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CN109734855A - Preparation method of L-cationic chiral amino acid methacrylate copolymer - Google Patents

Preparation method of L-cationic chiral amino acid methacrylate copolymer Download PDF

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CN109734855A
CN109734855A CN201811587688.2A CN201811587688A CN109734855A CN 109734855 A CN109734855 A CN 109734855A CN 201811587688 A CN201811587688 A CN 201811587688A CN 109734855 A CN109734855 A CN 109734855A
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amino acid
methacrylate
monomer
preparation
leu
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赵蕴慧
胡素利
胡文虹
李珍光
袁晓燕
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Tianjin University
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    • C08F122/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides or nitriles thereof
    • C08F122/10—Esters
    • C08F122/1006—Esters of polyhydric alcohols or polyhydric phenols, e.g. ethylene glycol dimethacrylate
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
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    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
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    • C08F2800/00—Copolymer characterised by the proportions of the comonomers expressed
    • C08F2800/20—Copolymer characterised by the proportions of the comonomers expressed as weight or mass percentages
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Abstract

本发明公开L‑阳离子手性氨基酸甲基丙烯酸酯共聚物的制备方法，以单体L‑赖氨酸甲基丙烯酸酯和L‑亮氨酸甲基丙烯酸酯进行嵌段共聚制备，首先将第一L型氨基酸单体进行均聚，以二硫代苯甲酸‑4‑氰基戊酸为链转移剂，偶氮二异丁腈为引发剂进行RAFT聚合，再将L构型氨基酸单体聚合物大分子链转移剂，偶氮二异丁腈为引发剂，加入第二L型氨基酸单体进行RAFT聚合以得到L‑赖氨酸甲基丙烯酸酯和L‑亮氨酸甲基丙烯酸酯嵌段共聚物。本发明的聚合物表现出优异的抗菌性能，能够明显的破坏细菌膜结构引起细菌的死亡；溶血实验和体外平滑肌细胞相容性实验表明其保持显著的生物相容性。

The invention discloses a preparation method of L-cationic chiral amino acid methacrylate copolymer. The monomers L-lysine methacrylate and L-leucine methacrylate are used for block copolymerization. One L-type amino acid monomer is homopolymerized, and dithiobenzoic acid-4-cyanovaleric acid is used as a chain transfer agent, and azobisisobutyronitrile is an initiator to carry out RAFT polymerization, and then L-configuration amino acid monomer is polymerized A macromolecular chain transfer agent, azobisisobutyronitrile is an initiator, and a second L-type amino acid monomer is added to carry out RAFT polymerization to obtain L-lysine methacrylate and L-leucine methacrylate. segmented copolymer. The polymer of the invention exhibits excellent antibacterial properties, and can obviously destroy the bacterial membrane structure to cause bacterial death; hemolysis experiments and in vitro smooth muscle cell compatibility experiments show that it maintains significant biocompatibility.

Description

The preparation method of L- cationic chiral amino acid methacrylate copolymer
The present invention is that parent application " answer by L- cationic chiral amino acid methacrylate copolymer and preparation and antibacterial With " divisional application, parent application application No. is 2016110295713, the applying date is on November 14th, 2016.
Technical field
The invention belongs to field of biomedical polymer materials, are related to the design synthesis side of Novel imitation antibacterial peptide polymer Method, and in particular to a kind of preparation and its antibacterial applications of cationic chiral amino acid methacrylate polymers.
Background technique
Currently, the appearance of antibiotic resistant bacteria brings great threat to public health, the adherency of bacterium, Proliferation and the biomembrane formed will lead to patient and infect and cause a series of complication or even jeopardize patient vitals.Although alternative The research of novel antibacterial polymer have been achieved with certain progress, but it still has pole on bio-medical and clinical application Big challenge.Alternative novel antibacterial polymer will not only have efficient anti-microbial property, while cannot influence body group Knit normal physiological action, in simple terms can the killing bacterium of selectivity and it is non-toxic to normal mammalian cell. Therefore, it has both high-efficiency antimicrobial activity and the antibacterial polymer of selectivity still requires study.
Summary of the invention
The purpose of the present invention is preparing a kind of cationic polymethacrylate polymer based on chiral amino acid, and It is studied in the application of antibiosis, to overcome the disadvantage that existing antibacterial polymer antibacterial activity is weaker, biocompatibility is poor.
The invention adopts the following technical scheme:
L-Leu methacrylate homopolymer, i.e. P (L-Leu-HEMA), with monomer D-Leu methacrylate Homopolymerization forms, and molecular formula schematic construction is as follows:
N is the degree of polymerization, and the number-average molecular weight of polymer is 10-23kDa, molecular weight distribution index 1.10-1.25.
The preparation method of above-mentioned homopolymer forms monomer L-Leu methacrylate homopolymerization, with two thio phenyl first Acid -4- cyanopentanoic acid is chain-transferring agent, and azodiisobutyronitrile is that initiator carries out RAFT polymerization, monomer L-Leu metering system The molar ratio of acid esters, chain-transferring agent and initiator is (45-60): 1:0.2, preferably (50-60): 1:0.2.
In the above-mentioned technical solutions, it is carried out under inert gas (nitrogen, helium or argon gas) protection, reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and environment.
In the above-mentioned technical solutions, monomer L-Leu methacrylate is prepared as steps described below: L- is bright Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein L-Leu, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), L-Leu be tertbutyloxycarbonyl protection L-Leu.
In the above-mentioned technical solutions, the homopolymer of preparation is deprotected under trifluoroacetic acid effect, preferably by homopolymer It is dissolved in methylene chloride, under trifluoroacetic acid effect, carries out deprotection reaction 2-4 hours under 20-25 degrees Celsius, after purification Dissolution dialysis, freeze-drying.
Due to acting on using trifluoroacetic acid, trifluoroacetic acid group is had in the polymer after deprotection reaction, due to Leu's Isoelectric point is pH 6.01, and the isoelectric point of Lys is pH 9.60, when adjusting pH is 7.2 ,-the NH of leu3 +Deprotonation occurs, it is raw At-NH2, shown in following chemical formula:
D-Leu methacrylate homopolymer, i.e. P (D-Leu-HEMA), with monomer D-Leu methacrylate Homopolymerization forms, and molecular formula schematic construction is as follows, and n is the degree of polymerization, and the number-average molecular weight of polymer is 10-23kDa, molecular weight distribution Coefficient is 1.10-1.25.
The preparation method of above-mentioned homopolymer forms monomer D-Leu methacrylate homopolymerization, with two thio phenyl first Acid -4- cyanopentanoic acid is chain-transferring agent, and azodiisobutyronitrile is that initiator carries out RAFT polymerization, monomer D-Leu metering system The molar ratio of acid esters, chain-transferring agent and initiator is (45-60): 1:0.2, preferably (50-60): 1:0.2.
In the above-mentioned technical solutions, it is carried out under inert gas (nitrogen, helium or argon gas) protection, reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and environment.
In the above-mentioned technical solutions, monomer D-Leu methacrylate is prepared as steps described below: D- is bright Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein D-Leu, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), D-Leu be tertbutyloxycarbonyl protection D-Leu.
In the above-mentioned technical solutions, the homopolymer of preparation is deprotected under trifluoroacetic acid effect, preferably by homopolymer It is dissolved in methylene chloride, under trifluoroacetic acid effect, carries out deprotection reaction 2-4 hours under 20-25 degrees Celsius, after purification Dissolution dialysis, freeze-drying.
Due to acting on using trifluoroacetic acid, trifluoroacetic acid group is had in the polymer after deprotection reaction, due to Leu's Isoelectric point is pH 6.01, and the isoelectric point of Lys is pH 9.60, when adjusting pH is 7.2 ,-the NH of leu3 +Deprotonation occurs, it is raw At-NH2, shown in following chemical formula:
L-lysine methacrylate homopolymer, i.e. P (L-Lys-HEMA), with monomer L-lysine methacrylate Homopolymerization forms, and molecular formula schematic construction is as follows, and n is the degree of polymerization, and the number-average molecular weight of polymer is 10-23kDa, molecular weight distribution Coefficient is 1.10-1.25.
The preparation method of above-mentioned homopolymer forms monomer L-lysine methacrylate homopolymerization, with two thio phenyl first Acid -4- cyanopentanoic acid is chain-transferring agent, and azodiisobutyronitrile is that initiator carries out RAFT polymerization, monomer L-lysine metering system The molar ratio of acid esters, chain-transferring agent and initiator is (45-60): 1:0.2, preferably (50-60): 1:0.2.
In the above-mentioned technical solutions, it is carried out under inert gas (nitrogen, helium or argon gas) protection, reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and environment.
In the above-mentioned technical solutions, monomer L-lysine methacrylate is prepared as steps described below: L- is relied Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein L-lysine, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), L-lysine be tertbutyloxycarbonyl protection L-lysine.
In the above-mentioned technical solutions, the homopolymer of preparation is deprotected under trifluoroacetic acid effect, preferably by homopolymer It is dissolved in methylene chloride, under trifluoroacetic acid effect, carries out deprotection reaction 2-4 hours under 20-25 degrees Celsius, after purification Dissolution dialysis, freeze-drying.
Due to acting on using trifluoroacetic acid, trifluoroacetic acid group is had in the polymer after deprotection reaction, adjusting pH is 7.2 lys is unaffected, shown in following chemical formula:
D-Lys methacrylate homopolymer, i.e. P (D-Leu-HEMA), with monomer D-Lys methacrylate Homopolymerization forms, and molecular formula schematic construction is as follows, and n is the degree of polymerization, and the number-average molecular weight of polymer is 10-23kDa, molecular weight distribution Coefficient is 1.10-1.25.
The preparation method of above-mentioned homopolymer forms monomer D-Lys methacrylate homopolymerization, with two thio phenyl first Acid -4- cyanopentanoic acid is chain-transferring agent, and azodiisobutyronitrile is that initiator carries out RAFT polymerization, monomer D-Lys metering system The molar ratio of acid esters, chain-transferring agent and initiator is (45-60): 1:0.2, preferably (50-60): 1:0.2.
In the above-mentioned technical solutions, it is carried out under inert gas (nitrogen, helium or argon gas) protection, reaction temperature is 60-80 degrees Celsius, the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and environment.
In the above-mentioned technical solutions, monomer D-Lys methacrylate is prepared as steps described below: D- is relied Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein D-Lys, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), D-Lys be tertbutyloxycarbonyl protection D-Lys.
In the above-mentioned technical solutions, the homopolymer of preparation is deprotected under trifluoroacetic acid effect, preferably by homopolymer It is dissolved in methylene chloride, under trifluoroacetic acid effect, carries out deprotection reaction 2-4 hours under 20-25 degrees Celsius, after purification Dissolution dialysis, freeze-drying.
Due to acting on using trifluoroacetic acid, trifluoroacetic acid group is had in the polymer after deprotection reaction, adjusting pH is 7.2, lys is unaffected, shown in following chemical formula:
D- cationic chiral amino acid methacrylate copolymer, it is bright with monomer D-Lys methacrylate and D- Propylhomoserin methacrylate carries out block copolymerization preparation, and molecular formula schematic construction is as follows, n, and m is the degree of polymerization of respective monomer, m/n For (0.8-1.2), the consistent m=n of the degree of polymerization of preferably two kinds monomers, the number-average molecular weight of polymer is 10-23kDa, molecular weight Breadth coefficient is 1.10-1.25.
The preparation method of above-mentioned copolymer, is prepared as steps described below:
First D type amino acid monomer is carried out homopolymerization by step 1., using dithiobenzoic acid -4- cyanopentanoic acid as chain tra nsfer Agent, azodiisobutyronitrile are that initiator carries out RAFT polymerization to obtain D amino acids monomer-polymer, the first D type ammonia The molar ratio of base acid monomers, chain-transferring agent and initiator is (45-60): 1:0.2, preferably (50-60): 1:0.2；
Step 2, D amino acids monomer-polymer Macromolecular chain transfer agent prepared by step 1, azodiisobutyronitrile are to draw Agent is sent out, the 2nd D type amino acid monomer is added and carries out RAFT polymerization to obtain D-Lys methacrylate and D-Leu first The molar ratio of base acrylate block copolymer, the 2nd 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-mentioned technical solutions, in step 1, it is carried out under inert gas (nitrogen, helium or argon gas) protection, instead Answering temperature is 60-80 degrees Celsius, and the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and ring Border.
In the above-mentioned technical solutions, in step 2, it is carried out under inert gas (nitrogen, helium or argon gas) protection, instead Answering temperature is 60-80 degrees Celsius, and the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and ring Border.
In the above-mentioned technical solutions, monomer D-Lys methacrylate is prepared as steps described below: D- is relied Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein D-Lys, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), D-Lys be tertbutyloxycarbonyl protection D-Lys.
In the above-mentioned technical solutions, monomer D-Leu methacrylate is prepared as steps described below: D- is bright Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein D-Leu, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), D-Leu be tertbutyloxycarbonyl protection D-Leu.
In the above-mentioned technical solutions, the copolymer of preparation is deprotected under trifluoroacetic acid effect, preferably by copolymer It is dissolved in methylene chloride, under trifluoroacetic acid effect, carries out deprotection reaction 2-4 hours under 20-25 degrees Celsius, after purification Dissolution dialysis, freeze-drying.
Due to acting on using trifluoroacetic acid, trifluoroacetic acid group is had in the polymer after deprotection reaction, due to Leu's Isoelectric point is pH 6.01, and the isoelectric point of Lys is pH 9.60, when adjusting pH is 7.2 ,-the NH of leu3 +Deprotonation occurs, it is raw At-NH2, adjusting pH is that 7.2, lys is unaffected, shown in following chemical formula:
L- cationic chiral amino acid methacrylate copolymer, it is bright with monomer L-lysine methacrylate and L- Propylhomoserin methacrylate carries out block copolymerization preparation, and molecular formula schematic construction is as follows, n, and m is the degree of polymerization of respective monomer, m/n For (0.8-1.2), the consistent m=n of the degree of polymerization of preferably two kinds monomers, the number-average molecular weight of polymer is 10-23kDa, molecular weight Breadth coefficient is 1.10-1.25.
The preparation method of above-mentioned copolymer, is prepared as steps described below:
First L-type amino acid monomer is carried out homopolymerization by step 1., using dithiobenzoic acid -4- cyanopentanoic acid as chain tra nsfer Agent, azodiisobutyronitrile are that initiator carries out RAFT polymerization to obtain D amino acids monomer-polymer, the first L-type ammonia The molar ratio of base acid monomers, chain-transferring agent and initiator is (45-60): 1:0.2, preferably (50-60): 1:0.2；
Step 2, L-configuration amino acid monomer polymer macromolecule chain-transferring agent prepared by step 1, azodiisobutyronitrile are to draw Agent is sent out, the second L-type amino acid monomer is added and carries out RAFT polymerization to obtain L-lysine methacrylate and L-Leu first The molar ratio of base acrylate block copolymer, 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-mentioned technical solutions, in step 1, it is carried out under inert gas (nitrogen, helium or argon gas) protection, instead Answering temperature is 60-80 degrees Celsius, and the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and ring Border.
In the above-mentioned technical solutions, in step 2, it is carried out under inert gas (nitrogen, helium or argon gas) protection, instead Answering temperature is 60-80 degrees Celsius, and the reaction time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and ring Border.
In the above-mentioned technical solutions, monomer L-lysine methacrylate is prepared as steps described below: L- is relied Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein L-lysine, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), L-lysine be tertbutyloxycarbonyl protection L-lysine.
In the above-mentioned technical solutions, monomer L-Leu methacrylate is prepared as steps described below: L- is bright Propylhomoserin and hydroxyethyl methacrylate are using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydration contracting Mixture reacts 30-60 minutes by solvent of methylene chloride in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein L-Leu, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate Mass ratio be 5:(0.2-0.25): (4-5): (2-3), L-Leu be tertbutyloxycarbonyl protection L-Leu.
In the above-mentioned technical solutions, the copolymer of preparation is deprotected under trifluoroacetic acid effect, preferably by copolymer It is dissolved in methylene chloride, under trifluoroacetic acid effect, carries out deprotection reaction 2-4 hours under 20-25 degrees Celsius, after purification Dissolution dialysis, freeze-drying.
Due to acting on using trifluoroacetic acid, trifluoroacetic acid group is had in the polymer after deprotection reaction, due to Leu's Isoelectric point is pH 6.01, and the isoelectric point of Lys is pH 9.60, when adjusting pH is 7.2 ,-the NH of leu3 +Deprotonation occurs, it is raw At-NH2, adjusting pH is that 7.2, lys is unaffected, shown in following chemical formula:
The present invention provides a kind of method of regulation copolymer overall configuration, i.e., is total to using the monomer of D configuration and L-configuration It is poly-, by D type/L-type monomer ingredient proportion, molar ratio between the two is changed, so that copolymer shows as D type or L-type, Application of the i.e. controllable living polymerization in regulation leucine-lysine methacrylate copolymer chirality.
Using reversible addion-fragmentation chain transfer polymerization by D type/L-type monomer ingredient proportion, change is between the two Molar ratio pass through the ingredient proportion of D type and L-type monomer so that copolymer shows as D type or L-type, change in copolymer Molar ratio between D type monomer and L-type monomer, so that copolymer overall performance is D type or L-type.
For above-mentioned four kinds of monomers, the polymer that homopolymerization obtains show with the consistent D of corresponding monomer and L-configuration, two The copolymer that a L-type monomer is copolymerized shows as L-type, and the copolymer that two D type monomers are copolymerized shows as D type, copolymerization Object is copolymerized using D type and L-type monomer, adjusts the relative populations of D type monomer and L-type monomer, and copolymer entirety can be realized Configuration shows controllable.
In the technical solution of the present invention, the control of (i.e. monomer molar ratio) of adding materials can be realized using living polymerization, Reaction condition is mild, and synthon yield is 70%-85%, polymerization conversion 50%-90%, homopolymer and copolymer Molecular weight it is controllable and breadth coefficient is relatively narrow.
The invention discloses based on chiral amino acid methacrylate polymers and preparation method thereof and antibacterial applications.Circle Dichroscope spectrum characterization result proves that above-mentioned obtained L-configuration and D configuration polymer have opposite deuterostrophies structure.Microdilution The minimum suppression of antibacterial experiment methacrylate polymers based on chiral amino acid after freeze-drying studies have shown that above-mentioned gained is dialysed Bacteria concentration (MIC) is 100~1000 μ gmL-1, excellent anti-microbial property is shown, field emission scanning electron microscope result figure is intuitive The death that bacterium membrane structure causes bacterium can significantly be destroyed by showing antibacterial polymer produced by the present invention；Hemolytic experiment and External smooth muscle cell compatibility experiments show cationic chiral amino acid methacrylate antimicrobial polymerizable prepared by the present invention Object can still keep significant biocompatibility under concentration needed for antibacterial, and wherein hemolysis rate is lower than 10%, and smooth muscle cell is living Property can reach 80% or more.
Detailed description of the invention
Fig. 1 is the mechanism of polymerization schematic diagram of reversible addion-fragmentation chain transfer polymerization (RAFT) in the present invention.
Fig. 2 be in the present invention monomer D-Leu (Boc)-HEMA in CDCl3In1H-NMR spectrum.
Fig. 3 be in the present invention monomer D-Lys (Boc)-HEMA in CDCl3In1H-NMR spectrum.
Fig. 4 is P (D-Leu (Boc)-HEMA) homopolymer (1) not being deprotected in the present invention, P (D-Lys (Boc)-HEMA) Homopolymer (2) and P (D-Leu (Boc)-HEMA)-b-P (D-Lys (Boc)-HEMA) block copolymer (3) are in CDCl3In1H- NMR spectra.
Fig. 5 is P (D-Leu-HEMA) homopolymer (1) after being deprotected in the present invention, P (D-Lys-HEMA) homopolymer homopolymer (2) and P (D-Leu-HEMA)-b-P (D-Lys-HEMA) block copolymer homopolymer (3) is in D2In O1H-NMR spectrum.
Fig. 6 is the circular dichroism spectra characterization result figure of homopolymer and copolymer in the present invention.
Fig. 7 is the stereoscan photograph of polymer antibacterial experiment in the present invention.
Fig. 8 is the antibacterial experiment effect curve figure (1) that polymer is directed to Escherichia coli in the present invention.
Fig. 9 is the antibacterial experiment effect curve figure (1) that polymer is directed to staphylococcus aureus in the present invention.
Figure 10 is the biocompatibility in vitro phenogram of Inventive polymers P (L-Leu-HEMA).
Figure 11 is the biocompatibility in vitro phenogram of Inventive polymers P (D-Leu-HEMA).
Figure 12 is the biocompatibility in vitro phenogram of Inventive polymers P (D-Leu-HEMA-b-D-Lys-HEMA).
Figure 13 is the biocompatibility in vitro phenogram of Inventive polymers P (L-Leu-HEMA-b-L-Lys-HEMA).
Specific embodiment
Technical solution of the present invention is further illustrated combined with specific embodiments below.The experimental raw and instrument that embodiment uses Device difference is as shown in the table:
(1) experimental raw
(2) laboratory apparatus
Polymerize in embodiments of the present invention using reversible addion-fragmentation chain transfer, RAFT polymerization be a kind of activity/controllable from It is polymerize by base, is applicable in the monomer containing double bond functional group.In RAFT polymerization, conventional initiator is thermally decomposed into primary group of free radicals I., and cause monomer polymerization into Propagating Radical Pn, the C=S key in Propagating Radical and chain-transferring agent carries out reversible addition shape At intermediate suspend mode kind, S-R key is broken in suspend mode kind, forms new reactive species free radical Rn, then cause monomer polymerization, react Mechanism is illustrated in fig. 1 shown below.It is different from conventional free radical polymerization, RAFT polymerize chain tra nsfer be reversible process, intermediate suspend mode kind and The balanced reaction of reversible addition and reversible fracture is carried out between growing chain free radical, so that it is guaranteed that all chains are raw with same probability It is long, the polymer of narrow ditribution is formed, number of free radical maintains a relative constant reduced levels in system, in inhibition system The biradical termination of free radical is reacted, so that polymerization activity is controllable.Initiator is used in the present invention and causes the first monomer, with the One monomer and chain-transferring agent carry out living polymerization, when second comonomer is added, supplement initiator to cause second comonomer, with activity Polymerizeing the first obtained monomer homopolymers is the living polymerization that Macromolecular chain transfer agent carries out second comonomer.
Embodiment 1-prepares monomer D-Leu hydroxyethyl methacrylate, L-Leu hydroxyethyl methacrylate, D- Lysine hydroxyethyl methacrylate and L-lysine hydroxyethyl methacrylate
The chiral amino acid monomer 5g for being dissolved in 22mL dry methylene chloride is added first into dry neck round bottom flask (respectively D-Leu, L-Leu, D-Lys and L-lysine select tertiary fourth for the activity for ensuring functional group in reacting The boc-protected above-mentioned four kinds of amino acid of oxygen carbonyl, sloughs Boc protecting group using trifluoroacetic acid after reacting), in magnetic agitation Logical nitrogen purification down, is then added the catalyst DMAP 0.24g for being dissolved in 1.5mL dry methylene chloride, reaction flask is placed in ice water In bath, it is slowly added to be dissolved in the dehydrating condensation agent DCC4.53g of 20mL dry methylene chloride dropwise, and exist under nitrogen protection 2.86g HEMA is added in 20min.30min is reacted under ice-water bath, under subsequent nitrogen atmosphere under 20-25 degrees Celsius of room temperature The reaction was continued 36h.It filters after reaction and removes white precipitate, obtained organic mixed solution 70mL distilled water and 100mL Methylene chloride extracts four times, and obtained organic layer is further successively extracted twice with 60mL0.1mol/L hydrochloric acid solution, and 60mL is full It is washed twice with sodium bicarbonate solution and 60mL saturated sodium chloride solution.It is finally dry with anhydrous sodium sulfate, it is stirred overnight, filters Afterwards, revolving removes solvent.
It is characterized using four kind monomers of the nuclear magnetic resonance to preparation, as a result as shown in Fig. 2 and 3, in view of four kinds of monomers Chemical composition be divided into two kinds (i.e. leucine methyl hydroxy-ethyl acrylates and lysine hydroxyethyl methacrylate), chemical structure It is divided into D and L, the nuclear magnetic spectrogram of the various configuration of the same chemical composition is almost the same, it is found that nuclear-magnetism is total as shown in two attached drawings The chemical shift of vibration corresponds to the hydrogen atom of the different chemical environments marked in chemical formula, exactly proves that four kinds of monomers are successfully prepared.
Four kinds of monomers that embodiment 2-is prepared using embodiment 1 are polymerize using reversible addion-fragmentation chain transfer and are made as raw material Standby four kinds of homopolymers
Cationic chiral amino acid hydroxyethyl methacrylate homopolymer is prepared using RAFT polymerization: with magnetic force In Schlenk bottles of 25mL of stirrer, be added 1.5g embodiment 1 made from monomer, CPADB 24.4mg, AIBN2.86mg and 1.5g anhydrous DMF solvent-is taken out-after melting the foreign gas that circulation removes in reaction system by freezing three times, is placed in 70 DEG C of oil bath In pot, react 10 hours under nitrogen protection.After the reaction was completed, exposure is placed in be quickly cooled down in ice-water bath in air and terminate Reaction, then with acetone/n-hexane repeated precipitation 5 times.Obtained product is placed in in 30 DEG C of vacuum oven dry 8h, is obtained The sample of every kind of homopolymer.Under the conditions of ice-water bath, the ratio of 20mL methylene chloride and 10mL trifluoroacetic acid is added according to every 1g sample Example, is added in reaction flask, then in room temperature reaction 3h, rotates to remove solvent.Products therefrom is added dropwise to anhydrous ether, instead Multiple precipitating is three times.Obtained product is placed in in 30 DEG C of vacuum oven dry 8h.Product after purification is dissolved in deionization In water, dialysis 72h removes remaining impurity, and pure spongy solid sample is obtained after freeze-drying.
It is characterized using four kind homopolymers of the nuclear magnetic resonance to preparation, as a result as shown in figs. 4 and 5, similarly based on bright There are the difference (D of chemical structure for the two kinds of polymer of propylhomoserin hydroxyethyl methacrylate and lysine hydroxyethyl methacrylate Type and L-type), the nuclear magnetic spectrogram of the various configuration of the same chemical composition is almost the same, it is found that nuclear-magnetism is total as shown in two attached drawings The chemical shift of vibration corresponds to the hydrogen atom of the different chemical environments marked in chemical formula, exactly proves that four kinds of homopolymers are successfully made It is standby, and block polymer does not generate variation after deprotection.
Embodiment 3-prepares D- cationic chiral amino acid hydroxyethyl methacrylate copolymer
(1) D-leucine homopolymer is prepared using the method for embodiment 2 first: having magnetic stir bar In 25mLSchlenk bottles, monomer D-leucine methyl hydroxy-ethyl acrylate of the addition preparation of 1.5g embodiment 1, CPADB24.4mg, AIBN 2.86mg and 1.5g anhydrous DMF solvent-are smoked-by freezing three times and are melted in circulation removing reaction system It after foreign gas, is placed in 70 DEG C of oil bath pan, reacts 10 hours under nitrogen protection, after reacting basic, without The termination of exposure air is reacted, and it is single as being added second to be to maintain D-leucine homopolymer end active group (CPADB) The Macromolecular chain transfer agent that body is reacted；
(2) in the reaction vessel, lysine methacrylate monomers (the i.e. D-lysine first of 3g identical configuration is added Base hydroxy-ethyl acrylate), AIBN 2.15mg and with 3g anhydrous DMF solvent, with the Macromolecular chain transfer agent 0.96g prepared before It is reacted ,-takes out-after melting the foreign gas that circulation removes in reaction system, be placed in 70 DEG C of oil bath pan by freezing three times, It is reacted 6 hours under nitrogen protection, obtained product is placed in dry 8h in 30 DEG C of vacuum oven after multiple deposition and purification；
(3) under the conditions of ice-water bath, 20mL methylene chloride is added according to every 1g sample (i.e. above-mentioned steps 2 obtained sample) It with the ratio of 10mL trifluoroacetic acid, is added in reaction flask, then in room temperature reaction 3h, rotates to remove solvent.Gained is produced Object is added dropwise to anhydrous ether, and repeated precipitation is three times.Obtained product is placed in in 30 DEG C of vacuum oven dry 8h.It will purifying Product afterwards is dissolved in deionized water, and dialysis 72h removes remaining impurity, and pure spongy solid sample is obtained after freeze-drying Product.Obtain the cationic chiral amino acid hydroxyethyl methacrylate copolymer of D configuration.
It is carried out using cationic chiral amino acid hydroxyethyl methacrylate copolymer of the nuclear magnetic resonance to the D configuration of preparation Characterization, as shown in figs. 4 and 5, the chemical shift of nuclear magnetic resonance corresponds to the hydrogen atom of the different chemical environments marked in chemical formula, Exactly prove that copolymer is successfully prepared, and block polymer does not generate variation after deprotection.
Embodiment 4-prepares L- cationic chiral amino acid methacrylate copolymer
(1) L-leucine homopolymer is prepared using the method for embodiment 2 first: in the 25mL for having magnetic stir bar In Schlenk bottles, be added 1.5g embodiment 1 prepare monomer L-leucine methyl hydroxy-ethyl acrylate, CPADB 24.4mg, AIBN 2.86mg and 1.5g anhydrous DMF solvent-is taken out-by freezing three times after melting the foreign gas that circulation removes in reaction system, It is placed in 70 DEG C of oil bath pan, reacts 10 hours under nitrogen protection, the end after reacting basic, without exposure air It only reacts, and is to maintain what L-leucine homopolymer end active group (CPADB) was reacted as addition second comonomer Macromolecular chain transfer agent；
(2) in the reaction vessel, lysine methacrylate monomers (the i.e. L-lysine first of 3g identical configuration is added Base hydroxy-ethyl acrylate), AIBN 2.15mg and with 3g anhydrous DMF solvent, with the Macromolecular chain transfer agent 0.96g prepared before It is reacted ,-takes out-after melting the foreign gas that circulation removes in reaction system, be placed in 70 DEG C of oil bath pan by freezing three times, It is reacted 6 hours under nitrogen protection, obtained product is placed in dry 8h in 30 DEG C of vacuum oven after multiple deposition and purification；
(3) under the conditions of ice-water bath, 20mL methylene chloride is added according to every 1g sample (i.e. above-mentioned steps 2 obtained sample) It with the ratio of 10mL trifluoroacetic acid, is added in reaction flask, then in room temperature reaction 3h, rotates to remove solvent.Gained is produced Object is added dropwise to anhydrous ether, and repeated precipitation is three times.Obtained product is placed in in 30 DEG C of vacuum oven dry 8h.It will purifying Product afterwards is dissolved in deionized water, and dialysis 72h removes remaining impurity, and pure spongy solid sample is obtained after freeze-drying Product.Obtain the cationic chiral amino acid hydroxyethyl methacrylate copolymer of L-configuration.
It is characterized using copolymer of the nuclear magnetic resonance to preparation, the cationic chiral ammonia of the D configuration prepared with embodiment 3 Base acids methacrylic acid hydroxyl ethyl ester copolymer is compared, and only there is different (L-types), the chemical environment of hydrogen atom basic one in configuration It causing, i.e. the chemical shift of D type and L-type is almost the same, as a result as shown in figs. 4 and 5, the corresponding chemistry of the chemical shift of nuclear magnetic resonance The hydrogen atom of the different chemical environments marked in formula exactly proves that copolymer is successfully prepared, and the bulk polymerization after deprotection Object does not generate variation.
Embodiment 5-prepares D-Lys/L-Leu methacrylate copolymer
(1) L-leucine homopolymer is prepared using the method for embodiment 2 first: in the 25mL for having magnetic stir bar In Schlenk bottles, be added 1.5g embodiment 1 prepare monomer L-leucine methyl hydroxy-ethyl acrylate, CPADB24.4mg, AIBN 2.86mg and 1.5g anhydrous DMF solvent-is taken out-by freezing three times after melting the foreign gas that circulation removes in reaction system, It is placed in 70 DEG C of oil bath pan, reacts 10 hours under nitrogen protection, the end after reacting basic, without exposure air It only reacts, and is to maintain what L-leucine homopolymer end active group (CPADB) was reacted as addition second comonomer Macromolecular chain transfer agent；
(2) in the reaction vessel, lysine methacrylate monomers (the i.e. D-lysine first of 3g various configuration is added Base hydroxy-ethyl acrylate), AIBN 2.15mg and with 3g anhydrous DMF solvent, with the Macromolecular chain transfer agent 0.96g prepared before It is reacted ,-takes out-after melting the foreign gas that circulation removes in reaction system, be placed in 70 DEG C of oil bath pan by freezing three times, It is reacted 6 hours under nitrogen protection, obtained product is placed in dry 8h in 30 DEG C of vacuum oven after multiple deposition and purification；
(3) under the conditions of ice-water bath, 20mL methylene chloride is added according to every 1g sample (i.e. above-mentioned steps 2 obtained sample) It with the ratio of 10mL trifluoroacetic acid, is added in reaction flask, then in room temperature reaction 3h, rotates to remove solvent.Gained is produced Object is added dropwise to anhydrous ether, and repeated precipitation is three times.Obtained product is placed in in 30 DEG C of vacuum oven dry 8h.It will purifying Product afterwards is dissolved in deionized water, and dialysis 72h removes remaining impurity, and pure spongy solid sample is obtained after freeze-drying Product obtain D-Lys/L-Leu methacrylate copolymer.
Embodiment 6-prepares D-Leu/L-lysine methacrylate copolymer
(1) D-leucine homopolymer is prepared using the method for embodiment 2 first: in the 25mL for having magnetic stir bar In Schlenk bottles, be added 1.5g embodiment 1 prepare monomer D-leucine methyl hydroxy-ethyl acrylate, CPADB 24.4mg, AIBN 2.86mg and 1.5g anhydrous DMF solvent-is taken out-by freezing three times after melting the foreign gas that circulation removes in reaction system, It is placed in 70 DEG C of oil bath pan, reacts 10 hours under nitrogen protection, the end after reacting basic, without exposure air It only reacts, and is to maintain what D-leucine homopolymer end active group (CPADB) was reacted as addition second comonomer Macromolecular chain transfer agent；
(2) in the reaction vessel, lysine methacrylate monomers (the i.e. L-lysine first of 3g various configuration is added Base hydroxy-ethyl acrylate), AIBN 2.15mg and with 3g anhydrous DMF solvent, with the Macromolecular chain transfer agent 0.96g prepared before It is reacted ,-takes out-after melting the foreign gas that circulation removes in reaction system, be placed in 70 DEG C of oil bath pan by freezing three times, It is reacted 6 hours under nitrogen protection, obtained product is placed in dry 8h in 30 DEG C of vacuum oven after multiple deposition and purification；
(3) under the conditions of ice-water bath, 20mL methylene chloride is added according to every 1g sample (i.e. above-mentioned steps 2 obtained sample) It with the ratio of 10mL trifluoroacetic acid, is added in reaction flask, then in room temperature reaction 3h, rotates to remove solvent.Gained is produced Object is added dropwise to anhydrous ether, and repeated precipitation is three times.Obtained product is placed in in 30 DEG C of vacuum oven dry 8h.It will purifying Product afterwards is dissolved in deionized water, and dialysis 72h removes remaining impurity, and pure spongy solid sample is obtained after freeze-drying Product.Obtain D-Leu/L-lysine methacrylate copolymer.
It is shown and the consistent D of corresponding monomer and L-configuration, two L-type monomers by the polymer that four kinds of monomer homopolymerizations obtain It is copolymerized obtained copolymer and shows as L-type, the copolymer that two D type monomers are copolymerized shows as D type, prepared by embodiment 5 and 6 Copolymer be copolymerized using D type and L-type monomer, rubbing between the two can be changed by D type/L-type monomer ingredient proportion That ratio passes through D type and L-type using reversible addion-fragmentation chain transfer polymerization so that copolymer shows as D type or L-type The ingredient proportion of monomer changes the molar ratio in copolymer between D type monomer and L-type monomer, so that copolymer overall performance is D Type or L-type.
7-homopolymer of embodiment, the test of copolymer property
As shown in fig. 6, the D configuration of preparation and L-configuration polymer are carried out circular dichroism spectra test, in identical wave-length coverage The upward wave crest of interior appearance and downward trough, the corresponding polymer of upward wave crest is L-configuration, to the corresponding polymer of lower wave trough For D configuration, antipodal helical conformation is shown.
Microdilution antibacterial experiment bibliography: 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；External human aorta Smooth muscle cell compatibility experiments bibliography: 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,2015,16,3845-3852；Hemolytic experiment bibliography: A.Pascual, J.P.K.Tan, A.Yuen,J.M.W.Chan,D.J.Coady,D.Mecerreyes,J.L.Hedrick,Y.Y.Yang,H.Sardon, Biomacromolecules,2015,16,1169-1178。
Select staphylococcus aureus for gram-positive bacteria representative, Escherichia coli are Gram-negative bacteria representative, first Bacterium bacterial strain is incubated overnight and reaches growth medium, is diluted to every milliliter 3 × 10 with PBS buffer solution8A Colony Forming Unit (CFU), Polymer Solution (aqueous solution of the polymer of preparation) is secondly used into aseptic liquid nutrient medium doubling dilution, takes 100 μ L The bacterial suspension diluted is added in 96 orifice plates in the Polymer Solution of 100 μ L, and the ultimate density for obtaining antibacterial polymer is 2-4096μg·mL-1.96 orifice plates are then placed in 37 DEG C of culture 18h, use absorbance of the microplate reader test microvia at 600nm Value.For bacterial cell culture solution using 200 μ L without antibacterial polymer as positive controls, 200 μ L pure culture solution is feminine gender Control group.Experiment is respectively provided with 4-6 parallel sample every time.A series of control group of concentration polymer solutions is set to exclude simultaneously Influence of the polymer solution to experimental group absorbance value.It then, will with the glutaraldehyde phosphate buffer of 2.5% percentage by volume Bacterium (20-25 degrees Celsius) the fixed 4h at room temperature of polymer solution culture contact 3h by 100 μ L lethal doses, and Twice with PBS buffer solution rinsing, it is successively then dehydrated 15min by the ethanol solution of gradient concentration, is centrifuged at 1000rpm 10min, it was evenly coated on silicon wafer, natural air drying, metal spraying 50s observes bacterium under field emission scanning electron microscope Surface breakdown pattern.
As shown in Fig. 7, antibacterial polymer produced by the present invention is intuitively shown from field emission scanning electron microscope result figure The death that bacterium membrane structure causes bacterium can be significantly destroyed, and is shown according to polymer prepared in the above embodiments and base This consistent performance.As shown in figs. 8 and 9, P (D-Leu-HEMA) homopolymer, P (D-Lys-HEMA) homopolymer and P (D-Leu- HEMA)-b-P (D-Lys-HEMA) block copolymer shows excellent anti-microbial property, the experimental results showed that, P (D-Leu- HEMA) minimum inhibitory concentration (MIC) of homopolymer is 798~820 μ gmL-1, the MIC of P (D-Lys-HEMA) homopolymer is 100 ~120 μ gmL-1, the MIC of P (D-Leu-HEMA)-b-P (D-Lys-HEMA) block copolymer is 180~200 μ gmL-1 (almost the same situation is shown for Escherichia coli and staphylococcus aureus)；Use the homopolymer based on L-type amino acid instead And copolymer, performance and the almost the same anti-microbial property of above-described embodiment, the slightly below homopolymer and copolymer of D type amino acid. The copolymer being copolymerized using D type and L-type monomer, by adjusting D type/L-type monomer ingredient proportion, copolymer shows as D Type or L-type, it is same have with the consistent anti-microbial property of respective configuration, lower than the anti-microbial property of respective configuration.
As shown in attached drawing 10-13, hemolytic experiment and external human aortic smooth muscle cell's compatibility experiments show this hair The polymer of bright preparation can still keep significant biocompatibility under concentration needed for antibacterial, and wherein hemolysis rate is lower than 10%, Smooth muscle cell activity can reach 80% or more.
Content carries out the preparation of above-mentioned polymer according to the present invention, and polymer shows the property almost the same with embodiment Energy.Illustrative description is done to the present invention above, it should which explanation is appointed in the case where not departing from core of the invention What simple deformation, modification or other skilled in the art can not spend the equivalent replacement of creative work to each fall within this The protection scope of invention.

Claims (9)

  1. The preparation method of 1.L- cationic chiral amino acid methacrylate copolymer, which is characterized in that as steps described below It is prepared:
    First L-type amino acid monomer is carried out homopolymerization by step 1., even using dithiobenzoic acid -4- cyanopentanoic acid as chain-transferring agent Nitrogen bis-isobutyronitrile is that initiator carries out RAFT polymerization to obtain D amino acids monomer-polymer, the first L-type amino acid list The molar ratio of body, chain-transferring agent and initiator is (45-60): 1:0.2；
    Step 2, L-configuration amino acid monomer polymer macromolecule chain-transferring agent prepared by step 1, azodiisobutyronitrile are to cause Agent is added the second L-type amino acid monomer and carries out RAFT polymerization to obtain L-lysine methacrylate and L-Leu methyl The molar ratio of acrylate block copolymer, the second L-type amino acid monomer, Macromolecular chain transfer agent and initiator is (80-100): 1:0.2.
  2. 2. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 1, special Sign is, in step 1, carries out under inert gas protection, and reaction temperature is 60-80 degrees Celsius, and the reaction time is that 6-10 is small When, solvent n,N-Dimethylformamide provides reaction atmosphere and environment；First L-type amino acid monomer, chain-transferring agent and initiator Molar ratio be (50-60): 1:0.2.
  3. 3. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 2, special Sign is that in step 1, inert gas is nitrogen, helium or argon gas.
  4. 4. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 1, special Sign is, in step 2, carries out under inert gas protection, and reaction temperature is 60-80 degrees Celsius, and the reaction time is that 6-10 is small When, solvent n,N-Dimethylformamide provides reaction atmosphere and environment；Second L-type amino acid monomer, Macromolecular chain transfer agent and The molar ratio of initiator is (85-90): 1:0.2.
  5. 5. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 4, special Sign is that in step 2, inert gas is nitrogen, helium or argon gas.
  6. 6. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 1, special Sign is that monomer L-lysine methacrylate is prepared as steps described below: by L-lysine and hydroxyethyl methacrylate Ethyl ester is using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydrating condensation agent, with methylene chloride be it is molten Agent is reacted 30-60 minutes in ice-water bath, is stirred to react under 20-25 degrees Celsius of room temperature 24-48 hours, wherein L- relies ammonia 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 are the L-lysine of tertbutyloxycarbonyl protection.
  7. 7. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 1, special Sign is that monomer L-Leu methacrylate is prepared as steps described below: by L-Leu and hydroxyethyl methacrylate Ethyl ester is using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide be dehydrating condensation agent, with methylene chloride be it is molten Agent is reacted 30-60 minutes in ice-water bath, is stirred to react 24-48 hours under 20-25 degrees Celsius of room temperature, wherein the bright ammonia of L- The mass ratio of acid, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and hydroxyethyl methacrylate is 5:(0.2- 0.25): (4-5): (2-3), L-Leu are the L-Leu of tertbutyloxycarbonyl protection.
  8. 8. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 1, special Sign is that the copolymer of preparation is deprotected under trifluoroacetic acid effect.
  9. 9. the preparation method of L- cationic chiral amino acid methacrylate copolymer according to claim 8, special Sign is, copolymer is dissolved in methylene chloride, and under trifluoroacetic acid effect, deprotection reaction is carried out under 20-25 degrees Celsius 2-4 hours, dissolution dialysis after purification, freeze-drying.
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SUN JINGJING等: ""Preparation of Functional Water-Soluble Low-Cytotoxic Poly(methacrylate)s With Pendant Cationic L-Lysines for Efficient Gene Delivery"", 《MACROMOLECULAR BIOSCIENCE》 *
SUN JINGJING等: ""Synthesis of diblock/statistical cationic glycopolymers with pendant galactose and lysine moieties: gene delivery application and intracellular behaviors"", 《JOURNAL OF MATERIALS CHEMISTRY B》 *

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