WO2019149121A1 - 一种支化聚氨基酸抑菌剂及应用 - Google Patents

一种支化聚氨基酸抑菌剂及应用 Download PDF

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WO2019149121A1
WO2019149121A1 PCT/CN2019/072812 CN2019072812W WO2019149121A1 WO 2019149121 A1 WO2019149121 A1 WO 2019149121A1 CN 2019072812 W CN2019072812 W CN 2019072812W WO 2019149121 A1 WO2019149121 A1 WO 2019149121A1
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derivatives
group
derivative
amino acid
bacteriostatic agent
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PCT/CN2019/072812
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English (en)
French (fr)
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季生象
刘骁
韩苗苗
刘亚栋
郭建伟
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中国科学院长春应用化学研究所
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Priority claimed from CN201810097056.1A external-priority patent/CN108184852A/zh
Priority claimed from CN201810096221.1A external-priority patent/CN108276572A/zh
Priority claimed from CN201810096212.2A external-priority patent/CN108129656A/zh
Application filed by 中国科学院长春应用化学研究所 filed Critical 中国科学院长春应用化学研究所
Priority to KR1020207024770A priority Critical patent/KR102541633B1/ko
Priority to EP19747183.2A priority patent/EP3747932A4/en
Priority to JP2020541485A priority patent/JP7291710B2/ja
Priority to US16/966,357 priority patent/US20200368270A1/en
Publication of WO2019149121A1 publication Critical patent/WO2019149121A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/54—1,3-Diazines; Hydrogenated 1,3-diazines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/28—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N<
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
    • A61K8/88—Polyamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • 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
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • 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
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/42—Polyamides containing atoms other than carbon, hydrogen, oxygen, and nitrogen
    • 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
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of bacteriostatic agents, in particular to a branched polyamino acid bacteriostatic agent and application thereof.
  • polymer bacteriostatic agents can be non-specifically combined with negatively charged bacterial membranes, and then inserted into the bacterial membrane, causing bacterial membrane rupture, thereby killing bacteria, so polymer bacteriostatic agents are less resistant to drug resistance. Sex. Therefore, the development and use of bacteriostatic polymer materials with high safety, good bacteriostatic effect, sustainable use and biodegradability is a significant topic of great significance.
  • Amino acid is a renewable resource, which is mainly synthesized from biomass (starch, cellulose, etc.) by hydrolysis and fermentation. The annual global output reaches a million tons. At present, amino acids are mainly used as food and feed additives with low added value. How to develop new high value-added products is an urgent problem to be solved in the amino acid industry. The application of branched polyamino acids in the field of bacteriostatic has not been reported so far.
  • the technical problem to be solved by the present invention is to provide a branched polyamino acid bacteriostatic agent and application thereof, and the branched polyamino acid polymer prepared by using amino acid as a raw material has excellent bacteriostatic performance.
  • the present invention provides a branched polyamino acid bacteriostatic agent, comprising a branched polyamino acid;
  • the branched polyamino acid is obtained by homopolymerization of one amino acid unit or by copolymerization of two or more amino acid units;
  • the amino acid unit has the structural formula of the formula I or a salt thereof:
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are independently selected from the group consisting of hydrogen, hydroxy, thiol, amino, carboxy, C1 to C18 (for example, 1 , 2 , 3 , 4 , 5 carbon atoms) , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18) alkyl groups and derivatives thereof, C6 to C30 (preferably having 6 to 18 carbon atoms) a base thereof and a derivative thereof, a C3 to C8 (for example, a carbon number of 3, 4, 5, 6, 7, 8) cycloalkyl group and a derivative thereof, C2 to C8 (for example, a carbon number of 2, 3) , 4, 5, 6, 7, 8) olefins and derivatives thereof, C2 to C8 (for example, 2, 3, 4, 5, 6, 7, 8 carbon atoms) and derivatives thereof, C1 a C8 (for example, 1, 2, 3, 4, 5, 6, 7, 8 carbon atoms) and a derivative thereof, C1 to
  • the above formula I is a structural formula of an amino acid unit which can be obtained by copolymerization of two or more kinds of amino acid units or by homopolymerization of one amino acid unit.
  • the branched polyamino acid is obtained by homopolymerization of an amino acid unit, at least one of T 1 , T 2 , T 3 , T 4 , T 5 , T 6 of the amino acid unit.
  • the branched polyamino acid is obtained by copolymerization of two or more amino acid units, and T 1 , T 2 , T 3 , T 4 , T of at least one of the amino acid units 5 , at least one of T 6 is selected from the group consisting of a hydroxyl group, an amino group, a decyl group, a carboxyl group, a C2 to C8 alkene and a derivative thereof, a C2 to C8 alkyne and a derivative thereof, a C1 to C8 alkoxy group and a derivative thereof, C1 -C8 alkylthio and its derivatives, carboxylic acids and derivatives thereof, amines and derivatives thereof, nitrogen heterocycles and derivatives thereof, oxygen heterocycles and derivatives thereof or sulfur heterocycles and derivatives thereof.
  • the branched polyamino acid has a number average molecular weight of from about 500 g/mol to 500,000 g/mol.
  • the molecular weight of the polymer of the present invention is obtained by testing by gel permeation chromatography (GPC).
  • GPC gel permeation chromatography
  • M n Polymer molecular weight
  • the branched homo- or co-polymeric amino acids according to the invention have a number average molecular weight of from about 500 g/mol to 500,000 g/mol, and/or a PDI of from about 1.0 to 4.0 (eg, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, Scopes of 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 and 4.0) Inside.
  • the salt may be an amino acid salt well known to those skilled in the art, preferably a hydrochloride, sulfate, phosphate, carbonate or nitrate.
  • a, b, c, d, e and f are independently an integer of 0 to 6, and 1 ⁇ a + b + c + d + e + f ⁇ 20.
  • the a+b+c+d+e+f 10
  • T 1 , T 2 , T 3 , T 4 , T 5 , T 6 in [] represent a random combination of functional groups.
  • the T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are independently selected from any of the following structures (in the present invention, the symbol or Represents the point of attachment of the indicated group/structure to the rest of Formula I):
  • g is an integer from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); wherein xx, yy, zz are independently selected from hydrogen, C1 ⁇ An alkyl group of C18 (preferably C1-C12, more preferably C1-C8, such as C1, C2, C3, C4, C5, C6, C7 or C8), C6-C30 (preferably C6-C18, more preferably C6-C12) An aryl group, a C3 to C18 (preferably C3-C12, more preferably C3-C8) cycloalkyl group, a carbonyl derivative; hh is independently selected from the group consisting of hydrogen, hydroxy, amino, halogen elements, C1 to C18 (preferably C1-C12) More preferably, C1-C8, such as C1, C2, C3, C4, C5, C6, C7 or C8) alkyl, C6-C30 (preferably C6-C18, more preferably C6-C12) aryl, C3
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are independently selected from the group consisting of hydrogen, hydroxy, thiol, amino, carboxy, C1-C18 alkyl and derivatives thereof.
  • T 1, T 2, T 3 , T 4, T 5, T 6 is at least one selected from hydroxyl, mercapto, amino, carboxy, C2 ⁇ C8 of Alkene and its derivatives, C2 to C8 alkyne and its derivatives, C1 to C8 alkoxy groups and derivatives thereof, C1 to C8 carboxylic acids and derivatives thereof, C1 to C8 amines and derivatives thereof, C2 a nitrogen heterocycle of -C
  • the above derivatives are preferably C1-C5 alkyl substituents, C1-C5 alkoxy substituents, halogen, hydroxy, decyl, nitro, cyano, C5-C8 aryl, C5-C8 heteroaryl, C3 ⁇ C5 cycloalkyl, carboxy, amino, amide substituent, or any one or more C atoms substituted by O or S.
  • said T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are independently selected from H, C1-C5 alkyl, or C1-C5 substituted alkyl; said substituted alkyl preferably comprises A hydroxy substituent, a mercapto substituent, an aryl substituent, a heteroaryl substituent, a carboxy substituent, a heterocyclic substituent, an amide substituent, an amino substituent, or a C atom is substituted by O or S.
  • the number of carbon atoms of the above aryl substituent, heteroaryl substituent or heterocyclic substituent is preferably 5 to 12, and more preferably 5 to 8.
  • the number of carbon atoms of the above-mentioned carboxyl substituent, amide substituent or amino substituent is preferably from 1 to 8, more preferably from 1 to 5.
  • the terminal groups of T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are independently selected from a carboxyl group, a hydroxyl group, an amino group, an amide group, a decyl group, a fluorenyl group or an N, S group or Heterocyclic group of O.
  • the N-containing heterocyclic group is preferably an imidazolyl group or a benzopyrrole.
  • the T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are independently selected from any of the following structures:
  • the obtained polyamino acid when T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are simultaneously H, the obtained polyamino acid is a linear structure; and in at least one amino acid unit, T 1 , T 2 T 3 , T 4 , T 5 and T 6 are not H at the same time, and the obtained polyamino acid is a branched structure; when the functionality of at least one amino acid unit is ⁇ 3, a branched polyamino acid can be obtained.
  • the amino acid unit preferably includes lysine, ornithine, arginine, glutamic acid, histidine, asparagine, glutamine, serine, tryptophan, citrulline, aspartic acid, sul Any one or more of amino acid, tyrosine, cysteine, glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, and methionine.
  • the branched polyamino acid is obtained by homopolymerization of an amino acid unit having a functionality of ⁇ 3.
  • the amino acid unit is preferably glutamic acid, lysine, arginine, ornithine, histidine, aspartic acid, tryptophan, serine, citrulline, tyrosine, cysteine, Asparagine, glutamine or threonine.
  • the amino acid unit is a basic amino acid; more preferably, the amino acid unit is lysine, arginine, ornithine or histidine.
  • the branched polyamino acid is obtained by copolymerization of two or more amino acid units, and the copolymerized unit contains at least one or more amino acids having a functionality of ⁇ 3;
  • the ratio of the amino acid unit of degree ⁇ 3 to the amino acid unit is ⁇ , 0 ⁇ ⁇ ⁇ 100%.
  • the amino acid unit having a functionality of ⁇ 3 provides a branched structure for the polyamino acid.
  • the copolymerized amino acid unit is preferably glutamic acid, lysine, ornithine, arginine, histidine, asparagine, glutamine, serine, tryptophan, aspartic acid, citrulline, Threonine, tyrosine or cysteine; preferably, the amino acid unit comprises at least one basic amino acid unit. More preferably, the amino acid unit includes at least one or more of lysine, ornithine, arginine and histidine.
  • the branched polyamino acid is obtained by copolymerization of arginine and alanine, or by copolymerization of ornithine and leucine, or by copolymerization of lysine and alanine. It is obtained either by copolymerization of histidine and phenylalanine or by copolymerization of lysine and arginine.
  • the comonomer is selected from the group consisting of arginine and alanine, ornithine and leucine, lysine and alanine, histidine and phenylalanine, ornithine and 6 - aminocaproic acid, lysine and arginine, phenylalanine, alanine and lysine, arginine and serine, arginine and glutamic acid, and histidine and serine.
  • the ratio ( ⁇ ) of the amino acid unit having a functionality of ⁇ 3 to the amino acid unit is required to be greater than 0 and less than or equal to 100%, that is, 0 ⁇ ⁇ ⁇ 100%.
  • the copolymerized amino acid is obtained by copolymerization of two amino acid units, and the molar ratio of the two amino acid units is in the range of 0.1:10 to 10:0.1; for the copolymerized amino acid obtained by copolymerization of the three amino acid units, the molar ratio of the three amino acid units In the range of 0.1-10:0.1-10:0.1-10.
  • the preparation method of the branched polyamino acid of the present invention is not particularly limited, and it can be prepared according to a method well known to those skilled in the art, and is preferably prepared according to the following method:
  • the amino acids are mixed and reacted in an inert gas atmosphere at 25 to 250 ° C for 1 min to 96 h to obtain a branched polyamino acid.
  • the inert gas is preferably nitrogen or argon.
  • the reaction temperature is preferably from 150 to 200 ° C, and the reaction time is preferably from 30 min to 24 h, more preferably from 2 h to 12 h.
  • the branched structure of the polyamino acid makes the substance have many reactive functional groups, can be further modified, has good biocompatibility, and the bacteriostatic agent does not produce drug resistance for long-term use.
  • the polyamino acid is modified by any one or more of the following:
  • amino group in the I, amino or amide group is modified to the following groups:
  • the sulfhydryl group is modified to -SR 3 ;
  • V a thiol group is modified to a group represented by the formula V-1;
  • X, Y, Z, and Q are independently selected from the group consisting of hydrogen, a C1-C18 alkyl group and a derivative thereof, a C6-C30 aryl group and a derivative thereof, a C3-C18 cycloalkyl group and a derivative thereof, and C2 ⁇ C18 olefins and derivatives thereof, C2 to C18 alkyne and derivatives thereof, C1 to C18 alkoxy groups and derivatives thereof, carboxylic acids and derivatives thereof, amines and derivatives thereof, nitrogen heterocycles and derivatives thereof , an oxygen heterocycle and its derivatives or a sulfur heterocycle and its derivatives;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are independently selected from H, C1 to C18 alkyl and derivatives thereof, C6 to C30 aryl and derivatives thereof, and C3 to C18 naphthenes And derivatives thereof, C2 to C18 olefins and derivatives thereof, C2 to C18 alkyne and derivatives thereof, C1 to C18 alkoxy groups and derivatives thereof, carboxylic acids and derivatives thereof, amines and derivatives thereof a nitrogen heterocycle and a derivative thereof, an oxygen heterocycle and a derivative thereof or a sulfur heterocycle and a derivative thereof; and R 1 , R 3 , R 5 and R 6 are not H.
  • the above derivatives are preferably C1-C5 alkyl substituents, C1-C5 alkoxy substituents, halogen, hydroxy, decyl, nitro, cyano, C5-C8 aryl, C5-C8 heteroaryl, C3 ⁇ C5 cycloalkyl, carboxy, amino, amide substituent, or any one or more C atoms substituted by O or S.
  • the X, Y, Z, and Q are independently selected from the group consisting of hydrogen, C1-C3 alkyl, C6-C8 aryl, C3-C6 cycloalkyl, C1-C3 alkoxy, C2-C5 aza a ring, a C2-C5 oxyheterocycle or a C2-C5 sulphur heterocycle;
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently selected from C1 to C3 alkyl, C6 to C8 aryl, C3 to C6 cycloalkyl, C1 to C3 alkoxy, C2 to C5.
  • X, Y, Z, Q, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are independently selected from the group consisting of hydrogen, methyl, ethyl, butyl. , isopropyl, acetyl, formyl and the like.
  • the number of N atoms in the nitrogen-containing heterocyclic group in the present invention is not particularly limited, and may be a nitrogen-containing hetero ring which is well known in the art, and the number of N atoms may be, but not limited to, 1 to 3.
  • the modified N atom may be all N atoms or may be modified by one or two N atoms.
  • the nitrogen-containing heterocyclic group is an imidazolyl group modified to a group of formula VI-1:
  • the polyamino acid of the present invention may be a thiol-modified, quaternary ammonium-modified, acetyl-modified, ether-modified, methyl ester-modified or hydroxyl-modified polyamino acid (homopolymerization) Or copolyamino acid), more preferably selected from the group consisting of thiol-modified hyperbranched polyornithine, quaternary ammonium salt modified hyperbranched polyornithine, acetyl-modified hyperbranched polylysine , thiol-modified ⁇ -polylysine, thiol-modified ⁇ -polylysine, ether-modified poly(arginine-serine), methyl ester-modified poly(arginine- Glutamate), hydroxyl-modified poly(ornithine-cysteine), ether-modified poly(histidine-serine), thiol-modified,
  • the above modification can improve the antibacterial property of the polyamino acid, and reduce the hemolysis ratio and cytotoxicity.
  • the method of the above modification of the present invention is not particularly limited, and a method known to those skilled in the art may be employed.
  • the bacteriostatic agent may further comprise an adjuvant, and the adjuvant is preferably present in an amount of from 0 to 99% by weight.
  • the type of the adjuvant is not particularly limited in the present invention, and may be an adjuvant suitable for the bacteriostatic agent well known to those skilled in the art.
  • the bacteriostatic adjuvant preferably comprises: [i] an inorganic bacteriostatic agent such as a metal, a metal ion, a metal salt or an oxide thereof; [ii] an organometallic, an organic halogenated substance, an anthracene, an organic nitro compound.
  • the bacteriostatic adjuvant is metal Ti, Ag + , Cu 2+ , Fe 3+ , Zn 2+ , quaternary ammonium salt, haloamine, polybiguanide, halogenated phenol, chitosan, fish One or more of protamine and natural bacteriostatin.
  • the adjuvant is polyhexamethylene biguanide.
  • the dosage form of the bacteriostatic agent in the present invention is not particularly limited and may be a solid, a solution, a suspension, an emulsion, a hydrogel, an oil gel, an aerosol, a coating or grafting onto a solid surface, and the like.
  • One or more states of material blending are used.
  • the preparation method of the bacteriostatic agent in the present invention is not particularly limited, and a branched polyamino acid and an auxiliary agent may be mixed.
  • the mixing process may or may not use a solvent.
  • the solvent may be water or an organic solvent.
  • the organic solvent is preferably methanol, ethanol, ethyl acetate, n-heptane, dimethylformamide, dimethylacetamide, tetrahydrofuran, chloroform, dichloromethane, carbon tetrachloride, acetonitrile, petroleum ether, n-hexane.
  • the bacteriostatic agent provided by the invention has simple preparation process, low equipment requirement, easy operation, easy availability of materials, low cost, industrial application prospect, and broad spectrum bacteriostasis.
  • the present invention also provides the use of the above bacteriostatic agent in the field of bacteriostatic, and the bacteriostatic range is not particularly limited, and may be a bacteriostatic range well known to those skilled in the art.
  • the bacteriostatic range preferably includes the use in inhibiting one or more of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes.
  • bacteriostatic agent can be applied to various fields of application well known to those skilled in the art, and is not particularly limited.
  • the field of application is preferably in the fields of food, cosmetics, medical supplies, health care products and the like.
  • Such as food preservatives, food preservatives, cosmetic additives, mouthwashes, disinfectants, multi-functional care solutions, preservatives in eye drops, swimming pool disinfectants, can also be used in toothpaste, facial cleansers, hand sanitizers, disinfecting soaps, Disinfection and preservatives for the storage of fruits and vegetables.
  • the present invention provides a branched polyamino acid bacteriostatic agent comprising a branched polyamino acid; the branched polyamino acid is obtained by homopolymerization of one amino acid unit, or two or more amino acid units Copolymerization; the amino acid unit has the structural formula shown in Formula I.
  • the raw material used in the invention is an amino acid, has no toxicity, has no side effects, and is a green and environmentally-friendly new bacteriostatic agent, which is easy for the user to accept.
  • the branched structure of the polyamino acid allows the substance to have many reactive functional groups, can be further modified, has good biocompatibility, and the bacteriostatic agent does not develop drug resistance for long-term use.
  • Example 1 is a nuclear magnetic resonance spectrum of branched polylysine prepared in Example 2 of the present invention.
  • Example 2 is a nuclear magnetic resonance spectrum of branched polyarginine prepared in Example 10 of the present invention.
  • Figure 3 is a nuclear magnetic resonance spectrum of a branched polyamino acid prepared in Example 25 of the present invention.
  • the branched polyamino acid bacteriostatic agent and application provided by the present invention are described in detail below with reference to the examples.
  • the reaction materials used in the following examples are commercially available, all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Sigma-Aldrich Chemical Reagent Co., Ltd., J&K BEHRINGER TECHNOLOGY CO., Ltd., Shanghai Maclean Biochemistry Co., Ltd. Technology Co., Ltd. or Sinopharm Chemical Reagent Co., Ltd.
  • Example 1-19 36 mg of the hyperbranched polyamino acid prepared in Example 1-19 was separately dissolved in 3 mL of sterile PBS to obtain a mother liquor of 12 mg/mL, and the antibacterial activity of the hyperbranched polyamino acid-based antibacterial agent was tested according to the following method. 1.
  • the antibacterial activity of the hyperbranched polyamino acid-based antibacterial agent was tested by 96-well plate method, and the ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the antibacterial ability and minimum inhibitory concentration of the obtained hyperbranched polyamino acid-based antibacterial agent.
  • MIC is defined as the lowest polymer concentration that inhibits 90% of bacterial growth compared to the control group.
  • a small number of strains were picked from the agar slant medium by using the inoculating loop in normal MH medium, and cultured at 37 ° C overnight to resuscitate the strain and reach exponential growth.
  • the bacterial solution was diluted to a concentration of 10 6 CFU/mL per well. 175 ⁇ L of bacterial solution and 25 ⁇ L of different concentrations of polymer solution were added, and the 96-well plate was incubated at 37 ° C for 20 hours, and the OD 600 value was measured with a microplate reader.
  • Example 1-19 36 mg of the hyperbranched polyamino acid prepared in Example 1-19 was separately dissolved in 3 mL of sterile PBS to obtain a mother liquor of 12 mg/mL, and the in vitro hemolytic activity of the hyperbranched polyamino acid-based antibacterial agent was tested according to the following method. 2.
  • the hyperbranched polyamino acid-based antibacterial agent was tested for in vitro hemolytic activity by a 96-well plate method, and ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the in vitro hemolytic activity of the obtained hyperbranched polyamino acid-based antibacterial agent.
  • the polymer was diluted with PBS buffer solution to prepare different concentrations of solution, added to a 96-well plate, and the PBS buffer solution alone was used as a negative control.
  • 0.2% Triton-X-100 was dissolved in water as a positive control for in vitro hemolytic activity detection.
  • the washed red blood cells (2% v/v, 50 ⁇ L) were added to a 96-well plate, thoroughly mixed and cultured. The absorbance at 540 nm was measured with a microplate reader.
  • This example was used to detect the acute toxicity of hyperbranched polyamino acid-based antibacterial agents in animals, and ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the acute toxicity of the obtained hyperbranched polyamino acid-based antibacterial agent in animals.
  • mice (Balb/C mice, purchased from Jilin University), male and female, weighing 21 ⁇ 3g, respectively, taking the hyperbranched polyamino-based antibacterial agent prepared in Examples 1-19 at a dose of 1mg/mL. Mice were intramuscularly administered once a day for 15 consecutive days to observe the toxicity of the mice. The results showed that after 21 days of intramuscular injection, except for the decrease of viability of individual mice, there was no obvious abnormal reaction, and all mice survived, which proved that the obtained hyperbranched polyamino acid-based antibacterial agent has a smaller body. toxicity.
  • the antibacterial activity of the hyperbranched polyamino acid-based antibacterial agent was tested by 96-well plate method, and the ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the antibacterial ability and minimum inhibitory concentration of the obtained hyperbranched polyamino acid-based antibacterial agent.
  • MIC is defined as the lowest polymer concentration that inhibits 90% of bacterial growth compared to the control group.
  • a small number of strains were picked from the agar slant medium by using the inoculating loop in normal MH medium, and cultured at 37 ° C overnight to resuscitate the strain and reach exponential growth.
  • the bacterial solution was diluted to a concentration of 10 6 CFU/mL per well. 175 ⁇ L of bacterial solution and 25 ⁇ L of different concentrations of polymer solution were added, and the 96-well plate was incubated at 37 ° C for 20 hours, and the OD 600 value was measured with a microplate reader.
  • the hyperbranched polyamino acid-based antibacterial agent was tested for in vitro hemolytic activity by a 96-well plate method, and ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the in vitro hemolytic activity of the obtained hyperbranched polyamino acid-based antibacterial agent.
  • the polymer was diluted with PBS buffer solution to prepare different concentrations of solution, added to a 96-well plate, and the PBS buffer solution alone was used as a negative control.
  • 0.2% Triton-X-100 was dissolved in water as a positive control for in vitro hemolytic activity detection.
  • the washed red blood cells (2% v/v, 50 ⁇ L) were added to a 96-well plate, thoroughly mixed and cultured. The absorbance at 540 nm was measured with a microplate reader.
  • This example was used to detect the acute toxicity of hyperbranched polyamino acid-based antibacterial agents in animals, and ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the acute toxicity of the obtained hyperbranched polyamino acid-based antibacterial agent in animals.
  • mice (Balb/C mice, purchased from Jilin University), male and female, weighing 21 ⁇ 3g, respectively, taking the hyperbranched polyamino-based antibacterial agent prepared in Examples 23-37 at a dose of 1mg/mL.
  • Mice were intramuscularly administered once a day for 15 consecutive days to observe the toxicity of the mice.
  • the experimental results showed that after 15 days of intramuscular injection, except for the decrease of viability of individual mice, there was no obvious abnormal reaction, and all the mice survived, which proved that the obtained hyperbranched polyamino acid-based antibacterial agent has a smaller body. toxicity.
  • Example 41 2 g of the hyperbranched polyornithine obtained in Example 41 was dissolved in 20 mL of N,N-dimethylformamide (DMF) by heating, 5 g of methyl iodide was added, and the reaction was stirred at 80 ° C for 24 hours, and then the heating was stopped. After cooling to room temperature, it was allowed to settle into ethyl acetate to obtain 2.4 g of a quaternary ammonium salt-modified hyperbranched polyornithine.
  • DMF N,N-dimethylformamide
  • Example 43 2 g of the hyperbranched polylysine obtained in Example 43 was dissolved in 5 mL of methanol, and acetyl chloride was slowly added dropwise at 0 ° C, and the temperature was raised to room temperature to continue the reaction for 12 hours, and then precipitated into ethyl acetate to obtain an acetyl group.
  • Sexual hyperbranched polylysine 2.3g.
  • Example 41 2 g of the hyperbranched polyornithine obtained in Example 41 was dissolved in 5 mL of methanol, 4.8 g of methylisothiourea hemisulfate and 5 mL of triethylamine were added, and reacted at 60 ° C for 12 h, then the heating was stopped and cooled to room temperature. The precipitate was poured into ethyl acetate to obtain 2.1 g of a thiol-modified hyperbranched polyornithine.
  • Example 47 2 g of ⁇ -polylysine obtained in Example 47 was dissolved in 5 mL of water, 5.1 g of 1H-pyrazole-1-carboxamidine hydrochloride and 5 mL of triethylamine were added, and reacted at 60 ° C for 12 h, then the heating was stopped and cooled. After standing at room temperature, it was allowed to settle into ethyl acetate to obtain 2.3 g of thiol-modified ?-polylysine.
  • Example 51 2 g of the polyamino acid obtained in Example 51 was dissolved in 20 mL of anhydrous methanol, and 0.9 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 0.1 g were added. 4-Dimethylaminopyridine (DMAP), stirred at room temperature for 10 h, the polymer was precipitated into diethyl ether to give a methyl ester-modified polyamino acid 2.1 g.
  • DMAP 4-Dimethylaminopyridine
  • Example 55 2 g of the polyamino acid obtained in Example 55 was dissolved in 20 mL of methanol, and 0.5 g of p-toluenesulfonic acid was added thereto, and the mixture was heated under reflux for 10 hours to precipitate a polymer into diethyl ether to obtain 2.2 g of an ether group-modified polyamino acid.
  • the minimum inhibitory concentration (MIC) was defined as the lowest polymer concentration that inhibited 90% of bacterial growth compared to the control group.
  • a small number of strains were picked from the agar slant medium by using the inoculating loop in normal MH medium, and cultured at 37 ° C overnight to resuscitate the strain and reach exponential growth.
  • the bacterial solution was diluted to a concentration of 10 6 CFU/mL per well. 175 ⁇ L of bacterial solution and 25 ⁇ L of different concentrations of polymer solution were added, and the 96-well plate was incubated at 37 ° C for 20 hours, and the OD 600 value was measured with a microplate reader.
  • the poly-amino acid-based antibacterial agent was tested for in vitro hemolytic activity by a 96-well plate method, and ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the in vitro hemolytic activity of the obtained polyamino-based antibacterial agent.
  • the polymer was diluted with PBS buffer solution to prepare different concentrations of solution, added to a 96-well plate, and the PBS buffer solution alone was used as a negative control.
  • 0.2% Triton-X-100 was dissolved in water as a positive control for in vitro hemolytic activity detection.
  • the washed red blood cells (2% v/v, 50 ⁇ L) were added to a 96-well plate, thoroughly mixed and cultured. The absorbance at 540 nm was measured with a microplate reader.
  • This example was used to detect acute toxicity in animals of polyamino acid-based antibacterial agents, and ⁇ -polylysine synthesized by fermentation was used as a control to evaluate the acute toxicity of the obtained polyamino-based antibacterial agent in animals.
  • mice (Balb/C mice, purchased from Jilin University), male and female, weighing 21 ⁇ 3g, respectively, taking the polyamino-based antibacterial agent prepared in Examples 41-56 at a dose of 1mg/mL, daily Mice were intramuscularly injected for 15 consecutive days to observe the toxicity of the mice. The results showed that after 15 days of intramuscular injection, except for the decrease of viability of individual mice, there was no obvious abnormal reaction, and all the mice survived, which proved that the obtained polyamino-based antibacterial agent had less in vivo toxicity.
  • Escherichia coli ATCC 8739
  • Staphylococcus aureus ATCC 25923
  • the sample membrane was transferred to a 48-well plate, and 1 mL of the bacterial suspension was added for 1 h, and then the culture solution was aspirated.
  • Escherichia coli was treated with 0.5% (wt/vol) glucose medium, and Staphylococcus aureus was continued at 37 °C using TSB medium. Static culture for 4h.
  • the sample membrane to which the bacteria were adhered was placed in 1 mL of fresh sterile PBS solution and ultrasonically washed for 1 min to desorb the bacteria on the surface.
  • the bacterial-containing PBS solution was diluted in a certain number and applied to a solid medium. After overnight incubation at 37 ° C, bacterial colonies formed on the surface of the solid medium were counted and counted.
  • the control polystyrene (PS) the bacterial adhesion of E. coli and S. aureus on the surface of HBPL-PS decreased by -95.5% and -98.8%, respectively.

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Abstract

本发明提供了一种支化聚氨基酸抑菌剂，包括支化聚氨基酸；所述支化聚氨基酸由一种氨基酸单元均聚得到，或者由两种或两种以上的氨基酸单元共聚得到；所述氨基酸单元具有式I所示结构通式。本发明所用的原料为氨基酸，无毒性，无副作用，是一种绿色环保型的新型抑菌剂，使用者易于接受。聚氨基酸的支化结构，使得该物质具有许多活性官能团，可以进一步进行修饰，具有良好的生物相容性，且该抑菌剂长期使用不会产生耐药性。

Description

一种支化聚氨基酸抑菌剂及应用 技术领域
本发明涉及抑菌剂技术领域，尤其涉及一种支化聚氨基酸抑菌剂及应用。
背景技术
抗生素的发明拯救了数以亿计人的生命，使人类免遭细菌感染的痛苦，可以说是医学上的一次革命。但是随着小分子抗生素的滥用，再加上细菌的短生命周期及基因水平转移特性，各种抗药细菌随之出现。致病性细菌的传播更是严重危及到人们正常的生活。一份最新研究显示，如果得不到控制，超级细菌到2050年将导致每年约1000万人丧命。耐药细菌已成为世界各国普遍关注的热点问题，关系到全球人类的健康、经济的发展以及社会的稳定。
与小分子抗生素相比，聚合物抑菌剂可以与带负电的细菌膜进行非特异性结合，进而插入细菌膜内部，导致细菌膜破裂，进而杀死细菌，所以聚合物抑菌剂不易产生耐药性。因此，开发和使用安全性高、抑菌效果好、可持续使用且可生物降解的抑菌聚合物材料是一个具有深远意义的重大课题。
氨基酸是一种可再生资源，主要由生物质(淀粉、纤维素等)原料经水解后发酵来合成，全球年产量达到百万吨级。目前，氨基酸主要作为食品和饲料添加剂，附加值较低。如何开发新的高附加值产品是氨基酸行业迫切需要解决的问题。支化聚氨基酸在抑菌领域的应用至今尚无报道。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种支化聚氨基酸抑菌剂及应用，以氨基酸为原料，制备的支化聚氨基酸聚合物具有优良的抑菌性能。
为解决以上技术问题，本发明提供了一种支化聚氨基酸抑菌剂，包括支化聚氨基酸；
所述支化聚氨基酸由一种氨基酸单元均聚得到，或者由两种或两种以上的氨基酸单元共聚得到；
所述氨基酸单元具有式I所示结构通式或其盐：
Figure PCTCN2019072812-appb-000001
其中，
a、b、c、d、e和f独立地为0～6的整数，且1≤a+b+c+d+e+f≤20(例如，a+b+c+d+e+f＝1，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19或20)，优选地，a+b+c+d+e+f≤10；
T 1、T 2、T 3、T 4、T 5和T 6独立地选自氢，羟基，巯基，氨基，羧基，C1～C18(例如，碳原子数为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17和18)的烷基及其衍生物，C6～C30(优选碳原子数为6～18)的芳基及其衍生物，C3～C8(例如，碳原子数为3、4、5、6、7、8)的环烷基及其衍生物，C2～C8(例如，碳原子数为2、3、4、5、6、7、8)的烯及其衍生物，C2～C8(例如，碳原子数为2、3、4、5、6、7、8)的炔及其衍生物，C1～C8(例如，碳原子数为1、2、3、4、5、6、7、8)的烷氧基及其衍生物，C1～C8(例如，碳原子数为1、2、3、4、5、6、7、8)的烷硫基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物。任选地，T 1、T 2、T 3、T 4、T 5和T 6不同时为H。
上述式I为氨基酸单元的结构通式，所述支化聚氨基酸可以由两种或两种以上的氨基酸单元共聚得到；或者由一种氨基酸单元均聚得到。
在本发明的一个优选实施方案中，所述支化聚氨基酸由一种氨基酸单元均聚得到，所述氨基酸单元的T 1、T 2、T 3、T 4、T 5、T 6中至少一个选自羟基，氨基，巯基，羧基，C2～C8的烯及其衍生物，C2～C8的炔及其衍生物，C1～C8的烷氧基及其衍生物，C1～C8的烷硫基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物。
在本发明的另一个优选实施方案中，所述支化聚氨基酸由两种或两种以上的氨基酸单元共聚得到，至少一个所述氨基酸单元的T 1、T 2、T 3、T 4、T 5、T 6中至少一个选自羟基，氨基，巯基，羧基，C2～C8的烯及其衍生物，C2～C8的炔及其衍生物，C1～C8的烷氧基及其衍生物，C1～C8的烷硫基及其衍生物， 羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物。
所述支化聚氨基酸的数均分子量为大约500g/mol-500,000g/mol。
优选地，本发明的聚合物的分子量是采用凝胶渗透色谱(GPC)进行测试获得的。如实施例中所示，一个具体测试方法为：聚合物分子量(M n)及其分布(PDI＝M w/M n)通过装备有Waters 2414干涉折射检测器的Waters 2414凝胶渗透色谱系统检测获得，0.2M醋酸/0.1M醋酸钠为流动相流速0.6mL/min，温度35℃，标准品为聚乙二醇。根据本发明的支化均聚或共聚氨基酸的数均分子量为大约500g/mol-500,000g/mol，和/或PDI在大约1.0至4.0(例如，1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9和4.0)的范围内。
所述盐可以为本领域技术人员熟知的氨基酸盐，优选为盐酸盐，硫酸盐，磷酸盐，碳酸盐或硝酸盐。
式I中，a、b、c、d、e和f独立地为0～6的整数，且1≤a+b+c+d+e+f≤20。
优选地，所述a+b+c+d+e+f≤10。
优选地，[]中的T 1、T 2、T 3、T 4、T 5、T 6代表官能团的无规组合。
在本发明的一个优选实施方案中，所述T 1、T 2、T 3、T 4、T 5和T 6独立地选自以下任一结构(在本发明中，符号
Figure PCTCN2019072812-appb-000002
或
Figure PCTCN2019072812-appb-000003
表示所示基团/结构与式I其余部分的连接点)：
Figure PCTCN2019072812-appb-000004
Figure PCTCN2019072812-appb-000005
及其盐，
Figure PCTCN2019072812-appb-000006
及其盐，
Figure PCTCN2019072812-appb-000007
Figure PCTCN2019072812-appb-000008
Figure PCTCN2019072812-appb-000009
其中，g为0至10的整数(例如，0、1、2、3、4、5、6、7、8、9、10)；其中，xx、yy、zz独立地选自氢，C1～C18(优选C1-C12，更优选C1-C8，例如C1、C2、C3、C4、C5、C6、C7或C8)的烷基，C6～C30(优选C6-C18，更优选C6-C12)的芳基，C3～C18(优选C3-C12，更优选C3-C8)的环烷基，羰基衍生物；hh独立地选自氢，羟基，氨基，卤族元素，C1～C18(优选C1-C12，更优选C1-C8，例如C1、C2、C3、C4、C5、C6、C7或C8)的烷基，C6～C30(优选C6-C18，更优选C6-C12)的芳基，C3～C18(优选C3-C12，更优选C3-C8)的环烷基，胺及其衍生物，烷氧基衍生物，烷巯基衍生物；ii，jj，kk独立地选自氢，C1～C18(优选C1-C12，更优选C1-C8，例如C1、C2、C3、C4、C5、C6、C7或C8)的烷基，C6～C30(优选C6-C18，更优选C6-C12)的芳基，C3～C18(优选C3-C12，更优选C3-C8)的环烷基，烷氧基及其衍生物；oo，pp，qq独立地选自氢，羧基，羟基，氨基，C1～C18(优选C1-C12，更优选C1-C8，例如C1、C2、C3、C4、C5、C6、C7或C8)的烷基，C6～C30(优选C6-C18，更优选C6-C12)的芳基，C3～C18(优选C3-C12，更优选C3-C8)的环烷基，卤族元素，胺及其衍生物，烷氧基衍生物，羰基衍生物；rr，tt独立地选自氢，C1～C18(优选C1-C12，更优选C1-C8，例如C1、C2、C3、C4、C5、C6、C7或C8)的烷基，C6～C30(优选C6-C18，更优选C6-C12)的芳基，C3～C18(优选C3-C12，更优选C3-C8)的环烷基，烷硫基衍生物，烷氧基衍生物，羰基衍生物；uu独立地选自以下化学式所代表的结构中的一种或几种：C nH 2n+1-hT h(n为0至10的整数，例如0、1、2、3、4、5、6、7、8、9或10)，C nH 2n-1-hT h(n为2至10的整数，例如2、3、4、5、6、7、8、9或10)，C nH 2n-3-hT h(n为2至10的整数，例如2、3、4、5、6、7、8、9或10)，C nH 2n-7-hT h(n为6至18的整数，例如6、7、8、9、10、11、12、13、14、15、16、17或18)，其中，h为0至3的整数(例如0、1、2或3)，T独立地选自卤族元素(例如，氟、氯、溴或碘)中的任意一种或几种。
在本发明的一个优选实施方案中，T 1、T 2、T 3、T 4、T 5和T 6独立地选自氢， 羟基，巯基，氨基，羧基，C1～C18的烷基及其衍生物，C6～C30的芳基及其衍生物，C3～C8的环烷基及其衍生物，C2～C8的烯及其衍生物，C2～C8的炔及其衍生物，C1～C8的烷氧基及其衍生物，C1～C8的羧酸及其衍生物，C1～C8的胺及其衍生物，C2～C8的氮杂环及其衍生物，C2～C8的氧杂环及其衍生物或C2～C8的硫杂环及其衍生物；且T 1、T 2、T 3、T 4、T 5、T 6中的至少一个选自羟基，巯基，氨基，羧基，C2～C8的烯及其衍生物、C2～C8的炔及其衍生物，C1～C8的烷氧基及其衍生物，C1～C8的羧酸及其衍生物，C1～C8的胺及其衍生物，C2～C8的氮杂环及其衍生物，C2～C8的氧杂环及其衍生物或硫杂环及其衍生物。
上述衍生物优选为C1～C5的烷基取代基，C1～C5的烷氧基取代基，卤素，羟基，巯基，硝基，氰基，C5～C8芳基，C5～C8杂芳基，C3～C5环烷基，羧基，氨基，酰胺基取代基，或者任意一个或多个C原子被O或S取代。
优选地，所述T 1、T 2、T 3、T 4、T 5和T 6独立地选自H，C1～C5烷基，或C1～C5的取代烷基；所述取代烷基优选含羟基取代基、巯基取代基、芳基取代基、杂芳基取代基、羧基取代基、杂环基取代基、酰胺基取代基、氨基取代基、或C原子被O或S取代。
上述芳基取代基、杂芳基取代基、杂环基取代基的碳原子个数优选为5～12个，更优选为5～8个。
上述羧基取代基、酰胺基取代基、氨基取代基的碳原子个数优选为1～8个，更优选为1～5个。
本发明优选地，所述T 1、T 2、T 3、T 4、T 5和T 6的端基独立地选自羧基、羟基、氨基、酰胺基、巯基、胍基或含N、S或O的杂环基。所述含N杂环基优选为咪唑基或苯并吡咯。
更优选地，所述T 1、T 2、T 3、T 4、T 5和T 6独立地选自以下任一结构：
Figure PCTCN2019072812-appb-000010
Figure PCTCN2019072812-appb-000011
上述聚氨基酸中，当T 1、T 2、T 3、T 4、T 5和T 6同时为H时，得到的聚氨基酸为直链结构；当至少一种氨基酸单元中，T 1、T 2、T 3、T 4、T 5和T 6不同时为H，得到的聚氨基酸为支链结构；当至少一种氨基酸单元的官能度≥3时，可以得到支化聚氨基酸。
所述氨基酸单元优选包括赖氨酸、鸟氨酸、精氨酸、谷氨酸、组氨酸、天冬酰胺、谷氨酰胺、丝氨酸、色氨酸、瓜氨酸、天冬氨酸、苏氨酸、酪氨酸、半胱氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸和蛋氨酸中的任意一种或多种。
在本发明的一个优选实施方案中，所述支化聚氨基酸由一种氨基酸单元均聚得到，所述氨基酸单元的官能度≥3。
所述氨基酸单元优选为谷氨酸、赖氨酸、精氨酸、鸟氨酸、组氨酸、天冬氨酸、色氨酸、丝氨酸、瓜氨酸、酪氨酸、半胱氨酸、天冬酰胺、谷氨酰胺或苏氨酸。优选地，所述氨基酸单元为碱性氨基酸；更优选地，所述氨基酸单元为赖氨酸、精氨酸、鸟氨酸或组氨酸。
在本发明的另一个优选实施方案中，所述支化聚氨基酸由两种或两种以上的氨基酸单元共聚得到，共聚单元中至少包含一种或多种官能度≥3的氨基酸；所述官能度≥3的氨基酸单元占氨基酸单元的比例为δ，0<δ≤100％。
在共聚过程中，所述官能度≥3的氨基酸单元为聚氨基酸提供支化结构。
所述共聚氨基酸单元优选为谷氨酸、赖氨酸、鸟氨酸、精氨酸、组氨酸、天冬酰胺、谷氨酰胺、丝氨酸、色氨酸、天冬氨酸、瓜氨酸、苏氨酸、酪氨酸或半胱氨酸；优选地，所述氨基酸单元中至少包括一种碱性氨基酸单元。更优选地，所述氨基酸单元中至少包括赖氨酸、鸟氨酸、精氨酸和组氨酸中的一种或多种。
在本发明的某些具体实施例中，所述支化聚氨基酸由精氨酸和丙氨酸共聚得到，或者由鸟氨酸和亮氨酸共聚得到，或者由赖氨酸和丙氨酸共聚得到，或者 由组氨酸和苯丙氨酸共聚得到，或者由赖氨酸和精氨酸共聚得到。更优选地，共聚单体选自以下组合：精氨酸和丙氨酸，鸟氨酸和亮氨酸，赖氨酸和丙氨酸，组氨酸和苯丙氨酸，鸟氨酸和6-氨基己酸，赖氨酸和精氨酸，苯丙氨酸、丙氨酸和赖氨酸，精氨酸和丝氨酸，精氨酸和谷氨酸，和组氨酸和丝氨酸。
关于共聚得到的支化聚氨基酸中各单体的摩尔比例，要求官能度≥3的氨基酸单元占氨基酸单元的比例(δ)大于0小于等于100％，即，0<δ≤100％。优选地，对于两种氨基酸单元共聚得到共聚氨基酸，两种氨基酸单元的摩尔比率在0.1:10～10:0.1范围内；对于由三种氨基酸单元共聚得到的共聚氨基酸，三种氨基酸单元的摩尔比率在0.1-10:0.1-10:0.1-10范围内。
本发明对所述支化聚氨基酸的制备方法并无特殊限定，可以按照本领域技术人员熟知的方法制备，优选按照以下方法制备：
将氨基酸混合，在惰性气体氛围中，在25～250℃反应1min～96h，得到支化聚氨基酸。
所述惰性气体优选为氮气或氩气。
所述反应温度优选为150～200℃，反应时间优选为30min～24h，更优选为2h～12h。
聚氨基酸的支化结构，使得该物质具有许多活性官能团，可以进一步进行修饰，具有良好的生物相容性，且该抑菌剂长期使用不会产生耐药性。
本发明优选地，对所述聚氨基酸进行以下任意一种或多种改性：
I、氨基或酰胺基中的氨基被改性为以下基团：
Figure PCTCN2019072812-appb-000012
Ⅱ、羟基被改性为-OR 1或-OC(＝O)R 2；
Ⅲ、巯基被改性为-SR 3；
Ⅳ、羧基被改性为-C(＝O)NHR 4或-C(＝O)OR 5；
Ⅴ、胍基被改性为式Ⅴ-1所示基团；
Ⅵ、含氮杂环基团中的NH被改性为NR 6；
Figure PCTCN2019072812-appb-000013
其中，X、Y、Z、Q独立地选自氢，C1～C18的烷基及其衍生物，C6～C30芳基及其衍生物，C3～C18的环烷基及其衍生物，C2～C18的烯及其衍生物，C2～C18的炔及其衍生物，C1～C18的烷氧基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物；
R 1、R 2、R 3、R 4、R 5、R 6独立地选自H、C1～C18的烷基及其衍生物，C6～C30芳基及其衍生物，C3～C18的环烷基及其衍生物，C2～C18的烯及其衍生物，C2～C18的炔及其衍生物，C1～C18的烷氧基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物；且R 1、R 3、R 5、R 6不为H。
上述衍生物优选为C1～C5的烷基取代基，C1～C5的烷氧基取代基，卤素，羟基，巯基，硝基，氰基，C5～C8芳基，C5～C8杂芳基，C3～C5环烷基，羧基，氨基，酰胺基取代基，或者任意一个或多个C原子被O或S取代。
更优选地，所述X、Y、Z、Q独立地选自氢、C1～C3烷基、C6～C8芳基、C3～C6环烷基、C1～C3烷氧基、C2～C5氮杂环、C2～C5氧杂环或C2～C5硫杂环；
R 1、R 2、R 3、R 4、R 5、R 6独立地选自C1～C3烷基、C6～C8芳基、C3～C6环烷基、C1～C3烷氧基、C2～C5氮杂环、C2～C5氧杂环或C2～C5硫杂环。
在本发明的某些具体实施例中，X、Y、Z、Q、R 1、R 2、R 3、R 4、R 5、R 6独立地选自氢、甲基、乙基、丁基、异丙基、乙酰基、甲酰基等。
本发明对上述含氮杂环基团中的N原子个数并无特殊限定，可以为本领域熟知的含氮杂环，N原子个数可以为但不限于1～3个。改性的N原子可以是全部N原子，也可以是1个或2个N原子的改性。
在本发明的某些具体实施例中，所述含氮杂环基团为咪唑基，被改性为式Ⅵ-1所示基团：
Figure PCTCN2019072812-appb-000014
上述X、Y的范围同上，在此不再赘述。
优选地，本发明的聚氨基酸可以是胍基改性的、季铵盐改性的、乙酰基改性的、醚基改性的、甲酯改性的或羟基改性的聚氨基酸(均聚或共聚氨基酸)，更优选选自由以下组成的组：胍基改性的超支化聚鸟氨酸，季铵盐改性的超支化聚鸟氨酸，乙酰基改性的超支化聚赖氨酸，胍基改性的ε-聚赖氨酸，胍基改性的α-聚赖氨酸，醚基改性的聚(精氨酸-丝氨酸)，甲酯改性的聚(精氨酸-谷氨酸)，羟基改性的聚(鸟氨酸-半胱氨酸)，醚基改性的聚(组氨酸-丝氨酸)，胍基改性的聚(鸟氨酸和亮氨酸)，胍基改性的聚(赖氨酸-丙氨酸)和季铵盐改性的聚(赖氨酸-丙氨酸)。
上述改性可以提高聚氨基酸的抑菌性能，降低溶血比率及细胞毒性。
本发明对上述改性的方法并无特殊限定，采用本领域技术人员公知的方法即可。
本发明优选地，所述抑菌剂还可包括辅剂，所述辅剂的含量优选为0～99wt％。
本发明对所述辅剂的种类并无特殊限定，可以为本领域技术人员熟知的抑菌剂适用的辅剂。
所述的抑菌剂辅剂优选包括：[i]金属、金属离子、金属盐及其氧化物等无机抑菌剂；[ii]有机金属类、有机卤代物，胍类，有机硝基化合物类，有机磷及有机砷类，呋喃及其衍生物类，吡咯类，咪唑类，酰基苯胺类，噻唑及其衍生物类，季铵盐类等有机抑菌剂；[iii]天然抑菌肽类及高分子糖类等天然抑菌剂中的一种或几种；[iv]甘油、PEG、高分子糖类、多肽类、塑料、陶瓷、玻璃、磷灰石、树脂、纤维、橡胶等无毒添加剂或载体。
更优选地，所述抑菌剂辅剂为金属Ti，Ag +，Cu 2+，Fe 3+，Zn 2+，季铵盐，卤胺，聚双胍类，卤代苯酚，壳聚糖，鱼精蛋白和天然抑菌肽等中的一种或多种。
在本发明的某些具体实施例中，所述辅剂为聚六亚甲基双胍。
本发明对所述抑菌剂的剂型并无特殊限定，可以为固体，溶液，悬浮液，乳浊液，水凝胶，油凝胶，气溶胶，涂覆或接枝到固体表面，与其他材料共混的 一种或多种状态使用。
本发明对所述抑菌剂的制备方法并无特殊限定，将支化聚氨基酸和辅剂混合即可。
所述混合过程可以使用溶剂也可以不使用溶剂。所述溶剂可以为水或有机溶剂。
所述有机溶剂优选为甲醇、乙醇、乙酸乙酯、正庚烷、二甲基甲酰胺、二甲基乙酰胺、四氢呋喃、氯仿、二氯甲烷、四氯化碳、乙腈、石油醚、正己烷、环己烷、二氧六环、二甲基亚砜、二甲苯、甲苯、苯、氯苯、溴苯、丙酮和离子液体中的一种或多种。
本发明提供的上述抑菌剂制备工艺简单，设备要求不高，易于操作，而且材料易得，成本低廉，具有工业化应用前景，且具有广谱抑菌性。
本发明还提供了上述抑菌剂在抑菌领域的应用，抑菌范围并无特殊限定，可以为本领域技术人员熟知的抑菌范围。
所述抑菌范围优选包括在抑制细菌、病毒、真菌、放线菌、立克次氏体、支原体、衣原体、螺旋体中的一种或多种中的应用。
上述抑菌剂可应用于本领域技术人员熟知的各种应用领域，并无特殊限定。
所述应用领域优选为食品、化妆品、医疗用品、保健品等领域。
比如食品防腐剂，食品保鲜剂，化妆品添加剂，漱口水，消毒液，多功能护理液，滴眼液中的防腐剂，游泳池消毒剂，还可用于牙膏、面部清洗剂、洗手液、消毒皂、果蔬储存的消毒防腐剂等。
与现有技术相比，本发明提供了一种支化聚氨基酸抑菌剂，包括支化聚氨基酸；所述支化聚氨基酸由一种氨基酸单元均聚得到，或者由两种以上的氨基酸单元共聚得到；所述氨基酸单元具有式I所示结构通式。本发明所用的原料为氨基酸，无毒性，无副作用，是一种绿色环保型的新型抑菌剂，使用者易于接受。特别地，聚氨基酸的支化结构，使得该物质具有许多活性官能团，可以进一步进行修饰，具有良好的生物相容性，且该抑菌剂长期使用不会产生耐药性。
附图说明
图1为本发明实施例2制备的支化聚赖氨酸的核磁氢谱；
图2为本发明实施例10制备的支化聚精氨酸的核磁氢谱；
图3为本发明实施例25制备的支化聚氨基酸的核磁氢谱。
具体实施方式
为了进一步说明本发明，下面结合实施例对本发明提供的支化聚氨基酸抑菌剂及应用进行详细描述。除非另外指出，下述实施例中所用到的反应原料均为市售商品，均采购于上海阿拉丁生化科技股份有限公司，Sigma-Aldrich化学试剂有限公司，J&K百灵威科技有限公司，上海麦克林生化科技有限公司或国药集团化学试剂有限公司。
下述实施例中聚合物分子量采用凝胶渗透色谱(GPC)进行测试，具体测试方法为：聚合物分子量(M n)及其分布(PDI＝M w/M n)通过装备有Waters 2414干涉折射检测器的Waters 2414凝胶渗透色谱系统检测获得，0.2M醋酸/0.1M醋酸钠为流动相流速0.6mL/min，温度35℃，标准品为聚乙二醇。
实施例1
将100克精氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应4h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚精氨酸82.7克，为淡黄色固体粉末，GPC表征：M n＝2200g/mol，PDI＝1.91。
实施例2
将91.32克赖氨酸盐酸盐和28.05克KOH加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，250℃下搅拌加热反应1分钟，停止加热，聚合物用甲醇溶解过滤除盐，浓缩后沉降到乙醚中，得到超支化聚赖氨酸84克，产品为淡黄色固体粉末，GPC表征：M n＝1100g/mol，PDI＝1.81。
合成的支化聚氨基酸核磁氢谱如图1所示。
实施例3
将50克丝氨酸和50mL的正己醇，加入500mL的圆底烧瓶中，连接分水装置，氮气氛围下，190℃下搅拌加热反应10h，停止加热，然后将反应体系冷却到室温，聚合物用乙醇溶解沉降到乙醚中，得到超支化聚丝氨酸28.5克，产品为淡黄色固体粉末，GPC表征：M n＝7800g/mol，PDI＝1.71。
实施例4
将91.32克赖氨酸盐酸盐、28.05克KOH及10mg三氧化二锑加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应96h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解过滤除盐，浓缩后沉降到乙醚中，得到超支化聚赖氨酸55.5克，产品为淡黄色固体粉末，GPC表征：M n＝500000g/mol，PDI＝2.36。
实施例5
将80克赖氨酸、20克赖氨酸盐酸盐和6.14g KOH加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，100℃下搅拌加热反应10h，停止加热，然后将反应体系冷却到室温，聚合物用乙醇溶解过滤除盐，浓缩后沉降到乙醚中，得到超支化聚赖氨酸68.5克，产品为褐色固体粉末，GPC表征：M n＝800g/mol，PDI＝1.66。
实施例6
将50克半胱氨酸和100mL的DMF，加入500mL的圆底烧瓶中，连接分水装置，氮气氛围下，180℃下搅拌加热反应10h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚半胱氨酸33.5克。产品为黄色固体粉末，GPC表征：M n＝1900g/mol，PDI＝2.07。
实施例7
将50克谷氨酸和100mL的乙二醇，加入500mL的圆底烧瓶中，连接分水装置，氮气氛围下，200℃下搅拌加热反应1分钟，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚谷氨酸31.5克，产品为浅黄色固体粉末，GPC表征：M n＝2100g/mol，PDI＝1.86。
实施例8
将50克精氨酸和100mL的乙二醇，加入500mL的圆底烧瓶中，连接分水装置，氮气氛围下，150℃下搅拌加热反应96h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙酸乙酯中，得到超支化聚精氨酸34.5克，为淡黄色固体粉末，GPC表征：M n＝1800g/mol，PDI＝2.18。
实施例9
将100克赖氨酸和0.1克磷酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，100℃下搅拌加热反应 96h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚赖氨酸78.5克，为黄褐色固体粉末，GPC表征：M n＝6800g/mol，PDI＝1.97。
实施例10
将100克精氨酸和200克乙二醇加入500mL的圆底烧瓶中，连接分水装置，N 2鼓泡30min，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，170℃下搅拌加热反应8h，停止加热，然后将反应体系冷却到室温，分离乙二醇，聚合物沉降到乙醚中，得到超支化聚精氨酸71.2克，产品为淡黄色固体粉末，GPC表征：M n＝3100g/mol，PDI＝1.78。
合成的支化聚氨基酸核磁氢谱如图2所示。
实施例11
将100克组氨酸和200克乙二醇加入500mL的圆底烧瓶中，连接分水装置，N 2鼓泡30min，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应24h，停止加热，然后将反应体系冷却到室温，分离乙二醇，聚合物用乙醚洗涤5次，得到超支化聚组氨酸71.2克，产品为黄色固体粉末，GPC表征：M n＝1500g/mol，PDI＝1.71。
实施例12
将100克鸟氨酸和50g水加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，150℃下搅拌加热反应5小时，停止加热，将聚合物粉碎，得到超支化聚赖氨酸87克，产品为褐色固体粉末，GPC表征：M n＝3400g/mol，PDI＝1.77。
实施例13
将2g实施例10中的超支化聚精氨酸与0.2g硝酸银溶解在5mL的水中，搅拌分散均匀，然后冻干，得到2.18g超支化聚精氨酸与银离子的混合物。
实施例14
将100克赖氨酸和50g水加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应5小时，停止加热，将聚合物粉碎，得到超支化聚赖氨酸87克，产品为褐色固体粉末，GPC表征：M n＝2400g/mol，PDI＝1.77。
实施例15
将2g实施例14中的超支化聚赖氨酸与0.2g壳聚糖溶解在5mL的水中，搅拌分散均匀，然后冻干，得到2.18g超支化聚赖氨酸与壳聚糖的混合物。
实施例16
将100克瓜氨酸和50g水加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，190℃下搅拌加热反应5小时，停止加热，将聚合物用水溶解并用二次水进行透析，得到超支化聚瓜氨酸57克，产品为淡黄色固体粉末，GPC表征：M n＝5700g/mol，PDI＝1.27。
实施例17
将100克(2S,3R,4S)-α-(羧基环丙基)甘氨酸和50g水加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应5小时，停止加热，将聚合物粉碎，得到超支化聚氨基酸86.4克，产品为褐色固体粉末，GPC表征：M n＝7500g/mol，PDI＝1.87。
实施例18
将100克5-氨基-2-肼基戊酸(CAS:60733-16-6)和50g水加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应5小时，停止加热，将聚合物粉碎，得到超支化聚氨基酸83.4克，产品为褐色固体粉末，GPC表征：M n＝8400g/mol，PDI＝1.94。
实施例19
将100克4-氨基-3-羟基丁酸和50g水加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应5小时，停止加热，将聚合物用凝胶柱进行分类，得到超支化聚氨基酸78.4克，产品为褐色固体粉末，GPC表征：M n＝7300g/mol，PDI＝1.48。
实施例20
分别取实施例1-19制备的超支化聚氨基酸36mg，用3mL无菌PBS溶解，得到12mg/mL的母液，按照以下方法测试超支化聚氨基酸基抗菌剂的抑菌活力，部分实验结果参见表1。
以下实施例中所用到的各种菌株均购自中国生物制品检定所。
采用96孔板法对超支化聚氨基酸基抗菌剂进行抗菌活力检测，并用发酵法合成的ε-聚赖氨酸作为对照以评价所得的超支化聚氨基酸基抗菌剂的抗菌能力，最小抑菌浓度(MIC)定义为与对照组相比，抑制90％的细菌增长的最低聚合 物浓度。
用接种环从琼脂斜面培养基上挑取少量菌种于普通MH培养基中，37℃培养过夜使菌种复苏并达到指数生长，稀释菌液使菌液浓度为10 6CFU/mL，每孔中加入175μL菌液及25μL不同浓度的聚合物溶液，将96孔板置于37℃培养20h，用酶标仪检测OD 600值。
表1 不同抑菌聚合物对不同细菌的MIC值的比较
Figure PCTCN2019072812-appb-000015
实施例21
分别取实施例1-19制备的超支化聚氨基酸36mg，用3mL无菌PBS溶解， 得到12mg/mL的母液，按照以下方法测试超支化聚氨基酸基抗菌剂的体外溶血活性，部分实验结果参见表2。
采用96孔板法对超支化聚氨基酸基抗菌剂进行体外溶血活性检测，并用发酵法合成的ε-聚赖氨酸作为对照以评价所得的超支化聚氨基酸基抗菌剂的体外溶血活性。
2％(v/v)红细胞悬液的制备：取新鲜健康人血2mL，用10mL无内毒素的PBS缓冲溶液稀释，放入盛有玻璃珠的三角烧瓶中振摇10分钟，或用玻璃棒搅动血液，除去纤维蛋白质，使成脱纤血液，20℃条件下1000～1500r/min离心10～15分钟，除去上清液，沉淀的红细胞再用PBS缓冲溶液按上述方法洗涤4次，至上清液不显红色为止。将所得红细胞用生理盐水配成2％的混悬液，供后续试验使用。
聚合物用PBS缓冲溶液稀释配成不同浓度的溶液，加入到96孔板中，单独的PBS缓冲溶液作为阴性对照，在水中溶解0.2％的Triton-X-100作为体外溶血活性检测的阳性对照，将清洗过的红细胞(2％v/v,50μL)加入到96孔板中，充分混合并培养。用酶标仪检测540nm下的吸收。
表2 不同抑菌聚合物的体外溶血活性检测结果
Figure PCTCN2019072812-appb-000016
实施例22
本实施例用于检测超支化聚氨基酸基抗菌剂的动物体内急性毒性，并用发酵 法合成的ε-聚赖氨酸作为对照以评价所得的超支化聚氨基酸基抗菌剂的动物体内急性毒性。
取小白鼠(Balb/C小白鼠，购买自吉林大学)100只，雌雄各半，体重21±3g，分别取实施例1-19制备的超支化聚氨基酸基抗菌剂按1mg/mL的剂量，每日一次连续15天给小鼠进行肌肉注射，观察小鼠的毒性反应。实验结果表明，连续21天肌肉注射后，除个别小鼠活力下降以外，其余均无明显的异常反应，且所有小鼠均存活，证明所得到的超支化聚氨基酸基抗菌剂具有较小的体内毒性。
实施例23
将80克精氨酸和20克丙氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，160℃下搅拌加热反应5h，停止加热，然后将反应体系冷却到室温，聚合物用乙醇溶解沉降到乙醚中，得到超支化聚氨基酸78.7克,产品为浅黄色固体粉末，GPC表征：M n＝3100g/mol，PDI＝1.76。
实施例24
将50克鸟氨酸、50克亮氨酸及10mg三氟甲基磺酸钪加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应2h，停止加热，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚氨基酸81.5克，产品为深黄色固体粉末，GPC表征：M n＝500g/mol，PDI＝1.82。
实施例25
将91.32克赖氨酸盐酸盐、28.05克KOH和20克丙氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应10h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到支化聚氨基酸75.2克。产品为淡黄色固体粉末，GPC表征：M n＝14100g/mol，PDI＝2.72。
合成的支化聚氨基酸核磁氢谱如图3所示。
实施例26
将90克组氨酸、10克苯丙氨酸及10mg三氯化铁加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应2h，停止加热，然后将反应体系冷却到室温，聚合物用水溶解 沉降到四氢呋喃中，得到超支化聚氨基酸79.5克。产品为淡黄色固体粉末，GPC表征：M n＝1100g/mol，PDI＝1.62。
实施例27
先将80克鸟氨酸加入到500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，190℃下搅拌加热反应4h，然后向反应体系中加入20克6-氨基己酸，继续反应4h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到核壳结构的超支化聚氨基酸82.3克，产品为淡黄色固体粉末，GPC表征：M n＝11100g/mol，PDI＝1.94。
实施例28
将91.32克赖氨酸盐酸盐、20克NaOH和20克丙氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应36h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚氨基酸74.8克，产品为淡黄色固体粉末，GPC表征：M n＝81100g/mol，PDI＝3.98。
实施例29
将91.32克赖氨酸盐酸盐、20克NaOH和20克丙氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，100℃下搅拌加热反应96h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚氨基酸72.8克，产品为深黄色固体粉末，GPC表征：M n＝481100g/mol，PDI＝2.21。
实施例30
先将91.32克赖氨酸盐酸盐、20克NaOH加入到500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，250℃下搅拌加热反应0.5h，然后向反应体系中加入20克丙氨酸，继续反应0.5h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到核壳结构的超支化聚氨基酸73.1克，产品为淡黄色固体粉末，GPC表征：M n＝1200g/mol，PDI＝1.51。
实施例31
将50克赖氨酸和50克精氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应 4h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙酸乙酯中，得到支化聚氨基酸80.1克，产品为淡黄色固体粉末，GPC表征：M n＝3200g/mol，PDI＝1.73。
实施例32
将80克(2S,3R,4S)-α-(羧基环丙基)甘氨酸和20克丙氨酸加入500mL的圆底烧瓶中，加入80g二次水溶解，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应5小时，停止加热，将聚合物粉碎，得到超支化聚氨基酸86.4克，产品为褐色固体粉末，GPC表征：M n＝5000g/mol，PDI＝1.88。
实施例33
将80克5-氨基-2-肼基戊酸(CAS:60733-16-6)和20克丙氨酸加入500mL的圆底烧瓶中，加入80g二次水溶解，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，160℃下搅拌加热反应10小时，停止加热，将聚合物粉碎，得到超支化聚氨基酸84.5克，产品为褐色固体粉末，GPC表征：M n＝8700g/mol，PDI＝1.92。
实施例34
先将20克苯丙氨酸、10克丙氨酸加入到500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，100℃下搅拌加热反应30h，然后向反应体系中加入70克赖氨酸盐酸盐和20g KOH，继续反应70h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到核壳结构的超支化聚氨基酸68.5克，产品为淡黄色固体粉末，GPC表征：M n＝6200g/mol，PDI＝1.88。
实施例35
将80克4-氨基-3-羟基丁酸和20克甘氨酸加入500mL的圆底烧瓶中，加入80g二次水溶解，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，170℃下搅拌加热反应12小时，停止加热，将聚合物溶解于水中，沉降到四氢呋喃中，得到超支化聚氨基酸84.5克，产品为褐色固体粉末，GPC表征：M n＝13700g/mol，PDI＝1.72。
实施例36
将2g实施例28制备的超支化聚氨基酸与2g聚六亚甲基双胍溶解在5mL 的水中，搅拌分散均匀，然后冻干，得到4g超支化聚氨基酸与聚六亚甲基双胍的混合物。
实施例37
将2g实施例24制备的超支化聚氨基酸与2g聚六亚甲基双胍溶解在5mL的水中，搅拌分散均匀，然后冻干，得到4g超支化聚氨基酸与聚六亚甲基双胍的混合物。
实施例38
分别取实施例23-37制备的超支化聚氨基酸36mg，用3mL无菌PBS溶解，得到12mg/mL的母液，按照以下方法测试超支化聚氨基酸基抗菌剂的抗菌活力，实验结果参见表3-1和表3-2。
以下实施例中所用到的各种菌株均购自中国生物制品检定所。
采用96孔板法对超支化聚氨基酸基抗菌剂进行抗菌活力检测，并用发酵法合成的ε-聚赖氨酸作为对照以评价所得的超支化聚氨基酸基抗菌剂的抗菌能力，最小抑菌浓度(MIC)定义为与对照组相比，抑制90％的细菌增长的最低聚合物浓度。
用接种环从琼脂斜面培养基上挑取少量菌种于普通M-H培养基中，37℃培养过夜使菌种复苏并达到指数生长，稀释菌液使菌液浓度为10 6CFU/mL，每孔中加入175μL菌液及25μL不同浓度的聚合物溶液，将96孔板置于37℃培养20h，用酶标仪检测OD 600值。
表3-1 不同抗菌聚合物对不同细菌的MIC值的比较
Figure PCTCN2019072812-appb-000017
Figure PCTCN2019072812-appb-000018
表3-2 不同抗菌聚合物对不同细菌的MIC值的比较
Figure PCTCN2019072812-appb-000019
实施例39
分别取实施例23-37制备的超支化聚氨基酸36mg，用3mL无菌PBS溶解，得到12mg/mL的母液，按照以下方法测试超支化聚氨基酸基抗菌剂的抗体外溶血活性，实验结果参见表4-1和表4-2。
采用96孔板法对超支化聚氨基酸基抗菌剂进行体外溶血活性检测，并用发酵法合成的ε-聚赖氨酸作为对照以评价所得的超支化聚氨基酸基抗菌剂的体外溶血活性。
2％(v/v)红细胞悬液的制备：取新鲜健康人血2mL，用10mL无内毒素的PBS缓冲溶液稀释，放入盛有玻璃珠的三角烧瓶中振摇10分钟，或用玻璃棒搅动血液，除去纤维蛋白质，使成脱纤血液，20℃条件下1000～1500r/min离心10～15分钟，除去上清液，沉淀的红细胞再用PBS缓冲溶液按上述方法洗涤4次，至上清液不显红色为止。将所得红细胞用生理盐水配成2％的混悬液，供后续试验使用。
聚合物用PBS缓冲溶液稀释配成不同浓度的溶液，加入到96孔板中，单独的PBS缓冲溶液作为阴性对照，在水中溶解0.2％的Triton-X-100作为体外溶血活性检测的阳性对照，将清洗过的红细胞(2％v/v,50μL)加入到96孔板中，充分混合并培养。用酶标仪检测540nm下的吸收。
表4-1 不同抗菌聚合物的体外溶血活性检测结果
Figure PCTCN2019072812-appb-000020
Figure PCTCN2019072812-appb-000021
表4-2 不同抗菌聚合物的体外溶血活性检测结果
Figure PCTCN2019072812-appb-000022
实施例40
本实施例用于检测超支化聚氨基酸基抗菌剂的动物体内急性毒性，并用发酵法合成的ε-聚赖氨酸作为对照以评价所得的超支化聚氨基酸基抗菌剂的动物体内急性毒性。
取小白鼠(Balb/C小白鼠，购买自吉林大学)100只，雌雄各半，体重21±3g，分别取实施例23-37制备的超支化聚氨基酸基抗菌剂按1mg/mL的剂量，每日一次连续15天给小鼠进行肌肉注射，观察小鼠的毒性反应。实验结果表明，连续15天肌肉注射后，除个别小鼠活力下降以外，其余均无明显的异常反应，且所有小鼠均存活，证明所得到的超支化聚氨基酸基抗菌剂具有较小的体内毒性。
实施例41
将100克鸟氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应4h，停止加热， 然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚鸟氨酸82.7克。
实施例42
将2g实施例41中所得的超支化聚鸟氨酸氨酸加热溶解在20mL的N，N-二甲基甲酰胺(DMF)中，加入5g碘甲烷，80℃搅拌反应24h，然后停止加热，冷却至室温，沉降到乙酸乙酯中，得到季铵盐改性的超支化聚鸟氨酸2.4g。
实施例43
将91.32克赖氨酸盐酸盐、20克NaOH加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应6h，停止加热，聚合物用甲醇溶解沉降到乙醚中，得到超支化聚赖氨酸72.8克。
实施例44
将2g实施例43中所得的超支化聚赖氨酸溶解在5mL的甲醇中，0℃条件下缓慢滴加乙酰氯，升温至室温继续反应12h，然后沉降到乙酸乙酯中，得到乙酰基改性的超支化聚赖氨酸2.3g。
实施例45
将2g实施例41中所得的超支化聚鸟氨酸溶解在5mL的甲醇中，加入4.8g甲基异硫脲半硫酸盐及5mL三乙胺，60℃反应12h，然后停止加热，冷却至室温，沉降到乙酸乙酯中，得到胍基改性的超支化聚鸟氨酸2.1g。
实施例46
将2gε-聚赖氨酸溶解在5mL的水中，加入5.1g 1H-吡唑-1-甲脒盐酸盐及5mL三乙胺，60℃反应12h，然后停止加热，冷却至室温，沉降到乙酸乙酯中，得到胍基改性的ε-聚赖氨酸2.2g。
实施例47
称取5.6g N-苄氧羰基赖氨酸与250mL三口烧瓶中，加入100mL四氢呋喃，搅拌分散。小心称取2.5g三光气溶于30mL四氢呋喃中，在氮气的保护下缓慢滴入到反应体系中，搅拌下回流3h直至溶液完全澄清。反应结束后，加入大量的正己烷沉淀得到粗产品，用四氢呋喃-正己烷体系重结晶2次，真空干燥得到产物4.96g，产率88.6％。以DMF为溶剂，正丁胺为引发剂引发赖氨酸NCA开环，得到α-聚赖氨酸。
实施例48
将实施例47中所得的2gα-聚赖氨酸溶解在5mL的水中，加入5.1g 1H-吡唑-1-甲脒盐酸盐及5mL三乙胺，60℃反应12h，然后停止加热，冷却至室温，沉降到乙酸乙酯中，得到胍基改性的α-聚赖氨酸2.3g。
实施例49
将80克精氨酸和20克丝氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应4h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到支化聚氨基酸81.2克。
实施例50
将2g实施例49中所得到的聚氨基酸溶解到20mL甲醇中，加入0.5g对甲苯磺酸，加热回流10h，将聚合物沉降到乙醚中，得到醚基改性的聚氨基酸2.2g
实施例51
将80克精氨酸和20克谷氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应4h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到支化聚氨基酸80.2克。
实施例52
将2g实施例51中所得到的聚氨基酸溶解到20mL无水甲醇中，加入0.9g1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)和0.1g 4-二甲氨基吡啶(DMAP)，室温搅拌10h，将聚合物沉降到乙醚中，得到甲酯改性的聚氨基酸2.1g
实施例53
将80克鸟氨酸和20克半胱氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应4h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到支化聚氨基酸80.2克。
实施例54
将2g实施例53中所得到的聚氨基酸溶解到20mL无水DMF中，通氩气除氧30min，加入0.9g丙炔醇和0.1g DMAP，室温搅拌10min。将反应混合物 在紫外光(365nm)照射条件下室温反应120min，然后将聚合物沉降到乙醚中，得到羟基改性的聚氨基酸2.1g。
实施例55
将80克组氨酸和20g丝氨酸加入500mL的圆底烧瓶中，连接分水装置，抽换氮气三次，每次大于10分钟，最后保持氮气氛围，180℃下搅拌加热反应4h，停止加热，然后将反应体系冷却到室温，聚合物用甲醇溶解沉降到乙醚中，得到支化聚氨基酸80.2克。
实施例56
将2g实施例55中所得到的聚氨基酸溶解到20mL甲醇中，加入0.5g对甲苯磺酸，加热回流10h，将聚合物沉降到乙醚中，得到醚基改性的聚氨基酸2.2g。
实施例57
分别取实施例41-56中制备的聚氨基酸36mg，用3mL无菌PBS溶解，得到12mg/mL的母液，按照以下方法测试聚氨基酸基抗菌剂的抗菌活力，部分实验结果参见表5-1和表5-2。
以下实施例中所用到的各种菌株均购自中国生物制品检定所。
采用96孔板法对聚氨基酸基抗菌剂进行抗菌活力检测，并用发酵法合成的ε-聚赖氨酸(M n＝4000g/mol)作为对照以评价所得的聚氨基酸基抗菌剂的抗菌能力，最小抑菌浓度(MIC)定义为与对照组相比，抑制90％的细菌增长的最低聚合物浓度。
用接种环从琼脂斜面培养基上挑取少量菌种于普通M-H培养基中，37℃培养过夜使菌种复苏并达到指数生长，稀释菌液使菌液浓度为10 6CFU/mL，每孔中加入175μL菌液及25μL不同浓度的聚合物溶液，将96孔板置于37℃培养20h，用酶标仪检测OD 600值。
表5-1 不同抗菌聚合物对不同细菌的MIC值的比较
Figure PCTCN2019072812-appb-000023
Figure PCTCN2019072812-appb-000024
表5-2 不同抗菌聚合物对不同细菌的MIC值的比较
Figure PCTCN2019072812-appb-000025
Figure PCTCN2019072812-appb-000026
实施例58
分别取实施例41-56中制备的聚氨基酸36mg，用3mL无菌PBS溶解，得到12mg/mL的母液，按照以下方法测试聚氨基酸基抗菌剂的抗体外溶血活性，部分实验结果参见表6-1和表6-2。
采用96孔板法对聚氨基酸基抗菌剂进行体外溶血活性检测，并用发酵法合成的ε-聚赖氨酸作为对照以评价所得的聚氨基酸基抗菌剂的体外溶血活性。
2％(v/v)红细胞悬液的制备：取新鲜健康人血2mL，用10mL无内毒素的PBS缓冲溶液稀释，放入盛有玻璃珠的三角烧瓶中振摇10分钟，或用玻璃棒搅动血液，除去纤维蛋白质，使成脱纤血液，20℃条件下1000～1500r/min离心10～15分钟，除去上清液，沉淀的红细胞再用PBS缓冲溶液按上述方法洗涤4次，至上清液不显红色为止。将所得红细胞用生理盐水配成2％的混悬液，供后续试验使用。
聚合物用PBS缓冲溶液稀释配成不同浓度的溶液，加入到96孔板中，单独的PBS缓冲溶液作为阴性对照，在水中溶解0.2％的Triton-X-100作为体外溶血活性检测的阳性对照，将清洗过的红细胞(2％v/v,50μL)加入到96孔板中，充分混合并培养。用酶标仪检测540nm下的吸收。
表6-1 不同抗菌聚合物的体外溶血活性检测结果
Figure PCTCN2019072812-appb-000027
Figure PCTCN2019072812-appb-000028
表6-2 不同抗菌聚合物的体外溶血活性检测结果
Figure PCTCN2019072812-appb-000029
实施例59
本实施例用于检测聚氨基酸基抗菌剂的动物体内急性毒性，并用发酵法合成的ε-聚赖氨酸作为对照以评价所得的聚氨基酸基抗菌剂的动物体内急性毒性。
取小白鼠(Balb/C小白鼠，购买自吉林大学)100只，雌雄各半，体重21±3g，分别取实施例41-56制备的聚氨基酸基抗菌剂按1mg/mL的剂量，每日一次连续15天给小鼠进行肌肉注射，观察小鼠的毒性反应。实验结果表明，连续15天肌肉注射后，除个别小鼠活力下降以外，其余均无明显的异常反应，且所有小 鼠均存活，证明所得到的聚氨基酸基抗菌剂具有较小的体内毒性。
实施例60
在装备有氮气口、搅拌器和温度计的三口瓶中加入50mL苯乙烯、50mL氯甲基苯乙烯和偶氮二异丁腈(苯乙烯和氯甲基苯乙烯均经过一段中性氧化铝柱子除去阻聚剂)，搅拌并通入N 2鼓泡30min，保持氮气氛围，70℃反应24h，反应结束后将体系沉降到乙醇中得到氯甲基化聚苯乙烯。
在反应瓶中加入5g氯甲基化聚苯乙烯、0.5g实施例14中制备的超支化聚赖氨酸和30mL蒸馏水，N 2鼓泡30min后封瓶，氮气氛围下120℃反应10h。过滤并用大量蒸馏水冲洗固体样品，得到超支化聚赖氨酸修饰的聚苯乙烯(HBPL-PS)，将HBPL-PS高温下压成2cm×1.5cm的样品膜用于抗菌测试。
我们采用大肠杆菌和金黄色葡萄球菌对材料的抗细菌粘附性能进行研究。首先，大肠杆菌(ATCC8739)与金黄色葡萄球菌(ATCC25923)分别在LB和TSB培养基中37℃培养24h。然后，将含细菌的培养基在2700rmp离心10min，去除上层液，重新悬浮至浓度为1×10 8个细菌/mL。样品膜转移至48孔板中，分别加入1mL细菌悬浮液粘附1h后吸出培养液，之后大肠杆菌采用0.5％(wt/vol)葡萄糖培养基、金黄色葡萄球菌采用TSB培养基继续在37℃静态培养4h。将粘附有细菌的样品膜置于1mL新鲜的无菌PBS溶液中并超声清洗1min以使细菌在表面脱粘附。含细菌的PBS溶液按一定倍数稀释，并涂于固体培养基上。37℃过夜培养后，对固体培养基表面形成的细菌菌落进行计数统计。与对照组聚苯乙烯(PS)相比，大肠杆菌和金黄色葡萄球菌在HBPL-PS表面细菌粘附量分别降低了～95.5％和～98.8％。

Claims (15)

  1. 一种支化聚氨基酸抑菌剂，包括支化聚氨基酸；
    所述支化聚氨基酸由一种氨基酸单元均聚得到，或者由两种或两种以上的氨基酸单元共聚得到；
    所述氨基酸单元具有式I所示结构通式或其盐：
    Figure PCTCN2019072812-appb-100001
    其中，
    a、b、c、d、e和f独立地为0～6的整数，且1≤a+b+c+d+e+f≤20，优选地，a+b+c+d+e+f≤10；
    T 1、T 2、T 3、T 4、T 5和T 6独立地选自氢，羟基，巯基，氨基，羧基，C1～C18的烷基及其衍生物，C6～C30的芳基及其衍生物，C3～C8的环烷基及其衍生物，C2～C8的烯及其衍生物，C2～C8的炔及其衍生物，C1～C8的烷氧基及其衍生物，C1～C8的烷硫基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物。
  2. 根据权利要求1所述的抑菌剂，其中所述支化聚氨基酸由一种氨基酸单元均聚得到，所述氨基酸单元的T 1、T 2、T 3、T 4、T 5、T 6中至少一个选自羟基，氨基，巯基，羧基，C2～C8的烯及其衍生物，C2～C8的炔及其衍生物，C1～C8的烷氧基及其衍生物，C1～C8的烷硫基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物。
  3. 根据权利要求1所述的抑菌剂，其中所述支化聚氨基酸由两种或两种以上的氨基酸单元共聚得到，至少一个所述氨基酸单元的T 1、T 2、T 3、T 4、T 5、T 6中至少一个选自羟基，氨基，巯基，羧基，C2～C8的烯及其衍生物，C2～C8的炔及其衍生物，C1～C8的烷氧基及其衍生物，C1～C8的烷硫基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物。
  4. 根据权利要求1-3中任一项所述的抑菌剂，其中所述T 1、T 2、T 3、T 4、T 5和T 6独立地选自以下任一结构：
    Figure PCTCN2019072812-appb-100002
    Figure PCTCN2019072812-appb-100003
    Figure PCTCN2019072812-appb-100004
    及其盐，
    Figure PCTCN2019072812-appb-100005
    及其盐，
    Figure PCTCN2019072812-appb-100006
    Figure PCTCN2019072812-appb-100007
    Figure PCTCN2019072812-appb-100008
    其中，g为0至10的整数；其中，xx、yy、zz独立地选自氢，C1～C18的烷基，C6～C30的芳基，C3～C18的环烷基，羰基衍生物；hh独立地选自氢，羟基，氨基，卤族元素，C1～C18的烷基，C6～C30的芳基，C3～C18的环烷基，胺及其衍生物，烷氧基衍生物，烷巯基衍生物；ii，jj，kk独立地选自氢，C1～C18的烷基，C6～C30的芳基，C3～C18的环烷基，烷氧基及其衍生物；oo，pp，qq独立地选自氢，羧基，羟基，氨基，C1～C18的烷基，C6～C30的芳基，C3～C18的环烷基，卤族元素，胺及其衍生物，烷氧基衍生物，羰基衍生物；rr，tt独立地选自氢，C1～C18的烷基，C6～C30的芳基，C3～C18的环烷基，烷硫基衍生物，烷氧基衍生物，羰基衍生物；uu独立地选自以下化学式所代表的结构中的一种或几种：C nH 2n+1-hT h(n为0至10的整数)，C nH 2n-1-hT h(n为2至10的整数)，C nH 2n-3-hT h(n为2至10的整数)，C nH 2n-7-hT h(n为6至18的整数)，其中，h为0至3的整数，T独立地选自卤族元素中的任意一种或几种。
  5. 根据权利要求1-4中任一项所述的抑菌剂，其中所述T 1、T 2、T 3、T 4、T 5和T 6独立地选自以下任一结构：
    Figure PCTCN2019072812-appb-100009
  6. 根据权利要求1、2、4和5中任一项所述的抑菌剂，其中所述支化聚氨基酸由一种氨基酸单元均聚得到，所述氨基酸的官能度≥3。
  7. 根据权利要求6所述的抑菌剂，其中所述氨基酸单元为谷氨酸、赖氨酸、精氨酸、鸟氨酸、组氨酸、天冬氨酸、色氨酸、丝氨酸、瓜氨酸、酪氨酸、半胱氨酸、天冬酰胺、谷氨酰胺或苏氨酸，优选地，所述氨基酸单元为赖氨酸、精氨酸、鸟氨酸或组氨酸。
  8. 根据权利要求1、3、4和5中任一项所述的抑菌剂，其中所述支化聚氨基酸的共聚单元中至少包含一种或多种官能度≥3的氨基酸，其中官能度≥3的氨基酸单元占总氨基酸单元的比例为δ，0<δ≤100％。
  9. 根据权利要求8所述的抑菌剂，所述官能度≥3的氨基酸单元为谷氨酸、赖氨酸、鸟氨酸、精氨酸、组氨酸、天冬酰胺、谷氨酰胺、丝氨酸、色氨酸、天冬氨酸、瓜氨酸、苏氨酸、酪氨酸或半胱氨酸中的一种或多种；优选地，所述氨基酸单元至少包括赖氨酸、鸟氨酸、精氨酸和组氨酸中的一种或多种。
  10. 根据权利要求1所述的抑菌剂，其中所述聚氨基酸进行以下任意一种或多种改性：
    I、氨基或酰胺基中的氨基被改性为以下基团：
    Figure PCTCN2019072812-appb-100010
    Ⅱ、羟基被改性为-OR 1或-OC(＝O)R 2；
    Ⅲ、巯基被改性为-SR 3；
    Ⅳ、羧基被改性为-C(＝O)NHR 4或-C(＝O)OR 5；
    Ⅴ、胍基被改性为式Ⅴ-1所示基团；
    Ⅵ、含氮杂环基团中的NH被改性为NR 6；
    Figure PCTCN2019072812-appb-100011
    其中，X、Y、Z、Q独立地选自氢，C1～C18的烷基及其衍生物，C6～C30芳基及其衍生物，C3～C18的环烷基及其衍生物，C2～C18的烯及其衍生物，C2～C18的炔及其衍生物、C1～C18的烷氧基及其衍生物，羧酸及其衍生物，胺及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物；
    R 1、R 2、R 3、R 4、R 5、R 6独立地选自H、C1～C18的烷基及其衍生物，C6～C30的芳基及其衍生物，C3～C18的环烷基及其衍生物，C2～C18的烯及其衍生物C2～C18的炔及其衍生物，C1～C18的烷氧基及其衍生物，羧酸及其衍生物，氨基及其衍生物，氮杂环及其衍生物，氧杂环及其衍生物或硫杂环及其衍生物；且R 1、R 3、R 5、R 6不为H。
  11. 根据权利要求10所述的抑菌剂，其中所述X、Y、Z、Q独立地选自氢、C1～C3烷基、C6～C8芳基、C3～C6环烷基、C1～C3烷氧基、C2～C5氮杂环、C2～C5氧杂环或C2～C5硫杂环；
    R 1、R 2、R 3独立地选自C1～C3烷基、C6～C8芳基、C3～C6环烷基、C1～C3烷氧基、C2～C5氮杂环、C2～C5氧杂环或C2～C5硫杂环。
  12. 根据权利要求1-11中任一项所述的抑菌剂，其中所述支化聚氨基酸的 数均分子量范围为500g/mol-500,000g/mol。
  13. 根据权利要求1所述的抑菌剂，其还可包括辅剂。
  14. 根据权利要求1所述的抑菌剂，其中所述抑菌剂为固体，溶液，悬浮液，乳浊液，水凝胶，油凝胶，或气溶胶，涂覆或接枝到固体表面，与其他材料共混的一种或多种状态使用。
  15. 权利要求1-14任一项所述的抑菌剂在细菌、病毒、真菌、放线菌、立克次氏体、支原体、衣原体、螺旋体中的一种或多种中的应用。
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