2, the -NH of leu ₃ ⁺ is deprotonated to generate -NH ₂ , as shown in the following chemical formula:

L-lysine methacrylate homopolymer, that is, P(L-Lys-HEMA), is formed by the homopolymerization of monomer L-lysine methacrylate. The molecular formula shows the structure as follows, n is the degree of polymerization, The number average molecular weight of the polymer is 10-23kDa, and the molecular weight distribution coefficient is 1.10-1.25.

The preparation method of the above-mentioned homopolymer is formed by homopolymerizing monomer L-lysine methacrylate, using dithiobenzoic acid-4-cyanovaleric acid as a chain transfer agent, and azobisisobutyronitrile as Initiator for RAFT polymerization, the molar ratio of monomer L-lysine methacrylate, chain transfer agent and initiator is (45-60):1:0.2, preferably (50-60):1:0.2.

In the above technical scheme, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N,N-dimethylformamide provides the reaction Ambiance and environment.

In the above technical scheme, the monomer L-lysine methacrylate is prepared according to the following steps: L-lysine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydration condensation agent, reacted in an ice-water bath for 30-60 minutes with dichloromethane as a solvent, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein L-lysine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), L-lysine is L-lysine protected by tert-butoxycarbonyl.

In the above technical scheme, the prepared homopolymer is deprotected under the action of trifluoroacetic acid, preferably the homopolymer is dissolved in dichloromethane, and the deprotection reaction is carried out at 20-25 degrees Celsius under the action of trifluoroacetic acid After 2-4 hours, it can be dissolved and dialyzed after purification, then freeze-dried.

Due to the use of trifluoroacetic acid, the polymer after the deprotection reaction contains trifluoroacetic acid groups, the pH is adjusted to 7.2, and lys is not affected, as shown in the following chemical formula:

D-lysine methacrylate homopolymer, that is, P(D-Leu-HEMA), is formed by the homopolymerization of monomer D-lysine methacrylate. The molecular formula shows the structure as follows, n is the degree of polymerization, The number average molecular weight of the polymer is 10-23kDa, and the molecular weight distribution coefficient is 1.10-1.25.

The preparation method of the above-mentioned homopolymer is formed by homopolymerizing the monomer D-lysine methacrylate, using dithiobenzoic acid-4-cyanovaleric acid as the chain transfer agent, and azobisisobutyronitrile as Initiator for RAFT polymerization, the molar ratio of monomer D-lysine methacrylate, chain transfer agent and initiator is (45-60):1:0.2, preferably (50-60):1:0.2.

In the above technical scheme, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N,N-dimethylformamide provides the reaction Ambiance and environment.

In the above technical scheme, the monomer D-lysine methacrylate is prepared according to the following steps: D-lysine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydration condensation agent, and dichloromethane is used as a solvent to react in an ice-water bath for 30-60 minutes, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein D-lysine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), D-lysine is D-lysine protected by tert-butoxycarbonyl.

In the above technical scheme, the prepared homopolymer is deprotected under the action of trifluoroacetic acid, preferably the homopolymer is dissolved in dichloromethane, and the deprotection reaction is carried out at 20-25 degrees Celsius under the action of trifluoroacetic acid After 2-4 hours, it can be dissolved and dialyzed after purification, then freeze-dried.

Due to the use of trifluoroacetic acid, the polymer after the deprotection reaction contains trifluoroacetic acid groups, the pH is adjusted to 7.2, and lys is not affected, as shown in the following chemical formula:

D-cationic chiral amino acid methacrylate copolymer is prepared by block copolymerization of monomers D-lysine methacrylate and D-leucine methacrylate. The molecular formula is as follows, and n and m are The degree of polymerization of the respective monomers, m/n is (0.8-1.2), preferably the degree of polymerization of the two monomers is consistent m=n, the number average molecular weight of the polymer is 10-23kDa, and the molecular weight distribution coefficient is 1.10-1.25.

The preparation method of above-mentioned copolymer is prepared according to the following steps:

Step 1. Homopolymerize the first D-type amino acid monomer, use dithiobenzoic acid-4-cyanovaleric acid as the chain transfer agent, and azobisisobutyronitrile as the initiator to perform RAFT polymerization to obtain the D configuration Amino acid monomer polymer, the molar ratio of the first D-type amino acid monomer, chain transfer agent and initiator is (45-60):1:0.2, preferably (50-60):1:0.2;

Step 2, the D-configuration amino acid monomer polymer macromolecular chain transfer agent prepared in step 1, azobisisobutyronitrile is used as an initiator, and the second D-type amino acid monomer is added to carry out RAFT polymerization to obtain D-lysine methyl Base acrylate and D-leucine methacrylate block copolymer, the molar ratio of the second D-type amino acid monomer, macromolecular chain transfer agent and initiator is (80-100): 1: 0.2, preferably (85—90):1:0.2.

In the above technical scheme, in step 1, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N, N-dimethyl The base formamide provides the reaction atmosphere and environment.

In the above technical scheme, in step 2, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N, N-dimethyl The base formamide provides the reaction atmosphere and environment.

In the above technical scheme, the monomer D-lysine methacrylate is prepared according to the following steps: D-lysine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydration condensation agent, and dichloromethane is used as a solvent to react in an ice-water bath for 30-60 minutes, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein D-lysine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), D-lysine is D-lysine protected by tert-butoxycarbonyl.

In the above technical scheme, the monomer D-leucine methacrylate is prepared according to the following steps: D-leucine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydrating condensation agent, reacted in an ice-water bath with dichloromethane as a solvent for 30-60 minutes, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein D-leucine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), D-leucine is D-leucine protected by tert-butoxycarbonyl.

In the above technical scheme, the prepared copolymer is deprotected under the action of trifluoroacetic acid, preferably the copolymer is dissolved in methylene chloride, and under the action of trifluoroacetic acid, the deprotection reaction is carried out at 20-25 degrees Celsius 2- After 4 hours, after purification, it was dissolved and dialyzed, then freeze-dried.

Due to the use of trifluoroacetic acid, there are trifluoroacetic acid groups in the polymer after the deprotection reaction. Since the isoelectric point of Leu is pH 6.01, and the isoelectric point of Lys is pH 9.60, when the pH is adjusted to 7.2, the -NH of leu ₃ ⁺ is deprotonated to generate -NH ₂ , and the pH is adjusted to 7.2, and lys is not affected, as shown in the following chemical formula:

L-cationic chiral amino acid methacrylate copolymer is prepared by block copolymerization of monomers L-lysine methacrylate and L-leucine methacrylate. The molecular formula is as follows, and n and m are The degree of polymerization of the respective monomers, m/n is (0.8-1.2), preferably the degree of polymerization of the two monomers is consistent m=n, the number average molecular weight of the polymer is 10-23kDa, and the molecular weight distribution coefficient is 1.10-1.25.

The preparation method of above-mentioned copolymer is prepared according to the following steps:

Step 1. Homopolymerize the first L-type amino acid monomer, use dithiobenzoic acid-4-cyanovaleric acid as the chain transfer agent, and azobisisobutyronitrile as the initiator to perform RAFT polymerization to obtain the D configuration Amino acid monomer polymer, the molar ratio of the first L-type amino acid monomer, chain transfer agent and initiator is (45-60):1:0.2, preferably (50-60):1:0.2;

Step 2, the L-configuration amino acid monomer polymer macromolecular chain transfer agent prepared in step 1, azobisisobutyronitrile is used as an initiator, and the second L-type amino acid monomer is added to carry out RAFT polymerization to obtain L-lysine methyl acrylate and L-leucine methacrylate block copolymer, the molar ratio of the second L-type amino acid monomer, macromolecular chain transfer agent and initiator is (80-100):1:0.2, preferably (85—90):1:0.2.

In the above technical scheme, in step 1, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N, N-dimethyl The base formamide provides the reaction atmosphere and environment.

In the above technical scheme, in step 2, under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and the solvent N, N-dimethyl The base formamide provides the reaction atmosphere and environment.

In the above technical scheme, the monomer L-lysine methacrylate is prepared according to the following steps: L-lysine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydration condensation agent, reacted in an ice-water bath for 30-60 minutes with dichloromethane as a solvent, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein L-lysine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), L-lysine is L-lysine protected by tert-butoxycarbonyl.

In the above technical scheme, the monomer L-leucine methacrylate is prepared according to the following steps: L-leucine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, N,N '-Dicyclohexylcarbodiimide is used as a dehydration condensation agent, reacted in an ice-water bath with dichloromethane as a solvent for 30-60 minutes, and then stirred and reacted for 24-48 hours at a room temperature of 20-25 degrees Celsius, wherein L-leucine The mass ratio of acid, 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2—0.25):(4—5):(2—3 ), L-leucine is L-leucine protected by tert-butoxycarbonyl.

In the above technical scheme, the prepared copolymer is deprotected under the action of trifluoroacetic acid, preferably the copolymer is dissolved in methylene chloride, and under the action of trifluoroacetic acid, the deprotection reaction is carried out at 20-25 degrees Celsius 2- After 4 hours, after purification, it was dissolved and dialyzed, then freeze-dried.

Due to the use of trifluoroacetic acid, there are trifluoroacetic acid groups in the polymer after the deprotection reaction. Since the isoelectric point of Leu is pH 6.01, and the isoelectric point of Lys is pH 9.60, when the pH is adjusted to 7.2, the -NH of leu ₃ ⁺ is deprotonated to generate -NH ₂ , and the pH is adjusted to 7.2, and lys is not affected, as shown in the following chemical formula:

The invention provides a method for regulating the overall configuration of the copolymer, that is, adopting D-configuration and L-configuration monomers for copolymerization, and changing the molar ratio between the two through the feeding ratio of D-type/L-type monomers, To make the copolymer exhibit D-type or L-type, that is, the application of controllable living polymerization in regulating the chirality of leucine-lysine methacrylate copolymer.

Use the reversible addition-fragmentation chain transfer polymerization method to change the molar ratio between the two through the feeding ratio of D-type/L-type monomers, so that the copolymer behaves as D-type or L-type, that is, through D-type and L-type The feeding ratio of the monomers is to change the molar ratio between the D-type monomer and the L-type monomer in the copolymer, so that the overall performance of the copolymer is D-type or L-type.

As far as the above four monomers are concerned, the polymer obtained by homopolymerization shows the D and L configurations consistent with the corresponding monomers, the copolymer obtained by the copolymerization of two L-type monomers exhibits L-type, and the two D-type monomers The copolymer obtained by bulk copolymerization is D-type, the copolymer is copolymerized with D-type and L-type monomers, and the relative quantity of D-type monomer and L-type monomer can be adjusted to realize the controllability of the overall configuration of the copolymer .

In the technical scheme of the present invention, active polymerization can be used to control the added materials (i.e. the molar ratio of monomers), the reaction conditions are mild, the yield of synthetic monomers is 70%-85%, and the conversion rate of polymerization reaction is 50%-90% , the molecular weight of homopolymers and copolymers is controllable and the distribution coefficient is narrow.

The invention discloses a chiral amino acid methacrylate polymer, its preparation method and antibacterial application. The circular dichroism spectrum characterization results prove that the L-configuration and D-configuration polymers prepared above have opposite secondary helical structures. Microdilution antibacterial experiments showed that the minimum inhibitory concentration (MIC) of chiral amino acid-based methacrylate polymers obtained above after dialysis and freeze-drying was 100-1000 μg·mL ^-1 , showing excellent antibacterial properties. Emission scanning electron microscope result figure visually demonstrates that the antibacterial polymer prepared by the present invention can obviously destroy the bacterial membrane structure and cause the death of bacteria; hemolysis experiment and in vitro smooth muscle cell compatibility experiment show that the cationic chiral amino acid formazan prepared by the present invention The acrylate antibacterial polymer can still maintain remarkable biocompatibility at the concentration required for antibacterial, wherein the hemolysis rate is lower than 10%, and the activity of smooth muscle cells can reach more than 80%.

Description of drawings

Figure 1 is a schematic diagram of the polymerization mechanism of reversible addition-fragmentation chain transfer polymerization (RAFT) in the present invention.

Fig. 2 is the ¹ H-NMR spectrum of monomer D-Leu(Boc)-HEMA in CDCl ₃ in the present invention.

Fig. 3 is the ¹ H-NMR spectrum of monomer D-Lys(Boc)-HEMA in CDCl ₃ in the present invention.

Fig. 4 is undeprotected P (D-Leu (Boc)-HEMA) homopolymer (1) in the present invention, P (D-Lys (Boc)-HEMA) homopolymer (2) and P (D- ¹ H-NMR spectrum of Leu(Boc)-HEMA)-bP(D-Lys(Boc)-HEMA) block copolymer (3) in CDCl ₃ .

Fig. 5 is P (D-Leu-HEMA) homopolymer (1) after deprotection in the present invention, P (D-Lys-HEMA) homopolymer homopolymer (2) and P (D-Leu-HEMA) - ¹ H-NMR spectrum of bP(D-Lys-HEMA) block copolymer homopolymer (3) in D ₂ O.

Fig. 6 is a graph showing circular dichroism characterization results of homopolymers and copolymers in the present invention.

Fig. 7 is a scanning electron micrograph of polymer antibacterial experiments in the present invention.

Fig. 8 is a graph (1) of the antibacterial experiment effect of the polymer against Escherichia coli in the present invention.

Fig. 9 is a graph (1) of the antibacterial experimental effect of the polymer against Staphylococcus aureus in the present invention.

Fig. 10 is a graph showing the in vitro biocompatibility of the polymer P(L-Leu-HEMA) of the present invention.

Fig. 11 is a graph showing the in vitro biocompatibility of the polymer P(D-Leu-HEMA) of the present invention.

Fig. 12 is a graph showing the in vitro biocompatibility of the polymer P(D-Leu-HEMA-b-D-Lys-HEMA) of the present invention.

Fig. 13 is an in vitro biocompatibility characterization chart of the polymer P (L-Leu-HEMA-b-L-Lys-HEMA) of the present invention.

detailed description

The technical solutions of the present invention will be further described below in conjunction with specific embodiments. Experimental raw material and instrument that embodiment uses are shown in the following table respectively:

(1) Experimental raw materials

(2) Experimental equipment

In the embodiment of the present invention, reversible addition-fragmentation chain transfer polymerization is used. RAFT polymerization is a kind of living/controllable free radical polymerization, which is suitable for monomers containing double bond functional groups. In RAFT polymerization, the traditional initiator is thermally decomposed into the primary free radical I ^· , and initiates the polymerization of the monomer into the propagating free radical P _n ^· , and the propagating free radical undergoes reversible addition to the C=S bond in the chain transfer agent to form a dormant intermediate species, the SR bond in the dormant species breaks to form a new active species free radical R _n ^· , and then initiates monomer polymerization. The reaction mechanism is shown in Figure 1 below. Different from traditional free radical polymerization, RAFT polymer chain transfer is a reversible process, and there is a balanced reaction of reversible addition and reversible fragmentation between intermediate dormant species and growing chain free radicals, thus ensuring that all chains grow with equal probability and form a narrow distribution For polymers, the concentration of free radicals in the system is maintained at a relatively constant low level, which inhibits the double-radical termination reaction of free radicals in the system, making the polymerization activity controllable. In the present invention, an initiator is used to initiate the first monomer, and when the first monomer and the chain transfer agent are used for active polymerization, when the second monomer is added, the initiator is supplemented to initiate the second monomer, which is obtained by active polymerization The homopolymer of the first monomer is a macromolecular chain transfer agent to carry out living polymerization of the second monomer.

Example 1—Preparation of monomers D-leucine hydroxyethyl methacrylate, L-leucine hydroxyethyl methacrylate, D-lysine hydroxyethyl methacrylate and L-lysine methyl Hydroxyethyl Acrylate

First, add 5 g of chiral amino acid monomers (D-leucine, L-leucine, D-lysine, and L-lysine) dissolved in 22 mL of dry dichloromethane into a dry double-neck round bottom flask. acid, in order to ensure the activity of the functional group in the reaction, select the above four amino acids protected by tert-butoxycarbonyl Boc, and use trifluoroacetic acid to remove the Boc protecting group after the reaction), purify with nitrogen gas under magnetic stirring, and then add soluble in 1.5 0.24g of catalyst DMAP in mL of dry dichloromethane, put the reaction flask in an ice-water bath, slowly add 4.53g of dehydration condensation agent DCC dissolved in 20mL of dry dichloromethane dropwise, and add 2.86g of it within 20min under the protection of nitrogen HEMA. The reaction was carried out in an ice-water bath for 30 minutes, and then the reaction was continued for 36 hours at room temperature 20-25 degrees Celsius under a nitrogen atmosphere. After the reaction was finished, the white precipitate was removed by suction filtration, and the obtained organic mixed solution was extracted four times with 70mL distilled water and 100mL dichloromethane, and the obtained organic layer was further extracted twice with 60mL0.1mol/L hydrochloric acid solution, and then extracted twice with 60mL saturated sodium bicarbonate solution. And 60mL saturated sodium chloride solution washed twice. Finally, it was dried with anhydrous sodium sulfate, stirred overnight, filtered, and the solvent was removed by rotary evaporation.

Utilize NMR to characterize four kinds of monomers prepared, the results are as shown in accompanying drawing 2 and 3, in view of the chemical composition of four kinds of monomers is divided into two kinds (being leucine hydroxyethyl methacrylate and lysine hydroxyethyl methacrylate), the chemical configuration is divided into D and L, and the NMR spectra of different configurations of the same chemical composition are basically the same. As shown in the two drawings, the chemical shift of NMR corresponds to the mark in the chemical formula Hydrogen atoms in different chemical environments proved that the four monomers were successfully prepared.

Example 2—Using the four monomers prepared in Example 1 as raw materials, four homopolymers were prepared using reversible addition-fragmentation chain transfer polymerization

Prepare cationic chiral amino acid hydroxyethyl methacrylate homopolymer by RAFT polymerization method: in a 25mL Schlenk bottle with a magnetic stirrer, add 1.5g of the monomer prepared in Example 1, CPADB 24.4mg, AIBN 2.86mg and 1.5 g of anhydrous DMF solvent, after three freeze-pump-thaw cycles to remove impurity gases in the reaction system, placed in an oil bath at 70° C., and reacted for 10 hours under nitrogen protection. After the reaction was completed, it was exposed to the air and placed in an ice-water bath for rapid cooling to terminate the reaction, and then the acetone/n-hexane precipitation was repeated 5 times. The obtained product was dried in a vacuum oven at 30° C. for 8 hours to obtain a sample of each homopolymer. Under the condition of ice-water bath, according to the ratio of 20 mL of dichloromethane and 10 mL of trifluoroacetic acid per 1 g of sample, it was added into the reaction flask, then reacted at room temperature for 3 h, and then rotary evaporated to remove the solvent. The resulting product was added dropwise to anhydrous ether, and the precipitation was repeated three times. The obtained product was dried in a vacuum oven at 30° C. for 8 h. The purified product was dissolved in deionized water, dialyzed for 72 hours to remove residual impurities, and a pure spongy solid sample was obtained after freeze-drying.

Utilize NMR to characterize four kinds of homopolymers of preparation, the result is shown in accompanying drawing 4 and 5, similarly based on two kinds of polymerizations of leucine hydroxyethyl methacrylate and lysine hydroxyethyl methacrylate There are different chemical configurations (D-type and L-type) of compounds, and the NMR spectra of different configurations of the same chemical composition are basically consistent. As shown in the two figures, the chemical shifts of NMR correspond to the different marks in the chemical formula. The hydrogen atoms in the chemical environment just proved that the four homopolymers were successfully prepared, and the overall polymer did not change after deprotection.

Example 3—Preparation of D-cationic chiral amino acid hydroxyethyl methacrylate copolymer

(1) Adopt the method of embodiment 2 to prepare the homopolymer of D-leucine at first: in the 25mLSchlenk bottle with magnetic stirrer, add the monomer D-leucine methacrylic acid prepared by 1.5g embodiment 1 Hydroxyethyl ester, CPADB24.4mg, AIBN 2.86mg and 1.5g of anhydrous DMF solvent, after three freeze-pump-thaw cycles to remove impurity gases in the reaction system, placed in an oil bath at 70°C under nitrogen protection React for 10 hours, after the reaction is basically finished, do not carry out the termination reaction of exposing the air, but keep the active group (CPADB) at the end of the D-leucine homopolymer as a macromolecular chain transfer for adding the second monomer to react agent;

(2) In the reaction vessel, add 3g of the same configuration of lysine methacrylate monomer (i.e. D-lysine hydroxyethyl methacrylate), AIBN 2.15mg and 3g of anhydrous DMF solvent, and 0.96 g of the previously prepared macromolecular chain transfer agent was reacted. After three freeze-pump-thaw cycles to remove impurity gases in the reaction system, it was placed in an oil bath at 70°C and reacted for 6 hours under the protection of nitrogen. After the first precipitation purification, the obtained product was dried in a vacuum oven at 30°C for 8 hours;

(3) Under ice-water bath conditions, add 20 mL of dichloromethane to 10 mL of trifluoroacetic acid per 1 g of the sample (i.e. the sample obtained in the above step 2), add it to the reaction flask, and then react at room temperature for 3 h, then rotate to evaporate Solvent was removed. The resulting product was added dropwise to anhydrous ether, and the precipitation was repeated three times. The obtained product was dried in a vacuum oven at 30° C. for 8 h. The purified product was dissolved in deionized water, dialyzed for 72 hours to remove residual impurities, and a pure spongy solid sample was obtained after freeze-drying. A D-configuration cationic chiral amino acid hydroxyethyl methacrylate copolymer was obtained.

Utilize nuclear magnetic resonance to characterize the cationic chiral amino acid hydroxyethyl methacrylate copolymer of prepared D configuration, as shown in accompanying drawing 4 and 5, the chemical shift of nuclear magnetic resonance corresponds to the hydrogen atom of different chemical environments marked in the chemical formula , which just proves that the copolymer was successfully prepared and the overall polymer did not change after deprotection.

Example 4—Preparation of L-cationic chiral amino acid methacrylate copolymer

(1) Adopt the method of embodiment 2 to prepare the homopolymer of L-leucine at first: in the 25mLSchlenk bottle with magnetic stirring bar, add the monomer L-leucine methacrylic acid prepared by 1.5g embodiment 1 Hydroxyethyl ester, CPADB 24.4mg, AIBN 2.86mg and 1.5g of anhydrous DMF solvent, after three freeze-pump-thaw cycles to remove impurity gases in the reaction system, put them in an oil bath at 70°C under nitrogen protection React for 10 hours. After the reaction is basically finished, do not carry out the termination reaction of exposing the air, but keep the active group (CPADB) at the end of the L-leucine homopolymer as the macromolecular chain transfer for adding the second monomer to react. agent;

(2) In the reaction vessel, add 3g of the same configuration of lysine methacrylate monomer (i.e. L-lysine hydroxyethyl methacrylate), AIBN 2.15mg and 3g of anhydrous DMF solvent, and 0.96 g of the previously prepared macromolecular chain transfer agent was reacted. After three freeze-pump-thaw cycles to remove impurity gases in the reaction system, it was placed in an oil bath at 70°C and reacted for 6 hours under the protection of nitrogen. After the first precipitation purification, the obtained product was dried in a vacuum oven at 30°C for 8 hours;

(3) Under ice-water bath conditions, add 20 mL of dichloromethane to 10 mL of trifluoroacetic acid per 1 g of the sample (i.e. the sample obtained in the above step 2), add it to the reaction flask, and then react at room temperature for 3 h, then rotate to evaporate Solvent was removed. The resulting product was added dropwise to anhydrous ether, and the precipitation was repeated three times. The obtained product was dried in a vacuum oven at 30° C. for 8 h. The purified product was dissolved in deionized water, dialyzed for 72 hours to remove residual impurities, and a pure spongy solid sample was obtained after freeze-drying. The L-configuration cationic chiral amino acid hydroxyethyl methacrylate copolymer was obtained.

Utilize NMR to characterize the copolymer prepared, compared with the cationic chiral amino acid hydroxyethyl methacrylate copolymer of D configuration prepared in Example 3, there is only difference (L type) in the configuration, the hydrogen atom The chemical environment is basically the same, that is, the chemical shifts of the D-type and the L-type are basically the same. The results are shown in Figures 4 and 5. The chemical shifts of the nuclear magnetic resonance correspond to the hydrogen atoms in different chemical environments marked in the chemical formula, which just proves that the copolymer was successfully prepared. , and the overall polymer does not change after deprotection.

Embodiment 5—Preparation of D-lysine/L-leucine methacrylate copolymer

(1) Adopt the method of embodiment 2 to prepare the homopolymer of L-leucine at first: in the 25mLSchlenk bottle with magnetic stirring bar, add the monomer L-leucine methacrylic acid prepared by 1.5g embodiment 1 Hydroxyethyl ester, CPADB 24.4mg, AIBN 2.86mg and 1.5g of anhydrous DMF solvent, after three freeze-pump-thaw cycles to remove impurity gases in the reaction system, put them in an oil bath at 70°C under nitrogen protection React for 10 hours. After the reaction is basically finished, do not carry out the termination reaction of exposing the air, but keep the active group (CPADB) at the end of the L-leucine homopolymer as the macromolecular chain transfer for adding the second monomer to react. agent;

(2) In the reaction vessel, add 3g of different configurations of lysine methacrylate monomer (i.e. D-lysine hydroxyethyl methacrylate), AIBN 2.15mg and 3g of anhydrous DMF solvent, and 0.96 g of the previously prepared macromolecular chain transfer agent was reacted. After three freeze-pump-thaw cycles to remove impurity gases in the reaction system, it was placed in an oil bath at 70°C and reacted for 6 hours under the protection of nitrogen. After the first precipitation purification, the obtained product was dried in a vacuum oven at 30°C for 8 hours;

(3) Under ice-water bath conditions, add 20 mL of dichloromethane to 10 mL of trifluoroacetic acid per 1 g of the sample (i.e. the sample obtained in the above step 2), add it to the reaction flask, and then react at room temperature for 3 h, then rotate to evaporate Solvent was removed. The resulting product was added dropwise to anhydrous ether, and the precipitation was repeated three times. The obtained product was dried in a vacuum oven at 30° C. for 8 h. The purified product was dissolved in deionized water, dialyzed for 72 hours to remove residual impurities, and a pure spongy solid sample was obtained after freeze-drying to obtain a D-lysine/L-leucine methacrylate copolymer.

Embodiment 6—Preparation of D-leucine/L-lysine methacrylate copolymer

(1) Adopt the method of embodiment 2 to prepare the homopolymer of D-leucine at first: in the 25mLSchlenk bottle with magnetic stirrer, add the monomer D-leucine methacrylic acid prepared by 1.5g embodiment 1 Hydroxyethyl ester, CPADB 24.4mg, AIBN 2.86mg and 1.5g of anhydrous DMF solvent, after three freeze-pump-thaw cycles to remove impurity gases in the reaction system, put them in an oil bath at 70°C under nitrogen protection React for 10 hours, after the reaction is basically finished, do not carry out the termination reaction of exposing the air, but keep the active group (CPADB) at the end of the D-leucine homopolymer as a macromolecular chain transfer for adding the second monomer to react agent;

(2) In the reaction vessel, add 3g of different configurations of lysine methacrylate monomer (i.e. L-lysine hydroxyethyl methacrylate), AIBN 2.15mg and 3g of anhydrous DMF solvent, and 0.96 g of the previously prepared macromolecular chain transfer agent was reacted. After three freeze-pump-thaw cycles to remove impurity gases in the reaction system, it was placed in an oil bath at 70°C and reacted for 6 hours under the protection of nitrogen. After the first precipitation purification, the obtained product was dried in a vacuum oven at 30°C for 8 hours;

(3) Under ice-water bath conditions, add 20 mL of dichloromethane to 10 mL of trifluoroacetic acid per 1 g of the sample (i.e. the sample obtained in the above step 2), add it to the reaction flask, and then react at room temperature for 3 h, then rotate to evaporate Solvent was removed. The resulting product was added dropwise to anhydrous ether, and the precipitation was repeated three times. The obtained product was dried in a vacuum oven at 30° C. for 8 h. The purified product was dissolved in deionized water, dialyzed for 72 hours to remove residual impurities, and a pure spongy solid sample was obtained after freeze-drying. A D-leucine/L-lysine methacrylate copolymer was obtained.

The polymer obtained by the homopolymerization of four monomers exhibits the D and L configurations consistent with the corresponding monomers, the copolymer obtained by the copolymerization of two L-type monomers exhibits an L-type, and the copolymer obtained by the copolymerization of two D-type monomers Copolymer shows as D type, and the copolymer prepared in embodiment 5 and 6 adopts D type and L type monomer to carry out copolymerization, can pass through the feeding ratio of D type/L type monomer, change the molar ratio between the two, with Make the copolymer exhibit D-type or L-type, that is, use the reversible addition-fragmentation chain transfer polymerization method to change the ratio between the D-type monomer and the L-type monomer in the copolymer through the feeding ratio of the D-type and L-type monomers. Molar ratio, so that the overall performance of the copolymer is D-type or L-type.

Embodiment 7—homopolymer, copolymer property test

As shown in Figure 6, the prepared D-configuration and L-configuration polymers were tested for circular dichroism, and an upward peak and a downward trough appeared in the same wavelength range, and the polymer corresponding to the upward peak was the L configuration. , the polymer corresponding to the downward trough is in the D configuration, showing a completely opposite helical conformation.

Micro-dilution antibacterial experiment references: P.Li, C.C.Zhou, S.Rayatpisheh, K.Ye, Y.F.Poon, P.T.Hammond, H.W.Duan, M.B.Chan-Park, Adv.Mater., 2012, 24, 4130-4137; In vitro human aortic smooth muscle cell compatibility test References: S.E.Exley, L.C.Paslay, G.S.Sahukhal, B.A.Abel, T.D.Brown, C.L.McCormick, S.Heinhorst, V.Koul, V.Choudhary, M.O.Elasri, S.E.Morgan, Biomacromolecules . , 1169-1178.

Select Staphylococcus aureus as the representative of Gram-positive bacteria, and Escherichia coli as the representative of Gram-negative bacteria. Firstly, the bacterial strains were cultured overnight to reach the mid-growth stage, and diluted with PBS buffer to ³ ×108 colony-forming units per milliliter ( CFU), followed by doubling the polymer solution (the aqueous solution of the prepared polymer) with a sterile liquid medium, taking 100 μL of the diluted bacterial suspension, and adding it to 100 μL of the polymer solution in a 96-well plate to obtain an antibacterial The final concentration of the polymer is 2-4096 μg·mL ⁻¹ . Subsequently, the 96-well plate was incubated at 37° C. for 18 h, and the absorbance value of the microwell at 600 nm was tested using a microplate reader. 200 μL of bacterial cell culture solution without antibacterial polymer was used as positive control group, and 200 μL of pure culture solution was used as negative control group. For each experiment, 4-6 parallel samples were set up. At the same time, a control group of polymer solutions with a series of concentrations was set to exclude the influence of the polymer solution on the absorbance value of the experimental group. Subsequently, bacteria that had been exposed to 100 μL of a lethal dose of polymer solution for 3 hours were fixed at room temperature (20-25 degrees Celsius) for 4 hours with 2.5% by volume glutaraldehyde phosphate buffer solution, washed twice with PBS buffer solution, and then After successively dehydrating with ethanol solution with gradient concentration for 15 min, centrifuge at 1000 rpm for 10 min, spread it evenly on the silicon wafer, air dry naturally, spray gold for 50 s, and observe the surface destruction morphology of bacteria under a field emission scanning electron microscope.

As shown in accompanying drawing 7, from the field emission scanning electron microscope result figure, intuitively demonstrates that the antibacterial polymer that the present invention makes can obviously destroy the bacterial membrane structure and cause the death of bacteria, and the polymer prepared according to the above-mentioned embodiment shows and Basically consistent performance. As shown in accompanying drawings 8 and 9, P(D-Leu-HEMA) homopolymer, P(D-Lys-HEMA) homopolymer and P(D-Leu-HEMA)-bP(D-Lys-HEMA) The block copolymer exhibits excellent antibacterial performance. The experimental results show that the minimum inhibitory concentration (MIC) of P(D-Leu-HEMA) homopolymer is 798～820μg·mL ^-1 , and P(D-Lys-HEMA ) homopolymer MIC is 100～120μg·mL ^-1 , the MIC of P(D-Leu-HEMA)-bP(D-Lys-HEMA) block copolymer is 180～200μg·mL ^-1 (for Escherichia coli and Staphylococcus aureus show basically the same situation); switch to homopolymers and copolymers based on L-type amino acids, and show antibacterial properties that are basically consistent with the above-mentioned examples, slightly lower than the homopolymers and copolymers of D-type amino acids things. Copolymers using D-type and L-type monomers for copolymerization, by adjusting the feeding ratio of D-type/L-type monomers, the copolymers will appear as D-type or L-type, and also have the same antibacterial properties as the corresponding configuration, which is lower than Antibacterial properties of the respective configurations.

As shown in accompanying drawing 10-13, hemolysis test and in vitro human aortic smooth muscle cell compatibility test show that the polymer prepared by the present invention can still maintain significant biocompatibility at the concentration required for antibacterial, wherein the hemolysis rate is low At 10%, the activity of smooth muscle cells can reach more than 80%.

According to the preparation of the above polymers according to the content of the present invention, the polymers all show the properties basically consistent with the examples. The present invention has been described as an example above, and it should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacements that can be made by those skilled in the art without creative labor all fall within the scope of this invention. protection scope of the invention.

Claims (9)

1. leucine methacrylate homopolymer, it is characterized in that, described leucine methacrylate homopolymer is L-leucine methacrylate homopolymer or D-leucine methyl Acrylate homopolymers, of which: L-leucine methacrylate homopolymer is formed by the homopolymerization of monomer L-leucine methacrylate. The molecular formula is as follows: n is the degree of polymerization; D-leucine methacrylate homopolymer is formed by the homopolymerization of monomer D-leucine methacrylate. The molecular formula is as follows: n is the degree of polymerization. 2. leucine methacrylate homopolymer according to claim 1, is characterized in that, the number average molecular weight of L-leucine methacrylate homopolymer is 10-23kDa, and molecular weight distribution coefficient is 1.10 -1.25; the number average molecular weight of D-leucine methacrylate homopolymer is 10-23kDa, and the molecular weight distribution coefficient is 1.10-1.25. 3. the preparation method of leucine methacrylate homopolymer is characterized in that, in the preparation method of L-leucine methacrylate homopolymer, monomer L-leucine methacrylate homopolymer Polymerization, using dithiobenzoic acid-4-cyanovaleric acid as chain transfer agent, azobisisobutyronitrile as initiator for RAFT polymerization, monomer L-leucine methacrylate, chain transfer agent The molar ratio of feeding and initiator is (45-60): 1:0.2, and the prepared homopolymer is deprotected under the action of trifluoroacetic acid; in the preparation method of D-leucine methacrylate homopolymer, The monomer D-leucine methacrylate is homopolymerized, and dithiobenzoic acid-4-cyanovaleric acid is used as the chain transfer agent, and azobisisobutyronitrile is used as the initiator for RAFT polymerization. The molar ratio of D-leucine methacrylate, chain transfer agent and initiator is (45—60):1:0.2, and the prepared homopolymer is deprotected under the action of trifluoroacetic acid. 4. the preparation method of leucine methacrylate homopolymer according to claim 3 is characterized in that, in the preparation method of L-leucine methacrylate homopolymer, monomer L-leucine The molar ratio of amino acid methacrylate, chain transfer agent and initiator is (50-60):1:0.2; under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 Celsius, the reaction time is 6-10 hours, the solvent N,N-dimethylformamide provides the reaction atmosphere and environment; the prepared homopolymer is deprotected under the action of trifluoroacetic acid, preferably the homopolymer is dissolved in dichloro In methane, under the action of trifluoroacetic acid, deprotection reaction is carried out at 20-25 degrees Celsius for 2-4 hours, after purification, it is dissolved and dialyzed, and then freeze-dried. 5. the preparation method of leucine methacrylate homopolymer according to claim 3 is characterized in that, in the preparation method of L-leucine methacrylate homopolymer, monomer L-leucine Amino acid methacrylate is prepared according to the following steps: L-leucine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, and N,N'-dicyclohexylcarbodiimide is dehydrated Condensing agent, react in an ice-water bath with dichloromethane as a solvent for 30-60 minutes, then stir and react at room temperature 20-25 degrees Celsius for 24-48 hours, wherein L-leucine, 4-dimethylaminopyridine, N, The mass ratio of N'-dicyclohexylcarbodiimide to hydroxyethyl methacrylate is 5: (0.2-0.25): (4-5): (2-3), and L-leucine is tert-butoxy Carbonyl protected L-leucine. 6. the preparation method of leucine methacrylate homopolymer according to claim 3 is characterized in that, in the preparation method of D-leucine methacrylate homopolymer, monomer D-leucine The molar ratio of amino acid methacrylate, chain transfer agent and initiator is (50-60):1:0.2; under the protection of inert gas (nitrogen, helium or argon), the reaction temperature is 60-80 Celsius, the reaction time is 6-10 hours, the solvent N,N-dimethylformamide provides the reaction atmosphere and environment; the prepared homopolymer is deprotected under the action of trifluoroacetic acid, preferably the homopolymer is dissolved in dichloro In methane, under the action of trifluoroacetic acid, deprotection reaction is carried out at 20-25 degrees Celsius for 2-4 hours, after purification, it is dissolved and dialyzed, and then freeze-dried. 7. the preparation method of leucine methacrylate homopolymer according to claim 3 is characterized in that, in the preparation method of D-leucine methacrylate homopolymer, monomer D-leucine Amino acid methacrylate is prepared according to the following steps: D-leucine and hydroxyethyl methacrylate are catalyzed by 4-dimethylaminopyridine, and N,N'-dicyclohexylcarbodiimide is dehydrated Condensing agent, react in an ice-water bath with dichloromethane as a solvent for 30-60 minutes, then stir and react at room temperature 20-25 degrees Celsius for 24-48 hours, wherein D-leucine, 4-dimethylaminopyridine, N, The mass ratio of N'-dicyclohexylcarbodiimide to hydroxyethyl methacrylate is 5: (0.2-0.25): (4-5): (2-3), and D-leucine is tert-butoxy Carbonyl protected D-leucine. 8. The application of leucine methacrylate homopolymer in antibacterial as claimed in claim 1 or 2, characterized in that the average minimum inhibitory concentration is 100-1000 μg·mL ^-1 , and the polymer can destroy bacteria The membrane structure causes the death of bacteria while maintaining remarkable biocompatibility. 9. The application according to claim 8, characterized in that the bacterium is Staphylococcus aureus or Escherichia coli.