WO2013039190A1 - Peptide-containing polymer, and method for peptide immobilization on fiber - Google Patents

Peptide-containing polymer, and method for peptide immobilization on fiber Download PDF

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Publication number
WO2013039190A1
WO2013039190A1 PCT/JP2012/073587 JP2012073587W WO2013039190A1 WO 2013039190 A1 WO2013039190 A1 WO 2013039190A1 JP 2012073587 W JP2012073587 W JP 2012073587W WO 2013039190 A1 WO2013039190 A1 WO 2013039190A1
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WIPO (PCT)
Prior art keywords
arg
peptide
leu
group
integer
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PCT/JP2012/073587
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French (fr)
Japanese (ja)
Inventor
允 中村
誠司 解野
鈴木　秀明
富郎 永野
悠介 河野
純 石橋
Original Assignee
和歌山県
Jitsubo株式会社
独立行政法人農業生物資源研究所
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Application filed by 和歌山県, Jitsubo株式会社, 独立行政法人農業生物資源研究所 filed Critical 和歌山県
Priority to JP2013533728A priority Critical patent/JP6074686B2/en
Priority to JP2013048724A priority patent/JP6090569B2/en
Publication of WO2013039190A1 publication Critical patent/WO2013039190A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
    • D01F6/02—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolymers obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • D01F6/16—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolymers obtained by reactions only involving carbon-to-carbon unsaturated bonds from polymers of unsaturated carboxylic acids or unsaturated organic esters, e.g. polyacrylic esters, polyvinyl acetate
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
    • D01F6/58—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products
    • D01F6/68—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products from polyaminoacids or polypeptides
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
    • D06M15/15—Proteins or derivatives thereof
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic

Definitions

  • the present invention relates to a novel peptide-containing polymer.
  • the present invention also relates to a novel method for immobilizing peptides on fibers.
  • Non-patent Document 1 a modified peptide derived from beetle defensin
  • a molecular chain called a spacer was bound to cotton, and amino acid synthesis was carried out starting from the terminal amino group, and a 9-residue modified peptide was synthesized on cotton.
  • MRSA showed strong antibacterial activity, and it was revealed that the activity was maintained even after repeated use
  • Patent Document 1 Non-Patent Document 2
  • this method involves complicated processing methods such as fiber pretreatment, and is difficult to implement with existing fiber processing techniques.
  • the condensation reaction of amino acids and the deprotection reaction of protecting groups are all carried out on cotton, the mechanical strength of the fiber itself is also lowered, so that it was necessary to develop a new processing method for practical use.
  • an antibacterial multiple antigen peptide in which an antibacterial peptide is linked to the N terminal of a branched peptide in which 15 Lys are linked which is a positively charged antibacterial peptide in an aqueous solution
  • Patent Document 2 has been reported.
  • Patent Document 2 There is a possibility that a part of the peptide fixed with the resin-based fixing agent may lose its activity when it is fixed, and it is necessary to fix more antibacterial peptides than necessary.
  • peptides that can be fixed with a resin-based fixing agent are limited.
  • a physiologically active substance-immobilized substrate characterized in that it has a polymer compound on the surface comprising a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having an aldehyde group or a maleimide group.
  • Patent Document 3 it is not immobilization of the peptide to the fiber.
  • An object of the present invention is to provide a polymer containing an antibacterial peptide (modified peptide derived from beetle defensin having antibacterial properties) that can be used for the production of antibacterial fibers.
  • the object of the present invention is also a new immobilization capable of immobilizing peptides, particularly antibacterial peptides, to fibers by relatively easy means, with less damage to the fibers, and capable of maintaining the immobilization of peptides even after repeated use. Is to provide a method.
  • the object of the present invention is also to provide an antimicrobial fiber having an antimicrobial peptide.
  • the present inventors can fix the peptide to the fiber by a relatively easy method, maintain the peptide activity on the fiber, and maintain the peptide activity even after repeated washing of the fiber. I found what I could do and completed the present invention.
  • the peptide-containing polymer of the present invention is used, the peptide can be immobilized on the fiber by a simple method. More specifically, by using the polymer of the present invention, the peptide can be immobilized on the fiber without complicated pretreatment of the fiber and without peptide synthesis on the fiber.
  • the present invention also relates to a method for immobilizing peptides on fibers. By using the method of the present invention, the peptide can be immobilized on the fiber by a simple method.
  • a polymer containing an antibacterial peptide (modified peptide derived from beetle defensin having antibacterial properties) is provided, and the polymer of the present invention can be immobilized on a fiber by a simple method.
  • a modified peptide derived from beetle defensin having antibacterial properties is conjugated with a linker in the polymer, such as a maleimide group, to prepare a peptide-containing polymer, and the antibacterial peptide is coated by coating it on a fiber. Can be immobilized on fiber.
  • the fiber is coated with a linker having a binding property to a peptide, such as a polymer having a maleimide group
  • the fiber is added to a solution containing a peptide, particularly a modified peptide derived from beetle defensin having antibacterial properties. So that the peptide can be immobilized on the fiber.
  • an antibacterial fiber in which a modified peptide derived from beetle defensin having antibacterial properties provided as described above is immobilized can be provided.
  • R 1 is a hydrogen atom or a methyl group
  • U 1 is a single bond, O or NH
  • V is a single bond or a spacer
  • W is a single bond or an amide group, a thioether group
  • Peptide is an amino acid sequence represented by the following formula: -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO-
  • X1 is Arg or Ala
  • X2 is Tyr, Arg or Leu
  • X3 is Arg or Ala
  • X4 is Ile, Leu or Val
  • X5 is Gly or Arg
  • X6 is
  • R 2 and R 3 are each independently a hydrogen atom or a methyl group, U 2 is a single bond, O or NH, U 3 is O or NH, and V is a single bond or A spacer, W 1 is a hydrogen atom or a group consisting of all or part of the linker W to which halogen may be added at the end, and Y has a single bond or a substituent having a molecular weight of 10 to 3000 It may be a linear or branched alkylene chain or polyoxyalkylene chain.
  • the antibacterial peptide-containing polymer according to (1) which comprises at least one of the repeating units represented by (3)
  • -W- represents the following formula:
  • Y may have a single bond or a substituent having a molecular weight of 10 to 3000, linear or branched
  • (4) -W-Peptide in the formula (1) is represented by the following formulas (11) to (27):
  • the spacer V may have a linear or branched polyethylene glycol having a weight average molecular weight of 50 to 1000, or a linear chain optionally having a substituent having a molecular weight of 10 to 500.
  • the antibacterial peptide-containing polymer according to any one of (1) to (5), which is a branched alkylene chain or a combination thereof.
  • An antibacterial peptide-containing polymer comprising a repeating unit represented by In the formula (1), U 1 is O and -VW-Peptide is -PEG-S-Cys-Peptide (where PEG is polyethylene glycol having a weight average molecular weight of 10 to 2000) or -CH 2 CHOHCH 2 -S-Cys-Peptide, or U 1 is NH, -VW-Peptide is -Peptide,-(CH 2 ) 3 -S-Cys-Peptide, -CH 2- (C 2 H 4 O) 3 —C 3 H 6 —NH—COCH 2 —S-Cys-Peptide and the following formula:
  • U 3 is O
  • -YH is-(CH 2 ) 11 -CH 3
  • -PEG-R 3 wherein PEG is a polyethylene having a weight average molecular weight of 10 to 2000
  • R 3 is OH or CH 3
  • U 3 is NH and —Y is a single bond.
  • the peptide is -HN-Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -NH-Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -NH -Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-Arg-CONH 2 , -HN-Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -HN-Arg -Leu-Tyr-Leu-Arg-Val-Gly-Arg-Arg-CONH 2 , -NH-Arg-Leu-Arg-Leu-Arg-Val-Gly-Arg-Arg-CONH 2 , -NH-Ala-Leu -Tyr-Leu-Ala-Val- Arg-Arg-Arg-Arg-CONH 2 , -NH-Ala-Leu -Ty
  • a method for immobilizing an antibacterial peptide on a fiber by coating the antibacterial peptide-containing polymer according to any one of (1) to (11) on the fiber.
  • R 1 is a hydrogen atom or a methyl group
  • U 1 is O or NH
  • V is a single bond or a spacer
  • Wo is a functional group that reacts with a peptide or a derivatized peptide. is there.
  • Peptide is an amino acid sequence represented by the following formula: -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxy
  • R 1 and R 3 are each independently a hydrogen atom or a methyl group
  • U 1 and U 3 are each independently O or NH
  • V is a single bond or a spacer
  • Wo is A functional group that reacts with a peptide or a derivatized peptide
  • Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000.
  • Peptide is an amino acid sequence represented by the following formula: -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxy
  • R 2 is a hydrogen atom or a methyl group.
  • Z is Cys.
  • Peptide is an amino acid sequence represented by the following formula: -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxy
  • the manufacturing method of the antimicrobial fiber which consists of.
  • R 1 and R 3 are each independently a hydrogen atom or a methyl group
  • U 1 and U 3 are each independently O or NH
  • V is a single bond or a spacer
  • Wo is A functional group that reacts with a peptide or a derivatized peptide
  • Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000.
  • Peptide is an amino acid sequence represented by the following formula: -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxy
  • the manufacturing method of the antimicrobial fiber which consists of.
  • R 2 is a hydrogen atom or a methyl group.
  • antimicrobial peptide-containing polymer of the present invention can also be expressed as follows.
  • n is a natural number
  • m1 is 0 or a natural number
  • R 1 , R 2 , U 1 , U 2 , V, W, W 1 , and Peptide are the same as described above.
  • n and m2 are natural numbers, and R 1 , R 3 , U 1 , U 3 , V, W, Y, and Peptide are the same as described above, or , The following formula (3a):
  • antimicrobial peptide-containing polymer of the present invention can also be expressed as follows. Following formula (4a):
  • n is a natural number
  • m1 is 0 or a natural number
  • Peptide is the same as described above.
  • n is a natural number
  • m2 is 0 or a natural number
  • R 1 , R 3 , U 1 , U 3 , V, Wo, and Y are the same as above.
  • Peptide is the same as above, and Z is a functional group that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker (for example, an azide group, a sulfhydryl group, a halogen group, an alkyne group, a carbonyl group). , A hydroxylamino group) and a molecular weight of 50 to 500.)
  • the method for producing the antibacterial fiber of the present invention can also be expressed as follows.
  • n is a natural number
  • m2 is 0 or a natural number
  • R 1 , R 3 , U 1 , U 3 , V, Wo, and Y are the same as above.
  • Peptide is the same as above, and Z is a functional group that reacts with a hydrogen atom, hydroxyl group, Cys or Wo to form a linker (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group). , A hydroxylamino group) and a molecular weight of 50 to 500.
  • an antibacterial peptide (modified beetle defensin-derived modified peptide) -containing polymer that can be used in the production of an antibacterial fiber can be provided.
  • a fiber in which a peptide is immobilized while retaining its activity can be provided by a relatively easy process using an antibacterial peptide-containing polymer.
  • a fiber on which a peptide, particularly an antibacterial peptide is immobilized can be provided by a relatively easy peptide immobilization method. The fiber provided in this way can maintain the activity of the peptide.
  • the provided fiber has antibacterial activity and is subjected to repeated washing and / or sterilization by autoclaving.
  • antibacterial activity can be maintained.
  • the fiber of the present invention is also excellent in safety, it can be used for medical purposes such as medical bandages, sheets, clothes and the like.
  • a to B means that A is included and is A or more, and B is included and is B or less.
  • fiber includes any of fibers as a raw material for yarn or fabric, yarn made from the fiber, fabric made from the yarn, or nonwoven fabric made from the fiber.
  • the term “coats a polymer on a fiber” means a fiber as a raw material for yarn or fabric.
  • fiber coated with polymer refers to either a fiber coated with a polymer or a fiber as a raw material of a fabric, a yarn made of a fiber coated with the polymer, or a fabric made of the yarn. Including.
  • a polypeptide and a peptide are used in the same meaning unless otherwise specified, and can be changed from each other.
  • S when it is expressed as -S-Cys-Peptide, S means S in cysteine.
  • Leu means a leucine residue.
  • a polymer having A and B in the polymer at random it can be represented by any of the following modes. Following formula:
  • the present invention provides an antibacterial peptide-containing polymer represented by the following formula.
  • R 1 and R 2 are each independently a hydrogen atom or a methyl group, n is a natural number, m1 is 0 or a natural number, and U 1 and U 2 are each independently O or NH.
  • V is a single bond or a spacer
  • W is a single bond or a linker having an amide group, a thioether group, a thioester group, an oxime group, a 1,2,3-triazole group or an ester group
  • W 1 is
  • Peptide is an amino acid sequence represented by the following formula: —NH—X1-Leu-X2-Leu-X3 -X4-X5-X6-X7-CONH 2 or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- (wherein X1 is Arg
  • R 1 and R 3 are each independently a hydrogen atom or a methyl group, n is a natural number, m2 is a natural number, U 1 is a single bond, O or NH, and U 3 is O or NH, V, W, and Peptide are the same as described above, and Y is a hydrogen atom or a linear or branched alkylene chain or polyoxy group that may have a substituent having a molecular weight of 10 to 3000.
  • An antibacterial peptide-containing polymer represented by the following formula (3a):
  • R 1 , R 2 and R 3 are each independently a hydrogen atom or a methyl group, n, m1 and m2 are natural numbers, and U 1 and U 2 are each independently a single bond, O or NH, U 3 is O or NH, and V, W, Peptide, and Y are the same as above.
  • the antibacterial peptide-containing polymer of the present invention may be a homopolymer or a copolymer.
  • Peptide may be bonded to all monomer units or to some monomer units.
  • the ratio of the unit to which Peptide is bonded in the polymer (referred to above as RC (CH 2 ) -CO-U 1 -VW-Peptide and referred to as “Peptide-containing unit” in this specification) is determined by coating the polymer on the cloth. (Coating) It is not particularly limited as long as the desired amount of peptide can be applied, and is 0.1 to 100 mol%, preferably 0.4 to 20 mol%, more preferably 0.4 to 2 mol. %.
  • the amount of polymer applied (coating) to the fabric can be controlled, and homogeneous and suitable antibacterial fibers having the desired antibacterial properties can be easily produced.
  • a unit in which Peptide in the polymer is not bonded meaning the above-mentioned RC (CH 2 ) —CO—U 2 —VW 1 and / or RC (CH 2 ) —CO—U 3 —Y, Appropriate physical properties such as viscosity and solubility can be obtained by appropriately selecting the ratio of “Peptide-free unit”.
  • the desired physical properties can be imparted to the polymer by appropriately selecting the Peptide-free unit (RC (CH 2 ) —CO—U 3 —Y).
  • Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000, preferably 100 to 2000.
  • the physical properties of the polymer can be changed by appropriately selecting the length and type of Y.
  • the peptide constituting the peptide is selected from a modified beetle defensin-derived modified peptide consisting of an amino acid sequence represented by the following formula, and the peptide can be bound to a linker at either the N-terminus or C-terminus.
  • the peptide is H 2 N-Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , H 2 N-Arg-Leu-Arg-Leu-Arg-Ile-Gly -Arg-Arg-CONH 2 , H 2 N-Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-CONH 2 , H 2 N-Arg-Leu-Leu-Leu-Arg-Ile-Gly -Arg-Arg-CONH 2 , H 2 N-Arg-Leu-Tyr-Leu-Arg-Val-Gly-Arg-Arg-CO 2 , H 2 N-Arg-Leu-Arg-Leu-Arg-Val-Gly -Arg-Arg-CO 2, H 2 N-Ala-Leu-Tyr-Leu-Ala-Val-Arg-Arg-Arg-Arg-CO 2 and Selected from 2 N-Arg-Leu-Leu -Leu-Arg-Val-G
  • modified peptides derived from beetle defensin that can be used as antibacterial peptides in the present invention include, for example, Japanese Patent No. 3273314, Japanese Patent Application Laid-Open No. 2010-184022, and Ishibashi et al., Eur. J. Biochem, 1999 Dec; 266 ( 2): 616-23, which are hereby incorporated by reference.
  • Peptide synthesis can be performed using any conventional peptide synthesis method (solid phase synthesis and / or liquid phase synthesis).
  • solid phase synthesis and / or liquid phase synthesis when the polymer and the peptide are bonded via Cys, it is possible to introduce Cys at the C-terminal or N-terminal of the synthetic polypeptide in peptide synthesis, which is convenient.
  • W in the above formula (1a) is a linker for immobilizing the peptide.
  • the linker is used to connect the N-terminal or C-terminal of the peptide to the spacer or directly to the side chain terminal of the polymer chain, and can be introduced at the N-terminal or C-terminal of the peptide or at the peptide binding site of the spacer. It is also possible to do.
  • the linker include a compound having a thioether group, a thioester group, an oxime group, a 1,2,3-triazole group or an ester group, or a group thereof, preferably a compound having an amide group or a thioether group. Or a group thereof, but is not limited thereto.
  • a peptide in which a peptide is modified for the purpose of the present invention such as a peptide into which a linker or a part thereof is introduced, is referred to as a peptide derivative.
  • a linker having reactivity with the functional group at the peptide end or spacer end to which the linker is not introduced is introduced at the peptide end or spacer end where the linker is introduced, and bonded.
  • the peptide can be bound to the spacer by carrying out the reaction.
  • a linker is introduced into both the peptide end and the spacer end, a part constituting the linker (linker part W X ) is introduced on one side and the other part constituting the linker (linker part W Y ) is provided on the other side.
  • a linker W is made from W x and W y .
  • a linker or linker part can be introduced at the side chain end of the polymer chain, and the peptide can be directly bonded to the side chain end of the polymer chain.
  • the linker is an amide group or an ester group.
  • the functional group to be a linker can be introduced at the end of the spacer, but a spacer having it at the end can also be used.
  • the linker site can be introduced into either the N-terminus or C-terminus of the peptide or the spacer terminus to serve as a linker. However, the linker site can be introduced into both the peptide terminus and the spacer terminus, It can also react to serve as a linker.
  • a thiol can be introduced at either the peptide end or the spacer end, a maleimide group can be introduced into the other, and the peptide can be bound to the spacer via a linker having a thioether group by the reaction of the thiol and the maleimide group.
  • the amino group at the N-terminal can be bonded to the spacer by reacting with a spacer having a functional group such as a carboxyl group, a halogen, an aldehyde group, or an isocyanate group.
  • a spacer having a functional group such as a carboxyl group, a halogen, an aldehyde group, or an isocyanate group.
  • the reaction between the amino group and these functional groups can be performed by a conventional method, and examples thereof include a condensation reaction and an addition reaction.
  • the peptide is bound to the N-terminal spacer by introducing a thiol group such as cysteine or thioglycolic acid as a linker site at the N-terminus, and reacting with the SH group such as maleimide group, halogen, thioester group, bromoacetyl group, etc. It can also be reacted with a spacer having a functional group to bind to the spacer. Thereby, a peptide and a spacer are couple
  • the reaction between the SH group and these functional groups can be performed by a conventional method.
  • the reaction with the maleimide group can be performed by a Michael addition reaction.
  • the reaction with halogen can be performed by nucleophilic substitution reaction, and the reaction with thioester is by transesterification.
  • propiolic acid, 3-butyric acid or the like having an alkyne as a linker site at the N-terminus of the peptide can be introduced and reacted with a spacer having an azide group that reacts with the alkyne to bind to the spacer.
  • the reaction between the alkyne and the azide group can be performed by a conventional method, for example, by a Huisgen reaction.
  • 3-oxyaminopropionic acid, 4-oxyaminobutyric acid or the like having an alkoxyamino group as a linker site at the N-terminal of the peptide is introduced and reacted with a spacer having a ketone that reacts with the alkoxyamino group to bind to the spacer.
  • the reaction between the alkoxyamino group and the ketone can be performed by a conventional method, for example, by an oximation reaction.
  • these linker sites can be joined by introducing a spacer and a peptide site in reverse.
  • the C-terminal carboxyl group can be bonded to the spacer by reacting with a spacer having a functional group such as an amino group or a hydroxyl group.
  • the reaction between the carboxyl group and these functional groups can be carried out by a conventional method, and examples thereof include a condensation reaction using a condensing agent.
  • the peptide is bound to the C-terminal spacer by introducing a thiol group, for example, cysteine or 2-amino-1-ethanethiol, as a linker site at the C-terminus and reacting with the SH group, a maleimide group, a halogen, a thioester group, It can also be reacted with a spacer having a functional group such as an aldehyde group or a bromoacetyl group to bind to the spacer.
  • a thiol group for example, cysteine or 2-amino-1-ethanethiol
  • the spacers in the above formulas (1a) to (3a) are not particularly limited as long as they can bind a polymer and a peptide.
  • the alkylene chain or the polyethylene glycol chain can be either linear or branched and may have a substituent.
  • the molecular weight of the alkylene chain is preferably 10 to 500, more preferably 20 to 200.
  • the polyethylene glycol chain has a weight average molecular weight of preferably 50 to 1000, more preferably 100 to 500, and still more preferably 100 to 300. Moreover, as long as it does not inhibit the other coupling
  • the spacer can be bonded to the polymer by synthesizing a polymer using a compound having a terminal functional group having reactivity with the carboxyl group of the monomer unit and then introducing it by reacting with the carboxyl group of the polymer, or the monomer. This can be achieved by polymerization after introduction at this point.
  • the bond between the carboxyl group of the polymer and the functional group of the spacer is not particularly limited, but the functional group having reactivity with the carboxyl group of the polymer is preferably an amino group or a hydroxyl group.
  • the spacer that binds to the polymer may have a linker moiety (functional group) for binding to the peptide at the end opposite to the binding end to the polymer, as long as the binding to the polymer is not affected.
  • Spacer A is
  • linker linker part
  • Linker introduction method At the time of monomer, the spacer part is deprotected, introduced by condensation, and then polymerized. [Ls-C]
  • Linker introduction method At the time of monomer, the spacer part is deprotected, introduced by condensation, and then polymerized.
  • Linker introduction method At the time of the monomer, the spacer part is deprotected, and a hydroxyamino group Boc protector is introduced by condensation and then polymerized. Thereafter, the hydroxyamino group is deprotected.
  • Linker introduction method After deprotecting the spacer part at the time of polymer, it is introduced by condensation.
  • Linker introduction method At the time of monomer, the spacer part is deprotected, introduced by condensation, and then polymerized.
  • Linker introduction method can be introduced into the peptide N-terminal.
  • the SH group-protected carboxylic acid is introduced into the protected peptide and then deprotected.
  • Linker introduction method can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide.
  • Linker introduction method can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide. [Lp-E]
  • Linker introduction method can be introduced at the N-terminus of the peptide.
  • a succinimidyl-type linker is introduced into the protected peptide for deprotection.
  • Linker introduction method can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide.
  • Linker introduction method can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide. [Lp-H]
  • Linker introduction method can be introduced at the N-terminus of the peptide.
  • a hydroxylamino group-protected linker is introduced into the protected peptide and then deprotected.
  • the method for synthesizing the polymer used in the present invention is not particularly limited.
  • a spacer may be introduced after the polymer is synthesized, or a polymer may be synthesized by polymerizing a monomer having a spacer.
  • bonds a peptide is preferable.
  • the monomer terminal of the unit to which the peptide is bonded is a primary amine
  • the monomer in which the primary amino group is previously protected with a protecting group is radical-copolymerized with a monomer having a spacer that is a unit to which the peptide does not bind, and then the protecting group is removed.
  • the antimicrobial polypeptide containing polymer of this invention can be manufactured by couple
  • monomer units that do not bind peptides include poly (ethylene glycol) methyl ether (meth) acrylates, poly (ethylene glycol) (meth) acrylates, (meth) acrylic acid, n-butyl (meth) ) Acrylate, iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl (meth) acrylate, iso-neopentyl (meth) acrylate, sec-neopentyl (meth) acrylate , Neopentyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) acrylate, heptyl (meth) acrylate, n-octyl (meth) acrylate, iso-octyl (meth) acrylate, 2-ethyl (
  • the monomer in which the primary amino group is previously protected with a protecting group is not particularly limited in structure, but the following formula (46) (wherein R 2 is a hydrogen atom or a methyl group, V is a spacer containing an alkylene glycol residue, T Represents a protective group), the (meth) acrylic group preferably has a structure via a spacer V containing an alkylene glycol residue.
  • the protecting group T is not particularly limited as long as it can protect an amino group, and any one can be used. Among them, a t-butoxycarbonyl group (Boc group) or a benzyloxycarbonyl group (Z group, Cbz group), 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like are preferable. Deprotection can be performed under general conditions using trifluoroacetic acid, hydrochloric acid, anhydrous hydrogen fluoride, or the like.
  • monomer examples include those represented by the following formula (47).
  • the polymer synthesis solvent used in the present invention is not particularly limited as long as each monomer can be dissolved.
  • alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol, Benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, Examples thereof include ethylene glycol monobutyl ether. These solvents may be used alone or in combination of two or more.
  • the polymerization initiator a normal radical initiator can be used.
  • a normal radical initiator for example, 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), etc.
  • Organic peroxides such as azo compounds, benzoyl peroxide and lauryl peroxide.
  • the weight average molecular weight of the polymer used in the present invention is preferably 3,000 or more. By setting the weight average molecular weight to 3,000 or more, separation from the monomer is facilitated, and the fixing force to the fiber is further increased. More preferably, it is 10,000 or more.
  • a linker site can be introduced into a polymer (including a spacer) for a binding reaction with a peptide.
  • a linker site can be introduced into a polymer (including a spacer) for a binding reaction with a peptide.
  • a functional group For example, a halogen, a carboxyl group, an aldehyde group, an isocyanate group, a thioester group, a bromoacetyl group, a maleimide group, a thiol group etc. can be mention
  • the linker to be introduced can be appropriately selected according to the functional group at the terminal of the peptide to be bound.
  • the bond destination is an amino group
  • the bond destination is a carboxyl group
  • an amino group and a hydroxyl group are preferable.
  • the bonding destination is a thiol group, for example, an SH group of cysteine, a halogen, a thioester group, an aldehyde group, a bromoacetyl group, or a maleimide group is preferable.
  • a cysteine residue is introduced at the peptide end as a linker site, it is preferable to introduce a maleimide group or a halogen group at the end of the spacer.
  • the peptide binding reaction to the spacer is not particularly limited and can be carried out by condensation reaction, thioetherification reaction, esterification reaction, Huisgen reaction, Michael addition reaction, oximation reaction, etc., but preferably the C-terminal of the peptide Alternatively, a Cys group or a thiol group is introduced at the N-terminus, and a maleimide group in the polymer is used, a maleimide-thiol Michael addition reaction, or a halogen group in the polymer is used. Can be done
  • the peptide-containing polymer is preferably prepared in a solvent that dissolves the polymer having a maleimide unit and is miscible with water.
  • a solvent that dissolves the polymer having a maleimide unit and is miscible with water.
  • a polymer by dissolving a polymer in alcohols such as methanol, ethanol, isopropanol, tetrahydrofuran, dioxane, N, N-dimethylformamide, dimethyl sulfoxide, etc., and reacting the peptide by adding a peptide solution dissolved in, for example, water. It can be carried out.
  • a maleimide group and a thiol group are used as linker sites, but the present invention is not limited to this.
  • Introduction of a maleimide group into a polymer can be performed using a compound having a maleimide group and a functional group capable of reacting with an amino group in one molecule.
  • maleimidopropionic acid N-succinimidyl, N- ( ⁇ -maleimidoacetoxy) succinimide ester, N- (4-maleimidobutyryloxy) succinimide, N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt, N- ( 6-maleimidocaproyloxy) succinimide, N- (8-maleimidocapryloxy) succinimide, N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt, N- (11-maleimidoundecanoyloxy) succinimide, N- ( 11-maleimidoundecanoyloxy) sulfosuccinimide sodium salt and the like.
  • These reagents may be used alone or in combination of two or more.
  • the antibacterial peptide-containing polymer of the present invention it is represented by the following formula (48) (wherein R 1 is a hydrogen atom or a methyl group, n is a natural number, and V is a spacer).
  • R 1 is a hydrogen atom or a methyl group
  • n is a natural number
  • V is a spacer.
  • the peptide is linked to the maleimide unit of the monomer unit via a cysteine at the C-terminus or N-terminus.
  • S in —S—Cys represents S in a cysteine residue.
  • the antibacterial peptide-containing polymer of the present invention may be all the monomer unit, but the monomer unit and the following formula (49) (R 1 is a hydrogen atom or a methyl group, n is a natural number, V May be a spacer).
  • the above formula (48) and the following formula (50) (n is a natural number, Y is a single bond or a linear or branched chain which may have a substituent having a molecular weight of 10 to 3000) Or an alkylene chain or a polyoxyalkylene chain, preferably a linear or branched alkylene chain or polyethylene glycol which may have a substituent, or a copolymer of the above formula (48) or the above formula ( 49) and the following formula (50) (n is a natural number).
  • a preferred polymer is a peptide-containing polymer represented by the following formula (51).
  • n1 is a natural number and m1 is 0 or a natural number.
  • the peptide can be bound to the polymer by adding a peptide solution in which the peptide is dissolved in a polymer solution in which the peptide is dissolved, using a Michael addition reaction.
  • the ratio of the peptide to the maleimide unit can be appropriately selected according to the purpose. For example, when the peptide is bound to all maleimide units, the coupling reaction is performed so that the molar ratio of the maleimide unit to the peptide is 1: 1. I do.
  • an antibacterial fiber in which the peptide-containing polymer of the present invention is coated on the fiber.
  • the polypeptide is immobilized on the fiber, the peptide is not easily detached in normal use, and the antibacterial property can be maintained even in repeated use.
  • the fibers are plant fibers such as cotton, hemp and silk, animal fibers such as wool, animal hair and silk, regenerated fibers such as rayon, semi-synthetic fibers such as acetate, and chemically synthetic fibers such as polyester, nylon and acrylic. , And complexes thereof, but are not particularly limited thereto.
  • a woven fabric or a knitted fabric made of staples, filament yarns and processed yarns thereof is used, but a fiber web may be used.
  • the fiber web refers to non-woven fabric, tissue paper, wet tissue, paper such as food wrapping paper, and the like.
  • the antibacterial fiber of the present invention can be used for various fiber products that require antibacterial properties, such as sanitary products. Since the antibacterial fiber of the present invention is also excellent in safety, it can be used for medical use and medical supplies such as medical bandages, sheets, gauze and clothes.
  • the peptide-containing polymer is fixed on the fiber, but the term “on the fiber” as used in the present invention means that a part of the peptide-containing polymer is a part of the fiber in addition to the case where the peptide-containing polymer is completely present on the surface of the fiber. It also includes the state of being in the middle.
  • the target peptide activity for example, the peptide is a modified peptide derived from beetle defensin, it is preferable that all or most of the peptide-containing polymer is present on the fiber surface in order to exhibit antibacterial activity.
  • the polymer coated on the cloth is preferably 10% by weight or less, more preferably 5% by weight or less, based on the weight of the cloth.
  • the peptide preferably the fabric 1 cm 0.001Myu mol 0.5 ⁇ mol per 2, most preferably 1 cm 0.03 microns mol per 2 from safety and economical point of view.
  • the peptide-containing polymer of the present invention can be fixed to the fiber by coating the fiber surface with the peptide-containing polymer, for example, using a fiber processing technique such as water repellent processing or antistatic processing. it can.
  • the immobilization of the polymerized peptide to the fiber can be performed using, for example, a method based on electrostatic interaction, a method of chemically bonding by a crosslinking reaction, or the like.
  • the method of immobilization by electrostatic interaction is simpler.
  • when chemically bonded by a cross-linking reaction there is an advantage of stronger immobilization, which can be arbitrarily selected according to the purpose. It is.
  • an emulsion containing the antimicrobial peptide-containing polymer of the present invention is provided.
  • a hydrophobic polymer (an peptide-containing polymer) having an immobilized peptide can be emulsified alone or with a surfactant or the like to form an emulsion that is a fiber processing agent.
  • the polymer emulsion can also be prepared by a conventionally known emulsion polymerization method.
  • a monomer can be dispersed in water with a surfactant, and emulsion polymerization can be carried out by adding a polymerization initiator, a chelating agent, an oxygen scavenger, a molecular weight modifier and the like.
  • a surfactant for example, a surfactant, a chelating agent, an oxygen scavenger, a molecular weight modifier and the like.
  • Conventionally known surfactants, polymerization initiators, chelating agents, oxygen scavengers, and molecular weight modifiers can be used.
  • the fiber in which the peptide of the present invention is immobilized can be easily produced by applying the emulsion thus prepared to the fiber and drying it.
  • a water-soluble polymer it can be sold or distributed in a state dissolved in water, and can be easily mixed with a cross-linking agent such as blocked isocyanate during processing and fixed to a cloth. .
  • Step (1) a method for producing an antibacterial fiber having an antibacterial peptide immobilized, comprising the following steps. Step (1); the following formula (1b):
  • R 1 and R 3 are each independently a hydrogen atom or a methyl group, n is a natural number, m 2 is 0 or a natural number, and U 2 and U 3 are each independently NH or O.
  • V is a single bond or a spacer
  • Wo is a functional group that reacts with a peptide or a derivatized peptide
  • Y is a single bond or a linear group that may have a substituent having a molecular weight of 10 to 3000 or A branched alkylene chain or a polyoxyalkylene chain
  • Peptide is an amino acid sequence represented by the following formula: -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.)
  • a polypeptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxylamino
  • the manufacturing method of the antimicrobial fiber which consists of.
  • the above binding reaction can be performed, for example, by reacting for 1 to 10 hours, preferably 2 to 10 hours, more preferably 3 to 6 hours.
  • concentration of the peptide solution used for the binding reaction can be appropriately selected according to the reaction conditions such as the type of linker and temperature, and is, for example, 0.1 to 100 mM, preferably 1 to 10 mM.
  • the polymer to be coated (applied) to the fiber used in the method of the present invention may be any of the polymers prior to binding the polypeptides described herein.
  • the following formula (6a) (wherein R 1 and R 3 are a hydrogen atom or a methyl group, n is a natural number, m is 0 or a natural number, U 1 and U 3 are , O or NH, and Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000).
  • R 1 and R 3 are a hydrogen atom or a methyl group
  • n is a natural number
  • m is 0 or a natural number
  • U 1 and U 3 are , O or NH
  • Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000.
  • 6a
  • Example 1 Peptide Synthesis Peptide A (RLYLRIGRR), Peptide 1 (CRLYLRIGRR) and Peptide 2 (RLYLRIGRRC) were synthesized by solid phase synthesis according to a conventional method.
  • Example 2 Synthesis of Monomer 1 To a two-necked flask (100 ml) equipped with a dropping funnel (3- ⁇ 2- [2- (3-aminopropyl) -ethoxy] -ethoxy ⁇ -propyl) carbamic acid-t-butyl ester (1060 mg, 3.31 mmol), triethylamine (401 mg, 3.97 mmol) and tetrahydrofuran (25 mL) were added, and the mixture was stirred under ice cooling. A mixed solution of methacrylic acid chloride (913 mg, 3.97 mmol) and tetrahydrofuran (25 mL) was added dropwise thereto, and the mixture was stirred for 3 hours under ice cooling.
  • methacrylic acid chloride 913 mg, 3.97 mmol
  • tetrahydrofuran 25 mL
  • the polymer was dissolved again in dichloromethane (10 ml), N-succinimidyl 3-maleimidopropionate (122.4 mg, 0.46 mmol) and triethylamine (46.5 mg, 0.46 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, purification was performed by gel permeation chromatography to obtain the following polymer 1.
  • Synthetic polymer 1 (3.5 mg, maleimide unit 8 ⁇ mol) of polymer 4 (peptide-containing polymer ) was dissolved in water (1.0 ml).
  • Peptide 2 (RLYLRIGRRC) (21 mg, 16 ⁇ mol) was dissolved in 1 ml of water, this was mixed with an aqueous solution of polymer 1 and reacted. This was dialyzed overnight with a dialysis tube (molecular weight cut off 8000) to remove excess peptide.
  • Example 7 Fabrication of processed cloths 1 to 3 Polymer 2DMF-water mixed solution was coated on a cotton JIS-attached white cloth and dried under reduced pressure to prepare processed cloths 1 to 3. The processed cloth 1 was adjusted so that the amount of peptide present was 0.3 ⁇ mol per cm 2 of cloth. The processed cloth 2 was produced in the same manner as the processed cloth 1 except that the amount of the peptide was adjusted to 0.1 ⁇ mol per 1 cm 2 of the cloth. The processed cloth 3 was produced in the same manner as the processed cloth 1 except that the amount of the peptide was adjusted to 0.03 ⁇ mol per 1 cm 2 of the cloth.
  • Example 8 Fabrication of work cloths 4 to 6 Fabrication of work cloths 4 to 6 was performed in the same manner as work cloth 1 except that polymer 3 was used as the polymer to be coated. The abundance of the peptide was 0.3 ⁇ mol per 1 cm 2 of the processed cloth 4, 0.1 ⁇ mol per 1 cm 2 of the processed cloth 5, and 0.03 ⁇ mol of the processed cloth 6 per 1 cm 2 of the cloth.
  • Example 9 Fabrication of work cloth 10 Fabrication of work cloth 10 was performed in the same manner as work cloth 1 except that polymer 4 was used as the polymer to be coated. The amount of peptide present was 0.3 ⁇ mol per cm 2 .
  • Example 10 Antibacterial activity test The antibacterial activity test was performed on the processed fabrics 1 to 10 produced as described above. A culture solution (1 cm x 1 cm) and a standard fabric each containing Staphylococcus aureus NBRC 12732 are added to the new culture solution in a volume of 1%. After culturing at 37 ° C. for 3 hours, the solution was put into 200 ⁇ l of a culture solution diluted to have an absorbance at a wavelength of 600 nm of 0.01), and cultured at 37 ° C. for 18 hours. It was shown as a logarithmic value obtained by dividing the number of viable bacteria in the standard cloth by the number of viable bacteria in the processed cloth after 18 hours of culture. The results are shown in Table 1.
  • the cloth is vortex washed with 40 ml of ultrapure water (maximum speed shaking for 10 seconds x 3 times) and sterilized by autoclave (121 ° C, 20 minutes), and the same processed cloth is used for the antibacterial test. Went. Further, this washing-sterilization operation-antibacterial test was repeated. The results are shown in Table 2 below.
  • the polymer was dissolved again in dichloromethane (10 ml), N-succinimidyl 3-maleimidopropionate (38 mg, 0.14 mmol) and triethylamine (14.1 mg, 0.14 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, it was purified by gel permeation chromatography. As a result of NMR, the substance ratio of maleimide unit: octadecyl unit was 1:14.
  • Example 12 Fabrication of processed fabrics 11 and 12
  • the polymer 5 produced in Example 11 was coated on a cotton JIS-attached white fabric so that the amount of maleimide units in the fabric was 0.03 ⁇ mol per cm 2 of fabric, and dried under reduced pressure.
  • This cloth was immersed in an aqueous solution (peptide concentration 3.6 mM) containing peptide 1 (CRLYLRIGRR) in the same mole as the maleimide unit, and left for 5 hours. Thereafter, the cloth was washed with ion-exchanged water to remove unreacted peptides, and a processed cloth 11 was produced.
  • a processed cloth 12 was produced in the same manner as the processed cloth 11 except that peptide 2 (RLYLRIGRRC) was used as a peptide to be bound to the maleimide unit.
  • RLYLRIGRRC peptide 2
  • Example 13 In the same manner as in Example 10, the antibacterial activity of the processed cloths 11 to 13 was measured. The results are shown in Table 3 below.
  • Example 17 Synthesis of polymer 6 N- (3-bromopropyl) -2-methacrylamide (monomer 2) (82 mg, 0.4 mmol) and poly (ethylene glycol) methyl ether methacrylate Mn300 (6.0 g, 20 mmol) were dissolved in 20 mL of ethanol. Initiator AIBN (67 mg, 0.4 mmol) was added. The inside of the container was purged with nitrogen, and then stirred under reflux for 6 hours. Dialysis was performed for 48 hours with a dialysis tube (molecular weight cut off 3500) to remove unreacted monomers, and polymer 6 was obtained. In the Beilstein reaction, a flame reaction derived from halogen was observed.
  • Synthetic monomer 3 of polymer 7 (305 mg, 1.04 mmol) and poly (ethylene glycol) methyl ether methacrylate Mn300 (15.6 g, 52 mmol) were dissolved in 50 mL of ethanol, and initiator AIBN (174 mg, 1.06 mmol) was added. The inside of the container was purged with nitrogen, and then stirred under reflux for 6 hours. Dialysis was performed for 48 hours with a dialysis tube (molecular weight cut off 3500) to remove unreacted monomers, and polymer 7 was obtained.
  • polymer 9 (peptide-containing polymer)
  • Polymer 7 (2.04 g, 0.13 mmol in terms of Br) and peptide 3 with CR linker (CRLLLRVGRR) (235.4 mg, 0.13 mmol) were dissolved in 10 ml of DMF, and tertiary butoxy potassium (87.5 mg, 0.78 mmol) was added. The mixture was heated and stirred at 40 ° C. for 7 hours. It was confirmed by TLC that the starting peptide had disappeared. The solution was diluted with 50 ml of DCM, and the precipitated salt was removed by filtration through celite. The filtrate was concentrated with an evaporator and vacuum-dried to obtain polymer 9, which is a polymer in which peptide 3 is bound to polymer 7.
  • Example 19 Preparation of Peptide-Containing Polymer Processing Agents A and B Water was added to the polymer 8 (4.8 g) prepared in Example 18 to make a total amount of 121 g. Stir overnight and completely disperse to prepare Processing Agent A. Processing agent A was diluted 10 times with distilled water to obtain processing agent B. In addition, water was added to polymer 7 (346 mg) to make the total amount 17.3 g, and the mixture was stirred overnight to be completely dispersed to obtain a processing agent C (a peptide-free polymer processing agent). This was used for reference experiments.
  • Example 20 Water was added to adjustment polymer 9 (2.08 g) of peptide-containing polymer processing agent D to make the total amount 500 g. Stir overnight and completely disperse to prepare Processing Agent D.
  • Fabrication of processed fabrics 14 to 16 Add 1.5 to 2.0 times the mass of processing agent A so that it penetrates the entire fabric, and remove excess processing agent with a mangle, and dry at room temperature. A work cloth 14 was produced. The amount of peptide present was 0.03 ⁇ mol per cm 2 . The processing agent was 4% based on the dough. In the ninhydrin reaction, it was confirmed that the dough was colored purple. A processing agent 15 having a mass of 1.5 to 2.0 times that of the cotton JIS-attached white cloth was added so as to penetrate the entire fabric, and the excess processing agent was removed with a mangle, followed by drying at room temperature to prepare a processed cloth 15. The amount of peptide present was 0.006 ⁇ mol / cm 2 .
  • the processing agent was 0.4% based on the dough. Add 1.5 to 2.0 times the mass of processing agent C (reference example) so that it penetrates the entire fabric, and remove excess processing agent with mangles and dry at room temperature. (Reference Example) was produced. The processing agent was 6.8% based on the dough. Fabrication of processed cloth 17 Add 1.5 to 2.0 times the mass of processing agent D so that it penetrates the entire fabric, and remove excess processing agent with a mangle, and dry at room temperature. 17 was produced. The amount of peptide present was 0.006 ⁇ mol / cm 2 . The processing agent was 0.8% based on the dough.
  • Example 22 Antibacterial test The processed fabric 17 was tested in the same manner as in Example 10. The results are shown in Table 4 below.
  • Example 23 Antibacterial activity test The antibacterial activity test was performed on the processed fabrics 14 to 16 produced as described above. The measurement was performed in accordance with the JIS L 1902 quantitative test method. The pour plate culture method (colony method) was used as a method for measuring the viable cell count after the culture. In addition, the repeated washing test for durability evaluation was conducted according to the method specified in Washing Method 103 of JIS L 0217 (washing at 40 ° C for 5 minutes and rinsing twice at 30 ° C for 2 minutes. After dehydration, direct sunlight The test was repeated 10 times, 20 times, and 50 times by screen drying in a non-contact location. At the time of washing, polyoxyethylene alkyl ether was used as a detergent.
  • the bacteriostatic activity value is (the common logarithm of the viable count of the standard cloth after 18 hours of culture-the common logarithm of the viable count of the standard cloth immediately after inoculation of the test bacteria)-(the processed cloth after 18 hours of culture)
  • the common logarithm of the number of viable bacteria in the product-the common logarithm of the number of viable bacteria in the processed cloth immediately after inoculation of the test bacteria The results are shown in Table 5 below.
  • Example 24 Safety test of antibacterial peptide-containing polymer Using the polymer 8 produced in Example 18, a safety test was conducted for each of the following items. 1. Acute oral toxicity test Polymer 8 was administered to 5 males and 5 females of the test animals, but survived during the test period of 2 weeks. Was not recognized. 2. Mutagenicity test Polymer 8 was used as a sample, and the mutagenicity test was performed in accordance with the mutagenicity test in the Industrial Safety and Health Act. As test strains, Salmonella typhimurium used TA1535, TA100, TA1537, TA98, and Escherichia coli used WP2uvrA. The result was negative.
  • the antibacterial peptide-containing polymer and antibacterial fiber of the present invention are safe and can be used in medical applications.

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Abstract

The purpose of the present invention is to provide a polypeptide-containing polymer that can be used to manufacture an antimicrobial fiber. The purpose of the present invention is also to provide a method for polypeptide immobilization on a fiber, whereby an antimicrobial polypeptide can be immobilized on a fiber by a relatively simple means, and whereby there is little damage to the fiber, and immobilization of the polypeptide can be maintained despite repeated use. The present invention provides an antimicrobial polypeptide-containing (beetle defensin-modified polypeptide-containing) polymer that can be used to manufacture an antimicrobial fiber. By the method of the present invention, an antimicrobial polypeptide can also be easily immobilized on a fiber.

Description

ペプチド含有ポリマー、及び繊維へのペプチド固定化方法Peptide-containing polymer and method for immobilizing peptide on fiber
　本発明は、新規なペプチド含有ポリマーに関する。本発明はまた、ペプチドを繊維に固定化する新規な方法に関する。 The present invention relates to a novel peptide-containing polymer. The present invention also relates to a novel method for immobilizing peptides on fibers.
　近年では、抗生物質に耐性を持った薬剤耐性菌が先進国を中心に蔓延しており、集団感染や院内感染を引き起こしている。特に、メチシリンに耐性を持つメチシリン耐性黄色ブドウ球菌（MRSA）による感染例は、国内で年間二万件以上報告されている。感染経路は、複雑であり特定は困難であるが病院内で使用するベッドシーツ、白衣なども感染リスク要因と考えられている。 In recent years, drug-resistant bacteria that are resistant to antibiotics have spread throughout developed countries, causing mass infections and nosocomial infections. In particular, more than 20,000 cases of infection with methicillin-resistant Staphylococcus aureus (MRSA) resistant to methicillin are reported annually in Japan. Infection routes are complex and difficult to identify, but bed sheets and lab coats used in hospitals are considered infection risk factors.
　発明者らは、これらの問題を解決するため、石橋らにより発明されたカブトムシディフェンシン由来の改変ペプチド（非特許文献１）を固定化した抗菌繊維を開発した。綿にスペーサーと呼ばれる分子鎖を結合し、その末端のアミノ基を起点としてアミノ酸合成を行い、綿上で９残基の改変ペプチドを合成した。抗菌試験の結果、MRSAに強い抗菌活性が認められ、繰り返し使用しても活性を維持していることが明らかになった（特許文献１、非特許文献２）。しかしながら、この方法は、繊維の前処理など加工方法が煩雑であり、既存の繊維加工技術で実施することは困難である。また、アミノ酸の縮合反応、保護基の脱保護反応を全て綿上で行うため、繊維自体の機械的強度も低下するため、実用化するには新たな加工方法の開発が必要であった。 In order to solve these problems, the inventors have developed an antibacterial fiber in which a modified peptide derived from beetle defensin (Non-patent Document 1) invented by Ishibashi et al. A molecular chain called a spacer was bound to cotton, and amino acid synthesis was carried out starting from the terminal amino group, and a 9-residue modified peptide was synthesized on cotton. As a result of the antibacterial test, MRSA showed strong antibacterial activity, and it was revealed that the activity was maintained even after repeated use (Patent Document 1, Non-Patent Document 2). However, this method involves complicated processing methods such as fiber pretreatment, and is difficult to implement with existing fiber processing techniques. In addition, since the condensation reaction of amino acids and the deprotection reaction of protecting groups are all carried out on cotton, the mechanical strength of the fiber itself is also lowered, so that it was necessary to develop a new processing method for practical use.
　また、水溶液中で陽性に荷電される抗菌ペプチドである、Lys１５個が連結した分岐型ペプチドのＮ末端に抗菌性ペプチドを連結した抗菌性マルチプルアンチゲンペプチドを樹脂系固着剤で繊維に固着した抗菌性繊維が報告されている（特許文献２）。しかしながら、樹脂系固着剤で固着したペプチドは、固着されることによって一部が活性を消失してしまう恐れがあり、必要以上の抗菌ペプチドを固着させることが必要である。また、樹脂系固着剤で固着できるペプチドも限られてしまう。 In addition, an antibacterial multiple antigen peptide in which an antibacterial peptide is linked to the N terminal of a branched peptide in which 15 Lys are linked, which is a positively charged antibacterial peptide in an aqueous solution, is fixed to the fiber with a resin-based fixing agent. Has been reported (Patent Document 2). However, there is a possibility that a part of the peptide fixed with the resin-based fixing agent may lose its activity when it is fixed, and it is necessary to fix more antibacterial peptides than necessary. In addition, peptides that can be fixed with a resin-based fixing agent are limited.
　そこで、繊維に固定されたペプチドの活性が使用において十分に維持され、かつ、繊維上でのアミノ酸の合成反応を伴わない、容易で簡便なペプチドの繊維への新たな固定化法が望まれていた。 Therefore, there is a demand for a new and simple method for immobilizing peptides on fibers, in which the activity of the peptides immobilized on the fibers is sufficiently maintained in use and does not involve an amino acid synthesis reaction on the fibers. It was.
　また、ホスホリルコリン基を有するユニット、疎水性基を有するユニット及びアルデヒド基又はマレイミド基を有するユニット、から構成される高分子化合物を表面に有することを特徴とする生理活性物質固定化基板が報告されている（特許文献３）。しかしながら、繊維へのペプチドの固定化ではない。 Also reported is a physiologically active substance-immobilized substrate characterized in that it has a polymer compound on the surface comprising a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having an aldehyde group or a maleimide group. (Patent Document 3). However, it is not immobilization of the peptide to the fiber.
特開２０１０－１８４０２２号公報JP 2010-184022 A 特開２０００－４５１８２号公報JP 2000-45182 A 特開２０１０－１１７１８９号公報JP 2010-117189 A 特許第３２７３３１４号公報号公報Japanese Patent No. 3273314 特開２０１０－１８４０２２号公報JP 2010-184022 A
　本発明の目的は、抗菌性繊維の製造に用いることができる抗菌性ペプチド（抗菌性を有するカブトムシディフェンシン由来の改変ペプチド）含有ポリマーを提供することである。本発明の目的はまた、比較的容易な手段により、繊維へのダメージが少なく、繰り返しの使用によってもペプチドの固定が維持できる、ペプチド、特には抗菌性ペプチドを繊維へ固定化できる新たな固定化方法を提供することである。本発明の目的はまた、抗菌性ペプチドを有する抗菌性繊維を提供することである。 An object of the present invention is to provide a polymer containing an antibacterial peptide (modified peptide derived from beetle defensin having antibacterial properties) that can be used for the production of antibacterial fibers. The object of the present invention is also a new immobilization capable of immobilizing peptides, particularly antibacterial peptides, to fibers by relatively easy means, with less damage to the fibers, and capable of maintaining the immobilization of peptides even after repeated use. Is to provide a method. The object of the present invention is also to provide an antimicrobial fiber having an antimicrobial peptide.
　本発明者らは、鋭意努力の結果、比較的容易な方法によりペプチドを繊維に固定でき、かつ繊維上でペプチドの活性も維持され、更には、繊維の洗浄を繰り返してもペプチドの活性が維持できることを見いだし、本発明を完成した。
　本発明のペプチド含有ポリマーを用いれば、簡易な方法によりペプチドを繊維に固定化することができる。より具体的には、本発明のポリマーを用いることにより、繊維を煩雑な前処理することなく、また、繊維上でペプチド合成をすることなく、繊維にペプチドを固定化することができる。
　本発明はまた、ペプチドを繊維に固定化する方法に関する。本発明の方法を用いることにより、簡便な方法で繊維にペプチドを固定化することができる。 As a result of diligent efforts, the present inventors can fix the peptide to the fiber by a relatively easy method, maintain the peptide activity on the fiber, and maintain the peptide activity even after repeated washing of the fiber. I found what I could do and completed the present invention.
If the peptide-containing polymer of the present invention is used, the peptide can be immobilized on the fiber by a simple method. More specifically, by using the polymer of the present invention, the peptide can be immobilized on the fiber without complicated pretreatment of the fiber and without peptide synthesis on the fiber.
The present invention also relates to a method for immobilizing peptides on fibers. By using the method of the present invention, the peptide can be immobilized on the fiber by a simple method.
　本発明の一つの態様においては、抗菌性ペプチド（抗菌性を有するカブトムシディフェンシン由来の改変ペプチド）を含有するポリマーが提供され、本発明のポリマーは、簡易な方法により繊維に固定化できる。
　本発明の一つの態様においては、抗菌性を有するカブトムシディフェンシン由来の改変ペプチドをポリマー中のリンカー、例えばマレイミド基と結合させてペプチド含有ポリマーを調製し、それを繊維にコーティングすることにより抗菌ペプチドを繊維に固定化できる。 In one embodiment of the present invention, a polymer containing an antibacterial peptide (modified peptide derived from beetle defensin having antibacterial properties) is provided, and the polymer of the present invention can be immobilized on a fiber by a simple method.
In one embodiment of the present invention, a modified peptide derived from beetle defensin having antibacterial properties is conjugated with a linker in the polymer, such as a maleimide group, to prepare a peptide-containing polymer, and the antibacterial peptide is coated by coating it on a fiber. Can be immobilized on fiber.
　本発明の別の態様においては、ペプチドとの結合性を有するリンカー、例えばマレイミド基を有するポリマーを繊維にコーティングした後、ペプチド、特には抗菌性を有するカブトムシディフェンシン由来の改変ペプチドを含む溶液に繊維を浸漬し、ペプチドを繊維に固定化することができる。
　本発明の別の態様においては、上記のようにして提供された抗菌性を有するカブトムシディフェンシン由来の改変ペプチドが固定化された抗菌性繊維が提供できる。 In another embodiment of the present invention, after the fiber is coated with a linker having a binding property to a peptide, such as a polymer having a maleimide group, the fiber is added to a solution containing a peptide, particularly a modified peptide derived from beetle defensin having antibacterial properties. So that the peptide can be immobilized on the fiber.
In another aspect of the present invention, an antibacterial fiber in which a modified peptide derived from beetle defensin having antibacterial properties provided as described above is immobilized can be provided.
　即ち、以下の発明が提供される。
（１）下記式（１） That is, the following invention is provided.
(1) The following formula (1)
Figure JPOXMLDOC01-appb-C000039
 （式中Ｒ1は、水素原子又はメチル基であり、Ｕ1は、単結合、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗは、単結合或いはアミド基、チオエーテル基、チオエステル基、オキシム基、１，２，３－トリアゾール基、カルボニル基又はエステル基を有するリンカーであり、Peptideは、下記式で表されるアミノ酸配列：
－ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－Ｘ４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ、Ｌｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６はＡｒｇ又はＬｙｓであり、Ｘ７はＡｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）
で表される反復単位を含む抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000039
 Wherein R 1 is a hydrogen atom or a methyl group, U 1 is a single bond, O or NH, V is a single bond or a spacer, W is a single bond or an amide group, a thioether group, A linker having a thioester group, an oxime group, a 1,2,3-triazole group, a carbonyl group or an ester group, and Peptide is an amino acid sequence represented by the following formula:
-NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile, Leu or Val, X5 is Gly or Arg, and X6 is Arg Or Lys and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) It is a peptide. )
The antibacterial peptide containing polymer containing the repeating unit represented by these.
（２）さらに、下記式（２）および下記式（３） (2) Further, the following formula (2) and the following formula (3)
Figure JPOXMLDOC01-appb-C000040
 （式中Ｒ2及びＲ3は、それぞれ独立に水素原子又はメチル基であり、Ｕ2は、単結合、Ｏ又はＮＨであり、Ｕ3は、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗ1は、水素原子又は末端にハロゲンを付加してもよい上記リンカーＷの全部又は一部からなる基であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）
で表される反復単位のうちの少なくとも一つを含む上記（１）に記載の抗菌性ペプチド含有ポリマー。
（３）前記式（１）～（３）において、－Ｗ－が、下記式：
Figure JPOXMLDOC01-appb-C000040
 (Wherein R 2 and R 3 are each independently a hydrogen atom or a methyl group, U 2 is a single bond, O or NH, U 3 is O or NH, and V is a single bond or A spacer, W 1 is a hydrogen atom or a group consisting of all or part of the linker W to which halogen may be added at the end, and Y has a single bond or a substituent having a molecular weight of 10 to 3000 It may be a linear or branched alkylene chain or polyoxyalkylene chain.)
The antibacterial peptide-containing polymer according to (1), which comprises at least one of the repeating units represented by
(3) In the above formulas (1) to (3), -W- represents the following formula:
Figure JPOXMLDOC01-appb-C000041
 （式中、ｔは、１～７の自然数である。）のいずれかを有するリンカーであり、かつ、Ｙが、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリエチレングリコール鎖である、上記（１）または（２）に記載の抗菌性ペプチド含有ポリマー。
（４）前記式（１）の－Ｗ－Peptideが、以下に表される式（１１）～（２７）：
Figure JPOXMLDOC01-appb-C000041
 (Wherein t is a natural number of 1 to 7), and Y may have a single bond or a substituent having a molecular weight of 10 to 3000, linear or branched The antibacterial peptide-containing polymer according to the above (1) or (2), which is an alkylene chain or a polyethylene glycol chain.
(4) -W-Peptide in the formula (1) is represented by the following formulas (11) to (27):
Figure JPOXMLDOC01-appb-C000042
（ここで、Peptideは前記と同じである。），
Figure JPOXMLDOC01-appb-C000042
(Where Peptide is the same as above),
Figure JPOXMLDOC01-appb-C000043
 （ここで、Peptideは前記と同じであり、ｉは１～１５の整数である。），
Figure JPOXMLDOC01-appb-C000043
 (Where Peptide is the same as described above, and i is an integer of 1 to 15),
Figure JPOXMLDOC01-appb-C000044
 （ここで、Peptideは前記と同じであり、ｉは１～７の整数である。），
Figure JPOXMLDOC01-appb-C000044
 (Here, Peptide is the same as described above, and i is an integer of 1 to 7.),
Figure JPOXMLDOC01-appb-C000045
 （ここで、Peptideは前記と同じであり、ｉは、１～７の整数であり、ｊは、１～１５の整数である。），
Figure JPOXMLDOC01-appb-C000045
 (Here, Peptide is the same as above, i is an integer of 1 to 7, and j is an integer of 1 to 15.),
Figure JPOXMLDOC01-appb-C000046
 （ここで、Peptideは前記と同じである。），
Figure JPOXMLDOC01-appb-C000046
 (Where Peptide is the same as above),
Figure JPOXMLDOC01-appb-C000047
 （ここで、Peptideは前記と同じであり、ｉは、１～１５の整数である。），
Figure JPOXMLDOC01-appb-C000047
 (Where Peptide is the same as described above, and i is an integer of 1 to 15),
Figure JPOXMLDOC01-appb-C000048
 （ここで、Peptideは前記と同じであり、ｉは１～８の整数であり、ｊは１～８の整数である。），
Figure JPOXMLDOC01-appb-C000048
 (Here, Peptide is the same as described above, i is an integer of 1 to 8, and j is an integer of 1 to 8.),
Figure JPOXMLDOC01-appb-C000049
 （ここで、Peptideは前記と同じであり、ｉは１～７の整数であり、ｊは１～７の整数である。），
Figure JPOXMLDOC01-appb-C000049
 (Here, Peptide is the same as above, i is an integer of 1 to 7, and j is an integer of 1 to 7.),
Figure JPOXMLDOC01-appb-C000050
 (ここで、Peptideは前記と同じである。），
Figure JPOXMLDOC01-appb-C000050
 (Where Peptide is the same as above),
Figure JPOXMLDOC01-appb-C000051
 （ここで、Peptideは前記と同じであり、ｉは２～７の整数である。），
Figure JPOXMLDOC01-appb-C000051
 (Here, Peptide is the same as described above, and i is an integer of 2 to 7.),
Figure JPOXMLDOC01-appb-C000052
 （ここで、Peptideは前記と同じであり、i は１～７の整数であり、j は２～７の整数である。），
Figure JPOXMLDOC01-appb-C000052
 (Where Peptide is the same as above, i is an integer from 1 to 7, and j is an integer from 2 to 7),
Figure JPOXMLDOC01-appb-C000053
 （ここで、Peptideは前記と同じであり、i は１～１５の整数であり、j は１～１０の整数である。），
Figure JPOXMLDOC01-appb-C000053
 (Where Peptide is the same as above, i is an integer of 1 to 15, and j is an integer of 1 to 10),
Figure JPOXMLDOC01-appb-C000054
 （ここで、Peptideは前記と同じであり、i は１～８の整数であり、j は１～７の整数である。），
Figure JPOXMLDOC01-appb-C000054
 (Where Peptide is the same as above, i is an integer of 1 to 8, and j is an integer of 1 to 7),
Figure JPOXMLDOC01-appb-C000055
 （ここで、Peptideは前記と同じであり、i は１～７の整数であり、j は１～７の整数である。），
Figure JPOXMLDOC01-appb-C000055
 (Where Peptide is the same as above, i is an integer of 1 to 7, and j is an integer of 1 to 7),
Figure JPOXMLDOC01-appb-C000056
 （ここで、Peptideは前記と同じであり、ｉは２～７の整数である。）
Figure JPOXMLDOC01-appb-C000056
 (Here, Peptide is the same as described above, and i is an integer of 2 to 7.)
Figure JPOXMLDOC01-appb-C000057
 （ここで、Peptideは前記と同じであり、i は１～１０の整数であり、j は１～１５の整数である。）, 
Figure JPOXMLDOC01-appb-C000057
 (Wherein Peptide is the same as above, i is an integer of 1 to 10, and j is an integer of 1 to 15),
Figure JPOXMLDOC01-appb-C000058
 （ここで、Peptideは前記と同じであり、i は２～７の整数である。）、及び
Figure JPOXMLDOC01-appb-C000058
 (Where Peptide is the same as above and i is an integer from 2 to 7), and
Figure JPOXMLDOC01-appb-C000059
 （ここで、Peptideは前記と同じであり、i は１～１０の整数である。）
からなる群から選ばれ、かつ、
前記式（２）の－Ｗ1が、下記式（３１）～（３８）：
Figure JPOXMLDOC01-appb-C000059
 (Here, Peptide is the same as described above, and i is an integer of 1 to 10.)
Selected from the group consisting of
In formula (2), -W 1 represents the following formulas (31) to (38):
Figure JPOXMLDOC01-appb-C000060
 （ここで、ｉは、１～７の整数であり、ｊは１～８の整数であり、ｋは１～１５の整数であり、ｌは０～８の整数であり、ｈは１～５の整数であり、Ｘは、Ｃｌ、Ｂｒ又はＩである）
からなる群から選ばれる、前記（２）に記載の抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000060
 (Where i is an integer from 1 to 7, j is an integer from 1 to 8, k is an integer from 1 to 15, l is an integer from 0 to 8, and h is from 1 to 5) And X is Cl, Br or I)
The antibacterial peptide-containing polymer according to (2), selected from the group consisting of:
上記抗菌性ペプチド含有ポリマーにおいて、前記式（１）中の－Ｗ－Peptideと前記式（２）中の－Ｗ1の好ましい組み合わせは、以下に示される組み合わせである。
式（１１）－式（３１）；式（１２）－式（３１）；式（１３）－式（３２）；式（１４）－式（３２）；式（１５）－式（３３）；式（１６）－式（３３）；式（１７）－式（３４）；式（１８）－式（３５）；式（１９）－式（３３）；式（２０）－式（３１）；式（２１）－式（３２）；式（２２）－式（３６）；式（２３）－式（３７）；式（２４）－式（３８）；式（２５）－式（３９）；式（２６）－式（３９）；式（２７）－式（３３）；式（２８）－式（３３）。 In the antibacterial peptide-containing polymer, a preferred combination of -W-Peptide in the formula (1) and -W 1 in the formula (2) is a combination shown below.
Formula (11) -Formula (31); Formula (12) -Formula (31); Formula (13) -Formula (32); Formula (14) -Formula (32); Formula (15) -Formula (33); Formula (16) -Formula (33); Formula (17) -Formula (34); Formula (18) -Formula (35); Formula (19) -Formula (33); Formula (20) -Formula (31); Formula (21) -Formula (32); Formula (22) -Formula (36); Formula (23) -Formula (37); Formula (24) -Formula (38); Formula (25) -Formula (39); Formula (26) -Formula (39); Formula (27) -Formula (33); Formula (28) -Formula (33).
（５）前記式（１）の－Ｗ－Peptideが、下記式： (5) -W-Peptide in the formula (1) is represented by the following formula:
Figure JPOXMLDOC01-appb-C000061
 （ここで、Peptideは前記と同じである。）であり、前記式（２）のＷ1が、下記式：
Figure JPOXMLDOC01-appb-C000061
 (Where Peptide is the same as above), and W 1 in the formula (2) is the following formula:
Figure JPOXMLDOC01-appb-C000062
 である前記（２）に記載の抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000062
 The antibacterial peptide-containing polymer as described in (2) above.
（６）前記スペーサーＶが、重量平均分子量が５０～１０００の置換基を有してもよい直鎖又は分岐状のポリエチレングリコール、又は分子量が１０～５００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖、或いはそれらの組合せである、前記（１）～（５）のいずれかに記載の抗菌性ペプチド含有ポリマー。 (6) The spacer V may have a linear or branched polyethylene glycol having a weight average molecular weight of 50 to 1000, or a linear chain optionally having a substituent having a molecular weight of 10 to 500. Alternatively, the antibacterial peptide-containing polymer according to any one of (1) to (5), which is a branched alkylene chain or a combination thereof.
（７）下記式（４） (7) Following formula (4)
Figure JPOXMLDOC01-appb-C000063
 で表される反復単位、及び下記式（５）
Figure JPOXMLDOC01-appb-C000063
 And a repeating unit represented by the following formula (5):
Figure JPOXMLDOC01-appb-C000064
で表される反復単位を含む抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000064
The antibacterial peptide containing polymer containing the repeating unit represented by these.
（８）下記式（１）、（２）及び（３） (8) The following formulas (1), (2) and (3)
Figure JPOXMLDOC01-appb-C000065
 で表される反復単位を含む抗菌性ペプチド含有ポリマーであって、
式（１）中、Ｕ1がＯで、－Ｖ－Ｗ－Peptideが、－ＰＥＧ－Ｓ－Ｃｙｓ－Peptide（ここで、ＰＥＧは、重量平均分子量が１０～２０００のポリエチレングリコールである。）又は-ＣＨ2ＣＨＯＨＣＨ2－Ｓ-Ｃｙｓ-Peptide、又は、Ｕ1がＮＨで、－Ｖ－Ｗ－Peptideが、-Peptide、－（ＣＨ2）3－Ｓ－Ｃｙｓ－Peptide、－ＣＨ2－（Ｃ2Ｈ4Ｏ）3－Ｃ3Ｈ6－ＮＨ－ＣＯＣＨ2－Ｓ－Ｃｙｓ－Peptide、及び下記式：
Figure JPOXMLDOC01-appb-C000065
 An antibacterial peptide-containing polymer comprising a repeating unit represented by
In the formula (1), U 1 is O and -VW-Peptide is -PEG-S-Cys-Peptide (where PEG is polyethylene glycol having a weight average molecular weight of 10 to 2000) or -CH 2 CHOHCH 2 -S-Cys-Peptide, or U 1 is NH, -VW-Peptide is -Peptide,-(CH 2 ) 3 -S-Cys-Peptide, -CH 2- (C 2 H 4 O) 3 —C 3 H 6 —NH—COCH 2 —S-Cys-Peptide and the following formula:
Figure JPOXMLDOC01-appb-C000066
 からなる群より選ばれ、かつ、
式（３）中、Ｕ3がＯで、－Ｙ－Ｈが、－（ＣＨ2）11－ＣＨ3、又は－ＰＥＧ－Ｒ3（ここで、ＰＥＧは、重量平均分子量が１０～２０００のポリエチレングリコールであり、Ｒ3はＯＨ又はＣＨ3である。）、又はＵ3がＮＨで、－Ｙが単結合である、請求項２に記載の抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000066
 Selected from the group consisting of and
In the formula (3), U 3 is O, -YH is-(CH 2 ) 11 -CH 3 , or -PEG-R 3 (wherein PEG is a polyethylene having a weight average molecular weight of 10 to 2000) The antibacterial peptide-containing polymer according to claim 2, wherein R 3 is OH or CH 3 ), or U 3 is NH and —Y is a single bond.
（９）前記ポリマー中に、前記Peptide含有単位を１～１００モル％の割合で含有する前記（１）～（８）のいずれかに記載の抗菌性ペプチド含有ポリマー。
（１０）前記ポリマー中に、前記Peptide含有単位を、０．４～２０モル％（より好ましくは、０．４～２モル％）の割合で含有する前記（９）に記載の抗菌性ペプチド含有ポリマー。 (9) The antibacterial peptide-containing polymer according to any one of (1) to (8), wherein the Peptide-containing unit is contained in the polymer at a ratio of 1 to 100 mol%.
(10) The antibacterial peptide content according to (9), wherein the Peptide-containing unit is contained in the polymer at a ratio of 0.4 to 20 mol% (more preferably 0.4 to 2 mol%). polymer.
（１１）前記ペプチドが、
－ＨＮ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｉｌｅ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＨＮ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＨＮ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｖａｌ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＨＮ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｉｌｅ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｖａｌ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯ－、及びＨ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－からなる群より選ばれるペプチドである前記（１）～（１０）のいずれかに記載の抗菌性ペプチド含有ポリマー。 (11) The peptide is
-HN-Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -NH-Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -NH -Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-CONH 2 , -HN-Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -HN-Arg -Leu-Tyr-Leu-Arg-Val-Gly-Arg-Arg-CONH 2 , -NH-Arg-Leu-Arg-Leu-Arg-Val-Gly-Arg-Arg-CONH 2 , -NH-Ala-Leu -Tyr-Leu-Ala-Val- Arg-Arg-Arg-CONH 2, -HN-Arg-Leu Leu-Leu-Arg-Val- Gly-Arg-Arg-CONH 2, H 2 N-Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-CO-, H 2 N-Arg-Leu- Arg-Leu-Arg-Ile-Gly-Arg-Arg-CO-, H 2 N-Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-CO-, H 2 N-Arg-Leu- Leu-Leu-Arg-Ile-Gly-Arg-Arg-CO-, H 2 N-Arg-Leu-Tyr-Leu-Arg-Val-Gly-Arg-Arg-CO-, H 2 N-Arg-Leu- Arg-Leu-Arg-Val- Gly-Arg-Arg-CO-, H 2 N-Ala-Leu-Tyr-Leu-Ala-Val-Arg-Ar -Arg-CO-, and H 2 N-Arg-Leu- Leu-Leu-Arg-Val-Gly-Arg-Arg-CO- wherein a peptide chosen from the group consisting of (1) either - (10) An antibacterial peptide-containing polymer according to claim 1.
（１２）前記（１）～（１１）のいずれかに記載の抗菌性ペプチド含有ポリマーが繊維上にコーティングされた抗菌性繊維。
（１３）前記抗菌性ペプチド含有ポリマーが、ペプチド存在量で、布１ｃｍ2あたり０．００１μモル以上０．３μモル以下存在する、前記（１２）に記載の抗菌性繊維。
（１４）医療用用途またはサニタリー用途における前記（１１）または（１２）に記載の抗菌性繊維。
（１５）前記（１）～（１１）のいずれかに記載の抗菌性ペプチド含有ポリマーを繊維上にコーティングすることにより、抗菌性ペプチドを繊維に固定化する方法。
（１６）前記（１）～（１１）のいずれかに記載の抗菌性ペプチド含有ポリマーを含有するポリマーエマルジョン。 (12) An antibacterial fiber in which the antibacterial peptide-containing polymer according to any one of (1) to (11) is coated on a fiber.
(13) The antibacterial fiber according to (12), wherein the antibacterial peptide-containing polymer is present in an amount of 0.001 μmol or more and 0.3 μmol or less per 1 cm 2 of the cloth.
(14) The antibacterial fiber according to the above (11) or (12) in medical use or sanitary use.
(15) A method for immobilizing an antibacterial peptide on a fiber by coating the antibacterial peptide-containing polymer according to any one of (1) to (11) on the fiber.
(16) A polymer emulsion containing the antibacterial peptide-containing polymer according to any one of (1) to (11).
（１７）下記式（９） (17) Formula (9) below
Figure JPOXMLDOC01-appb-C000067
 （式中Ｒ1は、水素原子又はメチル基であり、Ｕ1は、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチド又は誘導体化したペプチドと反応する官能基である。）
で表される反復単位を含むポリマーに、下記式（１０）：
Figure JPOXMLDOC01-appb-C000067
 Wherein R 1 is a hydrogen atom or a methyl group, U 1 is O or NH, V is a single bond or a spacer, Wo is a functional group that reacts with a peptide or a derivatized peptide. is there.)
In the polymer containing the repeating unit represented by the following formula (10):
Figure JPOXMLDOC01-appb-C000068
 （ここで、Peptideは、下記式で表されるアミノ酸配列：
－ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基（例えば、アジド基、スルフヒドリル基、ハロゲン基、アルキン基、カルボニル基、ヒドロキシルアミノ基）を有する分子量５０～５００の分子である。）
で表されペプチド又はその誘導体を反応させることにより、抗菌性ペプチド含有ポリマーを作製する方法。
Figure JPOXMLDOC01-appb-C000068
 (Here, Peptide is an amino acid sequence represented by the following formula:
-NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxylamino group) that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. 50 to 500 molecules. )
A method for producing an antibacterial peptide-containing polymer by reacting a peptide or a derivative thereof.
（１８）下記式（９）および（３） (18) The following formulas (9) and (3)
Figure JPOXMLDOC01-appb-C000069
 （式中Ｒ1及びＲ3は、それぞれ独立に水素原子又はメチル基であり、Ｕ1及びＵ3は、それぞれ独立にＯ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチド又は誘導体化したペプチドと反応する官能基であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）
で表される反復単位を含むポリマーに、下記式（１０）：
Figure JPOXMLDOC01-appb-C000069
 Wherein R 1 and R 3 are each independently a hydrogen atom or a methyl group, U 1 and U 3 are each independently O or NH, V is a single bond or a spacer, Wo is A functional group that reacts with a peptide or a derivatized peptide, and Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000.
In the polymer containing the repeating unit represented by the following formula (10):
Figure JPOXMLDOC01-appb-C000070
 （ここで、Peptideは、下記式で表されるアミノ酸配列：
－ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基（例えば、アジド基、スルフヒドリル基、ハロゲン基、アルキン基、カルボニル基、ヒドロキシルアミノ基）を有する分子量５０～５００の分子である。）
で表されペプチド又はその誘導体を反応させることにより、抗菌性ペプチド含有ポリマーを作製する方法。
Figure JPOXMLDOC01-appb-C000070
 (Here, Peptide is an amino acid sequence represented by the following formula:
-NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxylamino group) that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. 50 to 500 molecules. )
A method for producing an antibacterial peptide-containing polymer by reacting a peptide or a derivative thereof.
（１９）１前記式（９）で表される反復単位が下記式（６） (19) 1 The repeating unit represented by the formula (9) is represented by the following formula (6)
Figure JPOXMLDOC01-appb-C000071
 （ここで、式中Ｒ2は、水素原子又はメチル基である。）
であり、かつ、前記ペプチドが、前記式（１０）においてＺがＣｙｓである、前記（１７）又は（１８）に記載の方法。
Figure JPOXMLDOC01-appb-C000071
 (Here, R 2 is a hydrogen atom or a methyl group.)
And the peptide according to (17) or (18), wherein in the formula (10), Z is Cys.
（２０）以下の工程：
工程（１）：下記式（９） (20) The following steps:
Process (1): following formula (9)
Figure JPOXMLDOC01-appb-C000072
 （式中Ｒ1は、水素原子又はメチル基であり、Ｕ1は、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチドまたは誘導体化したペプチドと反応する官能基である。）
で表される反復単位を含むポリマーを含む溶液を繊維にコーティングする工程、及び
工程（２）：下記式：
Figure JPOXMLDOC01-appb-C000072
 (Wherein R 1 is a hydrogen atom or a methyl group, U 1 is O or NH, V is a single bond or a spacer, Wo is a functional group that reacts with a peptide or a derivatized peptide) is there.)
A step of coating a fiber with a solution containing a polymer containing a repeating unit represented by formula (2): and the following formula:
Figure JPOXMLDOC01-appb-C000073
 （ここで、Peptideは、下記式で表されるアミノ酸配列：
－ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基（例えば、アジド基、スルフヒドリル基、ハロゲン基、アルキン基、カルボニル基、ヒドロキシルアミノ基）を有する分子量５０～５００の分子である。）
で表される抗菌性ペプチド又はその誘導体を含む溶液に、前記ポリマー溶液をコーティングした繊維を浸漬する工程、及び
工程（３）：１～１０時間放置することによりポリマーにペプチドを結合させた後、該繊維を洗浄して未反応のペプチド又はその誘導体を除去するする工程、
からなる抗菌性繊維の製造方法。
Figure JPOXMLDOC01-appb-C000073
 (Here, Peptide is an amino acid sequence represented by the following formula:
-NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxylamino group) that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. 50 to 500 molecules. )
A step of immersing the fiber coated with the polymer solution in a solution containing the antibacterial peptide represented by the following: and step (3): after allowing the peptide to bind to the polymer by leaving it for 1 to 10 hours, Washing the fibers to remove unreacted peptides or derivatives thereof;
The manufacturing method of the antimicrobial fiber which consists of.
（２１）以下の工程：
工程（１）：下記式（９）および（３） (21) The following steps:
Step (1): Formulas (9) and (3) below
Figure JPOXMLDOC01-appb-C000074
 （式中Ｒ1及びＲ3は、それぞれ独立に水素原子又はメチル基であり、Ｕ1及びＵ3は、それぞれ独立にＯ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチド又は誘導体化したペプチドと反応する官能基であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）
で表される反復単位を含むポリマーを含む溶液を繊維にコーティングする工程、及び
工程（２）：下記式（１０）：
Figure JPOXMLDOC01-appb-C000074
 Wherein R 1 and R 3 are each independently a hydrogen atom or a methyl group, U 1 and U 3 are each independently O or NH, V is a single bond or a spacer, Wo is A functional group that reacts with a peptide or a derivatized peptide, and Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000.
A step of coating a fiber with a solution containing a polymer containing a repeating unit represented by formula (2): the following formula (10):
Figure JPOXMLDOC01-appb-C000075
 （ここで、Peptideは、下記式で表されるアミノ酸配列：
－ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基（例えば、アジド基、スルフヒドリル基、ハロゲン基、アルキン基、カルボニル基、ヒドロキシルアミノ基）を有する分子量５０～５００の分子である。）
で表される抗菌性ペプチド又はその誘導体を含む溶液に、前記ポリマー溶液をコーティングした繊維を浸漬する工程、及び
工程（３）：１～１０時間放置することによりポリマーにペプチドを結合させた後、該繊維を洗浄して未反応のペプチド又はその誘導体を除去するする工程、
からなる抗菌性繊維の製造方法。
Figure JPOXMLDOC01-appb-C000075
 (Here, Peptide is an amino acid sequence represented by the following formula:
-NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxylamino group) that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. 50 to 500 molecules. )
A step of immersing the fiber coated with the polymer solution in a solution containing the antibacterial peptide represented by the following: and step (3): after allowing the peptide to bind to the polymer by leaving it for 1 to 10 hours, Washing the fibers to remove unreacted peptides or derivatives thereof;
The manufacturing method of the antimicrobial fiber which consists of.
（２２）前記式（９）で表される反復単位が下記式（６） (22) The repeating unit represented by the formula (9) is represented by the following formula (6).
Figure JPOXMLDOC01-appb-C000076
 （ここで、式中Ｒ2は、水素原子又はメチル基である。）
であり、かつ、前記ペプチドが、前記式（１０）においてＺがＣｙｓである、前記（２０）又は（２１）に記載の抗菌性繊維の製造方法。
Figure JPOXMLDOC01-appb-C000076
 (Here, R 2 is a hydrogen atom or a methyl group.)
The method for producing an antibacterial fiber according to (20) or (21), wherein the peptide is Z in the formula (10) and Cys.
　本発明の抗菌性ペプチド含有ポリマーは、以下のように表すこともできる。
下記式（１ａ）： The antimicrobial peptide-containing polymer of the present invention can also be expressed as follows.
The following formula (1a):
Figure JPOXMLDOC01-appb-C000077
 （式中ｎは自然数であり、ｍ１は０又は自然数であり、Ｒ1、R2、Ｕ1、Ｕ2、Ｖ、Ｗ、Ｗ1、Peptideは、前記と同じである）で表される抗菌性ペプチド含有ポリマー、又は、
下記式（２ａ）：
Figure JPOXMLDOC01-appb-C000077
 Wherein n is a natural number, m1 is 0 or a natural number, and R 1 , R 2 , U 1 , U 2 , V, W, W 1 , and Peptide are the same as described above. A peptide-containing polymer, or
The following formula (2a):
Figure JPOXMLDOC01-appb-C000078
 （式中ｎ及びｍ２は自然数であり、Ｒ1、R3、Ｕ1、Ｕ3、Ｖ、Ｗ、Ｙ、Peptideは、前記と同じである。）で表される抗菌性ペプチド含有ポリマー、又は、
下記式（３ａ）：
Figure JPOXMLDOC01-appb-C000078
 Wherein n and m2 are natural numbers, and R 1 , R 3 , U 1 , U 3 , V, W, Y, and Peptide are the same as described above, or ,
The following formula (3a):
Figure JPOXMLDOC01-appb-C000079
 （式中ｎ、ｍ１及びｍ２は自然数であり、Ｒ1、R2、R3、Ｕ1、Ｕ2、Ｕ3、Ｖ、Ｗ、Ｗ1Ｙ、及びPeptideは、前記と同じである。）で表される抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000079
 (In the formula, n, m1 and m2 are natural numbers, and R 1 , R 2 , R 3 , U 1 , U 2 , U 3 , V, W, W 1 Y and Peptide are the same as described above.) An antibacterial peptide-containing polymer represented by
　本発明の抗菌性ペプチド含有ポリマーは、以下のように表すこともできる。
下記式（４ａ）： The antimicrobial peptide-containing polymer of the present invention can also be expressed as follows.
Following formula (4a):
Figure JPOXMLDOC01-appb-C000080
 （ここで、ｎは自然数であり、ｍ１は０又は自然数であり、Peptideは前記と同じである。）で表される抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000080
 (Where n is a natural number, m1 is 0 or a natural number, and Peptide is the same as described above).
　本発明の抗菌性ペプチド含有ポリマーを作製する方法は、以下のように表すこともできる。
工程（１）；下記式（１ｂ）： The method for producing the antibacterial peptide-containing polymer of the present invention can also be expressed as follows.
Step (1); the following formula (1b):
Figure JPOXMLDOC01-appb-C000081
 （式中、ｎは自然数であり、ｍ2は０又は自然数であり、Ｒ1、Ｒ3、Ｕ1、Ｕ3、Ｖ、Ｗo、Ｙは、上記と同じである。）
で表されるポリマーに、下記式（１０）：
Figure JPOXMLDOC01-appb-C000081
 (In the formula, n is a natural number, m2 is 0 or a natural number, and R 1 , R 3 , U 1 , U 3 , V, Wo, and Y are the same as above.)
In the polymer represented by the following formula (10):
Figure JPOXMLDOC01-appb-C000082
 （式中、Peptideは、上記と同じであり、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基（例えば、アジド基、スルフヒドリル基、ハロゲン基、アルキン基、カルボニル基、ヒドロキシルアミノ基）を有する分子量５０～５００の分子である。）
で表されるペプチド又はその誘導体を反応させることにより、抗菌性ペプチド含有ポリマーを作製する方法。
Figure JPOXMLDOC01-appb-C000082
 (Wherein Peptide is the same as above, and Z is a functional group that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker (for example, an azide group, a sulfhydryl group, a halogen group, an alkyne group, a carbonyl group). , A hydroxylamino group) and a molecular weight of 50 to 500.)
A method for producing an antibacterial peptide-containing polymer by reacting the peptide represented by
　本発明の抗菌性繊維を製造する方法は、以下のように表すこともできる。
下記式（１ｂ）： The method for producing the antibacterial fiber of the present invention can also be expressed as follows.
The following formula (1b):
Figure JPOXMLDOC01-appb-I000083
（式中、ｎは自然数であり、ｍ2は０又は自然数であり、Ｒ1、Ｒ3、Ｕ1、Ｕ3、Ｖ、Ｗo、Ｙは、上記と同じである。）
で表されるポリマーを含む溶液を繊維にコーティングする工程、及び
工程（２）；下記式（１０）：
Figure JPOXMLDOC01-appb-I000083
(In the formula, n is a natural number, m2 is 0 or a natural number, and R 1 , R 3 , U 1 , U 3 , V, Wo, and Y are the same as above.)
A step of coating a fiber with a solution containing a polymer represented by formula (2); and the following formula (10):
Figure JPOXMLDOC01-appb-C000084
 （ここで、Peptideは、上記と同じであり、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基（例えば、アジド基、スルフヒドリル基、ハロゲン基、アルキン基、カルボニル基、ヒドロキシルアミノ基）を有する分子量５０～５００の分子である。）
で表される抗菌性ペプチド又はその誘導体を含む溶液に、前記ポリマーをコーティングした繊維を浸漬する工程、及び
工程（３）；１～１０時間放置することによりポリマーにペプチドを結合させた後、該繊維を洗浄して未反応のペプチド又はその誘導体を除去するする工程、
からなる抗菌性繊維の製造方法。
Figure JPOXMLDOC01-appb-C000084
 (Here, Peptide is the same as above, and Z is a functional group that reacts with a hydrogen atom, hydroxyl group, Cys or Wo to form a linker (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group). , A hydroxylamino group) and a molecular weight of 50 to 500.)
A step of immersing the polymer-coated fiber in a solution containing the antibacterial peptide represented by the following formula: and step (3); after allowing the peptide to bind to the polymer by leaving it for 1 to 10 hours, Washing the fibers to remove unreacted peptides or derivatives thereof;
The manufacturing method of the antimicrobial fiber which consists of.
　本発明の一つの態様に従って、抗菌性繊維の製造に用いることができる抗菌性ペプチド（カブトムシディフェンシン由来改変ペプチド）含有ポリマーが提供できる。
　本発明の別の態様に従って、抗菌性ペプチド含有ポリマーを用いて、比較的容易な工程により、ペプチドがその活性を保持したまま固定化された繊維が提供できる。
　本発明の他の一つの態様に従って、比較的容易なペプチドの固定化方法により、ペプチド、特には抗菌性ペプチドが固定化された繊維が提供できる。
　このようにして提供される繊維は、ペプチドの活性を維持でき、特に、抗菌ペプチドを用いた場合は、提供される繊維は、抗菌活性を有し繰り返しの洗浄及び／又はオートクレーブによる滅菌処理を行っても抗菌活性を維持できる。
　本発明の繊維はまた安全性に優れているので、医療用用途、例えば、医療用包帯、シート、衣類等にも用いることができる。 According to one embodiment of the present invention, an antibacterial peptide (modified beetle defensin-derived modified peptide) -containing polymer that can be used in the production of an antibacterial fiber can be provided.
According to another aspect of the present invention, a fiber in which a peptide is immobilized while retaining its activity can be provided by a relatively easy process using an antibacterial peptide-containing polymer.
According to another embodiment of the present invention, a fiber on which a peptide, particularly an antibacterial peptide is immobilized, can be provided by a relatively easy peptide immobilization method.
The fiber provided in this way can maintain the activity of the peptide. In particular, when an antibacterial peptide is used, the provided fiber has antibacterial activity and is subjected to repeated washing and / or sterilization by autoclaving. However, antibacterial activity can be maintained.
Since the fiber of the present invention is also excellent in safety, it can be used for medical purposes such as medical bandages, sheets, clothes and the like.
　本明細書において、「Ａ～Ｂ」と表記する場合は、Ａを含みＡ以上であり、かつＢを含みＢ以下であることを意味する。
　本明細書において「繊維」とは、特に断りのない限り、糸または布地の原料としての繊維、その繊維から作られた糸、その糸から作られた布地あるいは繊維から作られた不織布のいずれも含む意味に用いられるが、「ポリマーを繊維にコーティングする」という場合は、糸または布地の原料としての繊維を意味する。一方、「ポリマーがコーティングされた繊維」という場合は、ポリマーがコーティングされた糸または布地の原料としての繊維、そのポリマーがコーティングされた繊維から作られた糸、その糸から作られた布地のいずれも含む。
　本明細書において、ポリペプチド及びペプチドは、特に断りのない限りは同じ意味で用いられ、互いに変更可能である。
　本明細書において、－Ｓ－Ｃｙｓ－Peptideと表記する場合は、Ｓはシステイン中のＳを意味する。
　本明細書において、ペプチドを表記する場合は、例えば、Ｌｅｕは、ロイシン残基を意味する。
　本明細書において、例えば、Ａ及びＢをポリマー内にランダムに有するポリマーを表す場合は、以下の何れの様式で表すこともできる。
　下記式： In this specification, “A to B” means that A is included and is A or more, and B is included and is B or less.
In this specification, unless otherwise specified, “fiber” includes any of fibers as a raw material for yarn or fabric, yarn made from the fiber, fabric made from the yarn, or nonwoven fabric made from the fiber. As used herein, the term “coats a polymer on a fiber” means a fiber as a raw material for yarn or fabric. On the other hand, the term “fiber coated with polymer” refers to either a fiber coated with a polymer or a fiber as a raw material of a fabric, a yarn made of a fiber coated with the polymer, or a fabric made of the yarn. Including.
In the present specification, a polypeptide and a peptide are used in the same meaning unless otherwise specified, and can be changed from each other.
In the present specification, when it is expressed as -S-Cys-Peptide, S means S in cysteine.
In the present specification, when a peptide is represented, for example, Leu means a leucine residue.
In the present specification, for example, when a polymer having A and B in the polymer at random is represented, it can be represented by any of the following modes.
Following formula:
Figure JPOXMLDOC01-appb-C000085
 及び下記式：
Figure JPOXMLDOC01-appb-I000086
の繰り返し単位を有するポリマー、又は
下記式：
Figure JPOXMLDOC01-appb-C000085
 And the following formula:
Figure JPOXMLDOC01-appb-I000086
Or a polymer having a repeating unit of the following formula:
Figure JPOXMLDOC01-appb-C000087
 （ここで、ｎおよびｍは自然数である。）
で表されるポリマー。
Figure JPOXMLDOC01-appb-C000087
 (Here, n and m are natural numbers.)
A polymer represented by
　本発明は、その一つの態様において、下記式で示される抗菌性ペプチド含有ポリマーを提供するものである。下記式（１ａ）： In one embodiment, the present invention provides an antibacterial peptide-containing polymer represented by the following formula. The following formula (1a):
Figure JPOXMLDOC01-appb-C000088
 （式中Ｒ1及びR2は、それぞれ独立に水素原子又はメチル基であり、ｎは自然数であり、ｍ１は０又は自然数であり、Ｕ1及びＵ2は、それぞれ独立にＯ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗは、単結合或いは、アミド基、チオエーテル基、チオエステル基、オキシム基、１，２，３－トリアゾール基又はエステル基を有するリンカーであり、Ｗ1は、水素原子又は末端にハロゲンを付加してもよいリンカーＷの全部又は一部からなる基であり、Peptideは、下記式で表されるアミノ酸配列：－ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するポリペプチドである。）で表される抗菌性ペプチド含有ポリマー、下記式（２ａ）：
Figure JPOXMLDOC01-appb-C000088
 Wherein R 1 and R 2 are each independently a hydrogen atom or a methyl group, n is a natural number, m1 is 0 or a natural number, and U 1 and U 2 are each independently O or NH. , V is a single bond or a spacer, W is a single bond or a linker having an amide group, a thioether group, a thioester group, an oxime group, a 1,2,3-triazole group or an ester group, and W 1 is , A hydrogen atom or a group consisting of all or part of a linker W to which halogen may be added to the terminal, and Peptide is an amino acid sequence represented by the following formula: —NH—X1-Leu-X2-Leu-X3 -X4-X5-X6-X7-CONH 2 or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- (wherein X1 is Arg or Ala, X2 Is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, X6 is Arg or Lys, and X7 is Arg or Lys. , Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.)) An antibacterial peptide-containing polymer represented by The following formula (2a):
Figure JPOXMLDOC01-appb-C000089
 （式中Ｒ1及びR3は、それぞれ独立に水素原子又はメチル基であり、ｎは自然数であり、ｍ２は自然数であり、Ｕ1は、単結合、Ｏ又はＮＨであり、Ｕ3は、Ｏ又はＮＨであり、Ｖ、Ｗ、及びPeptideは、上記と同じであり、Ｙは、水素原子又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）で表される抗菌性ペプチド含有ポリマー、又は下記式（３ａ）：
Figure JPOXMLDOC01-appb-C000089
 (Wherein R 1 and R 3 are each independently a hydrogen atom or a methyl group, n is a natural number, m2 is a natural number, U 1 is a single bond, O or NH, and U 3 is O or NH, V, W, and Peptide are the same as described above, and Y is a hydrogen atom or a linear or branched alkylene chain or polyoxy group that may have a substituent having a molecular weight of 10 to 3000. An antibacterial peptide-containing polymer represented by the following formula (3a):
Figure JPOXMLDOC01-appb-C000090
 （式中Ｒ1、Ｒ2及びR3は、それぞれ独立に水素原子又はメチル基であり、ｎ、ｍ１、及びｍ２は自然数であり、Ｕ1及びＵ2は、それぞれ独立に単結合、Ｏ又はＮＨであり、Ｕ3は、Ｏ又はＮＨでありＶ、Ｗ、Peptide、及びＹは、上記と同じである。）
で表される抗菌性ペプチド含有ポリマー。
Figure JPOXMLDOC01-appb-C000090
 Wherein R 1 , R 2 and R 3 are each independently a hydrogen atom or a methyl group, n, m1 and m2 are natural numbers, and U 1 and U 2 are each independently a single bond, O or NH, U 3 is O or NH, and V, W, Peptide, and Y are the same as above.)
An antibacterial peptide-containing polymer represented by
　本発明の抗菌性ペプチド含有ポリマーは、ホモポリマーであっても共重合体であっても良い。また、Peptideは、全てのモノマー単位に結合しても、一部のモノマー単位に結合しても良い。ポリマー中のPeptideが結合した単位（上記、R-C(CH2)-CO-U1-V-W-Peptideを意味し、本明細書では「Peptide含有単位」という。）の割合は、ポリマーを布にコーティング（塗布）した場合に目的の量のペプチドを塗布できる限り特に制限されず、０．１～１００モル％であるが、好ましくは０．４～２０モル％、より好ましくは０．４～２モル％である。ポリマー中のペプチド含有単位の割合を調整することにより、生地へのポリマーの付与（コーティング）量を制御でき、目的の抗菌性を持った均質で好適な抗菌性繊維を容易に製造できる。ポリマー中のPeptideが結合していない単位（上記、R-C(CH2)-CO-U2-V-W1及び／又はR-C(CH2)-CO-U3-Yを意味し、本明細書では「Peptide非含有単位」という。）の割合を適宜選択することにより、適当な物性、例えば粘度や溶解性を得ることができる。特には、共重合体において、Peptide非含有単位（R-C(CH2)-CO-U3-Y）を適宜選択することにより、目的の物性をポリマーに与えることができる。
　Ｙは、単結合又は分子量１０～３０００、好ましくは１００～２０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。Ｙの長さや種類を適宜選択することにより、ポリマーの物性を変更することができる。 The antibacterial peptide-containing polymer of the present invention may be a homopolymer or a copolymer. Peptide may be bonded to all monomer units or to some monomer units. The ratio of the unit to which Peptide is bonded in the polymer (referred to above as RC (CH 2 ) -CO-U 1 -VW-Peptide and referred to as “Peptide-containing unit” in this specification) is determined by coating the polymer on the cloth. (Coating) It is not particularly limited as long as the desired amount of peptide can be applied, and is 0.1 to 100 mol%, preferably 0.4 to 20 mol%, more preferably 0.4 to 2 mol. %. By adjusting the ratio of peptide-containing units in the polymer, the amount of polymer applied (coating) to the fabric can be controlled, and homogeneous and suitable antibacterial fibers having the desired antibacterial properties can be easily produced. A unit in which Peptide in the polymer is not bonded (meaning the above-mentioned RC (CH 2 ) —CO—U 2 —VW 1 and / or RC (CH 2 ) —CO—U 3 —Y, Appropriate physical properties such as viscosity and solubility can be obtained by appropriately selecting the ratio of “Peptide-free unit”. In particular, in the copolymer, the desired physical properties can be imparted to the polymer by appropriately selecting the Peptide-free unit (RC (CH 2 ) —CO—U 3 —Y).
Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000, preferably 100 to 2000. The physical properties of the polymer can be changed by appropriately selecting the length and type of Y.
　上記Peptideを構成するペプチドは、下記式で示されるアミノ酸配列からなるカブトムシディフェンシン由来改変ペプチドから選ばれ、該ペプチドはそのＮ末端又はＣ末端のいずれもリンカーに結合できる。
Ｈ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）。 The peptide constituting the peptide is selected from a modified beetle defensin-derived modified peptide consisting of an amino acid sequence represented by the following formula, and the peptide can be bound to a linker at either the N-terminus or C-terminus.
H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 (wherein X1 is Arg or Ala, X2 is Tyr, Arg or Leu, and X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys- CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.)
　特に好ましくは、上記ペプチドは、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、Ｈ2Ｎ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｉｌｅ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ2、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ2、Ｈ2Ｎ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｖａｌ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯ2及びＨ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2かららなる群より選ばれ、特に好ましくは、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2である。 Particularly preferably, the peptide is H 2 N-Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , H 2 N-Arg-Leu-Arg-Leu-Arg-Ile-Gly -Arg-Arg-CONH 2 , H 2 N-Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-CONH 2 , H 2 N-Arg-Leu-Leu-Leu-Arg-Ile-Gly -Arg-Arg-CONH 2 , H 2 N-Arg-Leu-Tyr-Leu-Arg-Val-Gly-Arg-Arg-CO 2 , H 2 N-Arg-Leu-Arg-Leu-Arg-Val-Gly -Arg-Arg-CO 2, H 2 N-Ala-Leu-Tyr-Leu-Ala-Val-Arg-Arg-Arg-CO 2 and Selected from 2 N-Arg-Leu-Leu -Leu-Arg-Val-Gly-Arg-Arg-CONH 2 Kararanaru group, particularly preferably, H 2 N-Arg-Leu -Leu-Leu-Arg-Val it is a -Gly-Arg-Arg-CONH 2 .
　本発明において抗菌性ペプチドとして用いることができるカブトムシディフェンシン由来の改変ペプチドは、例えば、特許第３２７３３１４号公報、特開２０１０－１８４０２２号公報、及び石橋ら、Eur. J. Biochem、1999 Dec; 266(2):616-23に記載されており、これらの文献は、引用することにより本明細書の一部である。 Examples of modified peptides derived from beetle defensin that can be used as antibacterial peptides in the present invention include, for example, Japanese Patent No. 3273314, Japanese Patent Application Laid-Open No. 2010-184022, and Ishibashi et al., Eur. J. Biochem, 1999 Dec; 266 ( 2): 616-23, which are hereby incorporated by reference.
　ペプチドの合成は、任意の慣用のペプチド合成法（固相合成、及び／又は液相合成）を用いて行うことができる。また、ポリマーとペプチドとをＣｙｓを介して結合する場合は、ペプチド合成において、合成ポリペプチドのＣ末端又はＮ末端にＣｙｓを導入することが可能であり、簡便である Peptide synthesis can be performed using any conventional peptide synthesis method (solid phase synthesis and / or liquid phase synthesis). In addition, when the polymer and the peptide are bonded via Cys, it is possible to introduce Cys at the C-terminal or N-terminal of the synthetic polypeptide in peptide synthesis, which is convenient.
　上記式（１ａ）中のＷは、ペプチドを固定化するためのリンカーである。リンカーは、ペプチドのＮ末端又はＣ末端をスペーサー又は直接ポリマー鎖の側鎖末端に結合するためのものであり、ペプチドのＮ末端又はＣ末端に導入することも、またスペーサーのペプチド結合部位に導入することも可能である。リンカーとしては、例えば、チオエーテル基、チオエステル基、オキシム基、１，２，３－トリアゾール基又はエステル基を有する化合物又はそれらの基そのものあげることができ、好ましくは、アミド基、チオエーテル基を有する化合物又はそれらの基であるが、これらに限定されない。本明細書では、リンカー又はその一部が導入されたペプチド等、本発明の目的に合わせてペプチドが修飾されたものを、ペプチド誘導体と言う。 W in the above formula (1a) is a linker for immobilizing the peptide. The linker is used to connect the N-terminal or C-terminal of the peptide to the spacer or directly to the side chain terminal of the polymer chain, and can be introduced at the N-terminal or C-terminal of the peptide or at the peptide binding site of the spacer. It is also possible to do. Examples of the linker include a compound having a thioether group, a thioester group, an oxime group, a 1,2,3-triazole group or an ester group, or a group thereof, preferably a compound having an amide group or a thioether group. Or a group thereof, but is not limited thereto. In the present specification, a peptide in which a peptide is modified for the purpose of the present invention, such as a peptide into which a linker or a part thereof is introduced, is referred to as a peptide derivative.
　ペプチド末端又はスペーサー末端の一方にリンカーを導入する場合は、リンカーを導入しないペプチド末端又はスペーサー末端の官能基と反応性を有するリンカーを、リンカーが導入されるペプチド末端又はスペーサー末端に導入し、結合反応を行うことによりペプチドをスペーサーに結合できる。
　ペプチド末端及びスペーサー末端の両者にリンカーを導入する場合は、一方にリンカーを構成する一部（リンカーパーツＷX）を導入し、他方にリンカーを構成する他の一部（リンカーパーツＷY）を導入し、両リンカーパーツを反応させることにより、ペプチドをスペーサーに結合できる。その結果、Ｗx及びＷyからリンカーＷが作られる。
　上記においてスペーサーを用いない場合は、ポリマー鎖の側鎖末端にリンカー又はリンカーパーツを導入して、ポリマー鎖の側鎖末端に直接ペプチドを結合することもできる。 When a linker is introduced at one of the peptide end or spacer end, a linker having reactivity with the functional group at the peptide end or spacer end to which the linker is not introduced is introduced at the peptide end or spacer end where the linker is introduced, and bonded. The peptide can be bound to the spacer by carrying out the reaction.
When a linker is introduced into both the peptide end and the spacer end, a part constituting the linker (linker part W X ) is introduced on one side and the other part constituting the linker (linker part W Y ) is provided on the other side. By introducing and reacting both linker parts, the peptide can be bound to the spacer. As a result, a linker W is made from W x and W y .
When no spacer is used in the above, a linker or linker part can be introduced at the side chain end of the polymer chain, and the peptide can be directly bonded to the side chain end of the polymer chain.
　ペプチドのＮ末端のアミノ基又はＣ末端のカルボキシル基と反応性を有する官能基を結合に用いる場合は、リンカーはアミド基又はエステル基となる。リンカーとなる当該官能基をスペーサーの末端に導入することもできるが、それを末端に有するスペーサーを用いることもできる。
　リンカー部位は、ペプチドのＮ末端又はＣ末端、或いはスペーサー末端のいずれかに導入してリンカーとしての機能を果たすことができるが、ペプチド末端及びスペーサー末端の両者に導入して、それぞれのリンカー部位が反応してリンカーとしての機能を果たすこともできる。例えば、ペプチド末端及びスペーサー末端のいずれかにチオールを導入し、他方にマレイミド基を導入して、チオールとマレイミド基の反応により、チオエーテル基を有するリンカーを介してペプチドをスペーサーに結合することもできる。 When a functional group having reactivity with the N-terminal amino group or the C-terminal carboxyl group of the peptide is used for bonding, the linker is an amide group or an ester group. The functional group to be a linker can be introduced at the end of the spacer, but a spacer having it at the end can also be used.
The linker site can be introduced into either the N-terminus or C-terminus of the peptide or the spacer terminus to serve as a linker. However, the linker site can be introduced into both the peptide terminus and the spacer terminus, It can also react to serve as a linker. For example, a thiol can be introduced at either the peptide end or the spacer end, a maleimide group can be introduced into the other, and the peptide can be bound to the spacer via a linker having a thioether group by the reaction of the thiol and the maleimide group. .
　ペプチドのＮ末端がスペーサーに結合する場合は、Ｎ末端のアミノ基を、カルボキシル基、ハロゲン、アルデヒド基、又はイソシアネート基等の官能基を有するスペーサーと反応させてスペーサーに結合することができる。アミノ基とこれらの官能基との反応は、常法により行うことができるが、例えば、縮合反応、付加反応等をあげることができる。
　ペプチドのＮ末端のスペーサーへの結合は、Ｎ末端にリンカー部位としてチオール基、例えば、システインやチオグリコール酸等を導入し、ＳＨ基と反応するマレイミド基、ハロゲン、チオエステル基、ブロモアセチル基等の官能基を有するスペーサーと反応させてスペーサーに結合することもできる。これにより、チオエーテル基を含むリンカーによりペプチドとスペーサーが結合される。ＳＨ基とこれらの官能基との反応は、常法により行うことができるが、例えば、マレイミド基との反応はマイケル付加反応により行うことができる。また、ハロゲンとの反応は求核置換反応により行うことができ、チオエステルとの反応は、エステル交換反応による。 When the N-terminal of the peptide is bonded to a spacer, the amino group at the N-terminal can be bonded to the spacer by reacting with a spacer having a functional group such as a carboxyl group, a halogen, an aldehyde group, or an isocyanate group. The reaction between the amino group and these functional groups can be performed by a conventional method, and examples thereof include a condensation reaction and an addition reaction.
The peptide is bound to the N-terminal spacer by introducing a thiol group such as cysteine or thioglycolic acid as a linker site at the N-terminus, and reacting with the SH group such as maleimide group, halogen, thioester group, bromoacetyl group, etc. It can also be reacted with a spacer having a functional group to bind to the spacer. Thereby, a peptide and a spacer are couple | bonded by the linker containing a thioether group. The reaction between the SH group and these functional groups can be performed by a conventional method. For example, the reaction with the maleimide group can be performed by a Michael addition reaction. The reaction with halogen can be performed by nucleophilic substitution reaction, and the reaction with thioester is by transesterification.
　また、ペプチドのＮ末端にリンカー部位としてアルキンを持つ、プロピオール酸、３－ブチン酸等を導入し、アルキンと反応するアジド基を有するスペーサーと反応させてスペーサーに結合することもできる。アルキンとアジド基との反応は、常法により行うことができるが、例えば、フイスゲン反応により行うことができる。
　また、ペプチドのＮ末端にリンカー部位としてアルコキシアミノ基を持つ、３-オキシアミノプロピオン酸、４－オキシアミノ酪酸等を導入し、アルコキシアミノ基と反応するケトンを有するスペーサーと反応させてスペーサーに結合することもできる。アルコキシアミノ基とケトンとの反応は、常法により行うことができるが、例えば、オキシム化反応により行うことができる。
　また、これらのリンカー部位はスペーサーとペプチドの部位を各々逆に導入し、結合させることも可能である。 Alternatively, propiolic acid, 3-butyric acid or the like having an alkyne as a linker site at the N-terminus of the peptide can be introduced and reacted with a spacer having an azide group that reacts with the alkyne to bind to the spacer. The reaction between the alkyne and the azide group can be performed by a conventional method, for example, by a Huisgen reaction.
In addition, 3-oxyaminopropionic acid, 4-oxyaminobutyric acid or the like having an alkoxyamino group as a linker site at the N-terminal of the peptide is introduced and reacted with a spacer having a ketone that reacts with the alkoxyamino group to bind to the spacer. You can also The reaction between the alkoxyamino group and the ketone can be performed by a conventional method, for example, by an oximation reaction.
In addition, these linker sites can be joined by introducing a spacer and a peptide site in reverse.
　ペプチドのＣ末端がスペーサーに結合する場合は、Ｃ末端のカルボキシル基を、アミノ基、水酸基等の官能基を有するスペーサーと反応させてスペーサーに結合することができる。カルボキシル基とこれらの官能基との反応は、常法により行うことができるが、例えば、縮合剤を用いた縮合反応をあげることができる。
　ペプチドのＣ末端のスペーサーへの結合は、Ｃ末端にリンカー部位としてチオール基、例えば、システインや２－アミノ－１－エタンチオール等を導入し、ＳＨ基と反応するマレイミド基、ハロゲン、チオエステル基、アルデヒド基、ブロモアセチル基等の官能基を有するスペーサーと反応させてスペーサーに結合することもできる。ＳＨ基とこれらの官能基との反応は、上記のような常法により行うことができる。 When the C-terminal of the peptide is bonded to the spacer, the C-terminal carboxyl group can be bonded to the spacer by reacting with a spacer having a functional group such as an amino group or a hydroxyl group. The reaction between the carboxyl group and these functional groups can be carried out by a conventional method, and examples thereof include a condensation reaction using a condensing agent.
The peptide is bound to the C-terminal spacer by introducing a thiol group, for example, cysteine or 2-amino-1-ethanethiol, as a linker site at the C-terminus and reacting with the SH group, a maleimide group, a halogen, a thioester group, It can also be reacted with a spacer having a functional group such as an aldehyde group or a bromoacetyl group to bind to the spacer. The reaction between the SH group and these functional groups can be performed by the conventional method as described above.
　上記式（１ａ）～（３ａ）中のスペーサーは、ポリマーとペプチドを結合できるものであれば特に制限はないが、例えば、アルキレン鎖、ポリエチレングリコール鎖、糖鎖、ポリペプチド、又は構造内にそれらら２つ以上含むものをあげることができるが、好ましくは、アルキレン鎖又はポリエチレングリコール鎖を主成分とするものである。アルキレン鎖又はポリエチレングリコール鎖は、直鎖状、分岐状のいずれを用いることもでき、また、置換基を有していても良い。アルキレン鎖は、分子量が、好ましくは１０～５００，より好ましくは２０～２００である。ポリエチレングリコール鎖は、重量平均分子量が、好ましくは５０～１０００，より好ましくは１００～５００，更に好ましくは１００～３００である。また、他の結合反応、例えば、ポリマーとスペーサー、スペーサーとリンカー、又はリンカーとペプチドの結合を阻害しない限り、官能基を有していても良い。 The spacers in the above formulas (1a) to (3a) are not particularly limited as long as they can bind a polymer and a peptide. For example, an alkylene chain, a polyethylene glycol chain, a sugar chain, a polypeptide, or those in the structure Among them, those containing two or more are preferable, but those having an alkylene chain or polyethylene glycol chain as the main component are preferred. The alkylene chain or the polyethylene glycol chain can be either linear or branched and may have a substituent. The molecular weight of the alkylene chain is preferably 10 to 500, more preferably 20 to 200. The polyethylene glycol chain has a weight average molecular weight of preferably 50 to 1000, more preferably 100 to 500, and still more preferably 100 to 300. Moreover, as long as it does not inhibit the other coupling | bonding reaction, for example, the coupling | bonding of a polymer and a spacer, a spacer and a linker, or a linker and a peptide, you may have a functional group.
　上記スペーサーのポリマーへの結合は、モノマー単位のカルボキシル基と反応性を有する官能基を末端に持つ化合物を用いて、ポリマーを合成した後にポリマーのカルボキシル基と反応させて導入することにより、又はモノマーの時点で導入した後に重合することにより達成できる。ポリマーのカルボキシル基とスペーサーの官能基の結合は、特に制限がないが、ポリマーのカルボキシル基と反応性を有する官能基としては、アミノ基又は水酸基が好ましい。ポリマーへ結合するスペーサーは、ポリマーとの結合に影響がない限り、ポリマーとの結合末端とは反対の末端に、ペプチドとの結合のためのリンカー部分（官能基）を持っていても良い。 The spacer can be bonded to the polymer by synthesizing a polymer using a compound having a terminal functional group having reactivity with the carboxyl group of the monomer unit and then introducing it by reacting with the carboxyl group of the polymer, or the monomer. This can be achieved by polymerization after introduction at this point. The bond between the carboxyl group of the polymer and the functional group of the spacer is not particularly limited, but the functional group having reactivity with the carboxyl group of the polymer is preferably an amino group or a hydroxyl group. The spacer that binds to the polymer may have a linker moiety (functional group) for binding to the peptide at the end opposite to the binding end to the polymer, as long as the binding to the polymer is not affected.
　好ましいスペーサーの具体例を以下にあげるが、これらに限定されるものではない。
スペーサーＡ： Specific examples of preferable spacers are listed below, but are not limited thereto.
Spacer A:
Figure JPOXMLDOC01-appb-C000091
 スペーサーＢ：
Figure JPOXMLDOC01-appb-C000091
 Spacer B:
Figure JPOXMLDOC01-appb-C000092
 スペーサーＣ：
Figure JPOXMLDOC01-appb-C000092
 Spacer C:
Figure JPOXMLDOC01-appb-C000093
 （式中、Ｘは、Ｃｌ又はＢｒである。）
Figure JPOXMLDOC01-appb-C000093
 (In the formula, X is Cl or Br.)
　以下、スペーサー側又はペプチド側に導入するリンカー（リンカーパーツ）及びその導入方法の具体例を示すが、これらに限定されない。 Hereinafter, specific examples of a linker (linker part) to be introduced on the spacer side or peptide side and a method for introducing the linker will be shown, but the present invention is not limited thereto.
１．スペーサー側リンカーパーツ
[Ls-A]： 1. Spacer side linker parts
[Ls-A]:
Figure JPOXMLDOC01-appb-C000094
 リンカー導入法：スペーサー部の脱保護を行った後、スクシンイミジル型のリンカーを導入。
[Ls-B]
Figure JPOXMLDOC01-appb-C000094
 Linker introduction method: After deprotecting the spacer part, a succinimidyl type linker is introduced.
[Ls-B]
Figure JPOXMLDOC01-appb-C000095
 （式中、Ｘは、Ｃｌ又はＢｒある。）
リンカー導入法：モノマー時点で、スペーサー部の脱保護を行い、縮合により導入後、ポリマー化する。
[Ls-C]
Figure JPOXMLDOC01-appb-C000095
 (Wherein X is Cl or Br)
Linker introduction method: At the time of monomer, the spacer part is deprotected, introduced by condensation, and then polymerized.
[Ls-C]
Figure JPOXMLDOC01-appb-C000096
 リンカー導入法：モノマー時点で、スペーサー部の脱保護を行い、縮合により導入後、ポリマー化する。
Figure JPOXMLDOC01-appb-C000096
 Linker introduction method: At the time of monomer, the spacer part is deprotected, introduced by condensation, and then polymerized.
[Ls-D] [Ls-D]
Figure JPOXMLDOC01-appb-C000097
 リンカー導入法：モノマー時点で、スペーサー部の脱保護を行い、ヒドロキシアミノ基Boc保護体を縮合により導入後、ポリマー化する。その後ヒドロキシアミノ基の脱保護をおこなう。
[Ls-E]
Figure JPOXMLDOC01-appb-C000097
 Linker introduction method: At the time of the monomer, the spacer part is deprotected, and a hydroxyamino group Boc protector is introduced by condensation and then polymerized. Thereafter, the hydroxyamino group is deprotected.
[Ls-E]
Figure JPOXMLDOC01-appb-C000098
 リンカー導入法：ポリマー時点で、スペーサー部の脱保護を行い、ＳＨ基Ｔｒｔ保護体を縮合により導入後、ＳＨ基の脱保護をおこなう。
[Ls-F]
Figure JPOXMLDOC01-appb-C000098
 Linker introduction method: At the time of polymer, the spacer part is deprotected, and after the SH group Trt protector is introduced by condensation, the SH group is deprotected.
[Ls-F]
Figure JPOXMLDOC01-appb-C000099
 リンカー導入法：ポリマー時点でスペーサー部の脱保護を行った後、縮合で導入。
Figure JPOXMLDOC01-appb-C000099
 Linker introduction method: After deprotecting the spacer part at the time of polymer, it is introduced by condensation.
[Ls-G] [Ls-G]
Figure JPOXMLDOC01-appb-C000100
 リンカー導入法：モノマー時点で、スペーサー部の脱保護を行い、縮合により導入後、ポリマー化する。
Figure JPOXMLDOC01-appb-C000100
 Linker introduction method: At the time of monomer, the spacer part is deprotected, introduced by condensation, and then polymerized.
２．ペプチド側リンカーパーツ
[Lp-A]
Cysteine：
リンカー導入法：ペプチド製造時に、ペプチドＮ末又はＣ末に導入。
[Lp-B] 2. Peptide side linker parts
[Lp-A]
Cysteine:
Linker introduction method: Introduced into peptide N-terminal or C-terminal during peptide production.
[Lp-B]
Figure JPOXMLDOC01-appb-C000101
 リンカー導入法：ペプチドＮ末に導入できる。ＳＨ基保護カルボン酸を保護ペプチドに導入後脱保護する。
[Lp-C]
Figure JPOXMLDOC01-appb-C000101
 Linker introduction method: can be introduced into the peptide N-terminal. The SH group-protected carboxylic acid is introduced into the protected peptide and then deprotected.
[Lp-C]
Figure JPOXMLDOC01-appb-C000102
 リンカー導入法：ペプチドＮ末端に導入できる。保護ペプチドに導入後脱保護する。
Figure JPOXMLDOC01-appb-C000102
 Linker introduction method: can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide.
[Lp-D] [Lp-D]
Figure JPOXMLDOC01-appb-C000103
 リンカー導入法：ペプチドＮ末端に導入できる。保護ペプチドに導入後脱保護する。
[Lp-E]
Figure JPOXMLDOC01-appb-C000103
 Linker introduction method: can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide.
[Lp-E]
Figure JPOXMLDOC01-appb-C000104
 リンカー導入法：ペプチドＮ末端に導入できる。保護ペプチドにスクシンイミジル型のリンカーを導入し脱保護する。
[Lp-Ｆ]
Figure JPOXMLDOC01-appb-C000104
 Linker introduction method: can be introduced at the N-terminus of the peptide. A succinimidyl-type linker is introduced into the protected peptide for deprotection.
[Lp-F]
Figure JPOXMLDOC01-appb-C000105
 （式中、Ｘは、Ｃｌ又はＢｒある。）
リンカー導入法：ペプチドのＮ末端に導入できる。保護ペプチドに導入後脱保護する。
Figure JPOXMLDOC01-appb-C000105
 (Wherein X is Cl or Br)
Linker introduction method: can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide.
[Lp-G] [Lp-G]
Figure JPOXMLDOC01-appb-C000106
 リンカー導入法：ペプチドのＮ末端に導入できる。保護ペプチドに導入後脱保護する。
[Lp-H]
Figure JPOXMLDOC01-appb-C000106
 Linker introduction method: can be introduced at the N-terminus of the peptide. Deprotection after introduction into the protected peptide.
[Lp-H]
Figure JPOXMLDOC01-appb-C000107
 リンカー導入法：ペプチドのＮ末端に導入できる。ヒドロキシルアミノ基保護リンカーを保護ペプチドに導入後脱保護する。
[Lp-I]
Figure JPOXMLDOC01-appb-C000107
 Linker introduction method: can be introduced at the N-terminus of the peptide. A hydroxylamino group-protected linker is introduced into the protected peptide and then deprotected.
[Lp-I]
Figure JPOXMLDOC01-appb-C000108
 ペプチドのＣ末端に、例えば、以下の反応により導入できる。
Figure JPOXMLDOC01-appb-C000108
 It can introduce | transduce into the C terminal of a peptide by the following reaction, for example.
Figure JPOXMLDOC01-appb-C000109
Figure JPOXMLDOC01-appb-C000109
スペーサーとそれぞれのリンカーパーツの組み合わせ、及びそれによって生成されるリンカー構造を以下の表１に示す。 The combinations of spacers and respective linker parts, and the linker structures generated thereby are shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000110
Figure JPOXMLDOC01-appb-T000110
　本発明に使用するポリマーの合成方法は、特に限定されるものではない。また、ポリマーの合成においては、ポリマーを合成した後スペーサーを導入しても、また、スペーサーを有するモノマーを重合してポリマーを合成しても良い。また、ペプチドが結合するユニットとペプチドが結合しないユニットから構成されるポリマーは、それぞれのモノマーを共重合させ、その後ペプチドを結合するユニットにリンカーを介してペプチドを結合させる方法が好ましい。ペプチドを結合させるユニットのモノマー末端が一級アミンの場合、一級アミノ基を予め保護基にて保護したモノマーを、ペプチドが結合しないユニットであるスペーサーを有するモノマーとラジカル共重合した後、保護基を除去する方法が好ましい。その後、リンカーを介して、スペーサーとペプチドを結合することにより本発明の抗菌性ポリペプチド含有ポリマーを製造できる。 The method for synthesizing the polymer used in the present invention is not particularly limited. In the synthesis of the polymer, a spacer may be introduced after the polymer is synthesized, or a polymer may be synthesized by polymerizing a monomer having a spacer. Moreover, the polymer comprised from the unit which a peptide couple | bonds, and the unit which a peptide does not couple | bond together, the method of copolymerizing each monomer and then couple | bonding a peptide via the linker to the unit which couple | bonds a peptide is preferable. When the monomer terminal of the unit to which the peptide is bonded is a primary amine, the monomer in which the primary amino group is previously protected with a protecting group is radical-copolymerized with a monomer having a spacer that is a unit to which the peptide does not bind, and then the protecting group is removed. Is preferred. Then, the antimicrobial polypeptide containing polymer of this invention can be manufactured by couple | bonding a spacer and a peptide through a linker.
　ペプチドを結合しないユニットのモノマーの具体的な例としては、ポリ（エチレングリコール）メチルエーテル（メタ）アクリレート類、ポリ（エチレングリコール）（メタ）アクリレート類、（メタ）アクリル酸、ｎ－ブチル（メタ）アクリレート、iso－ブチル（メタ）アクリレート、sec－ブチル（メタ）アクリレート、t－ブチル（メタ）アクリレート、ｎ－ネオペンチル（メタ）アクリレート、iso－ネオペンチル（メタ）アクリレート、sec－ネオペンチル（メタ）アクリレート、ネオペンチル（メタ）アクリレート、ｎ－ヘキシル（メタ）アクリレート、iso－ヘキシル（メタ）アクリレート、ヘプチル（メタ）アクリレート、ｎ－オクチル（メタ）アクリレート、iso－オクチル（メタ）アクリレート、２－エチルヘキシル（メタ）アクリレート、ｎ－ノニル（メタ）アクリレート、iso－ノニル（メタ）アクリレート、ｎ－デシル（メタ）アクリレート、iso－デシル（メタ）アクリレート、ｎ－ドデシル（メタ）アクリレート、iso－ドデシル（メタ）アクリレート、ｎ－トリデシル（メタ）アクリレート、iso－トリデシル（メタ）アクリレート、ｎ－テトラデシル（メタ）アクリレート、iso－テトラデシル（メタ）アクリレート、ｎ－ペンタデシル（メタ）アクリレート、iso－ペンタデシル（メタ）アクリレート、ｎ－ヘキサデシル（メタ）アクリレート、iso－ヘキサデシル（メタ）アクリレート、ｎ－オクタデシル（メタ）アクリレート、iso－オクタデシル（メタ）アクリレート、シクロヘキシル（メタ）アクリレート、イソボニル（メタ）アクリレート、（メタ）アクリルアミド、Ｎ－メチル（メタ）アクリルアミド、Ｎ－エチル（メタ）アクリルアミド、Ｎ－プロピル（メタ）アクリルアミド、Ｎ－イソプロピル（メタ）アクリルアミド、Ｎ，Ｎ－ジメチル（メタ）アクリルアミド、Ｎ，Ｎ－ジエチル（メタ）アクリルアミド、Ｎ－ヒドロキシメチル（メタ）アクリルアミド、Ｎ－（２－ヒドロキシエチル）（メタ）アクリルアミド、Ｎ，Ｎ－ビス（ヒドロキシメチル）（メタ）アクリルアミド、Ｎ，Ｎ－ビス（２－ヒドロキシエチル）（メタ）アクリルアミドなどがあげることができる。 Specific examples of monomer units that do not bind peptides include poly (ethylene glycol) methyl ether (meth) acrylates, poly (ethylene glycol) (meth) acrylates, (meth) acrylic acid, n-butyl (meth) ) Acrylate, iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl (meth) acrylate, iso-neopentyl (meth) acrylate, sec-neopentyl (meth) acrylate , Neopentyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) acrylate, heptyl (meth) acrylate, n-octyl (meth) acrylate, iso-octyl (meth) acrylate, 2-ethylhexyl (meth) Acu Relate, n-nonyl (meth) acrylate, iso-nonyl (meth) acrylate, n-decyl (meth) acrylate, iso-decyl (meth) acrylate, n-dodecyl (meth) acrylate, iso-dodecyl (meth) acrylate, n-tridecyl (meth) acrylate, iso-tridecyl (meth) acrylate, n-tetradecyl (meth) acrylate, iso-tetradecyl (meth) acrylate, n-pentadecyl (meth) acrylate, iso-pentadecyl (meth) acrylate, n- Hexadecyl (meth) acrylate, iso-hexadecyl (meth) acrylate, n-octadecyl (meth) acrylate, iso-octadecyl (meth) acrylate, cyclohexyl (meth) acrylate, isobornyl (meth) acrylate, (meth) acrylic Mido, N-methyl (meth) acrylamide, N-ethyl (meth) acrylamide, N-propyl (meth) acrylamide, N-isopropyl (meth) acrylamide, N, N-dimethyl (meth) acrylamide, N, N-diethyl ( (Meth) acrylamide, N-hydroxymethyl (meth) acrylamide, N- (2-hydroxyethyl) (meth) acrylamide, N, N-bis (hydroxymethyl) (meth) acrylamide, N, N-bis (2-hydroxyethyl) ) (Meth) acrylamide and the like.
　一級アミノ基をあらかじめ保護基にて保護したモノマーは、特に構造を限定しないが、下記式（４６）（式中、Ｒ2は水素原子またはメチル基、Ｖはアルキレングリコール残基を含むスペーサー、Ｔは保護基を示す。）で表されるように、（メタ）アクリル基がアルキレングリコール残基を含むスペーサーＶを介した構造であることが好ましい。 The monomer in which the primary amino group is previously protected with a protecting group is not particularly limited in structure, but the following formula (46) (wherein R 2 is a hydrogen atom or a methyl group, V is a spacer containing an alkylene glycol residue, T Represents a protective group), the (meth) acrylic group preferably has a structure via a spacer V containing an alkylene glycol residue.
Figure JPOXMLDOC01-appb-C000111
Figure JPOXMLDOC01-appb-C000111
　保護基Ｔとしてはアミノ基を保護基できるものであれば何ら制限されず、任意のものを用いることができるが、なかでもｔ－ブトキシカルボニル基（Ｂｏｃ基）やベンジロキシカルボニル基（Ｚ基、Ｃｂｚ基）、９－フルオレニルメトキシカルボニル基（Ｆｍｏｃ基）などが好ましい。脱保護化は、トリフルオロ酢酸や塩酸、無水フッ化水素等を用いて、一般的な条件で行うことができる。 The protecting group T is not particularly limited as long as it can protect an amino group, and any one can be used. Among them, a t-butoxycarbonyl group (Boc group) or a benzyloxycarbonyl group (Z group, Cbz group), 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like are preferable. Deprotection can be performed under general conditions using trifluoroacetic acid, hydrochloric acid, anhydrous hydrogen fluoride, or the like.
　具体的なモノマーの例としては、下記式（４７）で表されるものをあげることができる。 Specific examples of the monomer include those represented by the following formula (47).
Figure JPOXMLDOC01-appb-C000112
Figure JPOXMLDOC01-appb-C000112
　本発明で用いるポリマーの合成溶媒としては、それぞれの単量体が溶解するものであればよく、例えば、メタノール、エタノール、イソプロパノール、ｎ－ブタノール、ｔ－ブチルアルコール、ｎ－ペンタノール等アルコール類、ベンゼン、トルエン、テトラヒドロフラン、ジオキサン、ジクロロメタン、クロロホルム、シクロヘキサノン、N，N－ジメチルホルムアミド、ジメチルスルホキシド、酢酸メチル、酢酸エチル、酢酸ブチル、メチルエチルケトン、メチルブチルケトン、エチレングリコールモノエチルエーテル、エチレングリコールモノメチルエーテル、エチレングリコールモノブチルエーテル等をあげることができる。これらの溶媒は、単独または２種以上の組み合わせで用いてもよい。 The polymer synthesis solvent used in the present invention is not particularly limited as long as each monomer can be dissolved. For example, alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol, Benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, Examples thereof include ethylene glycol monobutyl ether. These solvents may be used alone or in combination of two or more.
　重合開始剤としては通常のラジカル開始剤を用いることができ、例えば、２，２’－アゾビスイソブチルニトリル（以下「ＡＩＢＮ」という）、１，１’－アゾビス（シクロヘキサン－１ －カルボニトリル）等のアゾ化合物、過酸化ベンゾイル、過酸化ラウリル等の有機過酸化物等を挙げることができる。
　また、本発明で用いるポリマーの重量平均分子量は、３，０００以上が好ましい。重量平均分子量を３，０００以上とすることにより、モノマーとの分離が容易になり、さらに繊維への固着力がより高まる。より好ましくは１０，０００以上である。 As the polymerization initiator, a normal radical initiator can be used. For example, 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), etc. Organic peroxides such as azo compounds, benzoyl peroxide and lauryl peroxide.
In addition, the weight average molecular weight of the polymer used in the present invention is preferably 3,000 or more. By setting the weight average molecular weight to 3,000 or more, separation from the monomer is facilitated, and the fixing force to the fiber is further increased. More preferably, it is 10,000 or more.
　ペプチドとの結合反応のために、（スペーサーを含む）ポリマーへリンカー部位（官能基）を導入することができる。官能基としては、特に制限はないが、例えば、ハロゲン、カルボキシル基、アルデヒド基、イソシアネート基、チオエステル基、ブロモアセチル基、マレイミド基、チオール基等をあげることができる。導入するリンカーは、結合するペプチドの末端の官能基に応じて適宜選択できる。結合先がアミノ基である場合は、ハロゲン、カルボキシル基、アルデヒド基又はイソシアネート基を導入するのが好ましい。結合先がカルボキシル基である場合は、アミノ基、水酸基が好ましい。結合先が、チオール基、例えば、システインのＳＨ基である場合は、ハロゲン、チオエステル基、アルデヒド基、ブロモアセチル基、又はマレイミド基が好ましい。特に、リンカー部位としてペプチド末端にシステイン残基を導入した場合は、スペーサーの末端にマレイミド基、ハロゲン基を導入することが好ましい。 A linker site (functional group) can be introduced into a polymer (including a spacer) for a binding reaction with a peptide. Although there is no restriction | limiting in particular as a functional group, For example, a halogen, a carboxyl group, an aldehyde group, an isocyanate group, a thioester group, a bromoacetyl group, a maleimide group, a thiol group etc. can be mention | raise | lifted. The linker to be introduced can be appropriately selected according to the functional group at the terminal of the peptide to be bound. When the bond destination is an amino group, it is preferable to introduce a halogen, a carboxyl group, an aldehyde group or an isocyanate group. When the bond destination is a carboxyl group, an amino group and a hydroxyl group are preferable. When the bonding destination is a thiol group, for example, an SH group of cysteine, a halogen, a thioester group, an aldehyde group, a bromoacetyl group, or a maleimide group is preferable. In particular, when a cysteine residue is introduced at the peptide end as a linker site, it is preferable to introduce a maleimide group or a halogen group at the end of the spacer.
　ペプチドのスペーサーへの結合反応は、特に限定されず、縮合反応、チオエーテル化反応、エステル化反応、Huisgen反応、マイケル付加反応、オキシム化反応などにより行うことができるが、好ましくは、ペプチドのＣ末端又はＮ末端にＣｙｓ基またはチオール基を導入し、ポリマー中のマレイミド基との間で、マレイミド－チオールのマイケル付加反応を用いて、または、ポリマー中のハロゲン基との間でチオエーテル化反応を用いて、行うことができる The peptide binding reaction to the spacer is not particularly limited and can be carried out by condensation reaction, thioetherification reaction, esterification reaction, Huisgen reaction, Michael addition reaction, oximation reaction, etc., but preferably the C-terminal of the peptide Alternatively, a Cys group or a thiol group is introduced at the N-terminus, and a maleimide group in the polymer is used, a maleimide-thiol Michael addition reaction, or a halogen group in the polymer is used. Can be done
　ペプチド含有ポリマーの作製は、例えば、ペプチドがＣ末端又はＮ末端にＣｙｓ基を有する場合は、マレイミドユニットを有するポリマーが溶解する溶媒でありかつ水と混ざり合う溶媒中で行うのが好ましい。例えば、メタノール、エタノール、イソプロパノール等アルコール類、テトラヒドロフラン、ジオキサン、N，N－ジメチルホルムアミド、ジメチルスルホキシド等にポリマーを溶解し、それにペプチドを、例えば、水、に溶解したペプチド溶液を加え反応させることにより行うことができる。 For example, when the peptide has a Cys group at the C-terminus or N-terminus, the peptide-containing polymer is preferably prepared in a solvent that dissolves the polymer having a maleimide unit and is miscible with water. For example, by dissolving a polymer in alcohols such as methanol, ethanol, isopropanol, tetrahydrofuran, dioxane, N, N-dimethylformamide, dimethyl sulfoxide, etc., and reacting the peptide by adding a peptide solution dissolved in, for example, water. It can be carried out.
　以下、リンカー部位としてマレイミド基とチオール基（システイン）を用いた例をもとに説明するが、本発明はこれに限定されるものではない。
　ポリマーへのマレイミド基の導入は、１分子内にアミノ基と反応できる官能基とマレイミド基を持つ化合物を用いて行うことができる。例えば、マレイミドプロピオン酸Ｎ-スクシンイミジル、Ｎ－（α－マレイミドアセトキシ）スクシンイミドエステル、Ｎ－（４－マレイミドブチリルオキシ）スクシンイミド、Ｎ－（４－マレイミドブチリルオキシ）スルホスクシンイミドナトリウム塩、Ｎ－（６－マレイミドカプロイルオキシ）スクシンイミド、Ｎ－（８－マレイミドカプリルオキシ）スクシンイミド、Ｎ－（８－マレイミドカプリルオキシ）スルホスクシンイミドナトリウム塩、Ｎ－（１１－マレイミドウンデカノイルオキシ）スクシンイミド、Ｎ－（１１－マレイミドウンデカノイルオキシ）スルホスクシンイミドナトリウム塩などがあげることができる。これらの試薬は、単独または２種以上の組み合わせで用いても良い。 The following description is based on an example in which a maleimide group and a thiol group (cysteine) are used as linker sites, but the present invention is not limited to this.
Introduction of a maleimide group into a polymer can be performed using a compound having a maleimide group and a functional group capable of reacting with an amino group in one molecule. For example, maleimidopropionic acid N-succinimidyl, N- (α-maleimidoacetoxy) succinimide ester, N- (4-maleimidobutyryloxy) succinimide, N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt, N- ( 6-maleimidocaproyloxy) succinimide, N- (8-maleimidocapryloxy) succinimide, N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt, N- (11-maleimidoundecanoyloxy) succinimide, N- ( 11-maleimidoundecanoyloxy) sulfosuccinimide sodium salt and the like. These reagents may be used alone or in combination of two or more.
　本発明の抗菌性ペプチド含有ポリマーの一つの態様においては、下記式（４８）（式中、Ｒ1は水素原子又はメチル基であり、ｎは自然数であり、Ｖはスペーサーである。）で示されように、モノマー単位のマレイミドユニットに、ペプチドが、そのC末端又はＮ末端においてシステインを介して結合している。式中、－Ｓ－ＣｙｓのＳは、システイン残基中のＳを表す。 In one embodiment of the antibacterial peptide-containing polymer of the present invention, it is represented by the following formula (48) (wherein R 1 is a hydrogen atom or a methyl group, n is a natural number, and V is a spacer). As described above, the peptide is linked to the maleimide unit of the monomer unit via a cysteine at the C-terminus or N-terminus. In the formula, S in —S—Cys represents S in a cysteine residue.
Figure JPOXMLDOC01-appb-C000113
Figure JPOXMLDOC01-appb-C000113
　本発明の抗菌性ペプチド含有ポリマーは、全てが上記モノマー単位であっても良いが、上記モノマー単位と下記式（４９）（Ｒ1は水素原子又はメチル基であり、ｎは自然数であり、Ｖはスペーサーである。）のモノマー単位の混合物であっても良い。 The antibacterial peptide-containing polymer of the present invention may be all the monomer unit, but the monomer unit and the following formula (49) (R 1 is a hydrogen atom or a methyl group, n is a natural number, V May be a spacer).
Figure JPOXMLDOC01-appb-C000114
Figure JPOXMLDOC01-appb-C000114
　本発明のポリマーにおいてはまた、上記式（４８）と下記式（５０）（ｎは自然数であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖、好ましくは、置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリエチレングリコールである。）の共重合体、又は上記式（４８）、上記式（４９）と下記式（５０）（ｎは自然数である）の共重合体とすることもできる。 In the polymer of the present invention, the above formula (48) and the following formula (50) (n is a natural number, Y is a single bond or a linear or branched chain which may have a substituent having a molecular weight of 10 to 3000) Or an alkylene chain or a polyoxyalkylene chain, preferably a linear or branched alkylene chain or polyethylene glycol which may have a substituent, or a copolymer of the above formula (48) or the above formula ( 49) and the following formula (50) (n is a natural number).
Figure JPOXMLDOC01-appb-C000115
Figure JPOXMLDOC01-appb-C000115
　好ましいポリマーとして以下の式（５１）で表されるペプチド含有ポリマーをあげることができる。ここで、ｎ１は自然数、ｍ１は０又は自然数である。 A preferred polymer is a peptide-containing polymer represented by the following formula (51). Here, n1 is a natural number and m1 is 0 or a natural number.
Figure JPOXMLDOC01-appb-C000116
Figure JPOXMLDOC01-appb-C000116
　上記の例においては、ペプチドのポリマーへの結合は、マイケル付加反応を用いて、溶媒に溶解したポリマー溶液にペプチドを溶解したペプチド溶液を加えて反応させることにより行うことができる。また、マレイミドユニットに対するペプチドの割合は、目的に応じて適宜選択でき、例えば、全てのマレイミドユニットにペプチドを結合する場合は、マレイミドユニットとペプチドのモル比が１：１になるようにして結合反応を行う。 In the above example, the peptide can be bound to the polymer by adding a peptide solution in which the peptide is dissolved in a polymer solution in which the peptide is dissolved, using a Michael addition reaction. Moreover, the ratio of the peptide to the maleimide unit can be appropriately selected according to the purpose. For example, when the peptide is bound to all maleimide units, the coupling reaction is performed so that the molar ratio of the maleimide unit to the peptide is 1: 1. I do.
　本発明の別の態様において、本発明のペプチド含有ポリマーが繊維上に塗布された抗菌性繊維が提供される。本発明の抗菌性繊維は、ポリペプチドが繊維に固定化され、通常の使用においてはペプチドが容易に脱離することがなく、繰り返しの使用においても抗菌性が維持できる。 In another aspect of the present invention, an antibacterial fiber is provided in which the peptide-containing polymer of the present invention is coated on the fiber. In the antibacterial fiber of the present invention, the polypeptide is immobilized on the fiber, the peptide is not easily detached in normal use, and the antibacterial property can be maintained even in repeated use.
　本発明において繊維は、綿、麻、絹などの植物繊維、羊毛、獣毛、絹などの動物繊維、レーヨンなどの再生繊維、アセテートなどの半合成繊維、ポリエステル、ナイロン、アクリルなどの化学合成繊維、及びそれらの複合体などを意味するが、特にこれらに限定されるものではない。
　また 繊維の形態としては、ステープル、フィラメント糸ならびにその加工糸などからなる織物、編物を言うが、さらには繊維ウエブ形態でも良い。繊維ウエブとは、不織布やティッシュペーパー、ウエットティッシュ、食品包装紙などの紙類などを言う。 In the present invention, the fibers are plant fibers such as cotton, hemp and silk, animal fibers such as wool, animal hair and silk, regenerated fibers such as rayon, semi-synthetic fibers such as acetate, and chemically synthetic fibers such as polyester, nylon and acrylic. , And complexes thereof, but are not particularly limited thereto.
Further, as the form of the fiber, a woven fabric or a knitted fabric made of staples, filament yarns and processed yarns thereof is used, but a fiber web may be used. The fiber web refers to non-woven fabric, tissue paper, wet tissue, paper such as food wrapping paper, and the like.
　本発明の抗菌性繊維は、抗菌性が求められる種々の繊維製品、例えばサニタリー用品に対して用いることができる。本発明の抗菌性繊維はまた、安全性に優れているので、医療用用途や医療用用品、例えば、医療用包帯、シーツ、ガーゼおよび衣類等に用いることができる。 The antibacterial fiber of the present invention can be used for various fiber products that require antibacterial properties, such as sanitary products. Since the antibacterial fiber of the present invention is also excellent in safety, it can be used for medical use and medical supplies such as medical bandages, sheets, gauze and clothes.
　本発明の方法において、ペプチド含有ポリマーは繊維上に固定されるが、本発明でいう繊維上とは、完全に繊維の表面上に存在する場合に加えて、ペプチド含有ポリマーの一部が繊維の中に入り込んでいる状態も含む意味である。目的のペプチド活性、例えば、ペプチドがカブトムシディフェンシン由来改変ペプチドの場合は、抗菌活性を呈するためには、ペプチド含有ポリマーの全て又は大部分が繊維表面上に存在することが好ましい。 In the method of the present invention, the peptide-containing polymer is fixed on the fiber, but the term “on the fiber” as used in the present invention means that a part of the peptide-containing polymer is a part of the fiber in addition to the case where the peptide-containing polymer is completely present on the surface of the fiber. It also includes the state of being in the middle. In the case where the target peptide activity, for example, the peptide is a modified peptide derived from beetle defensin, it is preferable that all or most of the peptide-containing polymer is present on the fiber surface in order to exhibit antibacterial activity.
　本発明においては、布にコーティングするポリマーは、布の重量に対して１０重量％以下が好ましく、より好ましくは５重量％以下である。この場合において、ペプチドは、布１ｃｍ2あたり０．００１μモル以上０．５μモル以下が好ましく、安全性および経済的な観点から１ｃｍ2あたり０．０３μモル以下が最も好ましい。 In the present invention, the polymer coated on the cloth is preferably 10% by weight or less, more preferably 5% by weight or less, based on the weight of the cloth. In this case, the peptide, preferably the fabric 1 cm 0.001Myu mol 0.5μ mol per 2, most preferably 1 cm 0.03 microns mol per 2 from safety and economical point of view.
　本発明のペプチド含有ポリマーの繊維への固定は、繊維表面にペプチド含有ポリマーをコーティングすることにより行うことができ、例えば、撥水加工や帯電防止加工のような繊維加工技術を用いて行うことができる。言い換えれば、ポリマー化ペプチドの繊維への固定化は、例えば、静電的な相互作用による方法、架橋反応により化学的に結合させる方法等を用いて行うことができる。静電的な相互作用により固定化する方法はより簡便であり、一方、架橋反応により化学的に結合させる場合は、より強固な固定化となるという利点があり、目的に応じて任意に選択可能である。 The peptide-containing polymer of the present invention can be fixed to the fiber by coating the fiber surface with the peptide-containing polymer, for example, using a fiber processing technique such as water repellent processing or antistatic processing. it can. In other words, the immobilization of the polymerized peptide to the fiber can be performed using, for example, a method based on electrostatic interaction, a method of chemically bonding by a crosslinking reaction, or the like. The method of immobilization by electrostatic interaction is simpler. On the other hand, when chemically bonded by a cross-linking reaction, there is an advantage of stronger immobilization, which can be arbitrarily selected according to the purpose. It is.
　本発明の他の態様においては、本発明の抗菌性ペプチド含有ポリマーを含有するエマルジョンが提供される。
　本発明においては、例えば、固定化されたペプチドを有する疎水性のポリマー（ペプチド含有ポリマー）を単独又は界面活性剤などを用いて乳化し、繊維の加工剤であるエマルジョンとすることができる。また、ポリマーエマルジョンは、従来公知の乳化重合法でも作製できる。例えば、モノマーを界面活性剤により水中に分散し、重合開始剤、キレート剤、酸素捕捉剤、分子量調整剤等が加えて乳化重合できる。使用する、界面活性剤、重合開始剤、キレート剤、酸素捕捉剤、分子量調整剤は従来公知のものを用いることができる。 In another aspect of the present invention, an emulsion containing the antimicrobial peptide-containing polymer of the present invention is provided.
In the present invention, for example, a hydrophobic polymer (an peptide-containing polymer) having an immobilized peptide can be emulsified alone or with a surfactant or the like to form an emulsion that is a fiber processing agent. The polymer emulsion can also be prepared by a conventionally known emulsion polymerization method. For example, a monomer can be dispersed in water with a surfactant, and emulsion polymerization can be carried out by adding a polymerization initiator, a chelating agent, an oxygen scavenger, a molecular weight modifier and the like. Conventionally known surfactants, polymerization initiators, chelating agents, oxygen scavengers, and molecular weight modifiers can be used.
　これにより、エマルジョン状態で、販売したりや流通させることも可能である。また、そのようにして調製したエマルジョンを繊維に塗布し、乾燥することにより、本発明のペプチドが固定化された繊維を容易に製造できる。また、水溶性のポリマーの場合は、水に溶解させた状態で販売したり流通させたりすることができ、加工時にブロックイソシアネートなどの架橋剤と混合し、布に固定化することが容易にできる。 This makes it possible to sell and distribute in the emulsion state. Moreover, the fiber in which the peptide of the present invention is immobilized can be easily produced by applying the emulsion thus prepared to the fiber and drying it. In the case of a water-soluble polymer, it can be sold or distributed in a state dissolved in water, and can be easily mixed with a cross-linking agent such as blocked isocyanate during processing and fixed to a cloth. .
　本発明の更に別の態様においては、以下の工程からなる、抗菌性ペプチドが固定化された抗菌性繊維の製造方法が提供される。
工程（１）；下記式（１ｂ）： In still another aspect of the present invention, there is provided a method for producing an antibacterial fiber having an antibacterial peptide immobilized, comprising the following steps.
Step (1); the following formula (1b):
Figure JPOXMLDOC01-appb-C000117
 （式中Ｒ1及びＲ3は、それぞれ独立に水素原子又はメチル基であり、ｎは自然数であり、ｍ2は０又は自然数であり、Ｕ2及びＵ3は、それぞれ独立にＮＨ又はＯであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチド又は誘導体化したペプチドと反応する官能基であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）で表されるポリマーを含む溶液を繊維にコーティングする工程、及び
工程（２）；下記式：
Figure JPOXMLDOC01-appb-C000117
 (Wherein R 1 and R 3 are each independently a hydrogen atom or a methyl group, n is a natural number, m 2 is 0 or a natural number, and U 2 and U 3 are each independently NH or O. , V is a single bond or a spacer, Wo is a functional group that reacts with a peptide or a derivatized peptide, and Y is a single bond or a linear group that may have a substituent having a molecular weight of 10 to 3000 or A branched alkylene chain or a polyoxyalkylene chain), a step of coating a fiber with a solution containing a polymer represented by formula (2);
Figure JPOXMLDOC01-appb-C000118
 （ここで、Peptideは、下記式で表されるアミノ酸配列：
－ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するポリペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基（例えば、アジド基、スルフヒドリル基、ハロゲン基、アルキン基、カルボニル基、ヒドロキシルアミノ基）を有する分子量５０～５００の分子である。）
で表され抗菌性ポリペプチド又はその誘導体を含む溶液中に、ポリマーを含む溶液をコーティングした繊維を浸漬する工程、及び
工程（３）；１～１０時間放置することによりポリマーにポリペプチドを結合させた後、繊維を洗浄して未反応のペプチド又はペプチド誘導体を除去する工程、
からなる抗菌性繊維の製造方法。
Figure JPOXMLDOC01-appb-C000118
 (Here, Peptide is an amino acid sequence represented by the following formula:
-NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) A polypeptide having Z is a molecular weight having a functional group (for example, azide group, sulfhydryl group, halogen group, alkyne group, carbonyl group, hydroxylamino group) that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. 50 to 500 molecules. )
A step of immersing fibers coated with a solution containing a polymer in a solution containing an antibacterial polypeptide or a derivative thereof, and step (3); allowing the polypeptide to bind to the polymer by leaving it for 1 to 10 hours. And then removing the unreacted peptide or peptide derivative by washing the fiber,
The manufacturing method of the antimicrobial fiber which consists of.
　上記結合反応は、例えば１～１０時間、好ましくは２～１０時間、更に好ましくは３～６時間反応させることにより行うことができる。また、結合反応に用いるペプチド溶液の濃度は、リンカーの種類や温度等の反応条件に応じて適宜選択可能であるが、例えば、０．１～１００ｍM、好ましくは１～１０ｍMである。 The above binding reaction can be performed, for example, by reacting for 1 to 10 hours, preferably 2 to 10 hours, more preferably 3 to 6 hours. The concentration of the peptide solution used for the binding reaction can be appropriately selected according to the reaction conditions such as the type of linker and temperature, and is, for example, 0.1 to 100 mM, preferably 1 to 10 mM.
　本発明の方法において用いる繊維にコーティング（塗布）するポリマーは、本明細書中に記載したポリペプチドを結合する前のポリマーのいずれであってもよい。例えば、好ましいポリマーとして、以下式（６ａ）（式中、Ｒ1及びＲ3は、水素原子又はメチル基であり、ｎは自然数であり、ｍは０又は自然数であり、Ｕ1及びＵ3は、Ｏ又はＮＨであり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である）で表されるポリマーをあげることができるが、これに限定されない。 The polymer to be coated (applied) to the fiber used in the method of the present invention may be any of the polymers prior to binding the polypeptides described herein. For example, as a preferred polymer, the following formula (6a) (wherein R 1 and R 3 are a hydrogen atom or a methyl group, n is a natural number, m is 0 or a natural number, U 1 and U 3 are , O or NH, and Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000). However, it is not limited to this.
Figure JPOXMLDOC01-appb-C000119
Figure JPOXMLDOC01-appb-C000119
　以下、実施例により本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
（実施例１）
ペプチドの合成
　ペプチドＡ（RLYLRIGRR）、ペプチド１（CRLYLRIGRR）及びペプチド２（RLYLRIGRRC）を、常法に従い固相合成法により合成した。 Example 1
Peptide Synthesis Peptide A (RLYLRIGRR), Peptide 1 (CRLYLRIGRR) and Peptide 2 (RLYLRIGRRC) were synthesized by solid phase synthesis according to a conventional method.
（実施例２）
モノマー１の合成
　滴下ロートを備えた二口フラスコ（100ml）に（３－｛２－[２－（３－アミノプロピル）－エトキシ]－エトキシ｝－プロピル）カルバミン酸－ｔ－ブチルエステル（1060mg、3.31mmol）とトリエチルアミン（401mg、3.97mmol）、テトラヒドロフラン（25mL）を加え、氷冷下で攪拌した。ここにメタクリル酸クロリド（913mg、3.97mmol）とテトラヒドロフラン（25mL）の混合溶液を滴下し、氷冷下で3時間攪拌した。その後、8時間室温で攪拌した。反応終了後、沈殿物を除去し、展開溶媒に酢酸エチルを用いたシリカゲルカラムクロマトグラフィーにて目的物を単離した。Rf値＝0.39 （シリカゲル、酢酸エチル）　収率５９％。
　1H-NMR; (CDCl3, 400MHz)δ：1.4-1.5 (9H, br, CH3), 1.7-1.8 (2H, m, NHCH2 CH 2 ), 1.8-1.9 (2H, m, NHCH2 CH 2 ), 2.05 (3H, s, OCCH 3 ), 3.20-3.25 (2H, q, J=6.24, NHCH 2 ), 3.42-3.47 (2H, q, J-5.56, NHCH 2 ), 3.51-3.54 (2H, t, J=5.96, OCH 2 ), 3.55-3.65 (12H, m, OCH 2 ) 5.29 (1H, s, CH3CCH 2 ), 5.71 (1H, s, CH3CCH 2 ) (Example 2)
Synthesis of Monomer 1 To a two-necked flask (100 ml) equipped with a dropping funnel (3- {2- [2- (3-aminopropyl) -ethoxy] -ethoxy} -propyl) carbamic acid-t-butyl ester (1060 mg, 3.31 mmol), triethylamine (401 mg, 3.97 mmol) and tetrahydrofuran (25 mL) were added, and the mixture was stirred under ice cooling. A mixed solution of methacrylic acid chloride (913 mg, 3.97 mmol) and tetrahydrofuran (25 mL) was added dropwise thereto, and the mixture was stirred for 3 hours under ice cooling. Then, it stirred at room temperature for 8 hours. After completion of the reaction, the precipitate was removed, and the target product was isolated by silica gel column chromatography using ethyl acetate as a developing solvent. Rf value = 0.39 (silica gel, ethyl acetate) Yield 59%.
1 H-NMR; (CDCl 3 , 400 MHz) δ: 1.4-1.5 (9H, br, CH 3 ), 1.7-1.8 (2H, m, NHCH 2 CH 2 ), 1.8-1.9 (2H, m, NHCH 2 CH 2 ), 2.05 (3H, s, OC CH 3 ), 3.20-3.25 (2H, q, J = 6.24, NH CH 2 ), 3.42-3.47 (2H, q, J-5.56, NH CH 2 ), 3.51- 3.54 (2H, t, J = 5.96, O CH 2 ), 3.55-3.65 (12H, m, O CH 2 ) 5.29 (1H, s, CH 3 C CH 2 ), 5.71 (1H, s, CH 3 C CH 2 )
Figure JPOXMLDOC01-appb-C000120
Figure JPOXMLDOC01-appb-C000120
（実施例３）
ポリマー１の合成
　窒素置換したシュレンク管（30mL）にモノマー１（900mg、2.32mmol）とAIBN（7.6mg、0.046mmol）とテトラヒドロフラン（1ml）を加え、還流攪拌した。反応終了後、ゲル浸透クロマトグラフィーにて低分子量物質を除去した。得られたポリマーをジクロロメタン（10ml）に溶解させ、トリフルオロ酢酸（5ml）を加え、室温で8時間攪拌した。反応終了後、溶媒を減圧下で留去した。再びポリマーをジクロロメタン（10ml）に溶解させ、3-マレイミドプロピオン酸N-スクシンイミジル（122.4mg、0.46mmol）とトリエチルアミン（46.5mg、0.46mmol）を加え、室温で3時間攪拌した。反応終了後、ゲル浸透クロマトグラフィーにて精製し、下記のポリマー１を得た。
1H-NMR; (CDCl3, 400MHz)δ：0.8-1.0 (3H, br, CH3), 1.4-1.9 (8H, br, CH2 ), 2.4-2.6 (2H, br, COCH 2 ), 3.1-3.4 (4H, br, NHCH 2 ), 3.4-3.7 (12H, br, OCH 2 ), 3.75-3.85 (2H, br, CONCH 2 ) 6.7-6.8 (2H, br, COCH)
（水、DMF、クロロホルム、THFに可溶） (Example 3)
Synthesis of Polymer 1 Monomer 1 (900 mg, 2.32 mmol), AIBN (7.6 mg, 0.046 mmol), and tetrahydrofuran (1 ml) were added to a nitrogen-substituted Schlenk tube (30 mL), and the mixture was stirred at reflux. After completion of the reaction, low molecular weight substances were removed by gel permeation chromatography. The obtained polymer was dissolved in dichloromethane (10 ml), trifluoroacetic acid (5 ml) was added, and the mixture was stirred at room temperature for 8 hours. After completion of the reaction, the solvent was distilled off under reduced pressure. The polymer was dissolved again in dichloromethane (10 ml), N-succinimidyl 3-maleimidopropionate (122.4 mg, 0.46 mmol) and triethylamine (46.5 mg, 0.46 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, purification was performed by gel permeation chromatography to obtain the following polymer 1.
1 H-NMR; (CDCl 3 , 400 MHz) δ: 0.8-1.0 (3H, br, CH 3 ), 1.4-1.9 (8H, br, CH 2 ), 2.4-2.6 (2H, br, CO CH 2 ), 3.1-3.4 (4H, br, NH CH 2 ), 3.4-3.7 (12H, br, O CH 2 ), 3.75-3.85 (2H, br, CON CH 2 ) 6.7-6.8 (2H, br, CO CH )
(Soluble in water, DMF, chloroform, THF)
Figure JPOXMLDOC01-appb-C000121
Figure JPOXMLDOC01-appb-C000121
（実施例４）
ポリマー２（ペプチド含有ポリマー）の合成
　実施例３で合成したポリマー１（15.8mg）をDMF（250μl）に溶解させた。ペプチド１（CRLYLRIGRR）（11.7mg、9.0mmol）を水1mlに溶解させ、これをポリマー１のDMF溶液と混合し、反応させた。ペプチドユニット：マレイミドユニット＝１：４ (Example 4)
Synthesis of Polymer 2 (Peptide-Containing Polymer) Polymer 1 (15.8 mg) synthesized in Example 3 was dissolved in DMF (250 μl). Peptide 1 (CRLYLRIGRR) (11.7 mg, 9.0 mmol) was dissolved in 1 ml of water, and this was mixed with a DMF solution of polymer 1 and reacted. Peptide unit: maleimide unit = 1: 4
Figure JPOXMLDOC01-appb-C000122
Figure JPOXMLDOC01-appb-C000122
（実施例５）
ポリマー３（ペプチド含有ポリマー）の合成
　反応させるペプチドにペプチド２（RLYLRIGRRC）を用いたこと以外は、ポリマー２と同様にして合成した。ペプチドユニット：マレイミドユニット＝１：４ (Example 5)
Polymer 3 (peptide-containing polymer) was synthesized in the same manner as polymer 2 except that peptide 2 (RLYLRIGRRC) was used as the peptide to be reacted. Peptide unit: maleimide unit = 1: 4
Figure JPOXMLDOC01-appb-C000123
Figure JPOXMLDOC01-appb-C000123
（実施例６）
ポリマー４（ペプチド含有ポリマー）の合成
　ポリマー１（3.5mg、マレイミドユニット8μmol）を水（1.0ml）に溶解させた。ペプチド2 (RLYLRIGRRC)（21mg, 16μmol）を水1mlに溶解させ、これをポリマー１水溶液と混合し、反応させた。これを透析チューブ（分画分子量8000）で、一晩透析し、過剰のペプチドを除去した。 (Example 6)
Synthetic polymer 1 (3.5 mg, maleimide unit 8 μmol) of polymer 4 (peptide-containing polymer ) was dissolved in water (1.0 ml). Peptide 2 (RLYLRIGRRC) (21 mg, 16 μmol) was dissolved in 1 ml of water, this was mixed with an aqueous solution of polymer 1 and reacted. This was dialyzed overnight with a dialysis tube (molecular weight cut off 8000) to remove excess peptide.
Figure JPOXMLDOC01-appb-C000124
Figure JPOXMLDOC01-appb-C000124
（実施例７）
加工布１～３の作製
　ポリマー２DMF-水混合溶液を綿のJIS添付白布にコーティングし、減圧下で乾燥させ、加工布１～３を作製した。
　加工布１は、ペプチドの存在量が布１cm2あたり0.3μmol存在するように調整した。
　加工布２は、ペプチドの存在量が布１cm2あたり0.1μmolになるように調整したこと以外は加工布１と同様にして作製した。
　加工布３は、ペプチドの存在量が布１cm2あたり0.03μmolになるように調整したこと以外は加工布１と同様にして作製した。 (Example 7)
Fabrication of processed cloths 1 to 3 Polymer 2DMF-water mixed solution was coated on a cotton JIS-attached white cloth and dried under reduced pressure to prepare processed cloths 1 to 3.
The processed cloth 1 was adjusted so that the amount of peptide present was 0.3 μmol per cm 2 of cloth.
The processed cloth 2 was produced in the same manner as the processed cloth 1 except that the amount of the peptide was adjusted to 0.1 μmol per 1 cm 2 of the cloth.
The processed cloth 3 was produced in the same manner as the processed cloth 1 except that the amount of the peptide was adjusted to 0.03 μmol per 1 cm 2 of the cloth.
（実施例８）
加工布４～６の作製
　コーティングするポリマーにポリマー３を用いたこと以外は、加工布１と同様にして作製した。ペプチドの存在量は、加工布４が布１cm2あたり0.3μmolであり、加工布５が布１cm2あたり0.1μmolであり、加工布６が布１cm2あたり0.03μmolであった。 (Example 8)
Fabrication of work cloths 4 to 6 Fabrication of work cloths 4 to 6 was performed in the same manner as work cloth 1 except that polymer 3 was used as the polymer to be coated. The abundance of the peptide was 0.3 μmol per 1 cm 2 of the processed cloth 4, 0.1 μmol per 1 cm 2 of the processed cloth 5, and 0.03 μmol of the processed cloth 6 per 1 cm 2 of the cloth.
（比較例１）
加工布７～９の作製
　コーティングするポリマーにポリマー１を用いたこと以外は、加工布１と同様にして作製した。マレイミドユニットの存在量は、加工布７が布１cm2あたり1.2μmolであり、加工布８が布１cm2あたり0.4μmolであり、加工布９が布１cm2あたり0.12μmolであった。 (Comparative Example 1)
Fabrication of work cloths 7 to 9 Fabrication of work cloths 7 to 9 was performed in the same manner as work cloth 1 except that polymer 1 was used as the polymer to be coated. Maleimide amounts present unit, the fabric 7 is 1.2μmol per fabric 1 cm 2, the fabric 8 is 0.4μmol per fabric 1 cm 2, the workpiece cloth 9 was 0.12μmol per fabric 1 cm 2.
（実施例９）
加工布１０の作製
　コーティングするポリマーにポリマー４を用いたこと以外は、加工布１と同様にして作製した。ペプチドの存在量は、１cm2あたり0.3μmolであった。 Example 9
Fabrication of work cloth 10 Fabrication of work cloth 10 was performed in the same manner as work cloth 1 except that polymer 4 was used as the polymer to be coated. The amount of peptide present was 0.3 μmol per cm 2 .
（実施例１０）
抗菌活性試験
　上記の如くして作製した加工布１～１０について、抗微生物活性試験を行った。加工布（1 cm×1 cm）および標準布をそれぞれ黄色ブドウ球菌（Staphylococcus aureus NBRC 12732）を含む培養液（0.8% NB培地で一昼夜培養した菌液を、新たな培養液に１％容量で加え、37℃で3時間培養した後、波長600nmにおける吸光度が0.01となるように希釈した培養液200μl）中に入れ、37℃で１８時間培養した後、生菌数を測定し、18時間培養後の標準布の生菌数を18時間培養後の加工布の生菌数で除した対数値として示した。結果を表１に示す。抗菌試験を実施した後、布を40ml超純水でボルテックス洗浄（最大スピード１０秒間振とう×３回）及びオートクレーブによる滅菌操作（121℃、20分）を行い、同じ加工布を用いて抗菌試験を行った。更に、この洗浄－滅菌操作－抗菌試験を繰り返し行った。結果を以下の表２に示す。 (Example 10)
Antibacterial activity test The antibacterial activity test was performed on the processed fabrics 1 to 10 produced as described above. A culture solution (1 cm x 1 cm) and a standard fabric each containing Staphylococcus aureus NBRC 12732 are added to the new culture solution in a volume of 1%. After culturing at 37 ° C. for 3 hours, the solution was put into 200 μl of a culture solution diluted to have an absorbance at a wavelength of 600 nm of 0.01), and cultured at 37 ° C. for 18 hours. It was shown as a logarithmic value obtained by dividing the number of viable bacteria in the standard cloth by the number of viable bacteria in the processed cloth after 18 hours of culture. The results are shown in Table 1. After the antibacterial test is performed, the cloth is vortex washed with 40 ml of ultrapure water (maximum speed shaking for 10 seconds x 3 times) and sterilized by autoclave (121 ° C, 20 minutes), and the same processed cloth is used for the antibacterial test. Went. Further, this washing-sterilization operation-antibacterial test was repeated. The results are shown in Table 2 below.
Figure JPOXMLDOC01-appb-T000125
Figure JPOXMLDOC01-appb-T000125
（実施例１１）
ポリマー５の合成
　窒素置換したシュレンク管（30mL）にモノマー１（54mg、0.14mmol）とN－オクタデシルアクリルアミド（931mg、2.88mmol）とAIBN（7.6mg、0.046mmol）とクロロホルム（1ml）を加え、還流攪拌した。反応終了後、ゲル浸透クロマトグラフィーにて低分子量物質を除去した。得られたポリマーをジクロロメタン（10ml）に溶解させ、トリフルオロ酢酸（5ml）を加え、室温で8時間攪拌した。反応終了後、溶媒を減圧下で留去した。再びポリマーをジクロロメタン（10ml）に溶解させ、3-マレイミドプロピオン酸N-スクシンイミジル（38mg、0.14mmol）とトリエチルアミン（14.1mg、0.14mmol）を加え、室温で3時間攪拌した。反応終了後、ゲル浸透クロマトグラフィーにて精製した。NMRの結果、マレイミドユニット：オクタデシルユニットの物質比は1:14であった。 (Example 11)
Synthesis of Polymer 5 Monomer 1 (54 mg, 0.14 mmol), N-octadecylacrylamide (931 mg, 2.88 mmol), AIBN (7.6 mg, 0.046 mmol) and chloroform (1 ml) were added to a nitrogen-substituted Schlenk tube (30 mL) and refluxed. Stir. After completion of the reaction, low molecular weight substances were removed by gel permeation chromatography. The obtained polymer was dissolved in dichloromethane (10 ml), trifluoroacetic acid (5 ml) was added, and the mixture was stirred at room temperature for 8 hours. After completion of the reaction, the solvent was distilled off under reduced pressure. The polymer was dissolved again in dichloromethane (10 ml), N-succinimidyl 3-maleimidopropionate (38 mg, 0.14 mmol) and triethylamine (14.1 mg, 0.14 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, it was purified by gel permeation chromatography. As a result of NMR, the substance ratio of maleimide unit: octadecyl unit was 1:14.
Figure JPOXMLDOC01-appb-C000126
Figure JPOXMLDOC01-appb-C000126
　1H-NMR; (CDCl3, 400MHz)δ：0.86-0.90 (42H, t, J=7.04, CH 3 ), 1.26 (420H, br, CH 2 ), 1.47 (10H, br, CH 2 ), 1.76-2.70 (64H, br, CH2), 3.15 (30H, br, NHCH 2 ), 3.42-3.82 (14H, br, OCH 2 , NCH 2 ), 6.73 (2H, s, COCH) 1 H-NMR; (CDCl 3 , 400 MHz) δ: 0.86-0.90 (42H, t, J = 7.04, CH 3 ), 1.26 (420H, br, CH 2 ), 1.47 (10H, br, CH 2 ), 1.76 -2.70 (64H, br, CH 2 ), 3.15 (30H, br, NH CH 2 ), 3.42-3.82 (14H, br, O CH 2 , NCH 2 ), 6.73 (2H, s, COC H )
（実施例１２）
加工布１１及び１２の作製
　マレイミドユニットの物質量が布１cm2あたり0.03μmolになるようにして、実施例１１で作製したポリマー５を綿のJIS添付白布にコーティングし、減圧下で乾燥させた。この布を、マレイミドユニットと同モルのペプチド１（CRLYLRIGRR）を含む水溶液中（ペプチド濃度　3.6mM）に浸し、5時間放置した。その後、布をイオン交換水で洗浄し、未反応のペプチドを除去し、加工布１１を作製した。
　マレイミドユニットに結合させるペプチドとしてペプチド２（RLYLRIGRRC）を用いたこと以外は、加工布１１と同様にして、加工布１２を作製した。 Example 12
Fabrication of processed fabrics 11 and 12 The polymer 5 produced in Example 11 was coated on a cotton JIS-attached white fabric so that the amount of maleimide units in the fabric was 0.03 μmol per cm 2 of fabric, and dried under reduced pressure. This cloth was immersed in an aqueous solution (peptide concentration 3.6 mM) containing peptide 1 (CRLYLRIGRR) in the same mole as the maleimide unit, and left for 5 hours. Thereafter, the cloth was washed with ion-exchanged water to remove unreacted peptides, and a processed cloth 11 was produced.
A processed cloth 12 was produced in the same manner as the processed cloth 11 except that peptide 2 (RLYLRIGRRC) was used as a peptide to be bound to the maleimide unit.
（比較例２）
加工布１３の作製
　マレイミドユニットに結合させるものとして、マレイミドユニットと同モルのシステインを含む水溶液を用いたこと以外は、加工布１１と同様にして作製した。 (Comparative Example 2)
Fabrication of work cloth 13 Fabrication was performed in the same manner as the work cloth 11 except that an aqueous solution containing the same mole of cysteine as the maleimide unit was used as a bond to the maleimide unit.
（実施例１３）
　実施例１０と同様にして、加工布１１～１３の抗菌活性を測定した。結果を以下の表３に示す。 (Example 13)
In the same manner as in Example 10, the antibacterial activity of the processed cloths 11 to 13 was measured. The results are shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000127
Figure JPOXMLDOC01-appb-T000127
　上記の結果、ポリペプチド含有ポリマーを塗布してペプチドを結合した加工布１～６及び１０では活性が認められたが、ペプチドを結合していない加工布７～９は全く活性が認められなかった。加工布１～３と４～６を比較すると僅かにＮ末端結合型の方が強い抗菌活性を持つがことが分かった。
　また、ポリマーを塗布した後ペプチドを反応させてペプチドを結合した加工布１１及び１２では活性が認められたが、ペプチドを結合していない加工布１３では活性が認められなかった。 As a result, activity was observed in the processed cloths 1 to 6 and 10 to which the peptide-containing polymer was applied and the peptide was bound, but the processed cloths 7 to 9 to which the peptide was not bound had no activity. . Comparing the processed fabrics 1 to 3 and 4 to 6, it was found that the N-terminal bond type has slightly stronger antibacterial activity.
In addition, the activity was recognized in the processed cloths 11 and 12 to which the peptide was reacted after the polymer was applied to bind the peptide, but the activity was not recognized in the processed cloth 13 to which the peptide was not bonded.
（実施例１４）
Ｃｙｓリンカー付ペプチドの合成
　Ｃｙｓリンカーが末端についたペプチド３（CRLLLRVGRR-NH2）を常法に従い固相合成法により合成した。 (Example 14)
Synthesis of peptide with Cys linker Peptide 3 terminated with a Cys linker (CRLLLRVGRR-NH 2 ) was synthesized by a solid phase synthesis method according to a conventional method.
（実際例１５）
モノマー２の合成
　3-ブロモプロピルアミン臭化水素酸塩(2.15g, 9.8mmol)をピリジン50mLに溶解させ、氷冷下で攪拌した。ここにメタクリル酸クロライド(1.54g, 14.7mmol)をTHF(30mL)に溶解させたものを滴下し、一晩攪拌した。反応終了後、ピリジンを乾固しない程度に留去し、水とクロロホルムで分離した。クロロホルム相を濃縮し、ゲル浸透クロマトグラフィーにて精製し、N-(3-ブロモプロピル)-2-メタクリルアミドを得た。収率３０％
1H-NMR; (CDCl3, 400MHz)δ：2.22 (3H, s, CH3), 2.22-2.30 (2H, quin, J=7.7, CH 2 ), 3.86-3.90 (2H, t, J=8.0, CH 2 ), 4.66-4.69 (2H, t, J=7.4, CH 2 ), 5.91 (1H, s, CH3CCH 2 ), 6.55 (1H, s, CH3CCH 2 ) (Actual example 15)
Synthesis of Monomer 2 3-Bromopropylamine hydrobromide (2.15 g, 9.8 mmol) was dissolved in 50 mL of pyridine and stirred under ice cooling. A solution of methacrylic acid chloride (1.54 g, 14.7 mmol) dissolved in THF (30 mL) was added dropwise thereto and stirred overnight. After completion of the reaction, pyridine was distilled off to such an extent that it did not dry and separated with water and chloroform. The chloroform phase was concentrated and purified by gel permeation chromatography to give N- (3-bromopropyl) -2-methacrylamide. Yield 30%
1 H-NMR; (CDCl 3 , 400 MHz) δ: 2.22 (3H, s, CH 3 ), 2.22-2.30 (2H, quin, J = 7.7, CH 2 ), 3.86-3.90 (2H, t, J = 8.0 , CH 2 ), 4.66-4.69 (2H, t, J = 7.4, CH 2 ), 5.91 (1H, s, CH 3 C CH 2 ), 6.55 (1H, s, CH 3 C CH 2 )
Figure JPOXMLDOC01-appb-C000128
Figure JPOXMLDOC01-appb-C000128
（実施例１６）
モノマー３の合成
　3-ブロモ-1-プロパノール(1.39g, 10mmol)を脱水したトルエン10mlに溶解させ、窒素雰囲気下で攪拌した。ここにメタクリル酸2-イソシアナトエチル(1.55g, 10mmol)を滴下し、60℃で一晩攪拌した。得られた副生成物をろ過で除去し、モノマー2を得た。
収率９５％
1H-NMR; (CDCl3, 400MHz)δ：1.95 (3H, s, CH3), 2.17 (2H, br, CH 2 ), 3.45-3.54 (4H, br, BrCH 2 , NCH 2 ), 4.20-4.25 (4H, br, OCH 2 ), 5.60 (1H, s, CH3CCH 2 ), 6.13 (1H, s, CH3CCH 2 ) (Example 16)
Synthesis of Monomer 3 3-Bromo-1-propanol (1.39 g, 10 mmol) was dissolved in 10 ml of dehydrated toluene and stirred under a nitrogen atmosphere. To this was added dropwise 2-isocyanatoethyl methacrylate (1.55 g, 10 mmol), and the mixture was stirred at 60 ° C. overnight. The obtained by-product was removed by filtration to obtain monomer 2.
Yield 95%
1 H-NMR; (CDCl 3 , 400 MHz) δ: 1.95 (3H, s, CH 3 ), 2.17 (2H, br, CH 2 ), 3.45-3.54 (4H, br, Br CH 2 , NC H 2 ), 4.20-4.25 (4H, br, O CH 2 ), 5.60 (1H, s, CH 3 C CH 2 ), 6.13 (1H, s, CH 3 C CH 2 )
Figure JPOXMLDOC01-appb-C000129
Figure JPOXMLDOC01-appb-C000129
（実施例１７）
ポリマー６の合成
　N-(3-ブロモプロピル)-2-メタクリルアミド(モノマー２)(82mg, 0.4mmol)とポリ(エチレングリコール)メチルエーテルメタクリラートMn300(6.0g, 20mmol)をエタノール20mLに溶解させ、開始剤のAIBN(67mg, 0.4mmol)を添加した。容器内を窒素置換した後、6時間還流攪拌を行った。透析チューブ（分画分子量3500）で、48時間透析し、未反応のモノマーを除去し、ポリマー６を得た。バイルシュタイン反応で、ハロゲン由来の炎色反応が認められた。 (Example 17)
Synthesis of polymer 6 N- (3-bromopropyl) -2-methacrylamide (monomer 2) (82 mg, 0.4 mmol) and poly (ethylene glycol) methyl ether methacrylate Mn300 (6.0 g, 20 mmol) were dissolved in 20 mL of ethanol. Initiator AIBN (67 mg, 0.4 mmol) was added. The inside of the container was purged with nitrogen, and then stirred under reflux for 6 hours. Dialysis was performed for 48 hours with a dialysis tube (molecular weight cut off 3500) to remove unreacted monomers, and polymer 6 was obtained. In the Beilstein reaction, a flame reaction derived from halogen was observed.
ポリマー７の合成
　モノマー３(305mg, 1.04mmol)とポリ(エチレングリコール)メチルエーテルメタクリラートMn300(15.6g, 52mmol)をエタノール50mLに溶解させ、開始剤のAIBN(174mg, 1.06mmol)を添加した。容器内を窒素置換した後、6時間還流攪拌を行った。透析チューブ（分画分子量3500）で、48時間透析し、未反応のモノマーを除去し、ポリマー７を得た。 Synthetic monomer 3 of polymer 7 (305 mg, 1.04 mmol) and poly (ethylene glycol) methyl ether methacrylate Mn300 (15.6 g, 52 mmol) were dissolved in 50 mL of ethanol, and initiator AIBN (174 mg, 1.06 mmol) was added. The inside of the container was purged with nitrogen, and then stirred under reflux for 6 hours. Dialysis was performed for 48 hours with a dialysis tube (molecular weight cut off 3500) to remove unreacted monomers, and polymer 7 was obtained.
（実施例１８）
ポリマー８（ペプチド含有ポリマー）の合成
　ポリマー６(8928mg, Br換算0.587mmol)とCysリンカー付きペプチド３（CRLLLRVGRR）(1062mg,0.587mmol)をDMF58.7mlに溶解させ、ターシャリーブトキシカリウム(395.2mg,3.522mmol)を添加した。40℃で4時間30分加熱撹拌した。TLCで原料ペプチドが消失したことを確認した。DCM500mlで希釈し、析出した塩をセライトろ過し取り除いた。ろ液をエバポレータで濃縮し、真空乾燥し、ポリマー６にペプチド３が結合したポリマーであるポリマー８を得た。 (Example 18)
Synthetic polymer 6 of polymer 8 (peptide-containing polymer) (8928 mg, 0.587 mmol in terms of Br) and peptide 3 with a Cys linker (CRLLLRVGRR) (1062 mg, 0.587 mmol) were dissolved in 58.7 ml of DMF, and tertiary butoxy potassium (395.2 mg, 3.522 mmol) was added. The mixture was heated and stirred at 40 ° C. for 4 hours and 30 minutes. It was confirmed by TLC that the starting peptide had disappeared. The solution was diluted with 500 ml of DCM, and the precipitated salt was removed by celite filtration. The filtrate was concentrated with an evaporator and vacuum-dried to obtain polymer 8, which is a polymer in which peptide 3 is bound to polymer 6.
ポリマー９（ペプチド含有ポリマー）の合成
　ポリマー７(2.04g,Br換算0.13mmol)とCysリンカー付きペプチド３（CRLLLRVGRR）(235.4mg,0.13mmol)をDMF10mlに溶解させ、ターシャリーブトキシカリウム(87.5mg,0.78mmol)を添加した。40℃で7時間加熱撹拌した。TLCで原料ペプチドが消失したことを確認した。DCM50mlで希釈し、析出した塩をセライトろ過し取り除いた。ろ液をエバポレータで濃縮し、真空乾燥し、ポリマー７にペプチド３が結合したポリマーであるポリマー９を得た。 Synthesis of polymer 9 (peptide-containing polymer) Polymer 7 (2.04 g, 0.13 mmol in terms of Br) and peptide 3 with CR linker (CRLLLRVGRR) (235.4 mg, 0.13 mmol) were dissolved in 10 ml of DMF, and tertiary butoxy potassium (87.5 mg, 0.78 mmol) was added. The mixture was heated and stirred at 40 ° C. for 7 hours. It was confirmed by TLC that the starting peptide had disappeared. The solution was diluted with 50 ml of DCM, and the precipitated salt was removed by filtration through celite. The filtrate was concentrated with an evaporator and vacuum-dried to obtain polymer 9, which is a polymer in which peptide 3 is bound to polymer 7.
（実施例１９）
ペプチド含有ポリマー加工剤Ａ及びＢの調整
　実施例１８で作製したポリマー８(4.8g)に水を加え全量を121gとした。一晩攪拌し完全に分散させ、加工剤Ａを調整した。
　加工剤Ａを蒸留水で10倍希釈し、加工剤Ｂを得た。
　なお、ポリマー７(346mg)に水を加え全量を17.3gとし、一晩攪拌し完全に分散させ、加工剤Ｃ（ペプチド非含有ポリマー加工剤）を得た。これを参照実験用として用いた。 (Example 19)
Preparation of Peptide-Containing Polymer Processing Agents A and B Water was added to the polymer 8 (4.8 g) prepared in Example 18 to make a total amount of 121 g. Stir overnight and completely disperse to prepare Processing Agent A.
Processing agent A was diluted 10 times with distilled water to obtain processing agent B.
In addition, water was added to polymer 7 (346 mg) to make the total amount 17.3 g, and the mixture was stirred overnight to be completely dispersed to obtain a processing agent C (a peptide-free polymer processing agent). This was used for reference experiments.
（実施例２０）
ペプチド含有ポリマー加工剤Ｄの調整
　ポリマー９(2.08g)に水を加え全量を500gとした。一晩攪拌し完全に分散させ、加工剤Ｄを調整した。 (Example 20)
Water was added to adjustment polymer 9 (2.08 g) of peptide-containing polymer processing agent D to make the total amount 500 g. Stir overnight and completely disperse to prepare Processing Agent D.
（実施例２１）
加工布１４～１６の作製
　綿のJIS添付白布に対し、1.5～2.0倍の質量の加工剤Ａを生地全体に浸透するように加え、マングルで余分な加工剤を除去し、室温で乾燥させ、加工布１４を作製した。ペプチドの存在量は、1cm2あたり0.03μmolであった。加工剤は生地に対して4％であった。ニンヒドリン反応では生地が紫色に呈色することを確認した。
　綿のJIS添付白布に対し、1.5～2.0倍の質量の加工剤Ｂを生地全体に浸透するように加え、マングルで余分な加工剤を除去し、室温で乾燥させ、加工布１５を作製した。ペプチドの存在量は、1cm2あたり0.006μmolであった。加工剤は生地に対して0.4％であった。
　綿のJIS添付白布に対し、1.5～2.0倍の質量の加工剤Ｃ（参照例）を生地全体に浸透するように加え、マングルで余分な加工剤を除去し、室温で乾燥させ、加工布１６（参照例）を作製した。加工剤は生地に対して6.8％であった。
加工布１７の作製
　綿のJIS添付白布に対し、1.5～2.0倍の質量の加工剤Ｄを生地全体に浸透するように加え、マングルで余分な加工剤を除去し、室温で乾燥させ、加工布１７を作製した。ペプチドの存在量は、1cm2あたり0.006μmolであった。加工剤は生地に対して0.8％であった。 (Example 21)
Fabrication of processed fabrics 14 to 16 Add 1.5 to 2.0 times the mass of processing agent A so that it penetrates the entire fabric, and remove excess processing agent with a mangle, and dry at room temperature. A work cloth 14 was produced. The amount of peptide present was 0.03 μmol per cm 2 . The processing agent was 4% based on the dough. In the ninhydrin reaction, it was confirmed that the dough was colored purple.
A processing agent 15 having a mass of 1.5 to 2.0 times that of the cotton JIS-attached white cloth was added so as to penetrate the entire fabric, and the excess processing agent was removed with a mangle, followed by drying at room temperature to prepare a processed cloth 15. The amount of peptide present was 0.006 μmol / cm 2 . The processing agent was 0.4% based on the dough.
Add 1.5 to 2.0 times the mass of processing agent C (reference example) so that it penetrates the entire fabric, and remove excess processing agent with mangles and dry at room temperature. (Reference Example) was produced. The processing agent was 6.8% based on the dough.
Fabrication of processed cloth 17 Add 1.5 to 2.0 times the mass of processing agent D so that it penetrates the entire fabric, and remove excess processing agent with a mangle, and dry at room temperature. 17 was produced. The amount of peptide present was 0.006 μmol / cm 2 . The processing agent was 0.8% based on the dough.
（実施例２２）
抗菌性試験
　実施例１０と同じ方法で加工布１７について試験した。その結果を以下の表４に示す。 (Example 22)
Antibacterial test The processed fabric 17 was tested in the same manner as in Example 10. The results are shown in Table 4 below.
Figure JPOXMLDOC01-appb-T000130
 （実施例２３）
抗菌活性試験
　上記の如くして作製した加工布１４～１６について、抗微生物活性試験を行った。JIS L 1902 の定量試験方法に準拠して行った。培養後の生菌数測定法には、混釈平板培養法（コロニー法）を用いた。また、耐久性評価のための繰り返し洗濯試験は、JIS L 0217の洗い方103に規定する方法（40℃で5分間洗い、30℃で2回すすぎ洗いを2分間行う。脱水後、直射日光の当たらない場所でスクリーン乾燥）で、10回、20回、50回の繰り返し試験を行った。洗濯時には、ポリオキシエチレンアルキルエーテルを洗剤として用いた。ただし、静菌活性値は、（18時間培養後の標準布の生菌数の常用対数値－試験菌接種直後の標準布の生菌数の常用対数値）－（18時間培養後の加工布の生菌数の常用対数値－試験菌接種直後の加工布の生菌数の常用対数値）として示した。結果を以下の表５に示す。
Figure JPOXMLDOC01-appb-T000130
 (Example 23)
Antibacterial activity test The antibacterial activity test was performed on the processed fabrics 14 to 16 produced as described above. The measurement was performed in accordance with the JIS L 1902 quantitative test method. The pour plate culture method (colony method) was used as a method for measuring the viable cell count after the culture. In addition, the repeated washing test for durability evaluation was conducted according to the method specified in Washing Method 103 of JIS L 0217 (washing at 40 ° C for 5 minutes and rinsing twice at 30 ° C for 2 minutes. After dehydration, direct sunlight The test was repeated 10 times, 20 times, and 50 times by screen drying in a non-contact location. At the time of washing, polyoxyethylene alkyl ether was used as a detergent. However, the bacteriostatic activity value is (the common logarithm of the viable count of the standard cloth after 18 hours of culture-the common logarithm of the viable count of the standard cloth immediately after inoculation of the test bacteria)-(the processed cloth after 18 hours of culture) The common logarithm of the number of viable bacteria in the product-the common logarithm of the number of viable bacteria in the processed cloth immediately after inoculation of the test bacteria). The results are shown in Table 5 below.
Figure JPOXMLDOC01-appb-T000131
Figure JPOXMLDOC01-appb-T000131
（実施例２４）
抗菌性ペプチド含有ポリマーの安全性試験
　実施例１８で作製した、ポリマー８を用いて、以下のそれぞれの項目について、安全性試験を行った。
１．急性経口毒性試験
　ポリマー８を、供試動物のマウス雌雄各五頭に対して投与したが、二週間の試験期間中生存し、その間の生育にも異常は見られず、解剖をしても異常は認められなかった。
２．変異原生試験
　ポリマー８を試料として用い、労働安全衛生法における変異原性試験に準拠して行った。試験菌株は、ネズミチフス菌がTA1535、TA100、TA1537、TA98、大腸菌はWP2uvrAを用いた。結果は陰性であった。また、この濃度で更に、5菌株に対して行った復帰突然変異試験でも変異原性は陰性であった。
３．皮膚刺激性試験
　ポリマー８を含む加工剤Ａを用いて試験を行った。「ウサギにおける生体材料の一時皮膚過敏症試験に関する標準実施基準ASTM F719-81-1996」に準じた方法で行った。PII 値が2.00(弱刺激)未満であった。
４．皮膚感作性試験
　ポリマー８を用い、「医療機器の生物学的安全性評価のための試験方法について」（厚生労働省、発翰番号事務連絡医療機器審査No.36）に準じ、モルモットを用いたマキシミゼーション法により実施した。感作率は０％であった。 (Example 24)
Safety test of antibacterial peptide-containing polymer Using the polymer 8 produced in Example 18, a safety test was conducted for each of the following items.
1. Acute oral toxicity test Polymer 8 was administered to 5 males and 5 females of the test animals, but survived during the test period of 2 weeks. Was not recognized.
2. Mutagenicity test Polymer 8 was used as a sample, and the mutagenicity test was performed in accordance with the mutagenicity test in the Industrial Safety and Health Act. As test strains, Salmonella typhimurium used TA1535, TA100, TA1537, TA98, and Escherichia coli used WP2uvrA. The result was negative. Further, the mutagenicity was negative in the reverse mutation test conducted for 5 strains at this concentration.
3. Skin irritation test A test was conducted using processing agent A containing polymer 8. It was carried out by a method in accordance with "Standard Practice ASTM F719-81 -1996 on the Temporary skin hypersensitivity testing biomaterials in rabbits." PII value was less than 2.00 (weak stimulation).
4). Skin sensitization test Using polymer 8, guinea pigs were used in accordance with "Test method for biological safety assessment of medical devices" (Ministry of Health, Labor and Welfare, Medical Number Examination No. 36, Medical Device Examination No. 36). This was performed by the maximization method. The sensitization rate was 0%.
　以上の結果より、本発明の抗菌性ペプチド含有ポリマー及び抗菌性繊維は、安全であり、医療用途においても用いることができることが示された。 From the above results, it was shown that the antibacterial peptide-containing polymer and antibacterial fiber of the present invention are safe and can be used in medical applications.
　上記の記載は、本発明の目的及び対象を単に説明するものであり、添付の特許請求の範囲を限定するものではない。添付の特許請求の範囲から離れることなしに、記載された実施態様に対しての、種々の変更及び置換は、本明細書に記載された教示より当業者にとって明らかであり、それらは全て本発明の範囲である。 The above description merely explains the objects and objects of the present invention, and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein, all without departing from the scope of the appended claims. Range.

Claims (22)

  1. 　下記式（１）
    Figure JPOXMLDOC01-appb-C000001
     （式中Ｒ1は、水素原子又はメチル基であり、Ｕ1は、単結合、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗは、単結合或いはアミド基、チオエーテル基、チオエステル基、オキシム基、１，２，３－トリアゾール基、カルボニル基又はエステル基を有するリンカーであり、Peptideは、下記式で表されるアミノ酸配列：
    －ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－Ｘ４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ、Ｌｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６はＡｒｇ又はＬｙｓであり、Ｘ７はＡｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）
    で表される反復単位を含む抗菌性ペプチド含有ポリマー。     Following formula (1)
    Figure JPOXMLDOC01-appb-C000001
     Wherein R 1 is a hydrogen atom or a methyl group, U 1 is a single bond, O or NH, V is a single bond or a spacer, W is a single bond or an amide group, a thioether group, A linker having a thioester group, an oxime group, a 1,2,3-triazole group, a carbonyl group or an ester group, and Peptide is an amino acid sequence represented by the following formula:
    -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile, Leu or Val, X5 is Gly or Arg, and X6 is Arg Or Lys and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) It is a peptide. )
    The antibacterial peptide containing polymer containing the repeating unit represented by these.
  2. 　さらに、下記式（２）および（３）
    Figure JPOXMLDOC01-appb-C000002
     （式中Ｒ2及びＲ3は、それぞれ独立に水素原子又はメチル基であり、Ｕ2は、単結合、Ｏ又はＮＨであり、Ｕ3は、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗ1は、水素原子又は末端にハロゲンを付加してもよい上記リンカーＷの全部又は一部からなる基であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）
    で表される反復単位のうちの少なくとも一つを含む請求項１に記載の抗菌性ペプチド含有ポリマー。     Further, the following formulas (2) and (3)
    Figure JPOXMLDOC01-appb-C000002
     (Wherein R 2 and R 3 are each independently a hydrogen atom or a methyl group, U 2 is a single bond, O or NH, U 3 is O or NH, and V is a single bond or A spacer, W 1 is a hydrogen atom or a group consisting of all or part of the linker W to which halogen may be added at the end, and Y has a single bond or a substituent having a molecular weight of 10 to 3000 It may be a linear or branched alkylene chain or polyoxyalkylene chain.)
    The antibacterial peptide-containing polymer according to claim 1, comprising at least one repeating unit represented by:
  3. 　前記式（１）～（３）において、－Ｗ－が、下記式：
    Figure JPOXMLDOC01-appb-C000003
     （式中、ｔは、１～７の自然数である。）のいずれかを有するリンカーであり、かつ、Ｙが、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリエチレングリコール鎖である、請求項１または２に記載の抗菌性ペプチド含有ポリマー。     In the above formulas (1) to (3), —W— represents the following formula:
    Figure JPOXMLDOC01-appb-C000003
     (Wherein t is a natural number of 1 to 7), and Y may have a single bond or a substituent having a molecular weight of 10 to 3000, linear or branched The antibacterial peptide-containing polymer according to claim 1, wherein the polymer is a chain-like alkylene chain or polyethylene glycol chain.
  4. 　前記式（１）の－Ｗ－Peptideが、以下に表される式：
    Figure JPOXMLDOC01-appb-C000004
    （ここで、Peptideは前記と同じである。），
    Figure JPOXMLDOC01-appb-C000005
     （ここで、Peptideは前記と同じであり、ｉは１～１５の整数である。），
    Figure JPOXMLDOC01-appb-C000006
     （ここで、Peptideは前記と同じであり、ｉは１～７の整数である。），
    Figure JPOXMLDOC01-appb-C000007
     （ここで、Peptideは前記と同じであり、ｉは、１～７の整数であり、ｊは、１～１５の整数である。），
    Figure JPOXMLDOC01-appb-C000008
     （ここで、Peptideは前記と同じである。），
    Figure JPOXMLDOC01-appb-C000009
     （ここで、Peptideは前記と同じであり、ｉは、１～１５の整数である。），
    Figure JPOXMLDOC01-appb-C000010
     （ここで、Peptideは前記と同じであり、ｉは１～８の整数であり、ｊは１～８の整数である。），
    Figure JPOXMLDOC01-appb-C000011
     （ここで、Peptideは前記と同じであり、ｉは１～７の整数であり、ｊは１～７の整数である。），
    Figure JPOXMLDOC01-appb-C000012
     (ここで、Peptideは前記と同じである。），
    Figure JPOXMLDOC01-appb-C000013
     （ここで、Peptideは前記と同じであり、ｉは２～７の整数である。），
    Figure JPOXMLDOC01-appb-C000014
     （ここで、Peptideは前記と同じであり、i は１～７の整数であり、j は２～７の整数である。），
    Figure JPOXMLDOC01-appb-C000015
     （ここで、Peptideは前記と同じであり、i は１～１５の整数であり、j は１～１０の整数である。），
    Figure JPOXMLDOC01-appb-C000016
     （ここで、Peptideは前記と同じであり、i は１～８の整数であり、j は１～７の整数である。），
    Figure JPOXMLDOC01-appb-C000017
     （ここで、Peptideは前記と同じであり、i は１～７の整数であり、j は１～７の整数である。），
    Figure JPOXMLDOC01-appb-C000018
     （ここで、Peptideは前記と同じであり、ｉは２～７の整数である。），
    Figure JPOXMLDOC01-appb-C000019
     （ここで、Peptideは前記と同じであり、i は１～１０の整数であり、j は１～１５の整数である。），
    Figure JPOXMLDOC01-appb-C000020
     （ここで、Peptideは前記と同じであり、i は２～７の整数である。），及び
    Figure JPOXMLDOC01-appb-C000021
     （ここで、Peptideは前記と同じであり、i は１～１０の整数である。）
    からなる群から選ばれ、かつ、
    前記式（２）の－Ｗ1が、以下に表される式（３１）～（３８）：
    Figure JPOXMLDOC01-appb-C000022
     （ここで、ｉは、１～７の整数であり、ｊは１～８の整数であり、ｋは１～１５の整数であり、ｌは０～８の整数であり、ｈは１～５の整数であり、Ｘは、Ｃｌ、Ｂｒ又はＩである）
    からなる群から選ばれる、請求項２に記載の抗菌性ペプチド含有ポリマー。     In the formula (1), -W-Peptide is represented by the following formula:
    Figure JPOXMLDOC01-appb-C000004
    (Where Peptide is the same as above),
    Figure JPOXMLDOC01-appb-C000005
     (Where Peptide is the same as described above, and i is an integer of 1 to 15),
    Figure JPOXMLDOC01-appb-C000006
     (Here, Peptide is the same as described above, and i is an integer of 1 to 7.),
    Figure JPOXMLDOC01-appb-C000007
     (Here, Peptide is the same as above, i is an integer of 1 to 7, and j is an integer of 1 to 15.),
    Figure JPOXMLDOC01-appb-C000008
     (Where Peptide is the same as above),
    Figure JPOXMLDOC01-appb-C000009
     (Where Peptide is the same as described above, and i is an integer of 1 to 15),
    Figure JPOXMLDOC01-appb-C000010
     (Here, Peptide is the same as described above, i is an integer of 1 to 8, and j is an integer of 1 to 8.),
    Figure JPOXMLDOC01-appb-C000011
     (Here, Peptide is the same as above, i is an integer of 1 to 7, and j is an integer of 1 to 7.),
    Figure JPOXMLDOC01-appb-C000012
     (Where Peptide is the same as above),
    Figure JPOXMLDOC01-appb-C000013
     (Here, Peptide is the same as described above, and i is an integer of 2 to 7.),
    Figure JPOXMLDOC01-appb-C000014
     (Where Peptide is the same as above, i is an integer from 1 to 7, and j is an integer from 2 to 7),
    Figure JPOXMLDOC01-appb-C000015
     (Where Peptide is the same as above, i is an integer of 1 to 15, and j is an integer of 1 to 10),
    Figure JPOXMLDOC01-appb-C000016
     (Where Peptide is the same as above, i is an integer of 1 to 8, and j is an integer of 1 to 7),
    Figure JPOXMLDOC01-appb-C000017
     (Where Peptide is the same as above, i is an integer of 1 to 7, and j is an integer of 1 to 7),
    Figure JPOXMLDOC01-appb-C000018
     (Here, Peptide is the same as described above, and i is an integer of 2 to 7.),
    Figure JPOXMLDOC01-appb-C000019
     (Where Peptide is the same as above, i is an integer of 1 to 10, and j is an integer of 1 to 15),
    Figure JPOXMLDOC01-appb-C000020
     (Where Peptide is the same as above and i is an integer of 2 to 7), and
    Figure JPOXMLDOC01-appb-C000021
     (Here, Peptide is the same as described above, and i is an integer of 1 to 10.)
    Selected from the group consisting of
    In formula (2), -W 1 represents the following formulas (31) to (38):
    Figure JPOXMLDOC01-appb-C000022
     (Where i is an integer from 1 to 7, j is an integer from 1 to 8, k is an integer from 1 to 15, l is an integer from 0 to 8, and h is from 1 to 5) And X is Cl, Br or I)
    The antibacterial peptide-containing polymer according to claim 2, which is selected from the group consisting of:
  5. 　前記式（１）の－Ｗ－Peptideが、下記式：
    Figure JPOXMLDOC01-appb-C000023
     （ここで、Peptideは前記と同じである。）であり、前記式（２）のＷ1が、下記式：
    Figure JPOXMLDOC01-appb-C000024
     である請求項２に記載の抗菌性ペプチド含有ポリマー。     In formula (1), -W-Peptide is represented by the following formula:
    Figure JPOXMLDOC01-appb-C000023
     (Where Peptide is the same as above), and W 1 in the formula (2) is the following formula:
    Figure JPOXMLDOC01-appb-C000024
     The antibacterial peptide-containing polymer according to claim 2.
  6. 　前記スペーサーＶが、重量平均分子量が５０～１０００の置換基を有してもよい直鎖又は分岐状のポリエチレングリコール、又は分子量が１０～５００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖、或いはそれらの組合せである、請求項１～５のいずれかに記載の抗菌性ペプチド含有ポリマー。 The spacer V may have a linear or branched polyethylene glycol which may have a substituent having a weight average molecular weight of 50 to 1000, or a linear or branched which may have a substituent having a molecular weight of 10 to 500. The antibacterial peptide-containing polymer according to any one of claims 1 to 5, which is an alkylene chain of the above, or a combination thereof.
  7. 　下記式（４）
    Figure JPOXMLDOC01-appb-C000025
     で表される反復単位、及び下記式（５）
    Figure JPOXMLDOC01-appb-C000026
     で表される反復単位を含む抗菌性ペプチド含有ポリマー。     Following formula (4)
    Figure JPOXMLDOC01-appb-C000025
     And a repeating unit represented by the following formula (5):
    Figure JPOXMLDOC01-appb-C000026
     The antibacterial peptide containing polymer containing the repeating unit represented by these.
  8. 下記式（１）、（２）及び（３）
    Figure JPOXMLDOC01-appb-C000027
     で表される反復単位を含む抗菌性ペプチド含有ポリマーであって、
    式（１）中、Ｕ1がＯで、－Ｖ－Ｗ－Peptideが、－ＰＥＧ－Ｓ－Ｃｙｓ－Peptide（ここで、ＰＥＧは、重量平均分子量が１０～２０００のポリエチレングリコールである。）又は-ＣＨ2ＣＨＯＨＣＨ2－Ｓ-Ｃｙｓ-Peptide、又は、Ｕ1がＮＨで、－Ｖ－Ｗ－Peptideが、-Peptide、－（ＣＨ2）3－Ｓ－Ｃｙｓ－Peptide、－ＣＨ2－（Ｃ2Ｈ4Ｏ）3－Ｃ3Ｈ6－ＮＨ－ＣＯＣＨ2－Ｓ－Ｃｙｓ－Peptide、及び下記式：
    Figure JPOXMLDOC01-appb-C000028
     からなる群より選ばれ、かつ、
    式（３）中、Ｕ3がＯで、－Ｙ－Ｈが、－（ＣＨ2）11－ＣＨ3、又は－ＰＥＧ－Ｒ3（ここで、ＰＥＧは、重量平均分子量が１０～２０００のポリエチレングリコールであり、Ｒ3はＯＨ又はＣＨ3である。）、又はＵ3がＮＨで、－Ｙが単結合である、請求項２に記載の抗菌性ペプチド含有ポリマー。     The following formulas (1), (2) and (3)
    Figure JPOXMLDOC01-appb-C000027
     An antibacterial peptide-containing polymer comprising a repeating unit represented by
    In the formula (1), U 1 is O and -VW-Peptide is -PEG-S-Cys-Peptide (where PEG is polyethylene glycol having a weight average molecular weight of 10 to 2000) or -CH 2 CHOHCH 2 -S-Cys-Peptide, or U 1 is NH, -VW-Peptide is -Peptide,-(CH 2 ) 3 -S-Cys-Peptide, -CH 2- (C 2 H 4 O) 3 —C 3 H 6 —NH—COCH 2 —S-Cys-Peptide and the following formula:
    Figure JPOXMLDOC01-appb-C000028
     Selected from the group consisting of and
    In the formula (3), U 3 is O, -YH is-(CH 2 ) 11 -CH 3 , or -PEG-R 3 (wherein PEG is a polyethylene having a weight average molecular weight of 10 to 2000) The antibacterial peptide-containing polymer according to claim 2, wherein R 3 is OH or CH 3 ), or U 3 is NH and —Y is a single bond.
  9. 　前記ポリマー中に、前記Peptide含有単位を１～１００モル％の割合で含有する請求項１～８のいずれかに記載の抗菌性ペプチド含有ポリマー。 The antibacterial peptide-containing polymer according to any one of claims 1 to 8, wherein the Peptide-containing unit is contained in the polymer in a proportion of 1 to 100 mol%.
  10. 　前記ポリマー中に、前記Peptide含有単位を、０．４～２０モル％の割合で含有する請求項９に記載の抗菌性ペプチド含有ポリマー。 The antibacterial peptide-containing polymer according to claim 9, wherein the Peptide-containing unit is contained in the polymer at a ratio of 0.4 to 20 mol%.
  11. 　前記ペプチドが、
    －ＨＮ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｉｌｅ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＨＮ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＨＮ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＮＨ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｖａｌ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、－ＨＮ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯＮＨ2、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｉｌｅ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－、Ｈ2Ｎ－Ａｌａ－Ｌｅｕ－Ｔｙｒ－Ｌｅｕ－Ａｌａ－Ｖａｌ－Ａｒｇ－Ａｒｇ－Ａｒｇ－ＣＯ－、及びＨ2Ｎ－Ａｒｇ－Ｌｅｕ－Ｌｅｕ－Ｌｅｕ－Ａｒｇ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－Ａｒｇ－ＣＯ－からなる群より選ばれるペプチドである請求項１～１０のいずれかに記載の抗菌性ペプチド含有ポリマー。     The peptide is
    -HN-Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -NH-Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -NH -Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-CONH 2 , -HN-Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-CONH 2 , -HN-Arg -Leu-Tyr-Leu-Arg-Val-Gly-Arg-Arg-CONH 2 , -NH-Arg-Leu-Arg-Leu-Arg-Val-Gly-Arg-Arg-CONH 2 , -NH-Ala-Leu -Tyr-Leu-Ala-Val- Arg-Arg-Arg-CONH 2, -HN-Arg-Leu Leu-Leu-Arg-Val- Gly-Arg-Arg-CONH 2, H 2 N-Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-CO-, H 2 N-Arg-Leu- Arg-Leu-Arg-Ile-Gly-Arg-Arg-CO-, H 2 N-Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-CO-, H 2 N-Arg-Leu- Leu-Leu-Arg-Ile-Gly-Arg-Arg-CO-, H 2 N-Arg-Leu-Tyr-Leu-Arg-Val-Gly-Arg-Arg-CO-, H 2 N-Arg-Leu- Arg-Leu-Arg-Val- Gly-Arg-Arg-CO-, H 2 N-Ala-Leu-Tyr-Leu-Ala-Val-Arg-Ar -Arg-CO-, and according to any one of H 2 N-Arg-Leu- Leu-Leu-Arg-Val-Gly-Arg-Arg-CO- is a peptide selected from the group consisting of claims 1 to 10, Antibacterial peptide-containing polymer.
  12. 　請求項１～１１のいずれかに記載の抗菌性ペプチド含有ポリマーが繊維上にコーティングされた抗菌性繊維。 An antibacterial fiber comprising a fiber coated with the antibacterial peptide-containing polymer according to any one of claims 1 to 11.
  13. 　前記抗菌性ペプチド含有ポリマーが、ペプチド存在量で、布１ｃｍ2あたり０．００１μモル以上０．３μモル以下存在する、請求項１２に記載の抗菌性繊維。 The antibacterial fiber according to claim 12, wherein the antibacterial peptide-containing polymer is present in an amount of 0.001 μmol or more and 0.3 μmol or less per 1 cm 2 of the fabric in a peptide abundance.
  14. 　医療用用途またはサニタリー用途における請求項１２または１３に記載の抗菌性繊維の使用。 Use of the antibacterial fiber according to claim 12 or 13 for medical use or sanitary use.
  15. 　請求項１～１１のいずれかに記載の抗菌性ペプチド含有ポリマーを繊維上にコーティングすることにより、抗菌性ペプチドを繊維に固定化する方法。 A method for immobilizing an antibacterial peptide on a fiber by coating the fiber with the antibacterial peptide-containing polymer according to any one of claims 1 to 11.
  16. 　請求項１～１１のいずれかに記載の抗菌性ペプチド含有ポリマーを含有するポリマーエマルジョン。 A polymer emulsion containing the antibacterial peptide-containing polymer according to any one of claims 1 to 11.
  17. 　下記式（９）
    Figure JPOXMLDOC01-appb-C000029
     （式中Ｒ1は、水素原子又はメチル基であり、Ｕ1は、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチド又は誘導体化したペプチドと反応する官能基である。）
    で表される反復単位を含むポリマーに、下記式（１０）：
    Figure JPOXMLDOC01-appb-C000030
     （ここで、Peptideは、下記式で表されるアミノ酸配列：
    －ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基を有する分子量５０～５００の分子である。）
    で表されるペプチド又はその誘導体を反応させることにより、抗菌性ペプチド含有ポリマーを作製する方法。     Following formula (9)
    Figure JPOXMLDOC01-appb-C000029
     Wherein R 1 is a hydrogen atom or a methyl group, U 1 is O or NH, V is a single bond or a spacer, Wo is a functional group that reacts with a peptide or a derivatized peptide. is there.)
    In the polymer containing the repeating unit represented by the following formula (10):
    Figure JPOXMLDOC01-appb-C000030
     (Here, Peptide is an amino acid sequence represented by the following formula:
    -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecule having a molecular weight of 50 to 500 having a functional group that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. )
    A method for producing an antibacterial peptide-containing polymer by reacting the peptide represented by
  18. 下記式（９）および（３）
    Figure JPOXMLDOC01-appb-C000031
     （式中Ｒ1及びＲ3は、それぞれ独立に水素原子又はメチル基であり、Ｕ1及びＵ3は、それぞれ独立にＯ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチド又は誘導体化したペプチドと反応する官能基であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）
    で表される反復単位を含むポリマーに、下記式（１０）：
    Figure JPOXMLDOC01-appb-C000032
     （ここで、Peptideは、下記式で表されるアミノ酸配列：
    －ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷｏと反応しリンカーを形成する官能基を有する分子量５０～５００の分子である。）
    で表されるペプチド又はその誘導体を反応させることにより、抗菌性ペプチド含有ポリマーを作製する方法。     The following formulas (9) and (3)
    Figure JPOXMLDOC01-appb-C000031
     Wherein R 1 and R 3 are each independently a hydrogen atom or a methyl group, U 1 and U 3 are each independently O or NH, V is a single bond or a spacer, Wo is A functional group that reacts with a peptide or a derivatized peptide, and Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000.
    In the polymer containing the repeating unit represented by the following formula (10):
    Figure JPOXMLDOC01-appb-C000032
     (Here, Peptide is an amino acid sequence represented by the following formula:
    -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecule having a molecular weight of 50 to 500 having a functional group that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. )
    A method for producing an antibacterial peptide-containing polymer by reacting the peptide represented by
  19. 　前記式（９）で表される反復単位が下記式（６）
    Figure JPOXMLDOC01-appb-C000033
     （ここで、式中Ｒ2は、水素原子又はメチル基である。）
    であり、かつ、前記ペプチドが、前記式（１０）においてＺがＣｙｓである、請求項１７又は１８に記載の方法。     The repeating unit represented by the formula (9) is represented by the following formula (6).
    Figure JPOXMLDOC01-appb-C000033
     (Here, R 2 is a hydrogen atom or a methyl group.)
    The method according to claim 17 or 18, wherein the peptide is Z in the formula (10) is Cys.
  20. 　以下の工程：
    工程（１）：下記式（９）
    Figure JPOXMLDOC01-appb-C000034
     （式中Ｒ1は、水素原子又はメチル基であり、Ｕ1は、Ｏ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチドまたは誘導体化したペプチドと反応する官能基である。）
    で表される反復単位を含むポリマーを含む溶液を繊維にコーティングする工程、及び
    工程（２）：下記式：
    Figure JPOXMLDOC01-appb-C000035
     （ここで、Peptideは、下記式で表されるアミノ酸配列：
    －ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基を有する分子量５０～５００の分子である。）
    で表される抗菌性ペプチド又はその誘導体を含む溶液に、前記ポリマー溶液をコーティングした繊維を浸漬する工程、及び
    工程（３）：１～１０時間放置することによりポリマーにペプチドを結合させた後、該繊維を洗浄して未反応のペプチド又はその誘導体を除去するする工程、
    からなる抗菌性繊維の製造方法。     The following steps:
    Process (1): following formula (9)
    Figure JPOXMLDOC01-appb-C000034
     (Wherein R 1 is a hydrogen atom or a methyl group, U 1 is O or NH, V is a single bond or a spacer, Wo is a functional group that reacts with a peptide or a derivatized peptide) is there.)
    A step of coating a fiber with a solution containing a polymer containing a repeating unit represented by formula (2): and the following formula:
    Figure JPOXMLDOC01-appb-C000035
     (Here, Peptide is an amino acid sequence represented by the following formula:
    -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecule having a molecular weight of 50 to 500 having a functional group that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. )
    A step of immersing the fiber coated with the polymer solution in a solution containing the antibacterial peptide represented by the following: and step (3): after allowing the peptide to bind to the polymer by leaving it for 1 to 10 hours, Washing the fibers to remove unreacted peptides or derivatives thereof;
    The manufacturing method of the antimicrobial fiber which consists of.
  21. 　以下の工程：
    工程（１）：下記式（９）および（３）
    Figure JPOXMLDOC01-appb-C000036
     （式中Ｒ1及びＲ3は、それぞれ独立に水素原子又はメチル基であり、Ｕ1及びＵ3は、それぞれ独立にＯ又はＮＨであり、Ｖは、単結合又はスペーサーであり、Ｗoは、ペプチド又は誘導体化したペプチドと反応する官能基であり、Ｙは、単結合又は分子量１０～３０００の置換基を有してもよい直鎖又は分岐状のアルキレン鎖又はポリオキシアルキレン鎖である。）
    で表される反復単位を含むポリマーを含む溶液を繊維にコーティングする工程、及び
    工程（２）：下記式：
    Figure JPOXMLDOC01-appb-C000037
     （ここで、Peptideは、下記式で表されるアミノ酸配列：
    －ＮＨ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯＮＨ2、又はＨ2Ｎ－Ｘ１－Ｌｅｕ－Ｘ２－Ｌｅｕ－Ｘ３－X４－Ｘ５－Ｘ６－Ｘ７－ＣＯ－（式中、Ｘ１はＡｒｇ又はＡｌａであり、Ｘ２はＴｙｒ、Ａｒｇ又はＬｅｕであり、Ｘ３はＡｒｇ又はＡｌａであり、X４はＩｌｅ又はＬｅｕ又はＶａｌであり、Ｘ５はＧｌｙ又はＡｒｇであり、Ｘ６は、Ａｒｇ又はＬｙｓであり、Ｘ７は、Ａｒｇ又はＬｙｓである。ここで、Ａｒｇ－ＣＯＮＨ2又はＬｙｓ－ＣＯＮＨ2は、Ａｒｇ又はＬｙｓのカルボキシル基がアミド化していることを示す。）より選ばれる抗菌活性を有するペプチドである。）より選ばれ、Ｚは、水素原子、水酸基、Ｃｙｓ又はＷoと反応しリンカーを形成する官能基を有する分子量５０～５００の分子である。）
    で表される抗菌性ペプチド又はその誘導体を含む溶液に、前記ポリマー溶液をコーティングした繊維を浸漬する工程、及び
    工程（３）：１～１０時間放置することによりポリマーにペプチドを結合させた後、該繊維を洗浄して未反応のペプチド又はその誘導体を除去するする工程、
    からなる抗菌性繊維の製造方法。     The following steps:
    Step (1): Formulas (9) and (3) below
    Figure JPOXMLDOC01-appb-C000036
     Wherein R 1 and R 3 are each independently a hydrogen atom or a methyl group, U 1 and U 3 are each independently O or NH, V is a single bond or a spacer, Wo is A functional group that reacts with a peptide or a derivatized peptide, and Y is a single bond or a linear or branched alkylene chain or polyoxyalkylene chain which may have a substituent having a molecular weight of 10 to 3000.
    A step of coating a fiber with a solution containing a polymer containing a repeating unit represented by formula (2): and the following formula:
    Figure JPOXMLDOC01-appb-C000037
     (Here, Peptide is an amino acid sequence represented by the following formula:
    -NH-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CONH 2 , or H 2 N-X1-Leu-X2-Leu-X3-X4-X5-X6-X7-CO- ( In the formula, X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, X4 is Ile or Leu or Val, X5 is Gly or Arg, and X6 is Arg or Lys, and X7 is Arg or Lys, where Arg-CONH 2 or Lys-CONH 2 indicates that the carboxyl group of Arg or Lys is amidated.) Is a peptide having Z is a molecule having a molecular weight of 50 to 500 having a functional group that reacts with a hydrogen atom, a hydroxyl group, Cys or Wo to form a linker. )
    A step of immersing the fiber coated with the polymer solution in a solution containing the antibacterial peptide represented by the following: and step (3): after allowing the peptide to bind to the polymer by leaving it for 1 to 10 hours, Washing the fibers to remove unreacted peptides or derivatives thereof;
    The manufacturing method of the antimicrobial fiber which consists of.
  22. 　前記式（９）で表される反復単位が下記式（６）
    Figure JPOXMLDOC01-appb-C000038
     （ここで、式中Ｒ2は、水素原子又はメチル基である。）
    であり、かつ、前記ペプチドが、前記式１０においてＺがＣｙｓである、請求項２０又は２１に記載の抗菌性繊維の製造方法。     The repeating unit represented by the formula (9) is represented by the following formula (6).
    Figure JPOXMLDOC01-appb-C000038
     (Here, R 2 is a hydrogen atom or a methyl group.)
    The method for producing an antimicrobial fiber according to claim 20 or 21, wherein the peptide is Z in the formula 10 and Cys.
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