WO2019170160A1 - 多肽化合物及其制备方法与应用 - Google Patents

多肽化合物及其制备方法与应用 Download PDF

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WO2019170160A1
WO2019170160A1 PCT/CN2019/077668 CN2019077668W WO2019170160A1 WO 2019170160 A1 WO2019170160 A1 WO 2019170160A1 CN 2019077668 W CN2019077668 W CN 2019077668W WO 2019170160 A1 WO2019170160 A1 WO 2019170160A1
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cyclic
peptide
poly
amino acid
polypeptide compound
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PCT/CN2019/077668
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English (en)
French (fr)
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韩苏
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韩苏
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Priority to CN201980018137.0A priority Critical patent/CN111819187B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the field of biomedicine, in particular to a polypeptide compound and a preparation method and application thereof.
  • antimicrobial peptides Since the isolation of 22 amino acids of the bell antibacterial peptide from the skin of the bell in 1962, a variety of antimicrobial peptides have been found in the skin of amphibians. In 1980, the first insect-derived antibacterial polypeptide was discovered in the American silkworm, and later, in the microorganisms, aquatic animals, vertebrates, and mammals, polypeptide molecules with antibacterial defense functions were also discovered. Peptide molecules that produce organisms against pathogenic microorganisms are collectively referred to as "antibacterial peptides" or as host “defensins.” "Antimicrobial peptides" or “defensins" constitute the first line of defense against host infection by foreign pathogens, protecting the host itself from pathogenic microorganisms.
  • An antibacterial peptide is characterized by a peptide molecule composed of amino acids having a number of amino acids of less than 100, often carrying a positive charge, and having a defined antibacterial activity.
  • the natural antibacterial peptides produced by the organisms currently found have the functions of inhibiting Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, parasites and the like.
  • the most attractive advantages of antimicrobial peptides include: Most antibacterial peptides have an amino acid number between 15 and 45 amino acids.
  • the target is the bacterial cell membrane, which is non-toxic or low-toxic to the host, has no drug resistance, and has good properties. Thermal stability and biosafety.
  • Nisin produced by the probiotic lactic acid bacteria associated with humans is the most well-known antibacterial peptide.
  • Nisin is a linear peptide molecule consisting of 34 amino acids, which is in the Gram-positive bacteria causing food spoilage.
  • Staphylococcus, streptococcus, and micrococci have good growth inhibition effects and have been approved by the Food and Drug Administration of the United States, the European Union, and China as food additives for food preservation.
  • the antibacterial peptides discovered in the past are natural antibacterial peptides produced by natural organisms. According to the reported polypeptide molecules which are mostly single-stranded, the same or different antimicrobial peptides in the natural world are connected by disulfide bonds between the peptide chains. Morphology, but the morphology of the antibacterial peptide molecules in nature exhibiting multivalent copolymerization has not been reported so far.
  • antibacterial peptides do not produce drug resistance, they have good biosafety and environmental safety, which attracts scientists to use various natural antibacterial peptides as lead compounds for molecular modification in various ways, in order to obtain more effective antibacterial effects and more. Long-term stability, better physiological adaptability, easier to apply to daily or clinical implementation, more convenient for artificial synthesis of antimicrobial peptides.
  • the poly-B m is a linear peptide chain formed by using a basic amino acid as a main component, and may be composed of the same amino acid or composed of different amino acids.
  • the basic amino acid is selected from one or more of arginine (Arg R), lysine (Lys K), and histidine (His H), preferably arginine (Arg R).
  • K-(Cyclic-A n ) is a cyclic peptide structure in which a hydrophobic amino acid is the main component, a linear peptide chain is formed first, and then the carboxy terminus of the terminal end is condensed with the Dde-protected amino group on the side chain of lysine K. .
  • the hydrophobic amino acid is selected from the group consisting of phenylalanine (Phe F), valine (Val V), leucine (Leu L), isoleucine (Ile I), methionine (Met M), tryptophan ( One or more of Trp W), Proline, Ala A, Gly G.
  • the polypeptide compound may be a salt compound formed with an organic acid or an inorganic acid; or
  • the hydroxyl group carried by the polypeptide compound may form, but is not limited to, a compound such as an ether, an ester, a glycoside or a hydrazine formed; or
  • the thiol group carried by the polypeptide compound may form, but is not limited to, the formed thioether, glucosinolate, or a disulfide-containing compound formed with a cysteine or a cysteine-containing peptide; or
  • the amino group carried by the polypeptide compound may form, but is not limited to, the formed acylate, a hydrocarbon compound, a glycoside formed with a saccharide substance, or the like; or
  • the carboxyl group carried by the polypeptide compound may form, but is not limited to, the ester, amide compound, etc. formed; or
  • the imino group carried by the polypeptide compound may form, but is not limited to, the glycoside, acylate, hydrocarbonate, etc. formed; or
  • the phenolic hydroxyl group carried by the polypeptide compound may form, but is not limited to, the formed ester, ether, glycoside, terpenoid, a salt compound formed with an organic base or an inorganic base; or
  • a hydrate or solvent formed by the polypeptide compound is a hydrate or solvent formed by the polypeptide compound.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above polypeptide compound, a geometric isomer thereof, a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a process for the preparation of the above polypeptide compound, which comprises the synthesis of the linear peptide poly-B m -K-Cyclic-A n and the preparation of the cyclic peptide K-(Cyclic-A n ).
  • the synthesis of the linear peptide poly-B m -K-Cyclic-A n is first carried out by manual solid phase Fmoc/tBu synthesis method, and the H-CTC resin (substitution value is about 0.6 mmol/g) as a starting material.
  • the amino acid is condensed one by one from the C-terminus to the N-terminus of the polypeptide, and the peptide chain is extended until the linear linear poly-B m -K-Cyclic-A n target peptide chain is synthesized.
  • the amino acid raw material used may be an L-form amino acid or may be D-type amino acid;
  • Fmoc-AA A2- OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide were grafted with H-CTC resin to introduce the second amino acid residue at the C-terminus, Fmoc-AA A2 -
  • the equivalents of OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide are three times that of H-CTC resin; thus repeating the sequential condensation of the fused amino acid Fmoc-AA An -OH to complete the linear Cyclic Synthesis of the -A n chain; followed by synthesis of the amino acid starting material in the order of Fmoc-Lys(Dde)-OH, followed by synthesis of the poly-B m peptide chain, Fmoc-AA B1 -OH, Fmoc-AA B2 -OH, Fmoc-AA Bm- OH until the completion of the synthesis of the poly-B m peptide chain,
  • the N-terminus of the poly-B m -K-Cyclic-A n -H-CTC resin is capped with Boc anhydride, followed by removal of the side chain Dde protecting group of the lysine (K, Lys) residue at the cyclization to release Lys side chain amino group; cleavage of the resin releases the linear peptide molecule from the resin, and the active side chain group of the N-terminus and other amino acid residues is protected, only lysine (K, Lys)
  • the side chain amino group and the carboxyl group at the C-terminus of the peptide chain exhibit a naked linear peptide molecule poly-B m -K-Cyclic-A n , undergo an intramolecular condensation cyclization reaction, and then remove the protective group of the side chain group, Crystallization gives the crude product of the target (poly-B m )-K-(Cyclic-A n ) heterocyclic peptide, and finally the crude product is pur
  • a polypeptide compound for the preparation of an antibacterial agent.
  • the antibacterial agent is to kill or inhibit the growth of pathogenic bacteria and fungi which are common in wounds causing burns or wounds.
  • the antibacterial includes killing bacteria in prokaryotic microorganisms (including cocci, bacilli, spirochetes, etc., or Gram-negative bacteria and positive bacteria), actinomycetes (including mycoplasma, chlamydia, rickettsia), cyanobacteria ( Including Chlorella, Nostoc, Algae, etc.) or inhibit its growth.
  • prokaryotic microorganisms including cocci, bacilli, spirochetes, etc., or Gram-negative bacteria and positive bacteria
  • actinomycetes including mycoplasma, chlamydia, rickettsia
  • cyanobacteria Including Chlorella, Nostoc, Algae, etc.
  • the antibacterial includes killing or inhibiting the growth of fungi (including yeast, mold, penicillium, slime, etc.) and native pathogenic microorganisms (including amoeba, etc.) in eukaryotic microorganisms.
  • fungi including yeast, mold, penicillium, slime, etc.
  • native pathogenic microorganisms including amoeba, etc.
  • the antibacterial activity is to kill Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii or inhibit its growth; in particular, killing Enterococcus faecium, Staphylococcus Aureus), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and other resistant bacteria or resistant fungi It grows.
  • polypeptide compound (poly-B m )-K-(Cyclic-A n ) is an antibacterial heterocyclic peptide, and the advantages thereof are as follows:
  • poly-B m )-K-(Cyclic-A n ) molecule is an amphiphilic molecule, has hydrogel properties at a certain concentration, and has good coating adhesion to wounds; molecular structure of polypeptide compound of the present invention It has good molecular stability and long-lasting residence time in wounds.
  • the polypeptide compound of the invention has broad-spectrum antibacterial property and does not produce drug resistance, and can be widely applied to Gram-positive pathogenic bacteria, Gram. Negative pathogenic bacteria, fungal pathogenic bacteria inhibit growth or kill.
  • the polypeptide compound of the invention belongs to pure peptide molecules, and does not cause pollution to the physiological environment and the natural environment; the synthesis process of the polypeptide compound of the invention is easy to implement, easy to industrialize and scale, and the prepared polypeptide compound has high purity.
  • the present invention relates to the design and preparation of a polypeptide compound (poly-B m )-K-(Cyclic-A n ) in which a linear peptide is linked to a cyclic peptide.
  • poly-B m is a linear peptide chain formed by basic amino acids; basic amino acids include arginine (Arg R), lysine (Lys K), and histidine (His H). Any other class of amino acids may be incorporated anywhere in the linear peptide chain, and the poly-B m peptide chain may also consist entirely of a single species of basic amino acid, or all of the different types of basic amino acids.
  • m is the number of amino acids contained in the poly-B m peptide chain, and is suitably in the range of 1 to 15, and the most preferable range is 6 to 12.
  • K represents the use of lysine Fmoc-Lys(Dde)-OH with two reactive amino groups in the synthesis process, the terminal Fmoc-protected amino group condenses with the basic amino acid to form poly-B m , and the other side chain Dde protected amino group Condensation with the carboxy terminus of the end of the Cyclic-A n sequence results in the formation of a cyclic peptide structure.
  • Cyclic-A n is composed of a hydrophobic amino acid as a main component, and a linear peptide chain is formed first, and then the carboxy terminus of the terminal end is condensed with the Dde-protected amino group on the side chain of lysine K to form a cyclic peptide structure.
  • Hydrophobic amino acids include: phenylalanine (Phe F), valine (Val V), leucine (Leu L), isoleucine (Ile I), methionine (Met M), tryptophan (Trp W One or more of Proline, Ala A, and Gly G.
  • the Cyclic-A n peptide chain composed mainly of hydrophobic amino acids may be incorporated into any other class of amino acids at any position.
  • the cyclic peptide formed by the Cyclic-A n peptide chain may be composed entirely of a single type of hydrophobic amino acid, or may be composed entirely of any kind of hydrophobic amino acid.
  • n is the number of amino acids contained in the K-(Cyclic-A n ) cyclic peptide, and is suitably in the range of 4-15, and the most preferable range is 6-12.
  • the heterocyclic peptide molecule provided by the invention has the function of killing or inhibiting bacteria (including cocci, bacillus, spirochetia, etc., or gram-negative bacteria and positive bacteria), actinomycetes (including mycoplasma, chlamydia, ricketts) in prokaryotic microorganisms.
  • bacteria including cocci, bacillus, spirochetia, etc., or gram-negative bacteria and positive bacteria
  • actinomycetes including mycoplasma, chlamydia, ricketts
  • cyanobacteria including chlorella, diatoms, diatoms, etc.
  • fungi including yeast, mold, penicillium, slime, etc.
  • pathogenic microorganisms including amoeba, etc.
  • the heterocyclic peptides described in the present invention not only have the ability to kill Staphylococcus aureus (quality control strain), Escherichia coli (quality control strain), which cause wound infection, Pseudomonas aeruginosa (quality control strain), Acinetobacter (quality control strain), etc., also inhibit or kill ESKEPE (Enterococcus faecium) Enterococcus faecium, Staphylococcus aureus Staphylococcus aureus, Klebsiella pneumoniae Klebsiella pneumoniae Bacteria, Acinetobacter baumannii, Acinetobacter baumannii, Ps
  • the preparation method of the heterocyclic peptide molecule of the invention is divided into two parts, the first part is the synthesis of the linear peptide poly-B m -K-Cyclic-A n , and the second part is the preparation of the K-(Cyclic-A n ) cyclic peptide.
  • the heterocyclic peptide synthesis process is as follows:
  • the amino acid raw material used may be an L-form amino acid or a D-form amino acid; the D-form and the L-form of the amino acid are mirror images of each other, that is, the carbon atom of the amino acid is centered, the carboxyl group is on the upper side, and the amino group is L-shaped on the left side.
  • Amino acids, amino groups are D-amino acids on the right, and amino acids present in nature are L-form amino acids.
  • the first amino acid residue at the C-terminus was introduced by grafting 1.5 times equivalent of Fmoc-AA A1 -OH, 3 eq DIPEA (Diiso-propylethylamine, diisopropylethylamine) with a resin for 1 hour.
  • the resin was washed 6 times with DMF, and after washing the resin, the unreacted active site on the resin was blocked with methanol.
  • the synthesis of the linear Cyclic-A n chain is accomplished by repeating the condensation of the amino acid Fmoc-AA An -OH in this order. Thereafter, the synthesized amino acid starting material is Fmoc-Lys(Dde)-OH, and then the poly-B m peptide chain is synthesized, Fmoc-AA B1 -OH, Fmoc-AA B2 -OH, ... Fmoc-AA Bm - OH until the synthesis of the poly-B m peptide chain is completed.
  • the resin was washed 6 times with pure DMF, and the condensation efficiency was measured by Kaiser Test after each condensation reaction was completed, and if the amino acid condensation reaction was incomplete, the condensation reaction was repeated once.
  • the resin was washed 6 times with pure DMF and then drained.
  • the resin was cleaved with 1% TFA (Trifloroacetic Acid, trifluoroacetic acid)/DCM (Dichloromethane, dichloromethane) (v/v) to cleave the linear peptide molecules from the resin. Released.
  • TFA Trifloroacetic Acid, trifluoroacetic acid
  • DCM Dichloromethane, dichloromethane
  • the N-terminus of the obtained linear peptide molecule poly-B m -K-Cyclic-A n and the active side chain group of other amino acid residues have been protected, and it is Boc-AA Bm -K-AA An , only lysine
  • the side chain amino group of the acid (Lys) K and the carboxyl group at the C terminal of the peptide chain are in a naked state, and it is suitable for the intramolecular condensation reaction. Since the obtained polypeptide molecule has high purity, it is directly used for the next cyclization reaction.
  • the protected linear peptide molecule prepared above was dissolved in a small amount of DMF, and its concentration was diluted to 10 -3 M or less by DCM. After the addition of 1.2 eq of BOP/HOBT, the solution was adjusted to basic with DIPEA and the cyclization process started. The cyclization reaction lasted for 6-12 hours, and the cyclization process was followed by mass spectrometry (cyclization to dehydration reaction, molecular weight reduction after cyclization was 18 Da) until the reaction was completed.
  • the cleavage solution was added with a large amount of cold anhydrous diethyl ether to precipitate a polypeptide, and the polypeptide was precipitated by centrifugation. The precipitate was washed with diethyl ether several times and dried to give a crude product of the desired (poly-B m )-K-(Cyclic-A n ) heterocyclic peptide.
  • the heterocyclic peptide was purified by HP1100 (Agilent, USA) reversed-phase high performance liquid chromatography. Column packing: Agela C18 (10 ⁇ m, 50 ⁇ 250mm). Chromatographic operating conditions: mobile phase A (aqueous solution containing 0.05% trifluoroacetic acid, 2% acetonitrile), mobile phase B (90% acetonitrile / water), flow rate 25 ml per minute, UV detection wavelength 220 nm. The peak portion of the polypeptide was collected and lyophilized to obtain a pure white fluffy heterocyclic peptide. The chemical structure was characterized by MALDI-TOF mass spectrometry and the purity was determined by analytical HPLC (Agela C18-10). ⁇ 250 mm, flow rate 1 ml per minute) was tested to confirm the structure of the heterocyclic peptide of the present invention.
  • Polypeptide synthesis is currently a routine technique.
  • the principle and operation of peptide synthesis and purification can be found in Chapter 3, "Chemical Synthesis and Purification of Polypeptides," in the book “Contemporary Theory and Application of Polypeptide Hormones", edited by Sheng Shuli and published by Science and Technology Literature Publishing House (1998).
  • the manner of synthesizing and preparing the polypeptide compound of the present invention can be referred to the above solid phase synthesis method, but is not limited to this synthesis mode.
  • the poly-B m is a linear peptide chain formed by a basic amino acid as a main component.
  • Basic amino acids include arginine (Arg R), lysine (Lys K), histidine (His H), and can be incorporated into any other class of amino acids at any position in the linear peptide chain, poly-B m peptide
  • the chains may also consist entirely of a single type of basic amino acid, or all of a different type of basic amino acid.
  • m is the number of amino acids contained in the poly-B m peptide chain, and is suitably in the range of 1 to 15, and the most preferable range is 6 to 12.
  • the H-CTC resin substitution value was about 0.6 mmol/g
  • the condensed amino acid was added one by one from the C-terminus to the N-terminus of the polypeptide to extend the peptide chain until the linearity was completed.
  • the amino acid raw material used may be an L-form amino acid or a D-form amino acid.
  • the synthesis of the linear Cyclic-A n chain is accomplished by repeating the condensation of the amino acid Fmoc-AA An -OH in this order. Thereafter, the synthesized amino acid starting material is Fmoc-Lys(Dde)-OH, and then the poly-B m peptide chain is synthesized, Fmoc-AA B1 -OH, Fmoc-AA B2 -OH, ... Fmoc-AA Bm - OH until the synthesis of the poly-B m peptide chain is completed.
  • the resin was washed 6 times with pure DMF, and the condensation efficiency was measured by Kaiser Test after each condensation reaction was completed, and if the amino acid condensation reaction was incomplete, the condensation reaction was repeated once.
  • the linear peptide fragment of the present invention was obtained by the above method, and the partially synthesized linear peptide fragment is shown in Table 1, and the mass spectrum molecular weight thereof was measured.
  • K-Cyclic-A n is composed of a hydrophobic amino acid as a main component, and a linear peptide chain is formed first, and then the carboxy terminus of the terminal end is condensed with the Dde-protected amino group on the side chain of lysine K to form a cyclic peptide structure.
  • Hydrophobic amino acids include: phenylalanine (Phe F), valine (Val V), leucine (Leu L), isoleucine (Ile I), methionine (Met M), tryptophan (Trp W One or more of Proline, Ala A, and Gly G.
  • the Cyclic-A n peptide chain composed mainly of hydrophobic amino acids may be incorporated into any other class of amino acids at any position.
  • the cyclic peptide formed by the Cyclic-A n peptide chain may be composed entirely of a single type of hydrophobic amino acid, or may be composed entirely of any kind of hydrophobic amino acid.
  • n is the number of amino acids contained in the K-(Cyclic-A n ) cyclic peptide, and is suitably in the range of 4-15, and the most preferable range is 6-12.
  • the resin was washed 6 times with pure DMF and then drained.
  • the resin was cleaved with 1% TFA/DCM (v/v) to cleave the linear peptide molecules from the resin.
  • the N-terminus of the obtained linear peptide molecule K-(Cyclic-A n ) and the active side chain group of other amino acid residues have been protected, as Boc-K-AA An , only lysine (K, Lys)
  • the side chain amino group and the carboxyl group at the C-terminus of the peptide chain are in a naked state suitable for intramolecular condensation reaction. Since the obtained polypeptide molecule has high purity, it is directly used for the next cyclization reaction.
  • the protected linear peptide molecule prepared above was dissolved in a small amount of DMF, and its concentration was diluted to 10 -3 M or less by DCM. After the addition of 1.2 eq of BOP/HOBT, the solution was adjusted to basic with DIPEA and the cyclization process started. The cyclization reaction lasted for 6-12 hours, and the cyclization process was followed by mass spectrometry (cyclization to dehydration reaction, molecular weight reduction after cyclization was 18 Da) until the reaction was completed.
  • the cyclic peptide of the present invention is obtained according to the above method, wherein only the amino acid sequence of the partially synthesized cyclic peptide fragment is listed in Table 2, since K in the cyclic peptide is formed by condensation with the carboxy terminal of the amino acid at the Cyclic-A n terminal, and K It is the point of attachment to the linear peptide poly-B m , so K is defaulted to the starting point of the cyclic peptide and its mass spectral molecular weight is measured.
  • Example 3-2 RRRHRR KM(M)MM 1571.04 >98%
  • Example 3-3 HRR KF(F)PAGMM 1359.68 >98%
  • Example 3-4 KKKKRRHRRHHH KVLPPMWWWGAAM 3240.92 >98%
  • Example 3-7 RRRKHHRRRKKH KVWLVV 2457.99 >98%
  • Example 3-8 RRRRRRRR KVWLVVVG 2130.61 >98%
  • Example 3-9 RRRRRRRRRRRRR KVVVVWW 3113.75 >98%
  • Example 3-10 RRRRKRRRRH KFVLPWLPVG 2,652.22 >98%
  • Amino acid abbreviations in parentheses indicate that the amino acid is a D-amino acid.
  • Each polypeptide compound (listed in Table 4) was lyophilized with 10 mg/tube, and 10 ml of the culture solution was added to prepare a 1 mg/ml mother liquor; the culture solution was LB culture solution, and the preparation method was as follows: Tryptone 10g Yeast extract 5 g, NaCl 10 g, adjusted to pH 7.4, and autoclaved at 121 ° C for 20 minutes.
  • step 3 Take 2ml of the mother liquor of step 1 and add it to the #1 culture tube. After mixing, take 2ml and add it to the #2 culture tube and mix it. Then do half-dilution by using this method: respectively, the concentration is 1000 ⁇ g/ml, 500 ⁇ g/ml. 250 ⁇ g/ml, 125 ⁇ g/ml, 62.5 ⁇ g/ml, 31.25 ⁇ g/ml sample;
  • Example 3-8 the polypeptide compound is shown in Table 4.
  • the minimum inhibitory concentration of the single linear polypeptide molecule and the single cyclic polypeptide molecule against Staphylococcus aureus was >500 ⁇ g/ml, and there was substantially no bacteriostatic effect.
  • the polypeptide compound of the present invention has a minimum inhibitory concentration (MIC) of not more than 62.5 ⁇ g/ml after binding the linear peptide fragment to the cyclic peptide molecule, and has a bacteriostatic action.
  • the minimum inhibitory concentration (MIC) of the polypeptide compounds listed in Table 3 of this Example 3 was not significantly different from the #7 polypeptide compound in Table 4, and will not be repeated here.
  • the nutrient agar medium is placed in a culture dish and coagulated for bacterial solid culture.
  • the sample obtained in the third step of the experiment example 1 is added to each culture dish, and the mark is made at the bottom of the plate, and each concentration is made. Cultivate test discs;
  • the minimum inhibitory concentration of the single linear polypeptide molecule and the single cyclic polypeptide molecule against Pseudomonas aeruginosa is >500 ⁇ g/ml, and there is substantially no bacteriostatic effect.
  • the polypeptide compound of the present invention has a minimum inhibitory concentration (MIC) of not more than 62.5 ⁇ g/ml after binding the linear peptide fragment to the cyclic peptide molecule, and has a bacteriostatic action.
  • the minimum inhibitory concentration (MIC) of the polypeptide compounds listed in Table 3 of this Example 3 was not significantly different from the #7 polypeptide compound in Table 4, and will not be repeated here.
  • the nutrient agar medium is placed in a culture dish and coagulated for bacterial solid culture.
  • the sample obtained in the third step of the experiment example 1 is added to each plate, and the mark is made at the bottom of the plate, each concentration (by gradient) Diluting samples of 1000 ⁇ g/ml, 500 ⁇ g/ml, 250 ⁇ g/ml, 125 ⁇ g/ml, 62.5 ⁇ g/ml, and 31.25 ⁇ g/ml to prepare two culture test discs;
  • polypeptide compound of the present invention also has an effect of inhibiting the production of resistant Staphylococcus aureus, Escherichia coli, and Acinetobacter baumannii.
  • the resistant strains were: Staphylococcus aureus ATCC 12600 T (Gram-positive bacteria), Acinetobacter baumannii ATCC 19606 T (Gram-negative bacteria).
  • LB solid medium formulation (1 L): Tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.4, steam sterilized at 121 ° C under high pressure for 20 min.
  • Preparation of resistant bacteria (1) Activate the two resistant bacteria from the seed-preserving tube, and after 16S sequencing, determine the pure strain, transfer to LB solid medium, and place it at 37 °C for constant temperature culture. The box is incubated for 1-2 days and is ready for use. (2) The drug resistance of two drug-resistant strains (penicillin 10 ⁇ g/tablet, vancomycin 30 ⁇ g/tablet) resistance test to determine the drug resistance of drug-resistant bacteria, of which Staphylococcus aureus and Bowman did not move Bacillus is resistant to vancomycin. (3) The two resistant bacteria cultured were taken out from the incubator, the turbidity of the bacterial suspension was adjusted to 0.5, and uniformly applied to fresh LB solid medium.
  • the heterocyclic peptide of the present invention has better bacteriostatic effects than the cyclic peptide or the linear peptide alone, and the difference is remarkable.
  • the heterocyclic peptides R8-C8, R15-C8, and R4-C8 of the present invention all have better bacteriostatic effects than the cyclic peptide C8 alone; compared with the cyclic peptide C8 alone, the heterocyclic peptide R8-C8 of the present invention is abalone.
  • the antibacterial effect of Acinetobacter baumannii and Staphylococcus aureus is very good, significantly better than C8.
  • the heterocyclic peptides R8-C8, R8-C15, and R8-C5 of the present invention all have better antibacterial activity than the linear peptide R8 alone; compared with the linear peptide R8 alone, the heterocyclic peptide R8-C8 of the present invention. It has excellent antibacterial effect against Acinetobacter baumannii and Staphylococcus aureus, and is significantly better than R8.
  • the nutrient agar medium was placed in a culture dish and coagulated for fungal growth.
  • the strain of Candida albicans was obtained from the clinical isolate of the burnt ward of the Fourth Medical Center of the People's Liberation Army General Hospital and the Candida albicans quality control strain ATCC90028.
  • the assay used LS1809 Candida color plate (Guangzhou Dijing Microbiology Technology Co., Ltd., batch number: CCP-81101D, 2-8 °C).
  • the sample obtained in the third step of the experimental example 1 was added dropwise to each culture dish, and the bottom of the plate was marked, and two culture test plates were prepared for each concentration of each strain;
  • the culture medium of the fungus is taken, uniformly coated on the surface of the solid culture plate, and then cultured in a 37 ° C incubator for 24 hours to observe the growth of the fungus in the dish.
  • Table 8 The Candida albicans obtained from clinical isolation has the same results as the Q. albicans ATCC90028).
  • the minimum inhibitory concentration of the single linear polypeptide molecule and the single cyclic polypeptide molecule against Candida albicans is >500 ⁇ g/ml, and there is substantially no bacteriostatic effect.
  • the polypeptide compound of the present invention has a minimal inhibitory concentration (MIC) of not more than 125 ⁇ g/ml after binding the linear peptide fragment to the cyclic peptide molecule, and has a bacteriostatic action.
  • the minimum inhibitory concentration (MIC) of the polypeptide compounds listed in Table 3 of this Example 3 was not significantly different from the #7 polypeptide compound in Table 8, and will not be repeated here.
  • the hair removal range is about 3 cm x 3 cm for each of the left and right sides.
  • the skin erythema and edema formation of the animals were scored according to Table 9, and the scores of the three animals were added at time points, divided by the number of animals, and the mean value of skin irritation response at different time points was obtained. (stimulus index). The highest skin irritation index was taken, and the level of irritation intensity of the sample on the skin of the animal was evaluated according to Table 10.
  • Sample the polypeptide compound solution in Table 3 of the present invention (the polypeptide compound in Table 3 of the present invention is separately dissolved in deionized water to prepare a solution having a concentration of 10 mg/ml);
  • the acute stimulatory responses of rabbit cornea, iris and conjunctiva were scored according to Table 11, and each animal was calculated at three different observation times (24h, 48h and 72h), corneal damage, iris damage, conjunctival hyperemia and conjunctiva.
  • the "average score" for the four aspects of edema ie, the sum of the 24h, 48h, and 72h scores for each animal divided by the number of observations 3).
  • the cornea, iris and conjunctival hyperemia, edema average score and recovery time were used respectively, and the eye irritation intensity was determined according to Table 12 and Table 13 eye stimulation reaction grading standards.
  • the polypeptide compound provided by the invention has broad-spectrum antibacterial property and does not produce drug resistance, and can be widely applied to inhibit growth or killing of Gram-positive pathogenic bacteria, Gram-negative pathogenic bacteria and fungal pathogenic bacteria. Suitable for industrial applications.

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Abstract

公开了一种多肽化合物及其制备方法与应用，该多肽化合物的结构式为：(poly-Bm)-K-(Cyclic-An)；其中，poly-Bm是直链肽链，K为赖氨酸(Lys,K)，K-(Cyclic-An)是环状肽。所提供的多肽化合物具有抑制致病微生物生长的作用，可用于对细菌的防护或临床抗感染。

Description

多肽化合物及其制备方法与应用 技术领域
本发明涉及生物医药领域，特别是涉及一种多肽化合物及其制备方法与应用。
背景技术
自从1962年从铃蟾皮肤中分离获得由22个氨基酸组成的铃蟾抗菌肽之后，人们已从两栖动物皮肤中发现了多种抗菌肽。1980年在美国天蚕体内发现了第一个昆虫来源的抗菌多肽，以后在微生物、水产动物、脊椎动物、哺乳动物中也陆续发现具有抗菌防御功能的多肽分子。生物体产生抵御致病微生物的肽类分子被统称为“抗菌肽”，或被称为宿主“防御素”。“抗菌肽”或“防御素”构成了宿主抵抗外来病原菌感染的第一道防线，保护宿主自身不受致病微生物的侵害。
抗菌肽(antibacterial peptide)的特征是由氨基酸组成的肽分子，氨基酸数目少于100，常携带正电荷，具有确定的抗菌活性。目前发现的生物体产生的天然抗菌肽有抑制革兰氏阳性菌、革兰氏阴性菌、真菌、病毒、寄生虫等功能。抗菌肽最为吸引人关注的优点包括：大多数抗菌肽的氨基酸数目在15-45个氨基酸之间，作用靶点为细菌的细胞膜，对宿主无毒或低毒，无耐药性，具有良好的热稳定性及生物安全性。与人类友好伴生的益生菌乳酸菌产生的乳链菌肽(Nisin)是目前研究得最为清楚的抗菌肽，Nisin是由34个氨基酸组成的直链肽分子，它对引起食品腐败的革兰氏阳性菌中的葡萄球菌、链球菌、微球菌具有良好的抑制生长作用，被美国、欧盟、中国的食品药品监督管理局批准作为食品添加剂应用于食品保存。
以往发现的抗菌肽均是自然界生物体产生的天然抗菌肽，根据报道多为单链形态的多肽分子，自然界中也偶出现相同或不同的抗菌肽通过肽链之间的二硫键相连接的形态，但自然界中的抗菌肽分子呈现共聚多价的形态至今未见报道。
由于抗菌肽不产生耐药性，具有良好的生物安全性、环境安全性等，从而吸引了科学家以天然抗菌肽为先导化合物进行各种方式的分子改良，以期获得更为高效的抗菌效果、更长久的稳定性、更良好的生理适应性，更易于应用于日常或临床实施，更便于人工合成的抗菌肽。
发明内容
本发明的目的是针对现有技术中存在的技术缺陷，第一方面，提供一种多肽化合物，其结构式为：(poly-B m)-K-(Cyclic-A n)，poly-B m是直链肽链，K为赖氨酸(Lys,K)，K-(Cyclic-A n)是环状肽；其中，m为poly-B m直链肽链包含的氨基酸数目，n为K- (Cyclic-A n)环状肽所包含的氨基酸数目；优选的，m为1-15，n为4-15；更优选的，m为4-15，n为4-15；再优选的，m为6-12，n为6-12；最优选的，m为8，n为8。
poly-B m是以碱性氨基酸为主要组成部分形成的直链肽链，可由同一氨基酸组成，或由不同氨基酸组成。
所述碱性氨基酸选自精氨酸(Arg R)、赖氨酸(Lys K)、组氨酸(His H)中的一种或几种，优选精氨酸(Arg R)。
K-(Cyclic-A n)是由疏水氨基酸为主要组成部分，先形成直链肽链，然后其末端的羧基端与赖氨酸K的侧链上Dde保护的氨基缩合最后形成的环肽结构。
所述疏水氨基酸选自苯丙氨酸(Phe F)、缬氨酸(Val V)、亮氨酸(Leu L)、异亮氨酸(Ile I)、蛋氨酸(Met M)、色氨酸(Trp W)、脯氨酸(Pro P)、丙氨酸(Ala A)、甘氨酸(Gly G)中的一种或几种。
所述多肽化合物可以是与有机酸或无机酸形成的盐类化合物；或
所述多肽化合物所带有的羟基可形成但不限于所形成的醚、酯、苷或甙等的化合物；或
所述多肽化合物所带有的巯基可形成但不限于所形成的硫醚、硫苷，或与半胱氨酸或含半胱氨酸的肽所形成的含有二硫键的化合物；或
所述多肽化合物所带有的氨基可形成但不限于所形成的酰化物、烃化物、与糖类物质所形成的苷类物质等；或
所述多肽化合物所带有的羧基可形成但不限于所形成的酯、酰胺类化合物等；或
所述多肽化合物所带有的亚氨基可形成但不限于所形成的苷、酰化物、烃化物等；或
所述多肽化合物所带有的酚羟基可形成但不限于所形成的酯、醚、苷、甙类化合物，与有机碱或无机碱所形成的盐类化合物；或
所述多肽化合物与金属离子所形成的配合物、络和物或螯合物；或
所述多肽化合物所形成的水合物或溶剂物。
第二方面，本发明提供一种药物组合物，其含有上述的多肽化合物、其几何异构体、其药学上可接受的盐或溶剂化合物以及可药用载体或赋形剂的药物组合物。
第三方面，本发明提供制备上述多肽化合物的方法，包括直链肽poly-B m-K-Cyclic-A n的合成，及环肽K-(Cyclic-A n)的制备。
所述直链肽poly-B m-K-Cyclic-A n的合成，首先采用手工固相Fmoc/tBu合成法，以H-CTC树脂(取代值为约0.6mmol/g)为起始原料，从多肽的C端向N端逐一缩合氨基酸，延长肽链直至完成线性直链poly-B m-K-Cyclic-A n目标肽链的合成，所用的氨基酸原料可以是L型氨基酸，也可以是D型氨基酸；
具体为：首先用1.5倍当量的Fmoc-AA A1-OH，3倍当量的二异丙基乙胺与H-CTC 树脂(取代值为约0.6mmol/g)进行接枝引入C端第一个氨基酸残基，洗涤含H-CTC树脂的反应液，洗净后用甲醇对H-CTC树脂上未反应的活性位点进行封闭；然后，去除N端Fmoc保护基使N端成为自由氨基，用Fmoc-AA A2-OH、1-羟基苯并三唑和N,N’-二异丙基碳二亚胺与H-CTC树脂接枝引入C端第二个氨基酸残基，Fmoc-AA A2-OH、1-羟基苯并三唑和N,N’-二异丙基碳二亚胺的当量均是H-CTC树脂的3倍；如此反复依次连接缩合氨基酸Fmoc-AA An-OH完成线性Cyclic-A n链的合成；其后采用合成的氨基酸原料顺序为Fmoc-Lys(Dde)-OH，然后进行poly-B m肽链的合成，Fmoc-AA B1-OH，Fmoc-AA B2-OH，……Fmoc-AA Bm-OH直至完成poly-B m肽链的合成，得到poly-B m-K-Cyclic-A n-H-CTC树脂。
所述环肽K-(Cyclic-A n)的制备具体为：
poly-B m-K-Cyclic-A n-H-CTC树脂的N端采用Boc酸酐进行封端，随后去除环化处赖氨酸(K，Lys)残基的侧链Dde保护基以释放出Lys侧链氨基；对树脂进行切割将直链肽分子从树脂上裂解释放出来，获得N端以及其它氨基酸残基的活泼侧链基团均已被保护、仅有赖氨酸(K，Lys)的侧链氨基及肽链C端的羧基呈现裸露状态的直链肽分子poly-B m-K-Cyclic-A n，进行分子内的缩合环化反应，然后将侧链基团的保护基去除，重结晶即得到目标(poly-B m)-K-(Cyclic-A n)杂环肽的粗品，最后采用高效液相色谱(HPLC)纯化所述粗品，获得纯度>98％的多肽化合物(poly-B m)-K-(Cyclic-A n)。
第四方面，多肽化合物在制备抗菌药物中的应用。
所述抗菌为杀灭引起烧伤或创伤的创面常见的感染致病菌及真菌或抑制其生长。
所述抗菌包括杀灭原核微生物中的细菌(包括球菌、杆菌、螺旋菌等，或革兰氏阴性菌和阳性菌)、放线菌(包括支原体、衣原体、立克次氏体)、蓝藻(包括蓝球藻、念珠藻、颤藻等)或抑制其生长。
所述抗菌包括杀灭真核微生物中的真菌(包括酵母菌，霉菌、青霉菌、黏菌等)和原生致病微生物(包括阿米巴变形虫等)或抑制其生长。
所述抗菌为杀灭金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌或抑制其生长；尤其是杀灭屎肠球菌(Enterococcus faecium)、金黄色葡萄球菌(Staphylococcus aureus)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、鲍曼不动杆菌(Acinetobacter baumannii)、铜绿假单胞菌(Pseudomonas aeruginosa)、大肠杆菌(Enterobacter species)等耐药细菌及耐药真菌或抑制其生长。
本发明提供的多肽化合物(poly-B m)-K-(Cyclic-A n)为一种抗菌杂环肽，其优点在于：
(poly-B m)-K-(Cyclic-A n)分子是两亲性分子，在一定浓度下具有水凝胶的特性，对创面具有良好的涂布附着性；本发明多肽化合物的分子结构特点使其具有良好的分 子稳定性，在创面的滞留时间较为持久；本发明多肽化合物具有广谱的抗菌性且不产生耐药性，可广泛应用于对革兰氏阳性致病菌、革兰氏阴性致病菌、真菌致病菌的抑制生长或杀灭。
本发明多肽化合物属于纯肽类分子，对生理环境及自然环境均不造成污染；本发明多肽化合物合成工艺易于实施，易于产业化、规模化，且制备得到的多肽化合物高纯度。
具体实施方式
本发明涉及设计和制备出一种直链肽与环状肽相连的多肽化合物(poly-B m)-K-(Cyclic-A n)。
其中，poly-B m是以碱性氨基酸为主要组成部分形成的直链肽链；碱性氨基酸包括精氨酸(Arg R)、赖氨酸(Lys K)、组氨酸(His H)，可在直链肽链中的任何位置参入任意其它类别的氨基酸，poly-B m肽链也可以全部由单一种类的碱性氨基酸组成，或全部由不同种类的碱性氨基酸组成。m为组成poly-B m肽链包含的氨基酸数目，适宜范围为1-15，最佳范围为6-12。
K代表在合成过程中采用具有两个活性氨基的赖氨酸Fmoc-Lys(Dde)-OH，其末端Fmoc保护的氨基与碱性氨基酸缩合构成poly-B m，另一个侧链Dde保护的氨基与Cyclic-A n序列末端的羧基端缩合使其形成环状肽结构。
Cyclic-A n是由疏水氨基酸为主要组成部分，先形成直链肽链，然后其末端的羧基端与赖氨酸K的侧链上Dde保护的氨基缩合最后形成环肽结构。疏水氨基酸包括：苯丙氨酸(Phe F)、缬氨酸(Val V)、亮氨酸(Leu L)、异亮氨酸(Ile I)、蛋氨酸(Met M)、色氨酸(Trp W)、脯氨酸(Pro P)、丙氨酸(Ala A)、甘氨酸(Gly G)中的一种或几种。由疏水氨基酸为主的Cyclic-A n肽链组成中可在其任意位置参入任意其它类别的氨基酸。Cyclic-A n肽链形成的环肽可全部由单一种类的疏水性氨基酸组成，也可以全部由任意种类的疏水性氨基酸混合组成。n为K-(Cyclic-A n)环肽所包含的氨基酸数目，适宜范围为4-15，最佳范围为6-12。
本发明提供的杂环肽分子具有杀灭或抑制原核微生物中的细菌(包括球菌、杆菌、螺旋菌等，或革兰氏阴性菌和阳性菌)、放线菌(包括支原体、衣原体、立克次氏体)、蓝藻(包括蓝球藻、念珠藻、颤藻等)的生长；还具有杀灭或抑制真核微生物中的真菌(包括酵母菌，霉菌、青霉菌、黏菌等)和原生致病微生物(包括阿米巴变形虫等)的生长；本发明阐述的杂环肽不仅具有杀灭引起创面感染的金黄色葡萄球菌(质控株)、大肠埃希菌(质控株)、铜绿假单胞菌(质控株)、不动杆菌(质控株)等，还具有抑制或杀灭ESKEPE(Enterococcus faecium)屎肠球菌、Staphylococcus aureus金黄色 葡萄球菌、Klebsiella pneumoniae肺炎克雷伯氏菌、Acinetobacter baumannii鲍曼不动杆菌、Pseudomonas aeruginosa铜绿假单胞菌、Enterobacter species大肠杆菌等耐药细菌，还能抑制这些菌的生长。
本发明杂环肽分子的制备方法分为两部分，第一部分为直链肽poly-B m-K-Cyclic-A n的合成，第二部分为K-(Cyclic-A n)环肽的制备。杂环肽合成过程如下：
一、直链肽poly-B m-K-Cyclic-A n的合成：
首先采用手工固相Fmoc/tBu合成法，以H-CTC树脂(取代值为约0.6mmol/g)为起始原料，从多肽的C端向N端逐一添加缩合氨基酸，延长肽链直至完成线性直链poly-B m-K-Cyclic-A n目标肽链的合成。所用的氨基酸原料可以是L型氨基酸，也可以是D型氨基酸；氨基酸的D型与L型互为镜像异构体，即以氨基酸的碳原子为中心，羧基在上，氨基在左为L型氨基酸，氨基在右为D性氨基酸，自然界存在的氨基酸均为L型氨基酸。
首先用1.5倍当量的Fmoc-AA A1-OH，3eq DIPEA(Diiso-propylethylamine，二异丙基乙胺)与树脂进行接枝引入C端第一个氨基酸残基，反应时长1小时。用DMF洗涤树脂6次，洗净树脂后用甲醇对树脂上未反应的活性位点进行封闭。然后，采用25％PIPE(Piperidine,六氢吡啶)/DMF(Dimethyl Fromamide，N,N-二甲基甲酰胺)(v/v)去除N端Fmoc保护基使N端成为自由氨基(2次，每次10分钟)。用3倍当量的Fmoc-AA A2-OH/HOBt(1-Hydroxybenzotriazole，1-羟基苯并三唑)/DIC(N,N’-Diisopropylcarbodiimide，N,N’-二异丙基碳二亚胺)与树脂接枝引入C端第二个氨基酸残基。如此反复依次连接缩合氨基酸Fmoc-AA An-OH完成线性Cyclic-A n链的合成。其后采用合成的氨基酸原料顺序为Fmoc-Lys(Dde)-OH，然后进行poly-B m肽链的合成，Fmoc-AA B1-OH，Fmoc-AA B2-OH，……Fmoc-AA Bm-OH直至完成poly-B m肽链的合成。
线性直链肽的每一步氨基酸缩合反应完成之后用纯DMF洗涤树脂6次，并且每次缩合反应完成之后采用Kaiser Test检测缩合效率，若氨基酸缩合反应显示不完全，重复缩合反应一次。
二、K-(Cyclic-A n)环肽的制备：
线性直链肽poly-B m-K-Cyclic-A n合成完成之后，其N端采用Boc酸酐进行封端(4eq Boc 2O，8eq DIPEA，30分钟)，随后采用2％肼/DMF(v/v)去除环化处赖氨酸(Lys)K残基的侧链Dde保护基以释放出Lys侧链氨基(2次，每次15分钟)。
树脂用纯DMF清洗6遍后抽干，用1％TFA(Trifloroacetic Acid,三氟乙酸)/DCM(Dichloromethane，二氯甲烷)(v/v)对树脂进行切割将直链肽分子从树脂上裂解释 放出来。获得的直链肽分子poly-B m-K-Cyclic-A n的N端以及其它氨基酸残基的活泼侧链基团均已被保护,呈Boc-AA Bm-K-AA An，仅有赖氨酸(Lys)K的侧链氨基及肽链C端的羧基呈现裸露状态适宜进行分子内的缩合反应。由于得到的多肽分子纯度较高直接用于下一步的环化反应。
用少量的DMF溶解以上制备的保护直链肽分子，通过DCM将其浓度稀释到10 -3M以下。加入1.2eq的BOP/HOBT后，用DIPEA将溶液调节至碱性，环化过程开始。环化反应持续6-12个小时，用质谱对环化过程进行跟踪(环化为脱水反应，环化后分子量减少18Da)，直至反应进行完全。通过旋蒸除去溶剂后，使用切割试剂(三氟乙酸：1,2-乙二硫醇：苯甲硫醚：苯酚：H 2O:三异丙基硅烷＝68.5:10:5:3.5:1，v/v)将多肽侧链保护基去除，30℃下切割3小时。切割溶液加入大量冷的无水乙醚使多肽沉淀析出，离心获得多肽沉淀。用乙醚洗涤沉淀数次后干燥即得到目标(poly-B m)-K-(Cyclic-A n)杂环肽的粗品。
杂环肽分子的纯化及表征：
采用HP1100型(美国安捷伦公司)反相高效液相色谱仪对杂环肽粗品进行纯化。色谱柱填料：Agela C18(10μm，
Figure PCTCN2019077668-appb-000001
50×250mm)。色谱操作条件：流动相A(含0.05％三氟乙酸，2％乙腈的水溶液)，流动相B(90％乙腈/水)，流速为每分钟25毫升，紫外检测波长为220nm。收集多肽流出峰部分，经冻干溶剂后得到白色蓬松状态的杂环肽纯品，其化学结构由MALDI-TOF质谱进行表征，而其纯度则由分析型高效液相色谱仪(Agela C18-10×250mm，流速每分钟1毫升)检测，确证其结构为本发明的杂环肽。
杂环肽分子的存储：
采用HPLC纯化得到的(poly-B m)-K-(Cyclic-A n)杂环肽精制纯品(通常纯度>95％)经过冷冻干燥后呈现白色蓬松絮状，将杂环肽产物密封在瓶中置于-20℃避光保存。
多肽合成目前成为常规技术。多肽合成及纯化的原理和操作参见由盛树力主编，科学技术文献出版社(1998年)出版的“多肽激素的当代理论和应用”一书的第三章“多肽的化学合成和纯化”。本发明合成制备多肽化合物的方式可以参照以上固相合成方式，但并不局限于此合成方式。
实施例1：(poly-B m)-K-(Cyclic-A n)杂环肽中的直链肽片段
poly-B m是以碱性氨基酸为主要组成部分形成的直链肽链。碱性氨基酸包括精氨酸(Arg R)、赖氨酸(Lys K)、组氨酸(His H)，可在直链肽链中的任何位置参入任意其它类别的氨基酸，poly-B m肽链也可以全部由单一种类的碱性氨基酸组成，或全部由不同种类的碱性氨基酸组成。m为组成poly-B m肽链包含的氨基酸数目，适宜范围为 1-15，最佳范围为6-12。
首先采用手工固相Fmoc/tBu合成法，以H-CTC树脂(取代值为约0.6mmol/g)为起始原料，从多肽的C端向N端逐一添加缩合氨基酸，延长肽链直至完成线性直链(poly-B m)-K-(Cyclic-A n)目标肽链的合成。所用的氨基酸原料可以是L型氨基酸，也可以是D型氨基酸。
首先用1.5倍当量的Fmoc-AA A1-OH，3 eq DIPEA与树脂进行接枝引入C端第一个氨基酸残基，反应时长1小时。用DMF洗涤树脂6次，洗净树脂后用甲醇对树脂上未反应的活性位点进行封闭。然后，采用25％PIPE/DMF(v/v)去除N端Fmoc保护基使N端成为自由氨基(2次，每次10分钟)。用3倍当量的Fmoc-AA A2-OH/HOBt/DIC与树脂接枝引入C端第二个氨基酸残基。如此反复依次连接缩合氨基酸Fmoc-AA An-OH完成线性Cyclic-A n链的合成。其后采用合成的氨基酸原料顺序为Fmoc-Lys(Dde)-OH，然后进行poly-B m肽链的合成，Fmoc-AA B1-OH，Fmoc-AA B2-OH，……Fmoc-AA Bm-OH直至完成poly-B m肽链的合成。
线性直链肽的每一步氨基酸缩合反应完成之后用纯DMF洗涤树脂6次，并且每次缩合反应完成之后采用Kaiser Test检测缩合效率，若氨基酸缩合反应显示不完全，重复缩合反应一次。
按照上述方法得到本发明的直链肽片段，表1中列出部分合成得到的直链肽片段，并测得其质谱分子量。
表1 直链肽片段poly-B m的各基团列表
Figure PCTCN2019077668-appb-000002
表1结果表明，本发明合成出的直链肽片段质谱分子量与理论分子量的误差在千分之一范围之内(‰)，说明该直链肽片段确证为对应实施例的直链肽片段。
此实施例部分是为了公开直链肽片段的内容，并不是对本发明的限制，实际合成时可按以下实施例中的说明进行。
实施例2：(poly-B m)-K-(Cyclic-A n)杂环肽中的K-(Cyclic-A n)环肽片段
K-Cyclic-A n是由疏水氨基酸为主要组成部分，先形成直链肽链，然后其末端的羧基端与赖氨酸K的侧链上Dde保护的氨基缩合最后形成环肽结构。疏水氨基酸包括：苯丙氨酸(Phe F)、缬氨酸(Val V)、亮氨酸(Leu L)、异亮氨酸(Ile I)、蛋氨酸(Met M)、色氨酸(Trp W)、脯氨酸(Pro P)、丙氨酸(Ala A)、甘氨酸(Gly G)中的一种或几种。由疏水氨基酸为主的Cyclic-A n肽链组成中可在其任意位置参入任意其它类别的氨基酸。Cyclic-A n肽链形成的环肽可全部由单一种类的疏水性氨基酸组成，也可以全部由任意种类的疏水性氨基酸混合组成。n为K-(Cyclic-A n)环肽所包含的氨基酸数目，适宜范围为4-15，最佳范围为6-12。
线性直链肽K-Cyclic-A n合成完成之后，其N端采用Boc酸酐进行封端(4eq Boc 2O，8eq DIPEA，30分钟)，随后采用2％肼/DMF(v/v)去除环化处赖氨酸(K，Lys)残基的侧链Dde保护基以释放出Lys侧链氨基(2次，每次15分钟)。
树脂用纯DMF清洗6遍后抽干，用1％TFA/DCM(v/v)对树脂进行切割将直链肽分子从树脂上裂解释放出来。获得的直链肽分子K-(Cyclic-A n)的N端以及其它氨基酸残基的活泼侧链基团均已被保护,呈Boc-K-AA An，仅有赖氨酸(K，Lys)的侧链氨基及肽链C端的羧基呈现裸露状态适宜进行分子内的缩合反应。由于得到的多肽分子纯度较高直接用于下一步的环化反应。
用少量的DMF溶解以上制备的保护直链肽分子，通过DCM将其浓度稀释到10 -3M以下。加入1.2eq的BOP/HOBT后，用DIPEA将溶液调节至碱性，环化过程开始。环化反应持续6-12个小时，用质谱对环化过程进行跟踪(环化为脱水反应，环化后分子量减少18Da)，直至反应进行完全。通过旋蒸除去溶剂后，使用切割试剂(三氟乙酸：1,2-乙二硫醇：苯甲硫醚：苯酚：H 2O:三异丙基硅烷＝68.5:10:5:3.5:1，v/v)将多肽侧链保护基去除，30℃下切割3小时。切割溶液加入大量冷的无水乙醚使多肽沉淀析出，离心获得多肽沉淀。用乙醚洗涤沉淀数次后干燥即得到目标K-(Cyclic-A n)杂环肽的粗品。
按照上述方法得到本发明的环肽，其中表2中仅列出部分合成得到的环肽片段的氨基酸序列，由于环肽中的K是与Cyclic-A n末端的氨基酸羧基端缩合形成，且K是与直链肽poly-B m的连接点，因此将K默认为环肽的起点，并测得其质谱分子量。
表2 环肽片段K-(Cyclic-A n)的各基团列表
Figure PCTCN2019077668-appb-000003
表2结果表明，本发明合成出的环肽片段质谱分子量与理论分子量的误差在千分之一范围之内(‰)，说明该环肽片段确证为对应实施例的环肽片段。
此实施例部分是为了公开环肽片段的内容，并不是对本发明的限制，实际合成时可按以下实施例中的说明进行。
为了更好地比较与说明杂环肽的抑菌功能，以上分别列举了合成制备杂环肽的直链部分与环肽部分，为了证明本发明杂环肽的效果，还检测了组成杂环肽的各部分肽片段的独立抑菌效果。
实施例3：(poly-B m)-K-(Cyclic-A n)杂环肽
按照本发明阐述的杂环肽分子通式(poly-B m)-K-(Cyclic-A n)，杂环肽的合成制备方式参照说明书表述的合成步骤，直链肽及环肽片段见表3。
表3 杂环肽分子通式(poly-B m)-K-(Cyclic-A n)的各基团列表
实施例 poly-B m K-(Cyclic-A n) 分子量 纯度
实施例3-1 KRHRRHHK KPPVLFFFAIMMW 2745.38 >98％
实施例3-2 RRRHRR KM(M)MM 1571.04 >98％
实施例3-3 HRR KF(F)PAGMM 1359.68 >98％
实施例3-4 KKKKRRHRRHHH KVLPPMWWWGAAM 3240.92 >98％
实施例3-5 HHHHHHHH KFFFFF 1961.16 >98％
实施例3-6 RHKR KVLVWLVVVG 1671.09 >98％
实施例3-7 RRRKHHRRRKKH KVWLVV 2457.99 >98％
实施例3-8 RRRRRRRR KVWLVVVG 2130.61 >98％
实施例3-9 RRRRRRRRRRRRR KVVVVWWW 3113.75 >98％
实施例3-10 RRRRKRRRRH KFVLPWLPVG 2652.22 >98％
备注：括号内的氨基酸缩写字母表示该氨基酸为D型氨基酸
实验例一：本发明多肽化合物对革兰氏阳性菌金黄色葡萄球菌的抑菌作用
采用包括本实施例3中表3列出的多肽化合物及表4中列出的多肽化合物对革兰氏阳性常见致病菌金黄色葡萄球菌(Staphylococcus aureus，来自ATCC29213的质控菌)进行最小抑菌浓度(MIC)的检测：
1、将各多肽化合物(列于表4中)冻干粉10mg/管，加入10ml培养液配成1mg/ml母液；培养液为LB培养液，配置方法为：取胰化蛋白胨(Tryptone)10g，酵母提取物(Yeast extract)5g，NaCl 10g，调pH至7.4，121℃高压蒸汽灭菌20分钟。
2、取无菌消毒培养管5个，分别加入2ml培养液，编号#1、#2、#3、#4、#5；
3、取步骤1的母液2ml加入#1培养管里，混匀后再取2ml加入#2培养管里混匀，以此类推做对半稀释：分别得到浓度为1000μg/ml，500μg/ml，250μg/ml，125μg/ml，62.5μg/ml,31.25μg/ml的样品；
4、用在酒精灯火焰上灼烧过的接种环挑取少许大肠杆菌的菌落置于含有1ml生理盐水的培养管中混匀后，得到菌液，用加样器吸取100μl菌液分别加入不同浓度的含有各多肽化合物的培养管中(即步骤3得到的样品中)；
5、37℃恒温培养箱中培养24小时，观察各个培养管中对细菌的抑制情况，以培养液呈清亮的最小浓度为该多肽化合物的最小抑菌浓度(MIC)，以实施例3-8的多肽化合物为例，检测结果见表4。
表4 各多肽化合物对金黄色葡萄球的最小抑菌浓度(MIC)
编号 分子结构与序列 MIC//杀菌
#1 K-(Cyclic-(V)W(L)V(V))简写：环肽C5 >500μg/ml
#2 K-(Cyclic-(V)W(L)V(V)VG)简写：环肽C7 >500μg/ml
#3 K-(Cyclic-VL(V)W(L)V(V)VG)简写：环肽C9 >500μg/ml
#4 RRRR简写：直链R4 >1000μg/ml
#5 RRRRRRRR简写：直链R8 >1000μg/ml
#6 RRRRRRRRRRRR简写：直链R12 >1000μg/ml
#7 实施例3-8多肽化合物简写：R8-C8 <62.5μg/ml
由表4可以看出，单独的直链多肽分子和单独的环状多肽分子对金黄色葡萄球菌的最小抑菌浓度均>500μg/ml，基本没有抑菌作用。本发明的多肽化合物将直链肽片段与环状肽分子结合起来后，最小抑菌浓度(MIC)不超过62.5μg/ml，具有抑菌作用。
本实施例3中表3列出的多肽化合物的最小抑菌浓度(MIC)与表4中#7多肽化合物无显著差异，在此不一一赘述。
实验例二：本发明多肽化合物对革兰氏阴性菌铜绿假单胞杆菌(绿脓杆菌)的抑菌作用
1、将营养琼脂培养基铺在培养盘中凝固供细菌固体培养使用，在每个培养盘中滴加实验例一中步骤3得到的样品，并在盘底做记号，每个浓度制作2个培养检测盘；
2、接种环在酒精灯火焰上灼烧冷却后，蘸取含有铜绿假单胞杆菌(来自ATCC27853的质控菌)的培养液，在固体培养盘上做均匀涂抹，然后放在37℃恒温培养箱中孵育培养24小时，观察盘中铜绿假单胞杆菌的生长，以实施例3-8的多肽化合物为例，结果见表5。
表5 各多肽化合物对铜绿假单胞杆菌(绿脓杆菌)的最小抑菌浓度(MIC)
编号 分子结构与序列 最小抑菌浓度(MIC)
#1 K-(Cyclic-(V)W(L)V(V))简写：环肽C5 >500μg/ml
#2 K-(Cyclic-(V)W(L)V(V)VG)简写：环肽C7 >500μg/ml
#3 K-(Cyclic-VL(V)W(L)V(V)VG)简写：环肽C9 >500μg/ml
#4 RRRR简写：直链R4 >1000μg/ml
#5 RRRRRRRR简写：直链R8 >1000μg/ml
#6 RRRRRRRRRRRR简写：直链R12 >1000μg/ml
#7 实施例3-8多肽化合物简写：R8-C8 <62.5μg/ml
由表5可以看出，单独的直链多肽分子和单独的环状多肽分子对铜绿假单胞杆菌的最小抑菌浓度均>500μg/ml，基本没有抑菌作用。本发明的多肽化合物将直链肽片段与环状肽分子结合起来后，最小抑菌浓度(MIC)不超过62.5μg/ml，具有抑菌作用。
本实施例3中表3列出的多肽化合物的最小抑菌浓度(MIC)与表4中#7多肽化合物无显著差异，在此不一一赘述。
实验例三：本发明多肽化合物对耐药菌的抑制作用
实验一、
以实施例3-8的多肽化合物为例，检测其对各种耐药菌的生长抑制作用。
1、将营养琼脂培养基铺在培养盘中凝固供细菌固体培养使用，在每个培养盘中滴加实验例一中步骤3得到的样品，并在盘底做记号，每个浓度(按梯度稀释得到1000μg/ml、500μg/ml、250μg/ml、125μg/ml、62.5μg/ml、31.25μg/ml的样品)制作2个培养检测盘；
2、接种环在酒精灯火焰上灼烧冷却后，蘸取表6列出的各种耐药菌(其中大肠杆菌对青霉素和万古霉素具有耐药性，金黄色葡萄球菌和鲍曼不动杆菌对万古霉素具有耐药性)的培养液，在固体培养盘表面做均匀涂抹，然后放在37℃培养箱中孵育培养24小时，观察盘中各种耐药菌的生长，结果见表6。
表6 实施例3-8的多肽化合物对耐药菌的最小抑菌浓度(MIC)
Figure PCTCN2019077668-appb-000004
表6的结果表明，本发明的多肽化合物对耐药的金黄色葡萄球菌、大肠杆菌、鲍曼不动杆菌也具有抑制其生产的作用。
实验二、
耐药菌株为：金黄色葡萄球菌Staphylococcus aureus ATCC 12600 T(革兰氏阳性菌)、鲍曼不动杆菌Acinetobacter baumannii ATCC 19606 T(革兰氏阴性菌)。
实验材料：药敏纸片(直径6mm)，商购得到：青霉素(Penicillin)10μg/片、万古霉素(Vancomycin)30μg/片。
LB固体培养基配方(1L)：含胰化蛋白胨(Tryptone)10g，酵母提取物(Yeast extract)5g，NaCl 10g，pH 7.4，高压下蒸汽灭菌121℃，20min。
实验步骤：
1、耐药菌的准备：(1)、将两种耐药菌从保种管活化出来，经16S测序，确定为纯菌株后，转接到LB固体培养基上，放置到37℃恒温培养箱，培养1-2天，待用。(2)、对两种耐药菌株进行药敏(青霉素10μg/片、万古霉素30μg/片)抗性实验， 以确定耐药菌的耐药性，其中金黄色葡萄球菌和鲍曼不动杆菌对万古霉素具有耐药性。(3)、将培养好的两种耐药菌从培养箱取出，将菌悬液浊度均调至0.5，均匀涂至新鲜的LB固体培养基上。
2、实验样品：将表7所列的多肽化合物8000r/min离心5分钟，然后用灭菌纯水进行溶解和梯度稀释，难溶的样品在用纯水溶解前，可加入30μl DMSO作为助溶剂进行溶解。
3、MIC值的测定：(1)、样品依次被无菌纯水稀释至浓度为1.0mg/ml、0.5mg/ml、0.25mg/ml、0.125mg/ml、0.1mg/ml、0.05mg/ml、0.025mg/ml。(2)、将稀释好的各样品吸取2μl分别滴加到涂有耐药菌的LB固体培养基中。(3)、将点好样的LB固体培养基平稳放置于37℃的恒温培养箱中培养，培养2天，结果见表7。
表7 本发明多肽化合物对耐药菌的最小抑菌浓度(MIC，mg/ml)
样品 鲍曼不动杆菌 金黄色葡萄球菌
KVWLVVVG：C8 >1.0 0.5
RRRR：R4 >1.0 >1.0
HHHHHHHH-KVWLVVVG：H8-C8 >1.0 >1.0
RRRRRRRR-KVWLVVVG：R8-C8 0.1 0.05
RRRRRRRRRRRRRRR-KVWLVVVG：R15-C8 0.25 0.25
RRRR-KVWLVVVG：R4-C8 0.5 0.05
RRRRRRRR-KVVPWLVGVVPVVLG：R8-C15 0.5 1.0
RRRRRRRR：R8 1.0 >1.0
RRRRRRRRRRRRRRR：R15 0.25 1.0
RRRRRRRR-KVWLV：R8-C5 0.25 0.5
由表7可以看出，本发明的杂环肽比单独的环肽或直链肽的抑菌效果都要好，且差异显著。如本发明的杂环肽R8-C8、R15-C8、R4-C8均比单独的环肽C8的抑菌效果好；与单独的环肽C8相比，本发明杂环肽R8-C8对鲍曼不动杆菌和金黄色葡萄球菌的抑菌效果非常优异，明显优于C8。如本发明的杂环肽R8-C8、R8-C15、R8-C5均比单独的直链肽R8的抑菌效果好；与单独的直链肽R8相比，本发明杂环肽R8-C8对鲍曼不动杆菌和金黄色葡萄球菌的抑菌效果非常优异，明显优于R8。
以上说明直链肽和环肽相结合能够加强多肽化合物的抑菌效果，比单独的直链肽和环肽的抑菌效果都要好。
实验例四：本发明多肽化合物对真菌的抑菌作用
1、将营养琼脂培养基铺在培养盘中凝固供真菌生长，白色念珠菌的菌株来自解放军总医院第四医学中心烧伤科病房从临床分离获得的菌株及白色念珠菌质控菌株ATCC90028。检定实验采用LS1809念珠菌显色平板(广州市迪景微生物科技有限公司出品，批号：CCP-81101D，2-8℃保存)。在每个培养盘中滴加实验例一中步骤3得到的样品，并在盘底做记号，每种菌株每个浓度制作2个培养检测盘；
2、接种环在酒精灯火焰上灼烧冷却后，蘸取真菌的培养液，在固体培养盘表面做均匀涂抹，然后放在37℃培养箱中孵育培养24小时，观察盘中真菌的生长，以实施例3-8的多肽化合物为例，结果见表8(从临床分离获得的白色念珠菌与质控白色念珠菌ATCC90028结果相同)。
表8 各多肽化合物对真菌的最小抑菌浓度(MIC)
编号 分子结构与序列 最小抑菌浓度(MIC)
#1 K-(Cyclic-(V)W(L)V(V))简写：环肽C5 >500μg/ml
#2 K-(Cyclic-(V)W(L)V(V)VG)简写：环肽C7 >500μg/ml
#3 K-(Cyclic-VL(V)W(L)V(V)VG)简写：环肽C9 >500μg/ml
#4 RRRR简写：直链R4 >1000μg/ml
#5 RRRRRRRR简写：直链R8 >1000μg/ml
#6 RRRRRRRRRRRR简写：直链R12 >1000μg/ml
#7 实施例3-8多肽化合物简写：R8-C8 <125μg/ml
由表8可以看出，单独的直链多肽分子和单独的环状多肽分子对白色念珠菌的最小抑菌浓度均>500μg/ml，基本没有抑菌作用。本发明的多肽化合物将直链肽片段与环状肽分子结合起来后，最小抑菌浓度(MIC)不超过125μg/ml，具有抑菌作用。
本实施例3中表3列出的多肽化合物的最小抑菌浓度(MIC)与表8中#7多肽化合物无显著差异，在此不一一赘述。
实验例五：本发明多肽化合物的生物安全性
实验一：一次完整皮肤刺激试验
1、实验材料及来源
家兔(来自解放军总医院第四医学中心动物实验室)，样品：本发明表3中多肽化合物溶液(将本发明表3中多肽化合物分别溶于去离子水中，配制成浓度为10mg/ml的溶液)；
2、实验步骤
(1)在试验前24h，用脱毛剂将家兔背部脊柱两侧的毛去掉，不损伤皮肤。去毛范围，左、右各约3cm×3cm。
(2)次日分别将本发明表3中多肽化合物溶液(浓度为10mg/ml)直接滴于面积为2.5cm×2.5cm的一侧去毛皮肤上，或滴于同样大小的2层-4层纱布上并敷贴在一侧去毛皮肤表面，然后用一层无刺激塑料膜或油纸覆盖，再用无刺激胶布固定。另一侧去毛皮肤作为空白对照(或溶剂对照)。敷贴时间为4h。试验结束后，用温水或无刺激性溶剂除去残留样品。
(3)分别于去除样品1h、24h和48h后观察皮肤局部反应，并按表9进行刺激反应评分。
表9 皮肤刺激反应评分标准
Figure PCTCN2019077668-appb-000005
按表9的评分标准，本发明表3中多肽化合物溶液处理过的所有家兔评分均为0分，未见红斑或水肿，表明本发明多肽化合物对于动物完整皮肤无刺激性。
实验二：一次破损皮肤刺激试验
1、实验材料及来源
家兔(来自解放军总医院第四医学中心动物实验室)，样品：本发明表3中多肽化合物溶液(将本发明表3中多肽化合物分别溶于去离子水中，配制成浓度为10mg/ml的溶液)；
2、实验步骤
(1)涂样品前，在2.5cm×2.5cm的去毛皮肤上，用75％酒精清洁、消毒暴露皮肤，待酒精挥发后，用灭菌刀片或注射针头在皮区内划一个“井”形的破损伤口，并 在该破损皮区内染毒，皮肤破损仅达表皮，不伤及真皮。
(2)涂样品前的皮肤准备、样品的涂抹和局部皮肤反应的观察，评分方法同实验一。在观察过程中需要鉴别感染和原发性刺激反应的区别，若有感染可疑，进行重复测试。
在各个观察时间点，按照表9对动物的皮肤红斑与水肿形成情况进行评分，并分别按时间点将3只动物的评分相加，除以动物数，获得不同时间点的皮肤刺激反应积分均值(刺激指数)。取其中最高皮肤刺激指数，按表10评定该样品对动物皮肤刺激强度的级别。
表10 皮肤刺激强度分级
皮肤刺激指数 刺激强度级别
0～0.5 无刺激性
0.5～2.0 轻刺激性
2.0～6.0 中等刺激性
6.0～8.0 强刺激性
按表10的强度分级，本发明表3中多肽化合物溶液处理过的所有家兔评分均为0分，未见红斑或水肿，表明本发明多肽化合物对于动物破损皮肤无刺激性。
实验三：急性眼刺激试验
1、实验材料及来源
家兔(来自解放军总医院第四医学中心动物实验室)，试验前检查家兔双眼，有异常者不能用于试验。
样品：本发明表3中多肽化合物溶液(将本发明表3中多肽化合物分别溶于去离子水中，配制成浓度为10mg/ml的溶液)；
2、实验步骤
(1)吸取样品0.1ml，滴入家兔一侧眼结膜囊内，另一侧眼滴以生理盐水作为正常对照。
(2)滴样品后，将眼被动闭合30s后用生理盐水冲洗。于滴眼1h、24h、48h、72h、7d、14d和21d后，肉眼观察家兔眼结膜、虹膜和角膜的损伤与恢复情况。如果72h内未出现刺激反应，或第7d或第14d，眼睛刺激反应完全恢复，即可提前终止试验。必要时，用2％荧光素钠溶液或裂隙灯、放大镜检查角膜及虹膜变化。
3、评价规定
按表11对家兔眼角膜、虹膜和结膜的急性刺激反应进行评分，并分别计算每只动 物在三个不同观察时间(24h、48h和72h)、在角膜损害、虹膜损害、结膜充血和结膜水肿四方面的“平均评分”(即每只动物的24h、48h和72h评分之和除以观察次数3)。分别以动物眼角膜、虹膜和结膜充血、水肿的平均评分和恢复时间进行，按表12、表13眼刺激反应分级标准判定样品对眼睛的刺激强度。
表11 家兔急性眼刺激反应的评分标准
Figure PCTCN2019077668-appb-000006
表12 眼刺激性反应分级标准
Figure PCTCN2019077668-appb-000007
Figure PCTCN2019077668-appb-000008
表13 眼刺激性反应分级标准
Figure PCTCN2019077668-appb-000009
按表11-13的分级标准，本发明表3中多肽化合物溶液处理过的所有家兔评分均为0分，未见红斑或水肿，表明本发明多肽化合物对于家兔角膜、虹膜和结膜无急性刺激性。
工业应用性
本发明提供的多肽化合物具有广谱的抗菌性且不产生耐药性，可广泛应用于对革兰氏阳性致病菌、革兰氏阴性致病菌、真菌致病菌的抑制生长或杀灭，适于工业应用。

Claims (17)

  1. 一种多肽化合物，其特征在于，其结构式为：(poly-B m)-K-(Cyclic-A n)，poly-B m是直链肽链，K为赖氨酸(Lys,K)，K-(Cyclic-A n)是环状肽；其中，m为poly-B m直链肽链包含的氨基酸数目，n为K-(Cyclic-A n)环状肽所包含的氨基酸数目；优选的，m为1-15，n为4-15；更优选的，m为4-15，n为4-15；再优选的，m为6-12，n为6-12；最优选的，m为8，n为8。
  2. 根据权利要求1所述的多肽化合物，其特征在于，poly-B m是由碱性氨基酸形成的直链肽链，可由同一碱性氨基酸组成，或由不同碱性氨基酸组成。
  3. 根据权利要求2所述的多肽化合物，其特征在于，所述碱性氨基酸选自精氨酸(Arg R)、赖氨酸(Lys K)、组氨酸(His H)中的一种或几种，优选精氨酸(Arg R)。
  4. 根据权利要求1或2或3所述的多肽化合物，其特征在于，K-(Cyclic-A n)是由疏水氨基酸形成的环状肽。
  5. 根据权利要求4所述的多肽化合物，其特征在于，所述环状肽是先合成直链肽链，直链肽链的一端为赖氨酸K；然后直链肽链一端的赖氨酸K上的氨基与另一端的氨基酸上的羧基进行氨基缩合形成环状肽。
  6. 根据权利要求4或5所述的多肽化合物，其特征在于，所述疏水氨基酸选自苯丙氨酸(Phe F)、缬氨酸(Val V)、亮氨酸(Leu L)、异亮氨酸(Ile I)、蛋氨酸(Met M)、色氨酸(Trp W)、脯氨酸(Pro P)、丙氨酸(Ala A)、甘氨酸(Gly G)中的一种或几种。
  7. 根据权利要求1-6任一所述的多肽化合物，其特征在于，所述多肽化合物可以是与有机酸或无机酸形成的盐类化合物；或
    所述多肽化合物所带有的羟基可形成但不限于所形成的醚、酯、苷或甙等的化合物；或
    所述多肽化合物所带有的巯基可形成但不限于所形成的硫醚、硫苷，或与半胱氨酸或含半胱氨酸的肽所形成的含有二硫键的化合物；或
    所述多肽化合物所带有的氨基可形成但不限于所形成的酰化物、烃化物、与糖类物质所形成的苷类物质等；或
    所述多肽化合物所带有的羧基可形成但不限于所形成的酯、酰胺类化合物等；或
    所述多肽化合物所带有的亚氨基可形成但不限于所形成的苷、酰化物、烃化物等；或
    所述多肽化合物所带有的酚羟基可形成但不限于所形成的酯、醚、苷、甙类化合物，与有机碱或无机碱所形成的盐类化合物；或
    所述多肽化合物与金属离子所形成的配合物、络和物或螯合物；或
    所述多肽化合物所形成的水合物或溶剂物。
  8. 药物组合物，其含有权利要求1-7任一所述的多肽化合物、其几何异构体、其药学上可接受的盐或溶剂化合物以及可药用载体或赋形剂的药物组合物。
  9. 制备权利要求1-7任一所述多肽化合物的方法，其特征在于，包括直链肽poly-B m-K-Cyclic-A n的合成，及环肽K-(Cyclic-A n)的制备。
  10. 根据权利要求9所述方法，其特征在于，所述直链肽poly-B m-K-Cyclic-A n的合成，首先采用手工固相Fmoc/tBu合成法，以H-CTC树脂(取代值为约0.6mmol/g)为起始原料，从多肽的C端向N端逐一缩合氨基酸，延长肽链直至完成线性直链poly-B m-K-Cyclic-A n目标肽链的合成，所用的氨基酸原料可以是L型氨基酸，也可以是D型氨基酸。
  11. 根据权利要求10所述方法，其特征在于，所述直链肽poly-B m-K-Cyclic-A n的合成具体为：首先用1.5倍当量的Fmoc-AA A1-OH，3倍当量的二异丙基乙胺与H-CTC树脂(取代值为约0.6mmol/g)进行接枝引入C端第一个氨基酸残基，洗涤含H-CTC树脂的反应液，洗净后用甲醇对H-CTC树脂上未反应的活性位点进行封闭；然后，去除N端Fmoc保护基使N端成为自由氨基，用Fmoc-AA A2-OH、1-羟基苯并三唑和N,N’-二异丙基碳二亚胺与H-CTC树脂接枝引入C端第二个氨基酸残基，Fmoc-AA A2-OH、1-羟基苯并三唑和N,N’-二异丙基碳二亚胺的当量均是H-CTC树脂的3倍；如此反复依次连接缩合氨基酸Fmoc-AA An-OH完成线性Cyclic-A n链的合成；其后采用合成的氨基酸原料顺序为Fmoc-Lys(Dde)-OH，然后进行poly-B m肽链的合成，Fmoc-AA B1-OH，Fmoc-AA B2-OH，……Fmoc-AA Bm-OH直至完成poly-B m肽链的合成，得到poly-B m-K-Cyclic-A n-H-CTC树脂。
  12. 根据权利要求9-11任一所述方法，其特征在于，所述环肽K-(Cyclic-A n)的制备具体为：
    poly-B m-K-Cyclic-A n-H-CTC树脂的N端采用Boc酸酐进行封端，随后去除环化处赖氨酸(K，Lys)残基的侧链Dde保护基以释放出Lys侧链氨基；对树脂进行切割将直链肽分子从树脂上裂解释放出来，获得N端以及其它氨基酸残基的活泼侧链基团均已被保护、仅有赖氨酸(K，Lys)的侧链氨基及肽链C端的羧基呈现裸露状态的直链肽分子poly-B m-K-Cyclic-A n，进行分子内的缩合环化反应，然后将侧链基团的保护基去除，重结晶即得到目标(poly-B m)-K-(Cyclic-A n)杂环肽的粗品，最后采用高效液相色谱(HPLC)纯化所述粗品，获得纯度>98％的多肽化合物(poly-B m)-K-(Cyclic-A n)。
  13. 权利要求1-7任一所述多肽化合物在制备抗菌药物中的应用。
  14. 根据权利要求13所述应用，其特征在于，所述抗菌为杀灭引起烧伤或创伤的创面常见的感染致病菌及真菌或抑制其生长。
  15. 根据权利要求13或14所述应用，其特征在于，所述抗菌包括杀灭原核微生物中的细菌(包括球菌、杆菌、螺旋菌等，或革兰氏阴性菌和阳性菌)、放线菌(包括支原体、衣原体、立克次氏体)、蓝藻(包括蓝球藻、念珠藻、颤藻等)或抑制其生长。
  16. 根据权利要求13或14所述应用，其特征在于，所述抗菌包括杀灭真核微生物中的真菌(包括酵母菌，霉菌、青霉菌、黏菌等)和原生致病微生物(包括阿米巴变形虫等)或抑制其生长。
  17. 根据权利要求13或14所述应用，其特征在于，所述抗菌为杀灭金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌或抑制其生长；尤其是杀灭屎肠球菌(Enterococcus faecium)、金黄色葡萄球菌(Staphylococcus aureus)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、鲍曼不动杆菌(Acinetobacter baumannii)、铜绿假单胞菌(Pseudomonas aeruginosa)、大肠杆菌(Enterobacter species)等耐药细菌及耐药真菌或抑制其生长。
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