CN109796549A - The preparation method of lysine methacrylate homopolymer - Google Patents
The preparation method of lysine methacrylate homopolymer Download PDFInfo
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Abstract
The present invention discloses the preparation method of lysine methacrylate homopolymer, monomer D type or L-type lysine methacrylate homopolymerization to be formed, using dithiobenzoic acid -4- cyanopentanoic acid as chain-transferring agent, azodiisobutyronitrile is that initiator carries out reversible addion-fragmentation chain transfer polymerization；Polymer of the invention shows excellent anti-microbial property, can significantly destroy the death that bacterium membrane structure causes bacterium；Hemolytic experiment and external smooth muscle cell compatibility experiments show that it keeps significant biocompatibility.
Description
The application is parent application " lysine methacrylate homopolymer and preparation method and the application in antibacterial "
Divisional application, parent application application No. is 2016110193156, 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, it reacts
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, AIBN 2.86mg 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: 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. 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 (7)
1. the preparation method of lysine methacrylate homopolymer, which is characterized in that equal in L-lysine methacrylate
In the preparation method of polymers, monomer L-lysine methacrylate homopolymerization is formed, with dithiobenzoic acid -4- cyanopentanoic acid
For chain-transferring agent, azodiisobutyronitrile is that initiator carries out RAFT polymerization, monomer L-lysine methacrylate, chain-transferring agent
Molar ratio with initiator is (45-60): 1:0.2, and the homopolymer of preparation is deprotected under trifluoroacetic acid effect,
Dissolution dialysis after purification, freeze-drying；In the preparation method of D-Lys methacrylate homopolymer, monomer D- relies
The molar ratio of propylhomoserin methacrylate, chain-transferring agent and initiator is (45-60): the homopolymer of 1:0.2, preparation exist
It is deprotected under trifluoroacetic acid effect, after purification dissolution dialysis, freeze-drying；The L-lysine methacrylate
The number-average molecular weight of homopolymer is 10-23kDa, molecular weight distribution index 1.10-1.25；The D-Lys methacrylic acid
The number-average molecular weight of ester homopolymer polymer is 10-23kDa, molecular weight distribution index 1.10-1.25.
2. the preparation method of lysine methacrylate homopolymer according to claim 1, which is characterized in that rely in L-
In the preparation method of propylhomoserin methacrylate homopolymer, monomer L-lysine methacrylate, chain-transferring agent and initiator
Molar ratio be (50-60): 1:0.2；It carries out under inert gas protection, reaction temperature is 60-80 degrees Celsius, reaction
Time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and environment；The homopolymer of preparation is in trifluoroacetic acid
It is deprotected under effect.
3. the preparation method of lysine methacrylate homopolymer according to claim 2, which is characterized in that rely in L-
In the preparation method of propylhomoserin methacrylate homopolymer, inert gas is nitrogen, helium or argon gas, and homopolymer is dissolved in two
In chloromethanes, under trifluoroacetic acid effect, carried out deprotection reaction 2-4 hours under 20-25 degrees Celsius.
4. the preparation method of lysine methacrylate homopolymer according to claim 2, which is characterized in that rely in L-
In the preparation method of propylhomoserin methacrylate homopolymer, monomer L-lysine methacrylate is made as steps described below
It is standby: by L-lysine and hydroxyethyl methacrylate using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide
It for dehydrating condensation agent, reacts in ice-water bath 30-60 minutes by solvent of methylene chloride, is stirred under 20-25 degrees Celsius of room temperature
Reaction 24-48 hours is mixed, wherein L-lysine, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and methacrylic acid
The mass ratio of hydroxyl ethyl ester is 5:(0.2-0.25): (4-5): (2-3), L-lysine are that the L- of tertbutyloxycarbonyl protection relies ammonia
Acid.
5. the preparation method of lysine methacrylate homopolymer according to claim 1, which is characterized in that rely in D-
In the preparation method of propylhomoserin methacrylate homopolymer, monomer D-Lys methacrylate, chain-transferring agent and initiator
Molar ratio be (50-60): 1:0.2；It carries out under inert gas protection, reaction temperature is 60-80 degrees Celsius, reaction
Time is 6-10 hours, and solvent n,N-Dimethylformamide provides reaction atmosphere and environment；The homopolymer of preparation is in trifluoroacetic acid
It is deprotected under effect.
6. the preparation method of lysine methacrylate homopolymer according to claim 5, which is characterized in that rely in D-
In the preparation method of propylhomoserin methacrylate homopolymer, inert gas is nitrogen, helium or argon gas；Homopolymer is dissolved in two
In chloromethanes, under trifluoroacetic acid effect, carried out deprotection reaction 2-4 hours under 20-25 degrees Celsius.
7. the preparation method of lysine methacrylate homopolymer according to claim 5, which is characterized in that rely in D-
In the preparation method of propylhomoserin methacrylate homopolymer, monomer D-Lys methacrylate is made as steps described below
It is standby: by D-Lys and hydroxyethyl methacrylate using 4-dimethylaminopyridine as catalyst, N, N '-dicyclohexylcarbodiimide
It for dehydrating condensation agent, reacts in ice-water bath 30-60 minutes by solvent of methylene chloride, is stirred under 20-25 degrees Celsius of room temperature
Reaction 24-48 hours is mixed, wherein D-Lys, 4-dimethylaminopyridine, N, N '-dicyclohexylcarbodiimide and methacrylic acid
The mass ratio of hydroxyl ethyl ester is 5:(0.2-0.25): (4-5): (2-3), D-Lys are that the D- of tertbutyloxycarbonyl protection relies ammonia
Acid.
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