CN108329467A - A kind of preparation method of novel hyperbranched antibacterial peptide polymer - Google Patents
A kind of preparation method of novel hyperbranched antibacterial peptide polymer Download PDFInfo
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- CN108329467A CN108329467A CN201810138737.8A CN201810138737A CN108329467A CN 108329467 A CN108329467 A CN 108329467A CN 201810138737 A CN201810138737 A CN 201810138737A CN 108329467 A CN108329467 A CN 108329467A
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- carboxylic acid
- acid anhydrides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/028—Polyamidoamines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/043—Proteins; Polypeptides; Degradation products thereof
- A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
- C08G83/002—Dendritic macromolecules
- C08G83/005—Hyperbranched macromolecules
- C08G83/006—After treatment of hyperbranched macromolecules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
Abstract
The invention discloses a kind of preparation method of novel hyperbranched antibacterial peptide polymer, step is：1), the synthesis of amine-terminated hyperbrancedization daiamid：The low molecular weight over-branched polyamidoamine of Amino End Group is prepared for by the Michael addition reaction of methyl acrylate and diethylenetriamine；2), lysine N carboxylic acid anhydrides and the synthesis of valine N carboxylic acid anhydrides：Using over-branched polyamidoamine as core, lysine N carboxylic acid anhydrides and valine N carboxylic acid anhydrides are obtained by the reaction with triphosgene by benzyloxycarbonyl group lysine and valine respectively；3), hyperbranched antibacterial peptide Macroscopic single crystal：Lysine N carboxylic acid anhydrides and valine N carboxylic acid anhydrides are grafted to by the polypeptide chain that ring-opening polymerisation is formed on the Amino End Group of over-branched polyamidoamine, and the antibacterial peptide polymer with dissaving structure is prepared after amino is deprotected and is dialysed.The beneficial effects of the invention are as follows preparation method simplicity, raw material are easy to get, and synthesis cost is relatively low, convenient for large-scale production.
Description
Technical field
The invention belongs to the preparing technical fields of antibiotic property biomaterial, more specifically to a kind of novel over-expense
Change the preparation method of antibacterial peptide polymer.
Background technology
In recent years since the life that the abuse of antibiotic causes the generation of drug resistance superbacteria to seriously threaten the mankind is pacified
Entirely.Antibacterial peptide is that living organism generated one kind in the defense reaction for resisting pathogenic microorganism is antimicrobial pernicious with some
The small peptide of cell.Since natural biological antibacterial peptide is a part for eucaryote immune system, itself has broad spectrum antibacterial,
Especially have a lethal effect to multi-drug resistant bacteria, and antibacterial activity have high efficiency, stability, quickly, be not easy to produce resistance
The features such as, the utilization of natural biological antibacterial peptide are expected to become the mankind and break away from the another new of drug-fast bacteria crisis and antineoplaston
Approach, therefore the potential using value of antibacterial peptide receives the extensive concern of scholar.Although biologic antibiotic peptide is in infection control side
Face has a extensive future, but practical clinical is very restricted.Compared with traditional antibiotic, the production of biologic antibiotic peptide
Cost is high, this is to limit its widely used major obstacle.In addition, larger concentration biologic antibiotic peptide can induce mammal thin
Born of the same parents generate toxicity.
Cationic polymer can show higher antibacterial activity by selectively design, and synthesis is relatively cheap,
Large-scale production can be achieved.The design of these antibacterial polymers, dispersible cationic and hydrophobic grouping usually on main polymer chain
Or with alkylation quaternary ammonium group on hydrophobic long-chain.Studies have found that can by ring-opening polymerisation synthesize have N- carboxylic acid anhydrides sun from
The pdef polypeptide of sub (lysine) and the amino acid residue of hydrophobicity (alanine, phenylalanine, leucine), this kind of pdef polypeptide have
Similar to the biodegradability and antibacterial activity of biologic antibiotic peptide.But at higher concentrations, this kind of polymer has stronger molten
Courage and uprightness are not suitable for systemic administration.
The hyperbranched antibacterial peptide polymer prepared in the present invention has excellent inhibiting effect to drug resistance Escherichia coli,
Still there is notable fungistatic effect under low concentration.In addition it is also greatly improved on hemolytic, there is very low hemolysis rate.On the one hand
Production cost is reduced, on the other hand improves biocompatibility again, major obstacle has been cleared away for its practical application.
Invention content
It is poly- in view of the deficiencies of the prior art, the present invention intends to provide a kind of novel hyperbranched antibacterial peptide
The preparation method of object is closed, reasonable design, obtained hyperbranched antibacterial peptide polymer antimicrobial concentration is low, has good bactericidal effect
With lower hemolysis rate, in addition, its simple synthetic method, cost is relatively low, convenient for large-scale production, has very high potential answer
With value.
To achieve the above object, the present invention provides following technical solutions：
A kind of preparation method of novel hyperbranched antibacterial peptide polymer, step are：
1), the synthesis of amine-terminated hyperbrancedization daiamid：Pass through the Michael's addition of methyl acrylate and diethylenetriamine
Reaction is prepared for the low molecular weight over-branched polyamidoamine of Amino End Group；
2), lysine-N- carboxylic acid anhydrides and the synthesis of valine-N- carboxylic acid anhydrides：Using over-branched polyamidoamine as core, pass through benzyl
Lysine-N- carboxylic acid anhydrides and valine-N- carboxylic acid anhydrides is obtained by the reaction in oxygen carbonyl lysine and valine with triphosgene respectively；
3), hyperbranched antibacterial peptide Macroscopic single crystal：Lysine-N- carboxylic acid anhydrides and valine-N- carboxylic acid anhydrides are poly- by open loop
It closes the polypeptide chain formed to be grafted on the Amino End Group of over-branched polyamidoamine, be prepared after amino is deprotected and is dialysed with super
The antibacterial peptide polymer of branched structure.
As a kind of technical solution of optimization, structure composition is：The core and external polypeptide that dissaving polymer is constituted
Polymer long-chain.
As a kind of technical solution of optimization, the end group of over-branched polyamidoamine is amino in step 1), and number is opposite
Molecular weight is in 1000-8000, range of molecular weight distributions 1.2-4.0.
As a kind of technical solution of optimization, benzyloxycarbonyl group lysine and valine are reacted with triphosgene respectively in step 2)
Rate of charge range 1.2:1-2.0:1；50-60 DEG C of range of reaction temperature；Reaction time 20-60min.
As a kind of technical solution of optimization, the polymerization of lysine-N- carboxylic acid anhydrides and valine-N- carboxylic acid anhydrides in step 3)
Mode is ring-opening polymerisation, and the proportional region of the two is 1 in the copolymer:1-5:1.
As a kind of technical solution of optimization, over-branched polyamidoamine in step 3)：Lysine-N- carboxylic acid anhydrides：Valine-
The rate of charge of N- carboxylic acid anhydrides ranging from 1:10:5-1:100:50, wherein be based on lysine-N- carboxylic acid anhydrides：Valine-N- carboxylic acids
The rate of charge of acid anhydride is 2:1.
As a kind of technical solution of optimization, dialysis molecular cut off is 3500Da in step 3), dialysis time 4d, more
It is secondary to change water.
As a kind of technical solution of optimization, the relative molecular weight range of hyperbranched antibacterial peptide polymer is in 10000-
100000, molecular weight distribution is in 1.1-5.0.
By adopting the above-described technical solution, compared with prior art, it is easy that the beneficial effects of the invention are as follows preparation methods,
Raw material are easy to get, and synthesis cost is relatively low, convenient for large-scale production, meanwhile, the hyperbranched antibacterial peptide polymerization prepared in the present invention
Object have good antibacterial effect and biocompatibility, also for practical application lay a good foundation drug delivery, treatment of wounds and
The fields such as medical embedded material are potential to be widely applied.
With reference to drawings and examples, the present invention will be further described.
Description of the drawings
Fig. 1 is the preparation route of the hyperbranched antibacterial peptide polymer of an embodiment of the present invention；
Fig. 2 is the antibacterial area using Odontothrips loti to the hyperbranched antibacterial peptide polymer prepared in embodiment 1 in the present invention
Domain test figure；
Fig. 3 is the hemolytic experiment comparison diagram of the hyperbranched antibacterial peptide polymer prepared in the present invention.
Specific implementation mode
Embodiment 1
As shown in Figure 1, a kind of preparation method of novel hyperbranched antibacterial peptide polymer, step are：
1), amine-terminated hyperbrancedization daiamid (H-PAMAM) synthesizes：A certain amount of diethylenetriamine (DETA) is taken to be dissolved in nothing
Water methanol leads to nitrogen protection, adds magnetic agitation.A certain amount of methacrylate is added dropwise into above-mentioned solution by syringe
(MA), wherein the molar ratio to feed intake is DETA：MA=1.2：1.Then reaction unit is transferred in ice-water bath, is stirred to react
2h is then moved to and is reacted 48h at room temperature.Finally system is transferred in oil bath pan, along with vacuumize successively 60 DEG C/80 DEG C/
1h/1h/1h/1.5h is reacted at 100 DEG C/120 DEG C respectively, removes solvent and unreacted monomer.It uses after reaction a large amount of
Ice ether precipitated product obtain clear yellow viscous product, as H-PAMAM three times.
2), lysine-N- carboxylic acid anhydrides (Lys NCA) and valine-N- carboxylic acid anhydrides (Val NCA) synthesis：Pass through benzyloxy carbonyl
Base lysine and valine are obtained by the reaction with triphosgene respectively, benzyloxycarbonyl group lysine and valine and triphosgene molar ratio
It is 1.5:1.The preparation process of the two is similar, illustrates by taking the synthesis of Lys NCA as an example.Take a certain amount of benzyloxycarbonyl group lysine point
It dissipates in anhydrous tetrahydro furan, adds magnetic agitation, lead to nitrogen protection.The triphosgene of corresponding proportion is taken to be dissolved in a small amount of anhydrous tetrahydrochysene furan
It mutters, is added drop-wise in reaction system dropwise using syringe, be warming up to 50 DEG C, reaction 0.5h to system, which becomes, to be clarified.Using a large amount of
Ice n-hexane precipitated product, and use n-hexane/tetrahydrofuran (v/v=15:1) mixed solvent recrystallization three times, finally filters
Obtain white product Lys NCA.Same procedure can also synthesize Val NCA transparent sheet-like crystal.
3), hyperbranched antibacterial peptide polymer (HAPP) synthesis：H-PAMAM：Lys NCA：Val NCA three's rate of charges are 1：
20:10.It weighs a certain amount of Lys NCA and Val NCA is dissolved in anhydrous n,N-Dimethylformamide, add magnetic agitation, lead to nitrogen
Protection.It takes the H-PAMAM of corresponding proportion to be dissolved in a small amount of anhydrous n,N-Dimethylformamide, is added drop-wise to reaction dropwise using syringe
In system, react at room temperature for 24 hours.A small amount of n-butanol is then added dropwise, the reaction was continued 1h, product is precipitated with anhydrous ether, after centrifugation
Vacuum drying.Deprotection processing is carried out to the product after drying, product is dissolved in containing trifluoroacetic acid (200mg/mL) and 33%
It in the glacial acetic acid mixed solution of HBr (20mL/g precipitations), is stirred to react at room temperature for 24 hours, production is precipitated using the ether that decaploid is accumulated
Object.Product is dissolved in HCl solution (0.2M, 0.2mL/mg are precipitated) again after drying after centrifugation, uses molecular cut off 3500Da's
After bag filter is dialysed 4 days, freeze-drying obtains final product.
Embodiment 2
A kind of preparation method of novel hyperbranched antibacterial peptide polymer, step are：
1), amine-terminated hyperbrancedization daiamid (H-PAMAM) synthesizes：A certain amount of diethylenetriamine (DETA) is taken to be dissolved in nothing
Water methanol leads to nitrogen protection, adds magnetic agitation.A certain amount of methacrylate is added dropwise into above-mentioned solution by syringe
(MA), wherein the molar ratio to feed intake is DETA：MA=1.2：1.Then reaction unit is transferred in ice-water bath, is stirred to react
2h is then moved to and is reacted 48h at room temperature.Finally system is transferred in oil bath pan, along with vacuumize successively 60 DEG C/80 DEG C/
1h/1h/1h/1.5h/2h is reacted at 100 DEG C/120 DEG C/140 DEG C respectively, removes solvent and unreacted monomer.After reaction
Clear yellow viscous product, as H-PAMAM are obtained three times using a large amount of ice ether precipitated product.
2), lysine-N- carboxylic acid anhydrides (Lys NCA) and valine-N- carboxylic acid anhydrides (Val NCA) synthesis：Pass through benzyloxy carbonyl
Base lysine and valine are obtained by the reaction with triphosgene respectively, benzyloxycarbonyl group lysine and valine and triphosgene molar ratio
It is 1.5:1.The preparation process of the two is similar, illustrates by taking the synthesis of Lys NCA as an example.Take a certain amount of benzyloxycarbonyl group lysine point
It dissipates in anhydrous tetrahydro furan, adds magnetic agitation, lead to nitrogen protection.The triphosgene of corresponding proportion is taken to be dissolved in a small amount of anhydrous tetrahydrochysene furan
It mutters, is added drop-wise in reaction system dropwise using syringe, be warming up to 50 DEG C, reaction 0.5h to system, which becomes, to be clarified.Using a large amount of
Ice n-hexane precipitated product, and use n-hexane/tetrahydrofuran (v/v=15:1) mixed solvent recrystallization three times, finally filters
Obtain white product Lys NCA.Same procedure can also synthesize Val NCA transparent sheet-like crystal.
3), hyperbranched antibacterial peptide polymer (HAPP) synthesis：H-PAMAM：Lys NCA：Val NCA three's rate of charges are 1：
20:10.It weighs a certain amount of Lys NCA and Val NCA is dissolved in anhydrous n,N-Dimethylformamide, add magnetic agitation, lead to nitrogen
Protection.It takes the H-PAMAM of corresponding proportion to be dissolved in a small amount of anhydrous n,N-Dimethylformamide, is added drop-wise to reaction dropwise using syringe
In system, react at room temperature for 24 hours.A small amount of n-butanol is then added dropwise, the reaction was continued 1h, product is precipitated with anhydrous ether, after centrifugation
Vacuum drying.Deprotection processing is carried out to the product after drying, product is dissolved in containing trifluoroacetic acid (200mg/mL) and 33%
It in the glacial acetic acid mixed solution of HBr (20mL/g precipitations), is stirred to react at room temperature for 24 hours, production is precipitated using the ether that decaploid is accumulated
Object.Product is dissolved in HCl solution (0.2M, 0.2mL/mg are precipitated) again after drying after centrifugation, uses molecular cut off 3500Da's
After bag filter is dialysed 4 days, freeze-drying obtains final product.
Embodiment 3
A kind of preparation method of novel hyperbranched antibacterial peptide polymer, step are：
1), amine-terminated hyperbrancedization daiamid (H-PAMAM) synthesizes：A certain amount of diethylenetriamine (DETA) is taken to be dissolved in nothing
Water methanol leads to nitrogen protection, adds magnetic agitation.A certain amount of methacrylate is added dropwise into above-mentioned solution by syringe
(MA), wherein the molar ratio to feed intake is DETA：MA=1.2：1.Then reaction unit is transferred in ice-water bath, is stirred to react
2h is then moved to and is reacted 48h at room temperature.Finally system is transferred in oil bath pan, along with vacuumize successively 60 DEG C/80 DEG C/
1h/1h/1h/1.5h is reacted at 100 DEG C/120 DEG C respectively, removes solvent and unreacted monomer.It uses after reaction a large amount of
Ice ether precipitated product obtain clear yellow viscous product, as H-PAMAM three times.
2), lysine-N- carboxylic acid anhydrides (Lys NCA) and valine-N- carboxylic acid anhydrides (Val NCA) synthesis：Pass through benzyloxy carbonyl
Base lysine and valine are obtained by the reaction with triphosgene respectively, benzyloxycarbonyl group lysine and valine and triphosgene molar ratio
It is 1.5:1.The preparation process of the two is similar, illustrates by taking the synthesis of Lys NCA as an example.Take a certain amount of benzyloxycarbonyl group lysine point
It dissipates in anhydrous tetrahydro furan, adds magnetic agitation, lead to nitrogen protection.The triphosgene of corresponding proportion is taken to be dissolved in a small amount of anhydrous tetrahydrochysene furan
It mutters, is added drop-wise in reaction system dropwise using syringe, be warming up to 50 DEG C, reaction 0.5h to system, which becomes, to be clarified.Using a large amount of
Ice n-hexane precipitated product, and use n-hexane/tetrahydrofuran (v/v=15:1) mixed solvent recrystallization three times, finally filters
Obtain white product Lys NCA.Same procedure can also synthesize Val NCA transparent sheet-like crystal.
3), hyperbranched antibacterial peptide polymer (HAPP) synthesis：H-PAMAM：Lys NCA：Val NCA three's rate of charges are 1：
50:25.It weighs a certain amount of Lys NCA and Val NCA is dissolved in anhydrous n,N-Dimethylformamide, add magnetic agitation, lead to nitrogen
Protection.It takes the H-PAMAM of corresponding proportion to be dissolved in a small amount of anhydrous n,N-Dimethylformamide, is added drop-wise to reaction dropwise using syringe
In system, react at room temperature for 24 hours.A small amount of n-butanol is then added dropwise, the reaction was continued 1h, product is precipitated with anhydrous ether, after centrifugation
Vacuum drying.Deprotection processing is carried out to the product after drying, product is dissolved in containing trifluoroacetic acid (200mg/mL) and 33%
It in the glacial acetic acid mixed solution of HBr (20mL/g precipitations), is stirred to react at room temperature for 24 hours, production is precipitated using the ether that decaploid is accumulated
Object.Product is dissolved in HCl solution (0.2M, 0.2mL/mg are precipitated) again after drying after centrifugation, uses molecular cut off 3500Da's
After bag filter is dialysed 4 days, freeze-drying obtains final product.
Embodiment 4
A kind of preparation method of novel hyperbranched antibacterial peptide polymer, step are：
1), amine-terminated hyperbrancedization daiamid (H-PAMAM) synthesizes：A certain amount of diethylenetriamine (DETA) is taken to be dissolved in nothing
Water methanol leads to nitrogen protection, adds magnetic agitation.A certain amount of methacrylate is added dropwise into above-mentioned solution by syringe
(MA), wherein the molar ratio to feed intake is DETA：MA=1.2：1.Then reaction unit is transferred in ice-water bath, is stirred to react
2h is then moved to and is reacted 48h at room temperature.Finally system is transferred in oil bath pan, along with vacuumize successively 60 DEG C/80 DEG C/
1h/1h/1h/1.5h/2h is reacted at 100 DEG C/120 DEG C/140 DEG C respectively, removes solvent and unreacted monomer.After reaction
Clear yellow viscous product, as H-PAMAM are obtained three times using a large amount of ice ether precipitated product.
2), lysine-N- carboxylic acid anhydrides (Lys NCA) and valine-N- carboxylic acid anhydrides (Val NCA) synthesis：Pass through benzyloxy carbonyl
Base lysine and valine are obtained by the reaction with triphosgene respectively, benzyloxycarbonyl group lysine and valine and triphosgene molar ratio
It is 1.5:1.The preparation process of the two is similar, illustrates by taking the synthesis of Lys NCA as an example.Take a certain amount of benzyloxycarbonyl group lysine point
It dissipates in anhydrous tetrahydro furan, adds magnetic agitation, lead to nitrogen protection.The triphosgene of corresponding proportion is taken to be dissolved in a small amount of anhydrous tetrahydrochysene furan
It mutters, is added drop-wise in reaction system dropwise using syringe, be warming up to 50 DEG C, reaction 0.5h to system, which becomes, to be clarified.Using a large amount of
Ice n-hexane precipitated product, and use n-hexane/tetrahydrofuran (v/v=15:1) mixed solvent recrystallization three times, finally filters
Obtain white product Lys NCA.Same procedure can also synthesize Val NCA transparent sheet-like crystal.
3), hyperbranched antibacterial peptide polymer (HAPP) synthesis：H-PAMAM：Lys NCA：Val NCA three's rate of charges are 1：
50:25.It weighs a certain amount of Lys NCA and Val NCA is dissolved in anhydrous n,N-Dimethylformamide, add magnetic agitation, lead to nitrogen
Protection.It takes the H-PAMAM of corresponding proportion to be dissolved in a small amount of anhydrous n,N-Dimethylformamide, is added drop-wise to reaction dropwise using syringe
In system, react at room temperature for 24 hours.A small amount of n-butanol is then added dropwise, the reaction was continued 1h, product is precipitated with anhydrous ether, after centrifugation
Vacuum drying.Deprotection processing is carried out to the product after drying, product is dissolved in containing trifluoroacetic acid (200mg/mL) and 33%
It in the glacial acetic acid mixed solution of HBr (20mL/g precipitations), is stirred to react at room temperature for 24 hours, production is precipitated using the ether that decaploid is accumulated
Object.Product is dissolved in HCl solution (0.2M, 0.2mL/mg are precipitated) again after drying after centrifugation, uses molecular cut off 3500Da's
After bag filter is dialysed 4 days, freeze-drying obtains final product.
Testing experiment 1：
The hyperbranched antibacterial peptide polymer prepared in Example 1, it is respectively 512/256/128/64/ to be configured to concentration
The aqueous solution of 32/16/8/4/2/0 μ g/mL, as shown in Fig. 2, testing it on agar medium to large intestine bar using Odontothrips loti
The size in the antibacterial region of bacterium.By Oxford cup dispersion be placed on inoculation Escherichia coli agar medium on, be separately added into it is above-mentioned not
With the hyperbranched antibacterial peptide polymer solution of concentration, at 37 DEG C after constant temperature incubation 18h, each sample inhibition zone is measured with ruler
Diameter.
Testing experiment 2：
The hyperbranched antibacterial peptide polymer 10mg prepared in Example 1 is dissolved in 10mL physiological saline, is added thereto
Anti-freezing rabbit blood fresh 0.2mL, as experimental group.The fresh anti-freezing rabbit blood of 0.2mL is added using in 10mL physiological saline as the moon
Property control group, the fresh anti-freezing rabbit blood of 0.2mL is added using in 10mL distilled water as positive controls.Above-mentioned sample is gently shaken
After shaking uniformly, it is transferred in 37 DEG C of constant incubator and stands 1h.Sample is then centrifuged into 5min under 3000rpm rotating speeds, is adopted
The supernatant of each sample is detected with ultraviolet-visible spectrophotometer, extinction of the test each sample at 570nm wavelength
Degree, as shown in figure 3, calculating hemolysis rate according to following formula：
Wherein：ODnIt is the OD values of negative control group
ODpIt is the OD values of positive controls
ODsIt is the OD values of laboratory sample group.
Protection scope of the present invention is not limited merely to above-described embodiment, and all technical solutions belonged under thinking of the present invention are equal
It belongs to the scope of protection of the present invention.It should be pointed out that for those skilled in the art, not departing from the present invention
Several improvements and modifications under the premise of principle, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (8)
1. a kind of preparation method of novel hyperbranched antibacterial peptide polymer, it is characterised in that：The specific steps are：
1), the synthesis of amine-terminated hyperbrancedization daiamid：Pass through the Michael addition reaction of methyl acrylate and diethylenetriamine
It is prepared for the low molecular weight over-branched polyamidoamine of Amino End Group；
2), lysine-N- carboxylic acid anhydrides and the synthesis of valine-N- carboxylic acid anhydrides：Using over-branched polyamidoamine as core, pass through benzyloxy carbonyl
Lysine-N- carboxylic acid anhydrides and valine-N- carboxylic acid anhydrides is obtained by the reaction in base lysine and valine with triphosgene respectively；
3), hyperbranched antibacterial peptide Macroscopic single crystal：Lysine-N- carboxylic acid anhydrides and valine-N- carboxylic acid anhydrides pass through ring-opening polymerisation shape
At polypeptide chain be grafted on the Amino End Group of over-branched polyamidoamine, through amino be deprotected and dialyse after prepare with hyperbranched
The antibacterial peptide polymer of structure.
2. a kind of preparation method of novel hyperbranched antibacterial peptide polymer according to claim 1, it is characterised in that：Its
Structure composition is：The core and external polypeptide polymer long-chain that dissaving polymer is constituted.
3. a kind of preparation method of novel hyperbranched antibacterial peptide polymer according to claim 2, it is characterised in that：Step
It is rapid 1) in the end group of over-branched polyamidoamine be amino, count equal relative molecular weight in 1000-8000, range of molecular weight distributions is
1.2-4.0。
4. a kind of preparation method of novel hyperbranched antibacterial peptide polymer according to claim 3, it is characterised in that：Step
It is rapid 2) in the rate of charge range 1.2 reacted respectively with triphosgene of benzyloxycarbonyl group lysine and valine:1-2.0:1；Reaction temperature
50-60 DEG C of range；Reaction time 20-60min.
5. a kind of preparation method of novel hyperbranched antibacterial peptide polymer according to claim 4, it is characterised in that：Step
It is rapid 3) in the polymerization methods of lysine-N- carboxylic acid anhydrides and valine-N- carboxylic acid anhydrides be ring-opening polymerisation, ratio both in the copolymer
Example ranging from 1:1-5:1.
6. a kind of preparation method of novel hyperbranched antibacterial peptide polymer according to claim 5, it is characterised in that：Step
It is rapid 3) in over-branched polyamidoamine：Lysine-N- carboxylic acid anhydrides：The rate of charge ranging from 1 of valine-N- carboxylic acid anhydrides:10:5-1:
100:50, wherein be based on lysine-N- carboxylic acid anhydrides：The rate of charge of valine-N- carboxylic acid anhydrides is 2:1.
7. a kind of preparation method of novel hyperbranched antibacterial peptide polymer according to claim 6, it is characterised in that：Step
Rapid 3) middle dialysis molecular cut off is 3500Da, and dialysis time 4d repeatedly changes water.
8. a kind of preparation method of novel hyperbranched antibacterial peptide polymer according to claim 7, it is characterised in that：It is super
The relative molecular weight range of branched antibacterial peptide polymer is in 10000-100000, and molecular weight distribution is in 1.1-5.0.
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