WO2022065172A1 - Cell membrane-permeable molecule and use thereof, and method of improving cell membrane permeability of cell membrane-permeable molecule - Google Patents
Cell membrane-permeable molecule and use thereof, and method of improving cell membrane permeability of cell membrane-permeable molecule Download PDFInfo
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- WO2022065172A1 WO2022065172A1 PCT/JP2021/034020 JP2021034020W WO2022065172A1 WO 2022065172 A1 WO2022065172 A1 WO 2022065172A1 JP 2021034020 W JP2021034020 W JP 2021034020W WO 2022065172 A1 WO2022065172 A1 WO 2022065172A1
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- cell membrane
- peptide
- permeable molecule
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- molecule according
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
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- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- ZGEGCLOFRBLKSE-UHFFFAOYSA-N methylene hexane Natural products CCCCCC=C ZGEGCLOFRBLKSE-UHFFFAOYSA-N 0.000 description 1
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- UQDJGEHQDNVPGU-UHFFFAOYSA-N serine phosphoethanolamine Chemical compound [NH3+]CCOP([O-])(=O)OCC([NH3+])C([O-])=O UQDJGEHQDNVPGU-UHFFFAOYSA-N 0.000 description 1
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- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
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- 125000002813 thiocarbonyl group Chemical group *C(*)=S 0.000 description 1
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- NONOKGVFTBWRLD-UHFFFAOYSA-N thioisocyanate group Chemical group S(N=C=O)N=C=O NONOKGVFTBWRLD-UHFFFAOYSA-N 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
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Classifications
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- 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
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
-
- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Definitions
- the present invention relates to a cell membrane permeable molecule, a peptide complex containing the cell membrane permeable molecule and a method for producing the same, a peptide library containing the peptide complex, a method for screening a functional peptide using the peptide library, and a cell membrane permeation.
- the present invention relates to a method for improving cell membrane permeability of a sex molecule.
- Small molecule drugs with a molecular weight of up to about 500 have been developed as many drugs because of their low manufacturing cost and low immunogenicity.
- this small molecule drug has low specificity, and it has become increasingly difficult to develop in recent years due to the problem of side effects due to off-targeting.
- sales of high molecular weight drugs derived from antibodies and proteins having a molecular weight exceeding 10,000 are increasing because of their high specificity and few side effects.
- the problems are that it is expensive, that it may cause immunogenicity due to its large molecular weight, and that it generally does not migrate into the cell, so that the molecules that can be the target are limited.
- molecules with a molecular weight of about 500 to 5,000 such as peptides and nucleic acids, so-called medium-molecular-weight drugs, have the advantages of low-molecular-weight drugs that are inexpensive and have low immunogenicity, and their specificity to target molecules. It is expected to be a drug that has the advantages of high molecular weight drugs, such as high prices and few side effects.
- peptides as medium-molecular-weight drugs, we have created a peptide (polypeptide) library consisting of various amino acid sequences and have high affinity for specific target molecules such as disease-related proteins. A method for selecting peptides is widely used.
- a ribosome display method using a ribosome display complex containing a peptide chain, an mRNA molecule, and a ribosome is known (for example, patent). See Document 1).
- the RD method is extremely excellent and useful because it is possible to prepare a peptide library of 1012 or more kinds in a few minutes simply by mixing an in vitro translation system and mRNA.
- a medium-molecular-weight drug such as a peptide or nucleic acid targets an intracellular molecule
- a means for permeating the cell membrane to reach the desired intracellular target molecule is required.
- Many cells, such as cytoskeleton-related proteins and kinase-related factors, can be targeted in the treatment of various diseases. Therefore, the establishment of a useful intracellular introduction method leads to a significant expansion of the scope of application of medium-molecular-weight drugs.
- Techniques for incorporating the active ingredient into cells include, for example, a method of fusing the amino acid sequence of a cell membrane penetrating peptide containing a large amount of basic amino acids, and a dendrimer which is a dendritic polymer having a regularly branched structure from the center.
- the method to be used see, for example, Patent Document 2 and the like are known.
- the present invention includes a cell membrane penetrating molecule having a function of incorporating an active ingredient into a cell and having low toxicity to a living body, a peptide complex containing the cell membrane penetrating molecule, a method for producing the same, and the peptide complex.
- a peptide library a method for screening a functional peptide using the peptide library, and a cell membrane penetrating molecule that can efficiently take up an active ingredient into a cell even if the molecular weight is a small cell membrane penetrating molecule.
- the purpose is to provide a method for improving sex.
- a salt with an acid having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 It was found that it can be a cell membrane permeable molecule with low toxicity to the living body while having a function of taking up the active ingredient into the cell. Further, by using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid, the cell membrane becomes equal to or higher than the cell membrane-permeable molecule having a large number of structures contributing to cell membrane permeation. It was found that the permeability can be improved. (In the general formula (I), R represents a bond).
- the present invention is based on the above-mentioned findings of the present inventors, and the means for solving the above-mentioned problems are as follows. That is, ⁇ 1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid. (In the general formula (I), R represents a bond). ⁇ 2> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to ⁇ 1>. ⁇ 3> A peptide library comprising the peptide complex described in ⁇ 2> above.
- ⁇ 4> A method for producing a peptide complex, which comprises introducing the cell membrane-permeable molecule according to ⁇ 1> into a peptide.
- ⁇ 5> A method for screening a functional peptide, which comprises screening a functional peptide using the peptide library according to the above ⁇ 3>.
- ⁇ 6> A method for improving the cell membrane permeability of a cell membrane-permeable molecule, which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid. (In the general formula (I), R represents a bond).
- the cell membrane-permeable molecule which is a cell membrane-permeable molecule having a function of taking up an active ingredient into a cell and having a function of taking up the active ingredient into the cell, which can solve the conventional problems and achieve the object, and the cell membrane permeation.
- the cell membrane permeable molecule of the present invention has at least a structure represented by the following general formula (I) and is a salt with an acid having a pKa value of less than 4.7. (In the general formula (I), R represents a bond).
- the portion other than R in the structure represented by the general formula (I) is a portion that contributes to cell membrane permeability.
- the number of portions of the cell membrane-permeable molecule other than R in the structure represented by the general formula (I) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably one. .. That is, the cell membrane permeable molecule of the present invention preferably has one dendritic structure represented by the general formula (I).
- the R portion of the structure represented by the general formula (I) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably bonded to a heteroatom-containing group.
- the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose.
- a peptide linking group linked to the peptide (A), the peptide linking group and the general formula (I). An embodiment consisting of a linking group for linking with the structure represented by), (B) an embodiment consisting of a peptide linking group for linking with a peptide, and the like.
- the heteroatom-containing group may have a skeleton of a peptide or nucleic acid.
- the length of the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose.
- the lower limit of the number of atoms bonded linearly is preferably 1 atom or more.
- the upper limit is preferably 50 atoms or less, more preferably 25 atoms or less. Examples of the number of atoms bonded to the linear chain of 25 or less include heteroatom-containing groups in the cell membrane-permeable molecule G3-DCX described later.
- the hetero atom in the hetero atom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose. For example, at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. And so on.
- the heteroatom may be only one kind or two or more kinds.
- the content ratio of the heteroatom in the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it may be 10% or more in terms of increasing the water solubility of the cell membrane-permeable molecule. preferable.
- the upper limit of the content ratio of the heteroatom is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 60% or less, more preferably 50% or less.
- the range of the content ratio of the heteroatom in the heteroatom-containing group is preferably 10% or more and 60% or less, and more preferably 10% or more and 50% or less.
- the content ratio of the heteroatom in the heteroatom-containing group can be calculated as follows. It should be noted that all atoms include hydrogen atoms.
- Content ratio of heteroatoms in heteroatom-containing groups (%) ⁇ (number of heteroatoms in heteroatom-containing groups) / (total number of heteroatom-containing groups) ⁇ ⁇ 100
- the heteroatom-containing group preferably contains a repeating unit.
- the repeating unit is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include alkylene oxide, alkyleneimine and alkylene sulfide.
- the repeating unit may be only one kind or two or more kinds. Further, the repeating unit may have a substituent.
- alkylene oxide is preferable from the viewpoint of availability and stability.
- the alkylene oxide is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include at least one selected from the group consisting of methylene oxide, ethylene oxide and propylene oxide.
- the number of the repeating units is not particularly limited and may be appropriately selected depending on the intended purpose.
- the lower limit of the repeating units is preferably 1 or more, and the upper limit is preferably 10 or less.
- the substituent contained in the repeating unit is not particularly limited and may be appropriately selected depending on the intended purpose.
- substituents include alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl. Hydroxy, mercapto, amino, carbonyl, amide, thioamide, urea, sulfonamide, carboxy and nitrile are preferred.
- the peptide linking group contributes to the introduction of the cell membrane-permeable molecule into the peptide by reacting with the reactive amino acid residue in the peptide.
- the reactive amino acid residue is an amino acid residue that reacts with the peptide linking group, may be an amino acid residue that directly reacts with the peptide linking group, or can react with the peptide linking group. It may be an amino acid residue modified to.
- the group for linking the peptide is not particularly limited as long as it can be linked to the peptide, and can be appropriately selected depending on the intended purpose.
- the thiol group of the cysteine residue and the side chain amino of the lysine residue can be selected.
- examples thereof include a chain hydroxyl group (-OH), an ortho-position carbon of a tyrosine residue, and a group capable of forming a bond by reacting with a side chain sulfide group (-SMe) of a methionine residue.
- Specific examples of the peptide linking group include, for example, an alkyl halide group, an activated carbonyl group, and an unsaturated hydrocarbon group described in paragraphs [0067] to [0076] of International Publication No.
- Examples thereof include an epoxy group, a sulfonyl-containing group, an isocyanate group, a thioisocyanate group, a carben generating group, a disulfide bond-containing group, and a thiol group.
- the peptide linking group contributes to the cyclization of the peptide by reacting with the reactive amino acid residue in the peptide (hereinafter, may be referred to as "cyclizing group").
- the reactive amino acid residue that reacts with the cyclic group may be an amino acid residue that directly reacts with the cyclic group, or may be an amino acid residue modified so as to react with the cyclic group. May be good.
- the cyclic group is not particularly limited and may be appropriately selected depending on the intended purpose, but an electron-withdrawing group is preferable.
- the electron-withdrawing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it preferably has a halogen.
- the type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose, but for example, a chlorine atom, a bromine atom and an iodine atom are preferable.
- the number of halogens is not particularly limited and may be appropriately selected depending on the intended purpose. However, the lower limit of the number of halogens is preferably 2 or more, and the upper limit is preferably 5 or less and 4 or less.
- the electron-withdrawing group may have a substituent. If the electron-withdrawing group contains an unsaturated structure in its structure, the peptide is likely to be cyclized.
- the unsaturated structure is not particularly limited, but is, for example, an aromatic ring structure, a heterocyclic structure, an alicyclic hydrocarbon structure, an alkenyl structure, an alkynyl structure, a carbonyl structure, a thiocarbonyl structure, an oxime structure, a cyano structure, or an isocyanate structure.
- a benzyl halide is more preferable, and a 3,5-bis (halomethyl) benzyl group is particularly preferable.
- the type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a chlorine atom, a bromine atom and an iodine atom are preferable, and a chlorine atom is more preferable.
- Cyclization of a peptide with the cyclic group may be carried out by reacting the cyclic group with at least one group selected from the group consisting of a thiol group, an amino group and a hydroxy group contained in the peptide. preferable.
- peptide linking group when the peptide and the cell membrane permeable molecule are bound by the oxime ligation method, those represented by the following structural formula can also be used as the peptide linking group.
- the linking group is a group that links the peptide linking group and the structure represented by the general formula (I).
- the structure of the linking group is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a structure containing the above-mentioned repeating unit.
- the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
- cell membrane permeable molecule examples include compounds represented by the following structural formulas.
- a part of the structure of the cell membrane permeable molecule may be changed when it is in the form of a salt.
- the cell membrane permeable molecule G3-DCX represented by the following structural formula is a cyclic group which is a 3,5-bis (chloromethyl) benzyl group as a heteroatom-containing group in the R portion in the general formula (I). This is an example having a peptide linking group) and a linking group. Since the cell membrane-permeable molecule G3-DCX has a cyclizing group, it is possible to cyclize the peptide and impart cell membrane permeability to the peptide in one step.
- the cell membrane permeable molecule G3 represented by the following structural formula is an example in the general formula (I) having a peptide linking group capable of binding to a peptide by the oxime ligation method as a heteroatom-containing group in the R portion. Is.
- the cell membrane permeable molecule of the present invention is a salt with an acid having a pK a (acid dissociation constant) value of less than 4.7.
- the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
- the acid having a pK a value of less than 4.7 is not particularly limited and may be appropriately selected depending on the intended purpose, but trifluoroacetic acid (-0) in that it is less toxic to the living body.
- the lower limit of the pK a value is more than -8.0 (the pK a value is -8.) In that it is less toxic to the living body. (Greater than 0) is preferred, more than -3.0 (pK a value is greater than -3.0) is more preferred.
- the upper limit of the pK a value for an acid having a pK a value of less than 4.7 is preferably 3.5 or less, and more preferably less than 3.3.
- the cell membrane permeable molecule has a structure represented by the above-mentioned general formula (I), and is a salt with an acid having a pKa value of less than 4.7, as long as the effect of the present invention is not impaired. , Other configurations may be included.
- the method for producing the cell membrane-permeable molecule is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807.
- a method of forming a salt a crude product of a cell membrane permeable molecule that is not in the form of a salt is purified by HPLC using a mobile phase according to the type of the target salt, and the form of the target salt is obtained.
- the method of making the cell membrane permeable molecule of the above the cell membrane permeable molecule in the form of a salt is passed through an anion exchange resin in which the counter ion is replaced with the ion of the target salt, and is eluted to form the target salt.
- an anion exchange resin in which the counter ion is replaced with the ion of the target salt, and is eluted to form the target salt. Examples thereof include a method of making a cell membrane permeable molecule.
- the method for confirming whether or not the obtained cell membrane permeable molecule has a desired structure is not particularly limited, and a known analytical method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance can be selected. Analytical methods such as spectroscopy, carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, and liquid chromatography can be mentioned.
- the cell membrane permeabilizing molecule of the present invention can impart excellent cell membrane permeability to active ingredients such as peptides, nucleic acids, proteins and complexes thereof while reducing toxicity to living organisms.
- the peptide complex of the present invention comprises at least the peptide and the above-mentioned cell membrane permeable molecule of the present invention, and further contains other configurations as required.
- the peptide is not particularly limited as long as the cell membrane permeable molecule can be introduced, and can be appropriately selected depending on the intended purpose. However, the peptide is cyclized by introducing the cell membrane permeable molecule. Is preferable.
- the type of amino acid in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be a natural amino acid, an unnatural amino acid, or a D-form. It may be present or it may be L-form.
- the peptide may be a modified peptide such as a lipopeptide.
- the amino acid residue used for the introduction of the cell membrane permeable molecule (hereinafter, may be referred to as “reactive amino acid residue”) is not particularly limited and may be appropriately selected depending on the intended purpose, for example. , Cysteine residue, lysine residue, histidine residue, tryptophan residue, tyrosine residue, serine residue, threonine residue and the like.
- the reactive amino acid residue may be, for example, a cysteine residue, a lysine residue, a serine residue, or a threonine residue. And so on. Further, a hydroxy group, a mercapto group, or an amino group in the amino acid residue may be used.
- the reactive amino acid residue may be used alone or in combination of two or more.
- the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
- the number of the reactive amino acid residues in the peptide is preferably 2 or more.
- the upper limit of the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
- the position is not stable and it may be difficult to compare the characteristics of the peptide derived from the amino acid sequence, so 10 or less is preferable.
- the reactive amino acid residue may be separately introduced into the peptide. preferable.
- the position of the reactive amino acid residue in the peptide is not particularly limited and can be appropriately selected depending on the intended purpose.
- RNA chain an mRNA molecule, a peptide chain which is a translation thereof (hereinafter, also referred to as “polypeptide chain”), and a ribosome display complex containing ribosome (hereinafter, referred to as “RD complex”).
- RD complex a ribosome display complex containing ribosome
- it is a portion protruding from the exit tunnel (exit tunnel) of the ribosome, specifically, the second position from the N-terminal to the 30th position (N) from the C-terminal. It is preferable to use it between the position 2nd from the terminal and the position 30th from the C end) in that the modification reaction by the linker molecule can be less likely to be sterically inhibited by the ribosome.
- the position from the C-terminal the 50th position from the C-terminal is preferable, and the 100th position is more preferable.
- the position can be appropriately set according to the chain length of the peptide, and is, for example, the 2nd to 1,000th position from the N-terminal.
- the 2nd to 100th positions from the N-terminal are preferable, and the 2nd to 50th positions from the N-terminal are more preferable.
- the method for producing the RD complex is not particularly limited, and a known method can be appropriately selected. Examples thereof include the method described in International Publication No. 2017/213158. It can also be manufactured using a commercially available kit.
- the amino acid sequence of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, but one containing a random sequence at a specific position is preferable so as to be useful as a peptide library. From such a random sequence, a useful amino acid sequence can be identified according to a predetermined purpose.
- the position of the random sequence in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
- the position is not particularly limited. It is preferably between the 2nd position from the N-terminal to the 30th position from the C-terminal (including the 2nd position from the N-terminal and the 30th position from the C-terminal). That is, the reactive amino acid residue is preferably contained in a random sequence. Therefore, the preferred position of the random sequence can be set from the same range as the preferred position of the reactive amino acid residue.
- the number of the random sequences in the peptide may be one or two or more.
- the upper limit of the number of the random sequences is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 or less.
- the number of amino acid residues per random sequence is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 1 or more and 30 or less. The longer one random sequence and the larger the number of random sequences, the greater the diversity of the peptide library.
- the peptide may further contain a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.
- a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.
- the number of amino acid residues of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 10 or more and 5,000 or less.
- the lower limit of the number of amino acid residues of the peptide is preferably 150 or more, more preferably 200 or more.
- the upper limit of the number of amino acid residues of the peptide is preferably 800 or less, more preferably 600 or less, and particularly preferably 500 or less.
- the lower limit value and the upper limit value can be appropriately combined and selected.
- the method for synthesizing the peptide is not particularly limited, and a known method can be appropriately selected.
- the cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
- the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
- the other constitution of the peptide complex is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the intended purpose.
- a luminescent substance such as a fluorescent substance, a dye, a radioactive substance, and the like. Examples include drugs, toxins, nucleic acids, amino acids, sugars, lipids, various polymers and the like. These may be used alone or in combination of two or more.
- the fluorescent substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene and cyanine.
- the other constitution can be attached to the above-mentioned peptide, for example, directly or via a linking group or the like.
- the peptide complex of the present invention has excellent cell permeability and low toxicity to the living body. Therefore, for example, by preparing a peptide complex library containing a random sequence and performing screening, it is useful to have excellent cell permeability, low toxicity to a living body, and high affinity for a target substance.
- the amino acid sequence can be specified.
- the method for producing a peptide complex of the present invention includes at least an introduction step of introducing the cell membrane permeable molecule of the present invention into a peptide, and further includes other steps as necessary.
- the introduction step is a step of introducing the cell membrane-permeable molecule of the present invention into a peptide (hereinafter, may be referred to as "binding", “inserting", or “linking”).
- a peptide hereinafter, may be referred to as "binding", "inserting", or “linking”
- cell membrane permeability can be imparted to the peptide.
- the cell membrane permeable molecule having the above-mentioned cyclization group is introduced, the cyclization of the peptide and the impartation of the cell membrane permeability to the peptide can be performed at the same time.
- the cell membrane-permeable molecule may be introduced into at least one peptide in the reaction product, but it is preferable that the cell membrane-permeable molecule is introduced into all the peptides.
- the peptide is the same as that described in the ⁇ Peptide> section of the above (peptide complex).
- the peptide used in the introduction step may be an embodiment of a peptide library.
- the peptide in this peptide library is in a state in which the cell membrane-permeable molecule of the present invention has not been introduced.
- the cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
- the method of introduction is not particularly limited and may be appropriately selected depending on the intended purpose.
- a method of reacting a peptide linking group in the cell membrane permeable molecule with a reactive amino acid residue in the peptide For example, a method of reacting the cell membrane permeable molecule with the peptide in the presence of a reducing agent can be mentioned.
- the reducing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tris (2-carboxyethyl) phosphine hydrochloride and the like.
- the conditions such as the temperature and time of the reaction are not particularly limited and may be appropriately selected depending on the intended purpose.
- the method for forming the peptide complex in the form of a salt is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out.
- a known chemical synthesis technique can be appropriately selected and carried out.
- the same method as the method described in 1) can be mentioned.
- the method for confirming whether or not the obtained peptide complex has a desired structure is not particularly limited, and a known analytical method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected. Analytical methods such as method, carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, and liquid chromatography can be mentioned.
- the peptide library of the present invention contains at least the peptide complex of the present invention, and further contains other configurations as required. That is, the peptide library of the present invention contains a peptide complex into which the cell membrane permeable molecule of the present invention has been introduced.
- the peptide library may consist only of the peptide complex of the present invention, or may contain a peptide into which the cell membrane permeable molecule has not been introduced.
- the peptide library can be produced in the same manner as described above (method for producing a peptide complex).
- the method for screening a functional peptide of the present invention includes at least a step of screening a functional peptide using the peptide library of the present invention, and further includes other steps as necessary.
- the screening method is not particularly limited as long as the peptide library of the present invention is used, and a known method can be appropriately selected. For example, a desired target substance and the peptide library are mixed, a bound peptide complex (for example, an RD complex) is selected, RNA is dissociated from the RD complex, and DNA is prepared from the RNA.
- a screening method based on the ribosome display method which repeats the steps of transcribing to mRNA and then producing an RD complex library again to screen for a functional peptide having an affinity for the target substance, can be mentioned. Further, a screening method using a phage display method, an mRNA display method, a DNA display method, a one-bead one-compound method, or the like can also be mentioned.
- the screening method may include an introduction step of introducing the cell membrane-permeable molecule of the present invention into the selected peptide in a process of repeating the screening step or the like.
- the introduction step can be performed in the same manner as the ⁇ introduction step> in the above-mentioned (method for producing a peptide complex).
- the method for improving the cell membrane permeability of a cell membrane-permeable molecule of the present invention includes at least a salt forming step, and further includes other steps as necessary. ..
- the salt forming step in the method for improving membrane permeability of the present invention is a step of converting a cell membrane-permeable molecule having a structure represented by the following general formula (I) into a salt with an acid. (In the general formula (I), R represents a bond).
- the method for producing the cell membrane-permeable molecule having the structure represented by the general formula (I) is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807.
- the method of using a cell membrane-permeable molecule having a structure represented by the general formula (I) as a salt with an acid is not particularly limited, and is a known chemical synthesis technique. Can be selected as appropriate. For example, a method of purifying a crude product of a cell membrane-permeable molecule that is not in the form of a salt by HPLC using a mobile phase according to the type of the target salt to obtain a cell membrane-permeable molecule in the form of the target salt.
- a method of passing a cell membrane-permeable molecule in the form of a salt through an anion exchange resin in which a counterion is replaced with an ion of the target salt and eluting the molecule to obtain a cell membrane-permeable molecule in the form of the target salt, etc. Can be mentioned.
- the cell membrane-permeable molecule produced in the salt-forming step of the method for improving membrane permeability of the present invention is a salt with an acid.
- the type of the acid is not particularly limited and may be appropriately selected depending on the intended purpose. For example, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartrate acid, citric acid, acetic acid. , Various amino acids and the like. Among these, acids other than acidic amino acids are preferable, and acids having a pKa (acid dissociation constant) value of ⁇ 8.0 or more and 4.7 or less are more preferable.
- the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
- the acid having a pK a value of ⁇ 8.0 or more and 4.7 or less is not particularly limited and may be appropriately selected depending on the intended purpose.
- trifluoroacetic acid (-0.3), hydrochloric acid ( -8.0), sulfuric acid (-3.0), nitric acid (-1.3), phosphoric acid (2.1 (pK a1 )), mesylic acid (-2.6), tosylic acid (-2.8) ), Tartrate acid (3.2 (pK a1 )), citric acid (3.1 (pK a1 )), acetic acid (4.7) and the like.
- the number in parentheses after the acid name represents the value of pK a . In the case of the value of pK a1 , it is described as pK a1 after the numerical value.
- the cell membrane-permeable molecule produced by the method for improving membrane permeability of the present invention has a structure represented by the above-mentioned general formula (I) and is a salt with an acid as long as the effect of the present invention is not impaired. In, it may have other configurations.
- the method for confirming whether or not the cell membrane permeable molecule has a desired structure is not particularly limited, and a known analytical method can be appropriately selected.
- mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected.
- the method for improving membrane permeability of the present invention it is possible to improve the cell membrane permeability of a cell membrane-permeable molecule having a small molecular weight. Therefore, by introducing the cell membrane-permeable molecule into an active ingredient such as a peptide, nucleic acid, or protein, the efficiency of incorporation into these cells can be improved.
- the cell membrane permeable molecule is preferably one introduced into a peptide.
- the present invention is characterized in that the peptide complex into which a cell membrane permeable molecule having a structure represented by the general formula (I) is introduced is used as a salt with an acid. It is also related to the method of improving cell membrane permeability.
- the method for improving the cell membrane permeability of the peptide complex includes at least a salt forming step, and further includes other steps as necessary.
- the salt forming step is a step of converting a peptide complex into which a cell membrane-permeable molecule having a structure represented by the general formula (I) into a salt with an acid, and the cell membrane-permeable molecule becomes a peptide. Except for the fact that it has been introduced, it can be carried out in the same manner as the salt forming step in the method for improving the cell membrane permeability of the cell membrane permeable molecule described above.
- the peptide complex contains at least a peptide and a cell membrane-permeable molecule having a structure represented by the general formula (I), and further contains other configurations as necessary.
- the peptide in the method for improving the cell membrane permeability of the peptide complex is the same as that described in the item of ⁇ Peptide> in the above-mentioned (peptide complex).
- the cell membrane-permeable molecule in the method for improving the cell membrane permeability of the peptide complex is a cell membrane-permeable molecule having a structure represented by the general formula (I) in the above-mentioned method for improving the membrane permeability.
- the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
- binding also referred to as “binding”, “inserting”, or “linking”
- linking The method for introducing the cell membrane-permeable molecule into the peptide (hereinafter, also referred to as “binding”, “inserting”, or “linking”) is described in the above-mentioned (method for producing a peptide complex). It is the same as that described in the item of ⁇ Introduction process>.
- the peptide used in the peptide complex may be in the form of a peptide library. That is, as the peptide before the introduction of the above-mentioned cell membrane permeable molecule, a peptide in the form of a peptide library can also be used.
- the other steps in the method for improving the cell membrane permeability of the peptide complex are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
- Example A1 Synthesis of cell membrane permeable molecule G3-DHX trifluoroacetic acid salt
- G3-DHX trifluoroacetic acid salt which is an example of a cell membrane permeable molecule, was synthesized as follows.
- the compound C3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, a methylene chloride solution (15 mL) of compound C1 (500 mg, 0.42 mmol) was cooled to 0 ° C., and compound C2 (manufactured by Tokyo Kasei Kogyo Co., Ltd., product number A2293) (117.5 mg, 0.504 mmol) was cooled. ), 1-Hydroxybenzotriazole (HOBT) (85.1 mg, 0.630 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC / HCl) (120 mg, 0.630 mmol).
- HOBT 1-Hydroxybenzotriazole
- EDC / HCl 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
- the mixture was stirred at 25 ° C. for 17 hours.
- the identification data of the compound C3 by 1H NMR were as follows.
- the compound C4 represented by the above structural formula was synthesized by the above reaction formula. Specifically, nitrogen gas bubbling was performed on a solution consisting of compound C3 (533 mg, 0.379 mmol), compound DBXA (188 mg, 0.568 mmol) and THF (40 mL) to create a nitrogen atmosphere. A copper sulfate aqueous solution (200 mM; 1.89 mL, 0.379 mmol) and an ascorbic acid sodium aqueous solution (100 mM; 7.58 mL, 0.758 mmol) were added thereto, and the mixture was stirred at 25 ° C. for 3 hours.
- the compound G3-DCX represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C4 (497 mg, 0.286 mmol) and a 4N dioxane hydrochloride solution (30 mL) were mixed, and the mixture was stirred at 25 ° C. for 46 hours. After the reaction, a white solid was precipitated. The supernatant was removed to obtain a crude product of compound G3-DCX. Purification by preparative HPLC (high performance liquid chromatography) gave compound G3-DCX as a white solid (116 mg, 0.110 mmol, yield 54%).
- the identification data of the compound G3-DCX by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry were as follows.
- MALDI-TOF MS C 24 H 51 N 14 O 8 Calculated value ([M + H] + ) 1047.502, measured value 1047.953
- the cell membrane permeable molecule G3-DHX trifluoroacetate represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound G3-DCX (506 mg, 0.480 mmol) and H2O (6 mL) were mixed and stirred at 80 ° C. for 2 hours. The reaction solution was concentrated to obtain a crude product of compound G3-DHX. Purification by HPLC (high performance liquid chromatography) using a mobile phase containing trifluoroacetic acid (TFA) gave the cell membrane permeable molecule G3-DHX trifluoroacetate as a white solid (224.6 mg, 0.169 mmol, Yield 35%).
- HPLC high performance liquid chromatography
- TSA trifluoroacetic acid
- the identification data of the G3-DHX trifluoroacetic acid salt by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry were as follows. MALDI-TOF MS C 42 H 74 N 16 O 13 Calculated value ([M + H] + ) 1011.153, measured value 1011.550.
- G3-DHX hydrochloride which is an example of a cell membrane permeable molecule, was prepared as follows.
- An anion exchange resin in which G3-DHX trifluoroacetate (19.7 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (5 mg / mL) and counterions were replaced with chloride ions (19.7 mg). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX hydrochloride (17.8 mg) as a white to off-white powder.
- G3-DHX nitrate which is an example of a cell membrane permeable molecule
- An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with nitrate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX nitrate (0.9 mg) as a white to off-white powder.
- G3-DHX sulfate which is an example of a cell membrane permeable molecule
- An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with sulfate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX sulfate (1.0 mg) as a white to off-white powder.
- G3-DHX phosphate which is an example of a cell membrane permeable molecule
- An anion exchange resin in which G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and counterions were replaced with phosphate ions (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX phosphate (1.4 mg) as a white to off-white powder.
- G3-DHX mesylate which is an example of a cell membrane permeable molecule
- G3-DHX tosylate which is an example of a cell membrane permeable molecule
- G3-DHX tartrate which is an example of a cell membrane permeable molecule
- An anion exchange resin (diamond) in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with tartrate ion. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX tartrate (1.8 mg) as a pale yellow powder.
- G3-DHX citrate which is an example of a cell membrane permeable molecule
- Example A10 Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt
- a peptide complex G3-DCX-P trifluoroacetate which is an example of a complex of a cell membrane permeable molecule and a peptide, was synthesized as follows.
- the resin on which the peptide was formed was converted to TFA / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (volume ratio)). After soaking for 3 hours, the peptide was excised from the resin. The obtained peptide was purified by HPLC and freeze-dried to obtain a peptide having the above sequence (hereinafter, may be referred to as "P"; see the following structural formula).
- the identification data of the peptide P by electrospray ionization mass spectrometry (ESI-MS) were as follows. ESI-MS C 72 H 92 N 16 O 22 O 3 Calculated value ([M + 2H] 2+ ) 815.295, measured value 814.67
- the peptide complex G3-DCX-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula. Specifically, the peptide P (11.0 mg, 6.75 ⁇ mol) synthesized above was dissolved in a mixed solution of 20 mM ammonium bicarbonate buffer (11.8 mL) and acetonitrile (MeCN) (1.7 mL). To this solution was added tris (2-carboxyethyl) phosphine (TCEP) (500 mM in H2O ; 14.8 ⁇ L, 7.4 ⁇ mol) and stirred at 25 ° C. for 15 minutes.
- TCEP (2-carboxyethyl) phosphine
- Example A11 Preparation of peptide complex G3-DCX-P hydrochloride
- the peptide complex G3-DCX-P trifluoroacetate (1.1 mg) synthesized in the same manner as in Example A10 was dissolved in ultrapure water (0.5 mg / mL), and the counterion was converted to chloride ion.
- a substituted anion exchange resin (Diaion PA306S) was added. After shaking for 1 hour, the anion exchange resin was filtered off and eluted with ultrapure water. The obtained solution was freeze-dried to obtain G3-DCX-P hydrochloride (0.6 mg) as a white to off-white powder.
- mice Toxicity evaluation of cell membrane permeable molecule
- a 7-week-old mouse female, 15 to 25 g, Charles River Laboratories, Japan
- a single dose was administered, and general symptoms and life / death were observed over time for 30 minutes after administration, and then general symptoms and life / death were continuously observed at a pace of once / day for 7 days.
- Various acid salts of the cell membrane permeable molecule G3-DHX and placebo buffer control were administered to 3 mice in each group and observed.
- body weight was measured on the day of administration and 1, 2, and 7 days after administration to investigate a significant increase or decrease in body weight. Whenever a fatal case was found, an autopsy was performed.
- G3-DHX tosylate, G3-DHX mesylate, G3-DHX phosphate, G3-DHX citrate, or G3-DHX tartrate was administered at a dose of 10 mg / kg.
- no symptoms such as walking disorders or respiratory irregularities or significant increase or decrease in body weight were observed in any of the individuals.
- survival was confirmed for 7 days after administration.
- G3-DHX trifluoroacetic acid salt no significant increase or decrease in body weight was observed, such as gait disturbance and respiratory irregularity, regardless of the dose of 10 mg / kg or 20 mg / kg.
- the individual to which G3-DHX hydrochloride was administered had a slight gait disorder immediately after the administration, but recovered after 3 minutes. No symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight.
- Individuals treated with G3-DHX sulfate showed respiratory irregularities immediately after administration, but recovered 3 minutes later. Abnormal symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight.
- the individual to which G3-DHX acetate was administered at a dose of 10 mg / kg showed tremor immediately after the administration and died 2 minutes after the administration.
- G3-DHX acetate has the highest toxicity after single intravenous administration to mice and is comparable to octaarginine (see below for the structural formula) (Marcel Grogg et al., Cell Penetration, Herbicidal Activity, and in- vivo-Toxicity of Oligo-Arginine Radivatives and of Novel Guandinium-Rich Compounds Dived from the Biopolymer Cyanophycin, Heil.
- G3-DHX trifluoroacetate, G3-DHX hydrochloride, G3-DHX sulfate, G3-DHX phosphate, G3-DHX mesylate, G3-DHX tosylate, G3-DHX tartrate. , And G3-DHX citrate were found to be less toxic than G3-DHX acetate. From the pK a value of each acid and the toxicity result of each acid salt, it can be seen that acetic acid having a pK a value of 4.7 is the most toxic, and an acid having a pK a value smaller than acetic acid is less toxic. ..
- the cell membrane permeable molecule becomes less toxic by using a salt with an acid having a pKa value of less than 4.7.
- a salt with an acid having a pKa value of less than 4.7 In addition, with regard to G3-DHX hydrochloride and G3-DHX sulfate, slight symptoms were observed immediately after administration, and from the viewpoint of low toxicity, salts with acids exceeding pK a -8.0 were observed. It has been found to be preferable, and salts with acids above pK a -3.0 are more preferred.
- Test Example A2 Evaluation of cell membrane permeability of peptide complex
- HeLa cells Human cervix adenocarcinoma cell were cultured for 2 hours under the above conditions.
- FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used. Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected.
- the peptide P synthesized in Example A10 hardly permeates the membrane 2 hours after the start of culture, whereas the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 has no membrane permeation. It was permeated through the membrane. From this, it was demonstrated that the cell membrane permeability was obtained by introducing the cell membrane permeability molecule of the present invention into the peptide.
- Test Example A3 Evaluation of cell membrane permeability of peptide complex hydrochloride 5% in a cell culture medium containing 2 ⁇ M of either the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 or the peptide complex G3-DCX-P hydrochloride synthesized in Example A11.
- HeLa cells Human cervix adenocarcinoma cell
- FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used. Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected.
- the P hydrochloride was permeated to the same extent. From this, it was demonstrated that even if the salt morphology of the peptide complex into which the cell membrane-permeable molecule of the present invention was introduced was changed, the same degree of cell membrane permeability was exhibited.
- Example B1 Synthesis of cell membrane permeable molecule G3-DCX trifluoroacetic acid salt
- ⁇ Synthesis of compound G3-DCX> -Synthesis of compound C1- In the same manner as in-Synthesis of compound C1 in Example A1 described above, compound C1 represented by the above-mentioned structural formula was synthesized.
- Example B2 Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt
- the peptide complex G3-DCX-P trifluoroacetic acid salt was synthesized in the same manner as in Example A10 described above.
- Example B3 Preparation of peptide complex G3-DCX-P hydrochloride
- the peptide complex G3-DCX-P hydrochloride was synthesized in the same manner as in Example A11 described above.
- Example B4 Preparation of peptide complex G3-DCX-P acetate
- the peptide complex G3-DCX-P acetate was synthesized in the same manner as in Reference Example A2 described above.
- Example B5 Preparation of peptide complex G3-DCX-P nitrate
- the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with nitrate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P nitrate as a white to off-white powder.
- anion exchange resin diaion PA306S
- Example B6 Preparation of peptide complex G3-DCX-P sulfate
- the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with sulfate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P sulfate as a white to off-white powder.
- anion exchange resin diaion PA306S
- Example B7 Preparation of peptide complex G3-DCX-P phosphate
- the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which counterions were replaced with phosphate ions. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P phosphate as a white to off-white color powder.
- anion exchange resin diaion PA306S
- Example B8 Preparation of peptide complex G3-DCX-P mesylate
- the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with a mesylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P mesylate as a colorless liquid.
- anion exchange resin diaion PA306S
- Example B9 Preparation of peptide complex G3-DCX-P tosylate
- the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with tosylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tosylate as a white powder.
- anion exchange resin diaion PA306S
- Example B10 Preparation of peptide complex G3-DCX-P tartrate
- the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which the counterion was replaced with tartrate ion. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tartrate as a pale yellow powder.
- anion exchange resin diaion PA306S
- Example B11 Preparation of peptide complex G3-DCX-P citrate
- the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with citrate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P citrate as a pale yellow powder.
- anion exchange resin diaion PA306S
- Example B12 Synthesis of peptide complex G3-P trifluoroacetate
- the compound K1 represented by the following structural formula was synthesized according to the method described in The Journal of Organometallic Chemistry 2013, 17-24.
- “Fmoc” in the structural formula of a compound represents "9-fluorenylmethyloxycarbonyl group”.
- the compound K3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound K1 (50 mg, 0.159 mmol) was dissolved in methylene chloride (5 mL) and cooled to 0 ° C. Here, compounds K2 (284 mg, 0.239 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride synthesized by the same method as described in US Pat. No. 7,862,807 are used. Salt (EDC / HCl) (45.8 mg, 0.239 mmol) and 1-hydroxybenzotriazole (HOBT) (32.2 mg, 0.239 mmol) were added and stirred at 0 ° C. for 12 hours.
- EDC / HCl 45.8 mg, 0.239 mmol
- HOBT 1-hydroxybenzotriazole
- the identification data of the compound K4 by 1H NMR were as follows.
- the cell membrane permeable molecule G3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, a dioxane hydrochloride solution (4N; 1.0 mL) was added to compound K4 to prepare a solution, and the mixture was stirred at 25 ° C. for 2 hours. The precipitated white solid was obtained by centrifugation and further washed with diethyl ether (3 times at 3 mL) to obtain a cell membrane permeable molecule G3 (90 mg, quantitative yield).
- the identification data of the cell membrane-permeable molecule G3 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows. MALDI-TOF MS C 24 H 50 N 14 O 8 Calculated value ([M + H] + ) 663.401, measured value 663.402
- the resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5).
- TFA trifluoroacetic acid
- the peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
- the obtained peptide (55.0 mg, 30.0 ⁇ mol) was dissolved in DMF (2.5 mL), and 1,3-dibromo-2-propanol (20 mM DMF solution; 1.5 mL, 30 ⁇ mol) and N-methylmorpholine ( 10 mM DMF solution; 6.0 mL, 60 ⁇ mol) was added and stirred at 25 ° C. for 1 hour. Diethyl ether (100 mL) was added thereto to remove the supernatant, and the residue was washed with diethyl ether (100 mL) to obtain compound K5 as a white solid (54.6 mg, 28.9 ⁇ mol, yield 97%). ..
- CMLYIVPYFSVGC in the structural formula of compound K5 represents the amino acid sequence of the peptide.
- the identification data of the compound K5 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows. MALDI-TOF MS C 94 H 128 N 15 O 20 S 3 + Calculated value ([M + H] + ) 1882.862, Measured value 1883.140
- the compound Fmoc-G3-P represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound K5 (30 mg, 0.0159 mmol), cell membrane permeable molecule G3 (52.7 mg, 0.0796 mmol), DMF (0.5 mL) and water (0.05 mL) were mixed and 25 ° C. Was stirred for 24 hours. Diethyl ether (5 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (3 times with 3 mL) to obtain a crude product of compound Fmoc-G3-P. The compound Fmoc-G3-P (3.8 mg, 0.00150 mmol, yield 9%) was obtained by purification by reverse phase HPLC (high performance liquid chromatography) and freeze-drying.
- HPLC high performance liquid chromatography
- the peptide complex G3-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula. Specifically, the compound Fmoc-G3-P synthesized above and a 20% piperidine / DMF solution (1.0 mL) were mixed and stirred at 25 ° C. for 2 hours using a vortex mixer. The solvent was volatilized by spraying nitrogen gas. The residue was washed with diethyl ether (1 mL 3 times).
- fluorescein isothiocyanate (FITC) (0.8 mg, 0.002 mmol), diisopropylethylamine (DIPEA) (0.022 mL, 0.0124 mmol) and DMF (0.5 mL) are mixed and stirred at 25 ° C. for 20 hours. did. Diethyl ether (15 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (twice at 5 mL) to give the peptide complex G3-P crude product.
- DIPEA diisopropylethylamine
- DMF 0.5 mL
- the peptide complex G3-P trifluoroacetic acid salt (1.6 mg, 0.00059 mmol, yield 40%) was obtained as a white solid by purification by HPLC using a mobile phase containing TFA and freeze-drying.
- the identification data of the peptide complex G3-P trifluoroacetic acid salt by MALDI-TOF MS were as follows. MALDI-TOF MS C 124 H 177 N 30 O 30 S 4 Calculated value ([M + H] + ) 2694.212, measured value 2694.208
- CMLYIVPYFSVGC in the following structural formula represents the amino acid sequence of the peptide.
- CMLYIVPYFSVGC in the following structural formula represents the amino acid sequence of the peptide.
- Test Example B1 Evaluation of cell membrane permeability of peptide complex
- a cell culture medium under the conditions of 5% (v / v) CO 2 and 37 ° C.
- the cell membrane permeability of the peptide complex increases in the order of G3-P, G6-P and G9-P, and the larger the number of dendritic skeletons having guanidyl groups, the more the membrane.
- the permeability was high.
- the cell membrane permeability of G3-P trifluoroacetic acid salt 4 hours after the start of culture was significantly higher than that of G6-P and G9-P. From this, it was clarified that G3-P exhibits cell membrane permeability equal to or higher than that of G6-P and G9-P by taking the form of a salt with an acid.
- Molecules with a molecular weight of more than 4,000 Da may cause immunogenicity, and it is desirable that the cell membrane-permeable molecule used in combination with a peptide having a molecular weight of more than 1,000 has a smaller molecular weight. Therefore, in the manufacture of pharmaceutical products, it is preferable to use G3-P having a smaller molecular weight than G6-P or G9-P. Further, as described above, by forming G3-P in the form of a salt with an acid, it is possible to remarkably improve the membrane permeability, and overcome the problem of lack of cell membrane permeability that G3-P has been a problem. It can be said that it was possible.
- the method for improving the cell membrane permeability of the cell membrane-permeable molecule of the present invention is a useful method capable of improving the cell membrane permeability of the peptide complex containing the peptide as a pharmaceutical product.
- Examples of aspects of the present invention include the following.
- ⁇ 1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid. (In the general formula (I), R represents a bond).
- ⁇ 2> The cell membrane-permeable molecule according to ⁇ 1>, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group.
- ⁇ 3> The cell membrane-permeable molecule according to ⁇ 2>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
- ⁇ 4> The above-mentioned ⁇ 2> to ⁇ 3>, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a cell membrane permeable molecule.
- ⁇ 5> The cell membrane-permeable molecule according to any one of ⁇ 2> to ⁇ 4>, wherein the heteroatom-containing group contains a repeating unit.
- ⁇ 6> The cell membrane permeable molecule according to ⁇ 5>, wherein the repeating unit is an alkylene oxide.
- the acid having a pKa value of less than 4.7 is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartaric acid and citric acid.
- ⁇ 9> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to any one of ⁇ 1> to ⁇ 8>.
- a peptide library comprising the peptide complex according to ⁇ 9>.
- a method for producing a peptide complex which comprises introducing the cell membrane-permeable molecule according to any one of ⁇ 1> to ⁇ 8> into a peptide.
- a method for screening a functional peptide which comprises screening a functional peptide using the peptide library according to ⁇ 10>.
- ⁇ 14> The method for screening a functional peptide according to ⁇ 13>, which comprises introducing the cell membrane permeable molecule according to any one of ⁇ 1> to ⁇ 8> into the peptide.
- a method for improving the cell membrane permeability of a cell membrane-permeable molecule which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid. (In the general formula (I), R represents a bond).
- R represents a bond.
- ⁇ 17> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to ⁇ 16>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
- the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a method for improving the cell membrane permeability of a cell membrane permeability molecule.
- ⁇ 19> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of ⁇ 16> to ⁇ 18>, wherein the heteroatom-containing group contains a repeating unit.
- ⁇ 20> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to ⁇ 19>, wherein the repeating unit is an alkylene oxide.
- ⁇ 21> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to ⁇ 20>, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide, and propylene oxide.
- ⁇ 22> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of ⁇ 15> to ⁇ 21>, wherein the cell membrane-permeable molecule is introduced into a peptide.
Abstract
A cell membrane-permeable molecule which has a structure represented by general formula (I) and which is in the form of a salt with an acid having a pKa value of lower than 4.7; and a method of improving the cell membrane permeability of a cell membrane-permeable molecule, said method comprising converting the cell membrane-permeable molecule, which has a structure represented by general formula (I), into a salt with an acid.
Description
本発明は、細胞膜透過性分子、前記細胞膜透過性分子を含むペプチド複合体及びその製造方法、前記ペプチド複合体を含むペプチドライブラリ、及び前記ペプチドライブラリを用いた機能性ペプチドのスクリーニング方法、並びに細胞膜透過性分子の細胞膜透過性の向上方法に関する。
The present invention relates to a cell membrane permeable molecule, a peptide complex containing the cell membrane permeable molecule and a method for producing the same, a peptide library containing the peptide complex, a method for screening a functional peptide using the peptide library, and a cell membrane permeation. The present invention relates to a method for improving cell membrane permeability of a sex molecule.
分子量が５００程度までの低分子医薬品は、製造コストが安く免疫原性が低いため、これまで多くの医薬品として開発されてきた。しかし、この低分子医薬品は、特異性が低く、オフターゲットによる副作用の問題から近年は次第に開発が難しくなってきている。これに対し、分子量が１０，０００を超えるような抗体やタンパク質由来の高分子医薬品は、特異性が高く副作用も少ないことから売り上げが伸びている。しかし、高価であること、その大きな分子量から免疫原性を起こす可能性があること、一般に細胞内へ移行しないためその標的となりうる分子が限られることが課題となっている。
そこで近年、ペプチドや核酸などをはじめとする分子量が５００～５，０００程度の分子、いわゆる中分子医薬品が、安価で免疫原性が低いという低分子医薬品の利点と、標的分子への特異性が高く、副作用が少ないというという高分子医薬品の利点を併せ持つ医薬品として期待されている。 Small molecule drugs with a molecular weight of up to about 500 have been developed as many drugs because of their low manufacturing cost and low immunogenicity. However, this small molecule drug has low specificity, and it has become increasingly difficult to develop in recent years due to the problem of side effects due to off-targeting. On the other hand, sales of high molecular weight drugs derived from antibodies and proteins having a molecular weight exceeding 10,000 are increasing because of their high specificity and few side effects. However, the problems are that it is expensive, that it may cause immunogenicity due to its large molecular weight, and that it generally does not migrate into the cell, so that the molecules that can be the target are limited.
Therefore, in recent years, molecules with a molecular weight of about 500 to 5,000, such as peptides and nucleic acids, so-called medium-molecular-weight drugs, have the advantages of low-molecular-weight drugs that are inexpensive and have low immunogenicity, and their specificity to target molecules. It is expected to be a drug that has the advantages of high molecular weight drugs, such as high prices and few side effects.
そこで近年、ペプチドや核酸などをはじめとする分子量が５００～５，０００程度の分子、いわゆる中分子医薬品が、安価で免疫原性が低いという低分子医薬品の利点と、標的分子への特異性が高く、副作用が少ないというという高分子医薬品の利点を併せ持つ医薬品として期待されている。 Small molecule drugs with a molecular weight of up to about 500 have been developed as many drugs because of their low manufacturing cost and low immunogenicity. However, this small molecule drug has low specificity, and it has become increasingly difficult to develop in recent years due to the problem of side effects due to off-targeting. On the other hand, sales of high molecular weight drugs derived from antibodies and proteins having a molecular weight exceeding 10,000 are increasing because of their high specificity and few side effects. However, the problems are that it is expensive, that it may cause immunogenicity due to its large molecular weight, and that it generally does not migrate into the cell, so that the molecules that can be the target are limited.
Therefore, in recent years, molecules with a molecular weight of about 500 to 5,000, such as peptides and nucleic acids, so-called medium-molecular-weight drugs, have the advantages of low-molecular-weight drugs that are inexpensive and have low immunogenicity, and their specificity to target molecules. It is expected to be a drug that has the advantages of high molecular weight drugs, such as high prices and few side effects.
また、中分子医薬品としてのペプチドを取得する技術として、様々なアミノ酸配列から成るペプチド（ポリペプチド）ライブラリを作製しその中から、疾患関連タンパク質などの特定の標的分子に対して高い親和性を持つペプチドを選別する方法が汎用される。
In addition, as a technique for acquiring peptides as medium-molecular-weight drugs, we have created a peptide (polypeptide) library consisting of various amino acid sequences and have high affinity for specific target molecules such as disease-related proteins. A method for selecting peptides is widely used.
前記ペプチドライブラリの作製方法として、例えば、ｉｎ ｖｉｔｒｏ翻訳系の方法として、ペプチド鎖、ｍＲＮＡ分子、及びリボソームを含むリボソームディスプレイ複合体を用いるリボソームディスプレイ法（ＲＤ法）が知られている（例えば、特許文献１参照）。前記ＲＤ法は、ｉｎ ｖｉｔｒｏ翻訳系とｍＲＮＡさえあれば、それらを混合するだけで、１０１２種類以上のペプチドライブラリを数分で作製することができる非常に優れた有用なものである。
As a method for producing the peptide library, for example, as an in vitro translation system method, a ribosome display method (RD method) using a ribosome display complex containing a peptide chain, an mRNA molecule, and a ribosome is known (for example, patent). See Document 1). The RD method is extremely excellent and useful because it is possible to prepare a peptide library of 1012 or more kinds in a few minutes simply by mixing an in vitro translation system and mRNA.
一方、ペプチドや核酸などの中分子医薬品は細胞内の分子を標的とする場合、細胞膜を透過させて所望の細胞内標的分子に到達させる手段が必要となる。細胞内には、細胞骨格関連タンパク質やキナーゼ関連因子など種々の疾病治療に際して標的となりうるものが多い。このため、有用な細胞内導入法の確立は、中分子医薬品の適用範囲を大幅に拡大することに繋がる。
有効成分を細胞内に取り込む技術としては、例えば、塩基性アミノ酸を多く含む細胞膜透過ペプチドのアミノ酸配列を融合する方法や、中心から規則的に分枝した構造を有する樹状高分子であるデンドリマーを用いる方法（例えば、特許文献２参照）などが知られている。 On the other hand, when a medium-molecular-weight drug such as a peptide or nucleic acid targets an intracellular molecule, a means for permeating the cell membrane to reach the desired intracellular target molecule is required. Many cells, such as cytoskeleton-related proteins and kinase-related factors, can be targeted in the treatment of various diseases. Therefore, the establishment of a useful intracellular introduction method leads to a significant expansion of the scope of application of medium-molecular-weight drugs.
Techniques for incorporating the active ingredient into cells include, for example, a method of fusing the amino acid sequence of a cell membrane penetrating peptide containing a large amount of basic amino acids, and a dendrimer which is a dendritic polymer having a regularly branched structure from the center. The method to be used (see, for example, Patent Document 2) and the like are known.
有効成分を細胞内に取り込む技術としては、例えば、塩基性アミノ酸を多く含む細胞膜透過ペプチドのアミノ酸配列を融合する方法や、中心から規則的に分枝した構造を有する樹状高分子であるデンドリマーを用いる方法（例えば、特許文献２参照）などが知られている。 On the other hand, when a medium-molecular-weight drug such as a peptide or nucleic acid targets an intracellular molecule, a means for permeating the cell membrane to reach the desired intracellular target molecule is required. Many cells, such as cytoskeleton-related proteins and kinase-related factors, can be targeted in the treatment of various diseases. Therefore, the establishment of a useful intracellular introduction method leads to a significant expansion of the scope of application of medium-molecular-weight drugs.
Techniques for incorporating the active ingredient into cells include, for example, a method of fusing the amino acid sequence of a cell membrane penetrating peptide containing a large amount of basic amino acids, and a dendrimer which is a dendritic polymer having a regularly branched structure from the center. The method to be used (see, for example, Patent Document 2) and the like are known.
上記したように、有効成分を細胞内に取り込む技術として、特許文献２に記載した方法などが検討されている。一方で、医薬品の開発という観点からは、有効成分を細胞内に取り込む機能を有しつつ、生体に対する毒性が低いことも求められる。また、有効成分を細胞内により効率良く取り込むためには、例えば細胞膜透過性分子において、細胞膜透過に寄与する構造の数を増やすことが考えられる。しかしながら、単純に細胞膜透過に寄与する構造の数を増やした場合には、細胞膜透過性分子全体の分子量も大きくなり、分子量の増大によって免疫原性が引き起こされる可能性がある。そのため、より小さい分子量の細胞膜透過性分子を用いて有効成分を細胞内により効率良く取り込む方法の開発が強く求められている。
したがって、本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、有効成分を細胞内に取り込む機能を有しつつ、生体に対する毒性が低い細胞膜透過性分子、前記細胞膜透過性分子を含むペプチド複合体及びその製造方法、前記ペプチド複合体を含むペプチドライブラリ、及び前記ペプチドライブラリを用いた機能性ペプチドのスクリーニング方法、並びに分子量が小さな細胞膜透過性分子であっても、有効成分を細胞内に効率良く取り込むことができる、細胞膜透過性分子の細胞膜透過性の向上方法を提供することを目的とする。 As described above, as a technique for incorporating the active ingredient into cells, the method described in Patent Document 2 and the like have been studied. On the other hand, from the viewpoint of drug development, it is also required to have a function of taking up the active ingredient into cells and to have low toxicity to the living body. Further, in order to take up the active ingredient more efficiently in the cell, it is conceivable to increase the number of structures contributing to cell membrane permeation, for example, in the cell membrane permeabilizing molecule. However, if the number of structures that contribute to cell membrane permeability is simply increased, the molecular weight of the entire cell membrane-permeable molecule also increases, and the increase in molecular weight may cause immunogenicity. Therefore, there is a strong demand for the development of a method for more efficiently incorporating the active ingredient into cells using a cell membrane permeable molecule having a smaller molecular weight.
Therefore, it is an object of the present invention to solve the above-mentioned problems in the past and to achieve the following objects. That is, the present invention includes a cell membrane penetrating molecule having a function of incorporating an active ingredient into a cell and having low toxicity to a living body, a peptide complex containing the cell membrane penetrating molecule, a method for producing the same, and the peptide complex. A peptide library, a method for screening a functional peptide using the peptide library, and a cell membrane penetrating molecule that can efficiently take up an active ingredient into a cell even if the molecular weight is a small cell membrane penetrating molecule. The purpose is to provide a method for improving sex.
したがって、本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、有効成分を細胞内に取り込む機能を有しつつ、生体に対する毒性が低い細胞膜透過性分子、前記細胞膜透過性分子を含むペプチド複合体及びその製造方法、前記ペプチド複合体を含むペプチドライブラリ、及び前記ペプチドライブラリを用いた機能性ペプチドのスクリーニング方法、並びに分子量が小さな細胞膜透過性分子であっても、有効成分を細胞内に効率良く取り込むことができる、細胞膜透過性分子の細胞膜透過性の向上方法を提供することを目的とする。 As described above, as a technique for incorporating the active ingredient into cells, the method described in Patent Document 2 and the like have been studied. On the other hand, from the viewpoint of drug development, it is also required to have a function of taking up the active ingredient into cells and to have low toxicity to the living body. Further, in order to take up the active ingredient more efficiently in the cell, it is conceivable to increase the number of structures contributing to cell membrane permeation, for example, in the cell membrane permeabilizing molecule. However, if the number of structures that contribute to cell membrane permeability is simply increased, the molecular weight of the entire cell membrane-permeable molecule also increases, and the increase in molecular weight may cause immunogenicity. Therefore, there is a strong demand for the development of a method for more efficiently incorporating the active ingredient into cells using a cell membrane permeable molecule having a smaller molecular weight.
Therefore, it is an object of the present invention to solve the above-mentioned problems in the past and to achieve the following objects. That is, the present invention includes a cell membrane penetrating molecule having a function of incorporating an active ingredient into a cell and having low toxicity to a living body, a peptide complex containing the cell membrane penetrating molecule, a method for producing the same, and the peptide complex. A peptide library, a method for screening a functional peptide using the peptide library, and a cell membrane penetrating molecule that can efficiently take up an active ingredient into a cell even if the molecular weight is a small cell membrane penetrating molecule. The purpose is to provide a method for improving sex.
前記課題を解決するため、本発明者らは鋭意検討した結果、下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩とすることで、有効成分を細胞内に取り込む機能を有しつつ、生体に対する毒性が低い細胞膜透過性分子とすることができることを知見した。また、下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とすることで、細胞膜透過に寄与する構造の数が多い細胞膜透過性分子と同等以上にまで細胞膜透過性を向上させることができることを知見した。
（前記一般式（Ｉ）中、Ｒは結合手を表す。）。
As a result of diligent studies to solve the above problems, the present inventors have decided to use a salt with an acid having a structure represented by the following general formula (I) and having a pKa value of less than 4.7. It was found that it can be a cell membrane permeable molecule with low toxicity to the living body while having a function of taking up the active ingredient into the cell. Further, by using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid, the cell membrane becomes equal to or higher than the cell membrane-permeable molecule having a large number of structures contributing to cell membrane permeation. It was found that the permeability can be improved.
(In the general formula (I), R represents a bond).
本発明は、本発明者らの前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ 下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩であることを特徴とする細胞膜透過性分子である。
（前記一般式（Ｉ）中、Ｒは結合手を表す。）。
＜２＞ ペプチドと、前記＜１＞に記載の細胞膜透過性分子とを含むことを特徴とするペプチド複合体である。
＜３＞ 前記＜２＞に記載のペプチド複合体を含むことを特徴とするペプチドライブラリである。
＜４＞ ペプチドに、前記＜１＞に記載の細胞膜透過性分子を導入することを含むことを特徴とするペプチド複合体の製造方法である。
＜５＞ 前記＜３＞に記載のペプチドライブラリを用いて機能性ペプチドをスクリーニングすることを含むことを特徴とする機能性ペプチドのスクリーニング方法である。
＜６＞ 下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とすることを含むことを特徴とする細胞膜透過性分子の細胞膜透過性の向上方法である。
（前記一般式（Ｉ）中、Ｒは結合手を表す。）。
The present invention is based on the above-mentioned findings of the present inventors, and the means for solving the above-mentioned problems are as follows. That is,
<1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid.
(In the general formula (I), R represents a bond).
<2> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to <1>.
<3> A peptide library comprising the peptide complex described in <2> above.
<4> A method for producing a peptide complex, which comprises introducing the cell membrane-permeable molecule according to <1> into a peptide.
<5> A method for screening a functional peptide, which comprises screening a functional peptide using the peptide library according to the above <3>.
<6> A method for improving the cell membrane permeability of a cell membrane-permeable molecule, which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid.
(In the general formula (I), R represents a bond).
＜１＞ 下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩であることを特徴とする細胞膜透過性分子である。
＜２＞ ペプチドと、前記＜１＞に記載の細胞膜透過性分子とを含むことを特徴とするペプチド複合体である。
＜３＞ 前記＜２＞に記載のペプチド複合体を含むことを特徴とするペプチドライブラリである。
＜４＞ ペプチドに、前記＜１＞に記載の細胞膜透過性分子を導入することを含むことを特徴とするペプチド複合体の製造方法である。
＜５＞ 前記＜３＞に記載のペプチドライブラリを用いて機能性ペプチドをスクリーニングすることを含むことを特徴とする機能性ペプチドのスクリーニング方法である。
＜６＞ 下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とすることを含むことを特徴とする細胞膜透過性分子の細胞膜透過性の向上方法である。
<1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid.
<2> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to <1>.
<3> A peptide library comprising the peptide complex described in <2> above.
<4> A method for producing a peptide complex, which comprises introducing the cell membrane-permeable molecule according to <1> into a peptide.
<5> A method for screening a functional peptide, which comprises screening a functional peptide using the peptide library according to the above <3>.
<6> A method for improving the cell membrane permeability of a cell membrane-permeable molecule, which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid.
本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、有効成分を細胞内に取り込む機能を有しつつ、生体に対する毒性が低い細胞膜透過性分子、前記細胞膜透過性分子を含むペプチド複合体及びその製造方法、前記ペプチド複合体を含むペプチドライブラリ、及び前記ペプチドライブラリを用いた機能性ペプチドのスクリーニング方法、並びに分子量が小さな細胞膜透過性分子であっても、有効成分を細胞内に効率良く取り込むことができる、細胞膜透過性分子の細胞膜透過性の向上方法を提供することができる。
According to the present invention, the cell membrane-permeable molecule, which is a cell membrane-permeable molecule having a function of taking up an active ingredient into a cell and having a function of taking up the active ingredient into the cell, which can solve the conventional problems and achieve the object, and the cell membrane permeation. A peptide complex containing a sex molecule and a method for producing the same, a peptide library containing the peptide complex, a method for screening a functional peptide using the peptide library, and an active ingredient even for a cell membrane-permeable molecule having a small molecular weight. It is possible to provide a method for improving the cell membrane permeability of a cell membrane-permeable molecule, which can efficiently take up the peptide into the cell.
（細胞膜透過性分子）
本発明の細胞膜透過性分子は、少なくとも下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩である。
（前記一般式（Ｉ）中、Ｒは結合手を表す。）。
(Cell membrane permeable molecule)
The cell membrane permeable molecule of the present invention has at least a structure represented by the following general formula (I) and is a salt with an acid having a pKa value of less than 4.7.
(In the general formula (I), R represents a bond).
本発明の細胞膜透過性分子は、少なくとも下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩である。
The cell membrane permeable molecule of the present invention has at least a structure represented by the following general formula (I) and is a salt with an acid having a pKa value of less than 4.7.
＜一般式（Ｉ）で表される構造＞
前記一般式（Ｉ）で表される構造における前記Ｒ以外の部分は、細胞膜透過性に寄与する部分である。
前記細胞膜透過性分子における一般式（Ｉ）で表される構造のＲ以外の部分の数としては、特に制限はなく、目的に応じて適宜選択することができるが、１つであることが好ましい。即ち、本発明の細胞膜透過性分子は、前記一般式（Ｉ）で表される樹状構造を１つ有する態様が好ましい。 <Structure represented by the general formula (I)>
The portion other than R in the structure represented by the general formula (I) is a portion that contributes to cell membrane permeability.
The number of portions of the cell membrane-permeable molecule other than R in the structure represented by the general formula (I) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably one. .. That is, the cell membrane permeable molecule of the present invention preferably has one dendritic structure represented by the general formula (I).
前記一般式（Ｉ）で表される構造における前記Ｒ以外の部分は、細胞膜透過性に寄与する部分である。
前記細胞膜透過性分子における一般式（Ｉ）で表される構造のＲ以外の部分の数としては、特に制限はなく、目的に応じて適宜選択することができるが、１つであることが好ましい。即ち、本発明の細胞膜透過性分子は、前記一般式（Ｉ）で表される樹状構造を１つ有する態様が好ましい。 <Structure represented by the general formula (I)>
The portion other than R in the structure represented by the general formula (I) is a portion that contributes to cell membrane permeability.
The number of portions of the cell membrane-permeable molecule other than R in the structure represented by the general formula (I) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably one. .. That is, the cell membrane permeable molecule of the present invention preferably has one dendritic structure represented by the general formula (I).
前記一般式（Ｉ）で表される構造の前記Ｒの部分としては、特に制限はなく、目的に応じて適宜選択することができるが、ヘテロ原子含有基と結合していることが好ましい。
The R portion of the structure represented by the general formula (I) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably bonded to a heteroatom-containing group.
－ヘテロ原子含有基－
前記ヘテロ原子含有基としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、（Ａ）ペプチドと連結するペプチド連結用基と、前記ペプチド連結用基と前記一般式（Ｉ）で表す構造とを連結する連結基とからなる態様、（Ｂ）ペプチドと連結するペプチド連結用基からなる態様などが挙げられる。また、前記ヘテロ原子含有基は、ペプチドや核酸の骨格を有してもよい。 -Heteroatom-containing group-
The heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a peptide linking group linked to the peptide (A), the peptide linking group and the general formula (I). ), An embodiment consisting of a linking group for linking with the structure represented by), (B) an embodiment consisting of a peptide linking group for linking with a peptide, and the like. Further, the heteroatom-containing group may have a skeleton of a peptide or nucleic acid.
前記ヘテロ原子含有基としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、（Ａ）ペプチドと連結するペプチド連結用基と、前記ペプチド連結用基と前記一般式（Ｉ）で表す構造とを連結する連結基とからなる態様、（Ｂ）ペプチドと連結するペプチド連結用基からなる態様などが挙げられる。また、前記ヘテロ原子含有基は、ペプチドや核酸の骨格を有してもよい。 -Heteroatom-containing group-
The heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a peptide linking group linked to the peptide (A), the peptide linking group and the general formula (I). ), An embodiment consisting of a linking group for linking with the structure represented by), (B) an embodiment consisting of a peptide linking group for linking with a peptide, and the like. Further, the heteroatom-containing group may have a skeleton of a peptide or nucleic acid.
前記ヘテロ原子含有基の長さとしては、特に制限はなく、目的に応じて適宜選択することができるが、例えば直鎖上に結合している原子数の下限値としては１原子以上が好ましく、上限値としては５０原子以下が好ましく、２５原子以下がより好ましい。前記直鎖上に結合している原子数が２５原子以下の例としては、後述する細胞膜透過性分子Ｇ３－ＤＣＸにおけるヘテロ原子含有基などが挙げられる。
The length of the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose. However, for example, the lower limit of the number of atoms bonded linearly is preferably 1 atom or more. The upper limit is preferably 50 atoms or less, more preferably 25 atoms or less. Examples of the number of atoms bonded to the linear chain of 25 or less include heteroatom-containing groups in the cell membrane-permeable molecule G3-DCX described later.
前記ヘテロ原子含有基におけるヘテロ原子としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、酸素原子、窒素原子、硫黄原子及びリン原子からなる群から選択される少なくとも１種などが挙げられる。前記ヘテロ原子は、１種のみであってもよいし、２種以上であってもよい。
The hetero atom in the hetero atom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose. For example, at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. And so on. The heteroatom may be only one kind or two or more kinds.
前記ヘテロ原子含有基におけるヘテロ原子の含有割合としては、特に制限はなく、目的に応じて適宜選択することができるが、細胞膜透過性分子の水溶性が高まる点で、１０％以上であることが好ましい。前記ヘテロ原子の含有割合の上限としては、特に制限はなく、目的に応じて適宜選択することができるが、６０％以下が好ましく、５０％以下がより好ましい。前記ヘテロ原子含有基におけるヘテロ原子の含有割合の範囲としては、１０％以上６０％以下が好ましく、１０％以上５０％以下がより好ましい。
前記ヘテロ原子含有基におけるヘテロ原子の含有割合は、下記のようにして算出することができる。なお、全原子には、水素原子も含まれる。
ヘテロ原子含有基におけるヘテロ原子の含有割合（％）＝｛（ヘテロ原子含有基におけるヘテロ原子の数）／（ヘテロ原子含有基の全原子数）｝×１００ The content ratio of the heteroatom in the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it may be 10% or more in terms of increasing the water solubility of the cell membrane-permeable molecule. preferable. The upper limit of the content ratio of the heteroatom is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 60% or less, more preferably 50% or less. The range of the content ratio of the heteroatom in the heteroatom-containing group is preferably 10% or more and 60% or less, and more preferably 10% or more and 50% or less.
The content ratio of the heteroatom in the heteroatom-containing group can be calculated as follows. It should be noted that all atoms include hydrogen atoms.
Content ratio of heteroatoms in heteroatom-containing groups (%) = {(number of heteroatoms in heteroatom-containing groups) / (total number of heteroatom-containing groups)} × 100
前記ヘテロ原子含有基におけるヘテロ原子の含有割合は、下記のようにして算出することができる。なお、全原子には、水素原子も含まれる。
ヘテロ原子含有基におけるヘテロ原子の含有割合（％）＝｛（ヘテロ原子含有基におけるヘテロ原子の数）／（ヘテロ原子含有基の全原子数）｝×１００ The content ratio of the heteroatom in the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it may be 10% or more in terms of increasing the water solubility of the cell membrane-permeable molecule. preferable. The upper limit of the content ratio of the heteroatom is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 60% or less, more preferably 50% or less. The range of the content ratio of the heteroatom in the heteroatom-containing group is preferably 10% or more and 60% or less, and more preferably 10% or more and 50% or less.
The content ratio of the heteroatom in the heteroatom-containing group can be calculated as follows. It should be noted that all atoms include hydrogen atoms.
Content ratio of heteroatoms in heteroatom-containing groups (%) = {(number of heteroatoms in heteroatom-containing groups) / (total number of heteroatom-containing groups)} × 100
前記ヘテロ原子含有基は、繰返し単位を含むことが好ましい。
前記繰返し単位としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、アルキレンオキサイド、アルキレンイミン、アルキレンスルフィドなどが挙げられる。前記繰返し単位は、１種のみであってもよいし、２種以上であってもよい。また、前記繰返し単位は、置換基を有していてもよい。前記繰り返し単位としては、入手容易性、安定性の観点からアルキレンオキサイドが好ましい。 The heteroatom-containing group preferably contains a repeating unit.
The repeating unit is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include alkylene oxide, alkyleneimine and alkylene sulfide. The repeating unit may be only one kind or two or more kinds. Further, the repeating unit may have a substituent. As the repeating unit, alkylene oxide is preferable from the viewpoint of availability and stability.
前記繰返し単位としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、アルキレンオキサイド、アルキレンイミン、アルキレンスルフィドなどが挙げられる。前記繰返し単位は、１種のみであってもよいし、２種以上であってもよい。また、前記繰返し単位は、置換基を有していてもよい。前記繰り返し単位としては、入手容易性、安定性の観点からアルキレンオキサイドが好ましい。 The heteroatom-containing group preferably contains a repeating unit.
The repeating unit is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include alkylene oxide, alkyleneimine and alkylene sulfide. The repeating unit may be only one kind or two or more kinds. Further, the repeating unit may have a substituent. As the repeating unit, alkylene oxide is preferable from the viewpoint of availability and stability.
前記アルキレンオキサイドとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、メチレンオキサイド、エチレンオキサイド及びプロピレンオキサイドからなる群から選択される少なくとも１種などが挙げられる。
The alkylene oxide is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include at least one selected from the group consisting of methylene oxide, ethylene oxide and propylene oxide.
前記繰返し単位の数としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記繰り返し単位の下限としては１以上が好ましく、上限としては１０以下が好ましい。
The number of the repeating units is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the lower limit of the repeating units is preferably 1 or more, and the upper limit is preferably 10 or less.
前記繰り返し単位が有する置換基としては、特に制限はなく、目的に応じて適宜選択することができるが、前記置換基としては、例えばアルキル、アルケニル、アルキニル、アリール、ヘテロアリール、シクロアルキル、ヘテロシクリル、ヒドロキシ、メルカプト、アミノ、カルボニル、アミド、チオアミド、ウレア、スルホンアミド、カルボキシ、ニトリルが好ましい。
The substituent contained in the repeating unit is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the substituent include alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl. Hydroxy, mercapto, amino, carbonyl, amide, thioamide, urea, sulfonamide, carboxy and nitrile are preferred.
－－ペプチド連結用基－－
前記ペプチド連結用基は、前記ペプチドにおける反応性アミノ酸残基との反応により、細胞膜透過性分子のペプチドへの導入に寄与する。
前記反応性アミノ酸残基は、前記ペプチド連結用基と反応するアミノ酸残基であり、前記ペプチド連結用基と直接反応するアミノ酸残基であってもよいし、前記ペプチド連結用基と反応できるように修飾されたアミノ酸残基であってもよい。 --Peptide linking group ---
The peptide linking group contributes to the introduction of the cell membrane-permeable molecule into the peptide by reacting with the reactive amino acid residue in the peptide.
The reactive amino acid residue is an amino acid residue that reacts with the peptide linking group, may be an amino acid residue that directly reacts with the peptide linking group, or can react with the peptide linking group. It may be an amino acid residue modified to.
前記ペプチド連結用基は、前記ペプチドにおける反応性アミノ酸残基との反応により、細胞膜透過性分子のペプチドへの導入に寄与する。
前記反応性アミノ酸残基は、前記ペプチド連結用基と反応するアミノ酸残基であり、前記ペプチド連結用基と直接反応するアミノ酸残基であってもよいし、前記ペプチド連結用基と反応できるように修飾されたアミノ酸残基であってもよい。 --Peptide linking group ---
The peptide linking group contributes to the introduction of the cell membrane-permeable molecule into the peptide by reacting with the reactive amino acid residue in the peptide.
The reactive amino acid residue is an amino acid residue that reacts with the peptide linking group, may be an amino acid residue that directly reacts with the peptide linking group, or can react with the peptide linking group. It may be an amino acid residue modified to.
前記ペプチド連結用基としては、前記ペプチドと連結することができる限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、システイン残基のチオール基、リジン残基の側鎖アミノ基（－ＮＨ２）、ヒスチジン残基又はトリプトファン残基の側鎖アミノ基（＞ＮＨ）、トリプトファン残基のインドール環２位及び３位炭素、チロシン残基又はセリン残基又はスレオニン残基の側鎖水酸基（－ＯＨ）、チロシン残基のオルト位炭素、メチオニン残基の側鎖スルフィド基（－ＳＭｅ）と反応して結合を形成可能な基などが挙げられる。
前記ペプチド連結用基の具体例としては、例えば、国際公開第２０１７／２１３１５８号の段落〔００６７〕～〔００７６〕に記載されているハロゲン化アルキル基、活性化カルボニル基、不飽和炭化水素基、エポキシ基、スルホニル含有基、イソシアネート基、チオイソシアネート基、カルベン発生基、ジスルフィド結合含有基、チオール基などが挙げられる。 The group for linking the peptide is not particularly limited as long as it can be linked to the peptide, and can be appropriately selected depending on the intended purpose. For example, the thiol group of the cysteine residue and the side chain amino of the lysine residue can be selected. Side chain amino group (> NH) of group (-NH 2 ), histidine residue or tryptophan residue, indole ring 2 and 3 carbon of tryptophan residue, tyrosine residue or serine residue or threonine residue side Examples thereof include a chain hydroxyl group (-OH), an ortho-position carbon of a tyrosine residue, and a group capable of forming a bond by reacting with a side chain sulfide group (-SMe) of a methionine residue.
Specific examples of the peptide linking group include, for example, an alkyl halide group, an activated carbonyl group, and an unsaturated hydrocarbon group described in paragraphs [0067] to [0076] of International Publication No. 2017/213158. Examples thereof include an epoxy group, a sulfonyl-containing group, an isocyanate group, a thioisocyanate group, a carben generating group, a disulfide bond-containing group, and a thiol group.
前記ペプチド連結用基の具体例としては、例えば、国際公開第２０１７／２１３１５８号の段落〔００６７〕～〔００７６〕に記載されているハロゲン化アルキル基、活性化カルボニル基、不飽和炭化水素基、エポキシ基、スルホニル含有基、イソシアネート基、チオイソシアネート基、カルベン発生基、ジスルフィド結合含有基、チオール基などが挙げられる。 The group for linking the peptide is not particularly limited as long as it can be linked to the peptide, and can be appropriately selected depending on the intended purpose. For example, the thiol group of the cysteine residue and the side chain amino of the lysine residue can be selected. Side chain amino group (> NH) of group (-NH 2 ), histidine residue or tryptophan residue, indole ring 2 and 3 carbon of tryptophan residue, tyrosine residue or serine residue or threonine residue side Examples thereof include a chain hydroxyl group (-OH), an ortho-position carbon of a tyrosine residue, and a group capable of forming a bond by reacting with a side chain sulfide group (-SMe) of a methionine residue.
Specific examples of the peptide linking group include, for example, an alkyl halide group, an activated carbonyl group, and an unsaturated hydrocarbon group described in paragraphs [0067] to [0076] of International Publication No. 2017/213158. Examples thereof include an epoxy group, a sulfonyl-containing group, an isocyanate group, a thioisocyanate group, a carben generating group, a disulfide bond-containing group, and a thiol group.
また、前記ペプチド連結用基は、ペプチドにおける反応性アミノ酸残基との反応により、前記ペプチドの環状化に寄与するもの（以下、「環状化基」と称することがある。）であることが好ましい。
前記環状化基と反応する反応性アミノ酸残基は、前記環状化基と直接反応するアミノ酸残基であってもよいし、前記環状化基と反応できるように修飾されたアミノ酸残基であってもよい。
前記環状化基としては、特に制限はなく、目的に応じて適宜選択することができるが、電子吸引基が好ましい。 Further, it is preferable that the peptide linking group contributes to the cyclization of the peptide by reacting with the reactive amino acid residue in the peptide (hereinafter, may be referred to as "cyclizing group"). ..
The reactive amino acid residue that reacts with the cyclic group may be an amino acid residue that directly reacts with the cyclic group, or may be an amino acid residue modified so as to react with the cyclic group. May be good.
The cyclic group is not particularly limited and may be appropriately selected depending on the intended purpose, but an electron-withdrawing group is preferable.
前記環状化基と反応する反応性アミノ酸残基は、前記環状化基と直接反応するアミノ酸残基であってもよいし、前記環状化基と反応できるように修飾されたアミノ酸残基であってもよい。
前記環状化基としては、特に制限はなく、目的に応じて適宜選択することができるが、電子吸引基が好ましい。 Further, it is preferable that the peptide linking group contributes to the cyclization of the peptide by reacting with the reactive amino acid residue in the peptide (hereinafter, may be referred to as "cyclizing group"). ..
The reactive amino acid residue that reacts with the cyclic group may be an amino acid residue that directly reacts with the cyclic group, or may be an amino acid residue modified so as to react with the cyclic group. May be good.
The cyclic group is not particularly limited and may be appropriately selected depending on the intended purpose, but an electron-withdrawing group is preferable.
前記電子吸引基としては、特に制限はなく、目的に応じて適宜選択することができるが、ハロゲンを有することが好ましい。前記ハロゲンの種類としては、特に制限はなく、目的に応じて適宜選択することができるが、例えば塩素原子、臭素原子、ヨウ素原子が好ましい。前記ハロゲンの数としては、特に制限はなく、目的に応じて適宜選択することができるが、前記ハロゲン数の下限としては２以上が好ましく、上限としては、５以下、４以下が好ましい。
前記電子吸引基は、置換基を有していてもよい。前記電子吸引基は、その構造中に不飽和構造を含むとペプチドを環状化させやすい。前記不飽和構造としては、特に制限はないが、例えば芳香環構造、複素環構造、脂環式炭化水素構造、アルケニル構造、アルキニル構造、カルボニル構造、チオカルボニル構造、オキシム構造、シアノ構造、イソシアネート構造を挙げることができる。中でも前記電子吸引基としては、ベンジルハライドがより好ましく、３，５－ビス（ハロメチル）ベンジル基が特に好ましい。前記ハロゲンの種類としては、特に制限はなく、目的に応じて適宜選択することができるが、例えば塩素原子、臭素原子、ヨウ素原子が好ましく、塩素原子がより好ましい。 The electron-withdrawing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it preferably has a halogen. The type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose, but for example, a chlorine atom, a bromine atom and an iodine atom are preferable. The number of halogens is not particularly limited and may be appropriately selected depending on the intended purpose. However, the lower limit of the number of halogens is preferably 2 or more, and the upper limit is preferably 5 or less and 4 or less.
The electron-withdrawing group may have a substituent. If the electron-withdrawing group contains an unsaturated structure in its structure, the peptide is likely to be cyclized. The unsaturated structure is not particularly limited, but is, for example, an aromatic ring structure, a heterocyclic structure, an alicyclic hydrocarbon structure, an alkenyl structure, an alkynyl structure, a carbonyl structure, a thiocarbonyl structure, an oxime structure, a cyano structure, or an isocyanate structure. Can be mentioned. Among them, as the electron-withdrawing group, a benzyl halide is more preferable, and a 3,5-bis (halomethyl) benzyl group is particularly preferable. The type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a chlorine atom, a bromine atom and an iodine atom are preferable, and a chlorine atom is more preferable.
前記電子吸引基は、置換基を有していてもよい。前記電子吸引基は、その構造中に不飽和構造を含むとペプチドを環状化させやすい。前記不飽和構造としては、特に制限はないが、例えば芳香環構造、複素環構造、脂環式炭化水素構造、アルケニル構造、アルキニル構造、カルボニル構造、チオカルボニル構造、オキシム構造、シアノ構造、イソシアネート構造を挙げることができる。中でも前記電子吸引基としては、ベンジルハライドがより好ましく、３，５－ビス（ハロメチル）ベンジル基が特に好ましい。前記ハロゲンの種類としては、特に制限はなく、目的に応じて適宜選択することができるが、例えば塩素原子、臭素原子、ヨウ素原子が好ましく、塩素原子がより好ましい。 The electron-withdrawing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it preferably has a halogen. The type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose, but for example, a chlorine atom, a bromine atom and an iodine atom are preferable. The number of halogens is not particularly limited and may be appropriately selected depending on the intended purpose. However, the lower limit of the number of halogens is preferably 2 or more, and the upper limit is preferably 5 or less and 4 or less.
The electron-withdrawing group may have a substituent. If the electron-withdrawing group contains an unsaturated structure in its structure, the peptide is likely to be cyclized. The unsaturated structure is not particularly limited, but is, for example, an aromatic ring structure, a heterocyclic structure, an alicyclic hydrocarbon structure, an alkenyl structure, an alkynyl structure, a carbonyl structure, a thiocarbonyl structure, an oxime structure, a cyano structure, or an isocyanate structure. Can be mentioned. Among them, as the electron-withdrawing group, a benzyl halide is more preferable, and a 3,5-bis (halomethyl) benzyl group is particularly preferable. The type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a chlorine atom, a bromine atom and an iodine atom are preferable, and a chlorine atom is more preferable.
前記環状化基によるペプチドの環状化は、前記環状化基と、ペプチドに含まれるチオール基、アミノ基、及びヒドロキシ基からなる群から選択される少なくとも１種の基との反応により行われることが好ましい。
Cyclization of a peptide with the cyclic group may be carried out by reacting the cyclic group with at least one group selected from the group consisting of a thiol group, an amino group and a hydroxy group contained in the peptide. preferable.
また、例えば、オキシムライゲーション法により、前記ペプチドと前記細胞膜透過性分子とを結合する場合には、下記構造式で表されるものを前記ペプチド連結用基として用いることもできる。
Further, for example, when the peptide and the cell membrane permeable molecule are bound by the oxime ligation method, those represented by the following structural formula can also be used as the peptide linking group.
－－連結基－－
前記連結基は、前記ペプチド連結用基と前記一般式（Ｉ）で表す構造とを連結する基である。
前記連結基の構造としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、上記した繰返し単位を含む構造などが挙げられる。 －－Connecting group －－
The linking group is a group that links the peptide linking group and the structure represented by the general formula (I).
The structure of the linking group is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a structure containing the above-mentioned repeating unit.
前記連結基は、前記ペプチド連結用基と前記一般式（Ｉ）で表す構造とを連結する基である。
前記連結基の構造としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、上記した繰返し単位を含む構造などが挙げられる。 －－Connecting group －－
The linking group is a group that links the peptide linking group and the structure represented by the general formula (I).
The structure of the linking group is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a structure containing the above-mentioned repeating unit.
前記細胞膜透過性分子は、ペプチドに導入される際に、前記ペプチドにおける反応性アミノ酸残基と反応する部位の構造が変化してもよい。
When the cell membrane permeable molecule is introduced into a peptide, the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
前記細胞膜透過性分子の具体例としては、例えば、以下の構造式で表される化合物などが挙げられる。なお、前記細胞膜透過性分子は、塩の形態とする際に、その構造の一部が変化してもよい。
Specific examples of the cell membrane permeable molecule include compounds represented by the following structural formulas. In addition, a part of the structure of the cell membrane permeable molecule may be changed when it is in the form of a salt.
下記構造式で表される細胞膜透過性分子Ｇ３－ＤＣＸは、一般式（Ｉ）において、Ｒの部分にヘテロ原子含有基として、３，５－ビス（クロロメチル）ベンジル基である環状化基（ペプチド連結用基）と、連結基とを有している例である。前記細胞膜透過性分子Ｇ３－ＤＣＸは環状化基を有しているので、ペプチドの環状化と、ペプチドへの細胞膜透過性の付与とを１工程で行うことができる。
The cell membrane permeable molecule G3-DCX represented by the following structural formula is a cyclic group which is a 3,5-bis (chloromethyl) benzyl group as a heteroatom-containing group in the R portion in the general formula (I). This is an example having a peptide linking group) and a linking group. Since the cell membrane-permeable molecule G3-DCX has a cyclizing group, it is possible to cyclize the peptide and impart cell membrane permeability to the peptide in one step.
下記構造式で表される細胞膜透過性分子Ｇ３は、一般式（Ｉ）において、Ｒの部分にヘテロ原子含有基として、オキシムライゲーション法によりペプチドと結合可能なペプチド連結用基を有している例である。
The cell membrane permeable molecule G3 represented by the following structural formula is an example in the general formula (I) having a peptide linking group capable of binding to a peptide by the oxime ligation method as a heteroatom-containing group in the R portion. Is.
＜ｐＫａの値が４．７未満の酸＞
本発明の細胞膜透過性分子は、ｐＫａ（酸解離定数）の値が４．７未満の酸との塩である。
本発明において、酸が複数のｐＫａの値を有している場合には、１段階目の解離におけるｐＫａの値であるｐＫａ１の値をｐＫａの値とする。
前記ｐＫａの値が４．７未満の酸としては、特に制限はなく、目的に応じて適宜選択することができるが、生体に対してより低毒性である点で、トリフルオロ酢酸（－０．３）、塩酸（－８．０）、硫酸（－３．０）、硝酸（－１．３）、リン酸（２．１（ｐＫａ１））、メシル酸（－２．６）、トシル酸（－２．８）、酒石酸（３．２（ｐＫａ１））及びクエン酸（３．１（ｐＫａ１））からなる群から選択されるいずれかが好ましい。前記酸の名称の後のかっこ内の数値はｐＫａの値を表す。なお、ｐＫａ１の値の場合は、数値の後にｐＫａ１と記載した。
前記ｐＫａの値が４．７未満の酸の中でも、生体に対してより低毒性である点では、ｐＫａの値の下限としては、－８．０超（ｐＫａの値が－８．０よりも大きい）が好ましく、－３．０超（ｐＫａの値が－３．０よりも大きい）がより好ましい。また、前記ｐＫａの値が４．７未満の酸におけるｐＫａの値の上限としては３．５以下が好ましく、３．３未満がより好ましい。 <Acids with a pK a value of less than 4.7>
The cell membrane permeable molecule of the present invention is a salt with an acid having a pK a (acid dissociation constant) value of less than 4.7.
In the present invention, when the acid has a plurality of pK a values, the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
The acid having a pK a value of less than 4.7 is not particularly limited and may be appropriately selected depending on the intended purpose, but trifluoroacetic acid (-0) in that it is less toxic to the living body. .3), Hydrochloric acid (-8.0), Sulfuric acid (-3.0), Nitric acid (-1.3), Phosphoric acid (2.1 (pK a1 )), Mesic acid (-2.6), Tosyl Any one selected from the group consisting of acid (-2.8), tartrate acid (3.2 (pK a1 )) and citric acid (3.1 (pK a1 )) is preferred. The number in parentheses after the acid name represents the value of pK a . In the case of the value of pK a1 , it is described as pK a1 after the numerical value.
Among the acids having a pK a value of less than 4.7, the lower limit of the pK a value is more than -8.0 (the pK a value is -8.) In that it is less toxic to the living body. (Greater than 0) is preferred, more than -3.0 (pK a value is greater than -3.0) is more preferred. Further, the upper limit of the pK a value for an acid having a pK a value of less than 4.7 is preferably 3.5 or less, and more preferably less than 3.3.
本発明の細胞膜透過性分子は、ｐＫａ（酸解離定数）の値が４．７未満の酸との塩である。
本発明において、酸が複数のｐＫａの値を有している場合には、１段階目の解離におけるｐＫａの値であるｐＫａ１の値をｐＫａの値とする。
前記ｐＫａの値が４．７未満の酸としては、特に制限はなく、目的に応じて適宜選択することができるが、生体に対してより低毒性である点で、トリフルオロ酢酸（－０．３）、塩酸（－８．０）、硫酸（－３．０）、硝酸（－１．３）、リン酸（２．１（ｐＫａ１））、メシル酸（－２．６）、トシル酸（－２．８）、酒石酸（３．２（ｐＫａ１））及びクエン酸（３．１（ｐＫａ１））からなる群から選択されるいずれかが好ましい。前記酸の名称の後のかっこ内の数値はｐＫａの値を表す。なお、ｐＫａ１の値の場合は、数値の後にｐＫａ１と記載した。
前記ｐＫａの値が４．７未満の酸の中でも、生体に対してより低毒性である点では、ｐＫａの値の下限としては、－８．０超（ｐＫａの値が－８．０よりも大きい）が好ましく、－３．０超（ｐＫａの値が－３．０よりも大きい）がより好ましい。また、前記ｐＫａの値が４．７未満の酸におけるｐＫａの値の上限としては３．５以下が好ましく、３．３未満がより好ましい。 <Acids with a pK a value of less than 4.7>
The cell membrane permeable molecule of the present invention is a salt with an acid having a pK a (acid dissociation constant) value of less than 4.7.
In the present invention, when the acid has a plurality of pK a values, the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
The acid having a pK a value of less than 4.7 is not particularly limited and may be appropriately selected depending on the intended purpose, but trifluoroacetic acid (-0) in that it is less toxic to the living body. .3), Hydrochloric acid (-8.0), Sulfuric acid (-3.0), Nitric acid (-1.3), Phosphoric acid (2.1 (pK a1 )), Mesic acid (-2.6), Tosyl Any one selected from the group consisting of acid (-2.8), tartrate acid (3.2 (pK a1 )) and citric acid (3.1 (pK a1 )) is preferred. The number in parentheses after the acid name represents the value of pK a . In the case of the value of pK a1 , it is described as pK a1 after the numerical value.
Among the acids having a pK a value of less than 4.7, the lower limit of the pK a value is more than -8.0 (the pK a value is -8.) In that it is less toxic to the living body. (Greater than 0) is preferred, more than -3.0 (pK a value is greater than -3.0) is more preferred. Further, the upper limit of the pK a value for an acid having a pK a value of less than 4.7 is preferably 3.5 or less, and more preferably less than 3.3.
前記細胞膜透過性分子は、上記した一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩であって、本発明の効果を損なわない限りにおいて、その他の構成を有していてもよい。
The cell membrane permeable molecule has a structure represented by the above-mentioned general formula (I), and is a salt with an acid having a pKa value of less than 4.7, as long as the effect of the present invention is not impaired. , Other configurations may be included.
＜細胞膜透過性分子の製造方法＞
前記細胞膜透過性分子の製造方法としては、特に制限はなく、公知の化学合成の技術を適宜選択して行うことができる。例えば、米国特許第７，８６２，８０７号明細書などに記載の方法を参考にして、公知の化学合成の技術を適宜選択することにより製造することができる。例えば、塩の形態とする方法としては、塩の形態ではない細胞膜透過性分子の粗生成物を、目的の塩の種類に応じた移動相を用いたＨＰＬＣにより精製し、目的とする塩の形態の細胞膜透過性分子とする方法、塩の形態である細胞膜透過性分子を、対イオンを目的の塩のイオンへと置換した陰イオン交換樹脂に通じ、溶出することで目的とする塩の形態の細胞膜透過性分子とする方法などが挙げられる。 <Manufacturing method of cell membrane permeable molecule>
The method for producing the cell membrane-permeable molecule is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807. For example, as a method of forming a salt, a crude product of a cell membrane permeable molecule that is not in the form of a salt is purified by HPLC using a mobile phase according to the type of the target salt, and the form of the target salt is obtained. The method of making the cell membrane permeable molecule of the above, the cell membrane permeable molecule in the form of a salt is passed through an anion exchange resin in which the counter ion is replaced with the ion of the target salt, and is eluted to form the target salt. Examples thereof include a method of making a cell membrane permeable molecule.
前記細胞膜透過性分子の製造方法としては、特に制限はなく、公知の化学合成の技術を適宜選択して行うことができる。例えば、米国特許第７，８６２，８０７号明細書などに記載の方法を参考にして、公知の化学合成の技術を適宜選択することにより製造することができる。例えば、塩の形態とする方法としては、塩の形態ではない細胞膜透過性分子の粗生成物を、目的の塩の種類に応じた移動相を用いたＨＰＬＣにより精製し、目的とする塩の形態の細胞膜透過性分子とする方法、塩の形態である細胞膜透過性分子を、対イオンを目的の塩のイオンへと置換した陰イオン交換樹脂に通じ、溶出することで目的とする塩の形態の細胞膜透過性分子とする方法などが挙げられる。 <Manufacturing method of cell membrane permeable molecule>
The method for producing the cell membrane-permeable molecule is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807. For example, as a method of forming a salt, a crude product of a cell membrane permeable molecule that is not in the form of a salt is purified by HPLC using a mobile phase according to the type of the target salt, and the form of the target salt is obtained. The method of making the cell membrane permeable molecule of the above, the cell membrane permeable molecule in the form of a salt is passed through an anion exchange resin in which the counter ion is replaced with the ion of the target salt, and is eluted to form the target salt. Examples thereof include a method of making a cell membrane permeable molecule.
得られた細胞膜透過性分子が所望の構造を有するか否かを確認する方法としては、特に制限はなく、公知の分析方法を適宜選択することができ、例えば、質量分析法、プロトン核磁気共鳴分光法、炭素１３核磁気共鳴分光法、紫外分光法、赤外分光法、液体クロマトグラフィーなどの分析方法が挙げられる。
The method for confirming whether or not the obtained cell membrane permeable molecule has a desired structure is not particularly limited, and a known analytical method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance can be selected. Analytical methods such as spectroscopy, carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, and liquid chromatography can be mentioned.
本発明の細胞膜透過分子は、生体への毒性を低減しつつ、ペプチド、核酸、タンパク質及びこれらの複合体などの有効成分に対して優れた細胞膜透過性を付与することができる。
The cell membrane permeabilizing molecule of the present invention can impart excellent cell membrane permeability to active ingredients such as peptides, nucleic acids, proteins and complexes thereof while reducing toxicity to living organisms.
（ペプチド複合体）
本発明のペプチド複合体は、ペプチドと、上記した本発明の細胞膜透過性分子とを少なくとも含み、必要に応じて更にその他の構成を含む。 (Peptide complex)
The peptide complex of the present invention comprises at least the peptide and the above-mentioned cell membrane permeable molecule of the present invention, and further contains other configurations as required.
本発明のペプチド複合体は、ペプチドと、上記した本発明の細胞膜透過性分子とを少なくとも含み、必要に応じて更にその他の構成を含む。 (Peptide complex)
The peptide complex of the present invention comprises at least the peptide and the above-mentioned cell membrane permeable molecule of the present invention, and further contains other configurations as required.
＜ペプチド＞
前記ペプチドとしては、前記細胞膜透過性分子を導入することができる限り、特に制限はなく、目的に応じて適宜選択することができるが、前記細胞膜透過性分子を導入することにより環状化されるペプチドが好ましい。
前記ペプチドにおけるアミノ酸の種類としては、特に制限はなく、目的に応じて適宜選択することができ、天然のアミノ酸であってもよいし、非天然のアミノ酸であってもよく、また、Ｄ体であってもよいし、Ｌ体であってもよい。
前記ペプチドは、リポペプチドなどの修飾型ペプチドであってもよい。 <Peptide>
The peptide is not particularly limited as long as the cell membrane permeable molecule can be introduced, and can be appropriately selected depending on the intended purpose. However, the peptide is cyclized by introducing the cell membrane permeable molecule. Is preferable.
The type of amino acid in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be a natural amino acid, an unnatural amino acid, or a D-form. It may be present or it may be L-form.
The peptide may be a modified peptide such as a lipopeptide.
前記ペプチドとしては、前記細胞膜透過性分子を導入することができる限り、特に制限はなく、目的に応じて適宜選択することができるが、前記細胞膜透過性分子を導入することにより環状化されるペプチドが好ましい。
前記ペプチドにおけるアミノ酸の種類としては、特に制限はなく、目的に応じて適宜選択することができ、天然のアミノ酸であってもよいし、非天然のアミノ酸であってもよく、また、Ｄ体であってもよいし、Ｌ体であってもよい。
前記ペプチドは、リポペプチドなどの修飾型ペプチドであってもよい。 <Peptide>
The peptide is not particularly limited as long as the cell membrane permeable molecule can be introduced, and can be appropriately selected depending on the intended purpose. However, the peptide is cyclized by introducing the cell membrane permeable molecule. Is preferable.
The type of amino acid in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be a natural amino acid, an unnatural amino acid, or a D-form. It may be present or it may be L-form.
The peptide may be a modified peptide such as a lipopeptide.
前記細胞膜透過性分子の導入に利用するアミノ酸残基（以下、「反応性アミノ酸残基」と称することがある。）としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、システイン残基、リジン残基、ヒスチジン残基、トリプトファン残基、チロシン残基、セリン残基、スレオニン残基などが挙げられる。
前記ペプチドとして、細胞膜透過性分子を導入することにより環状化されるペプチドを用いる場合には、前記反応性アミノ酸残基としては、例えば、システイン残基、リジン残基、セリン残基、スレオニン残基などが挙げられる。
また、アミノ酸残基におけるヒドロキシ基、メルカプト基、又はアミノ基を利用してもよい。
前記反応性アミノ酸残基は、１種を単独で使用してもよいし、２種以上を併用してもよい。 The amino acid residue used for the introduction of the cell membrane permeable molecule (hereinafter, may be referred to as “reactive amino acid residue”) is not particularly limited and may be appropriately selected depending on the intended purpose, for example. , Cysteine residue, lysine residue, histidine residue, tryptophan residue, tyrosine residue, serine residue, threonine residue and the like.
When a peptide that is cyclized by introducing a cell membrane permeable molecule is used as the peptide, the reactive amino acid residue may be, for example, a cysteine residue, a lysine residue, a serine residue, or a threonine residue. And so on.
Further, a hydroxy group, a mercapto group, or an amino group in the amino acid residue may be used.
The reactive amino acid residue may be used alone or in combination of two or more.
前記ペプチドとして、細胞膜透過性分子を導入することにより環状化されるペプチドを用いる場合には、前記反応性アミノ酸残基としては、例えば、システイン残基、リジン残基、セリン残基、スレオニン残基などが挙げられる。
また、アミノ酸残基におけるヒドロキシ基、メルカプト基、又はアミノ基を利用してもよい。
前記反応性アミノ酸残基は、１種を単独で使用してもよいし、２種以上を併用してもよい。 The amino acid residue used for the introduction of the cell membrane permeable molecule (hereinafter, may be referred to as “reactive amino acid residue”) is not particularly limited and may be appropriately selected depending on the intended purpose, for example. , Cysteine residue, lysine residue, histidine residue, tryptophan residue, tyrosine residue, serine residue, threonine residue and the like.
When a peptide that is cyclized by introducing a cell membrane permeable molecule is used as the peptide, the reactive amino acid residue may be, for example, a cysteine residue, a lysine residue, a serine residue, or a threonine residue. And so on.
Further, a hydroxy group, a mercapto group, or an amino group in the amino acid residue may be used.
The reactive amino acid residue may be used alone or in combination of two or more.
前記反応性アミノ酸残基のペプチドにおける数としては、特に制限はなく、目的に応じて適宜選択することができる。
例えば、前記ペプチドとして、細胞膜透過性分子を導入することにより環状化されるペプチドを用いる場合には、前記反応性アミノ酸残基のペプチドにおける数は、２以上が好ましい。前記反応性アミノ酸残基のペプチドにおける数の上限値としては、特に制限はなく、目的に応じて適宜選択することができるが、反応点が多くなるとペプチドに結合する前記細胞膜透過性分子の数や位置が安定せず、アミノ酸配列に由来するペプチドの特性を比較し難くなる場合があるので、１０以下が好ましい。
なお、例えば、ペプチド中のシステイン残基がジスルフィド結合により前記ペプチドの高次構造の安定化に関与しているような場合には、別途、上記反応性アミノ酸残基を前記ペプチドに導入することが好ましい。 The number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
For example, when a peptide that is cyclized by introducing a cell membrane permeable molecule is used as the peptide, the number of the reactive amino acid residues in the peptide is preferably 2 or more. The upper limit of the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. The position is not stable and it may be difficult to compare the characteristics of the peptide derived from the amino acid sequence, so 10 or less is preferable.
For example, when the cysteine residue in the peptide is involved in the stabilization of the higher-order structure of the peptide by disulfide bond, the reactive amino acid residue may be separately introduced into the peptide. preferable.
例えば、前記ペプチドとして、細胞膜透過性分子を導入することにより環状化されるペプチドを用いる場合には、前記反応性アミノ酸残基のペプチドにおける数は、２以上が好ましい。前記反応性アミノ酸残基のペプチドにおける数の上限値としては、特に制限はなく、目的に応じて適宜選択することができるが、反応点が多くなるとペプチドに結合する前記細胞膜透過性分子の数や位置が安定せず、アミノ酸配列に由来するペプチドの特性を比較し難くなる場合があるので、１０以下が好ましい。
なお、例えば、ペプチド中のシステイン残基がジスルフィド結合により前記ペプチドの高次構造の安定化に関与しているような場合には、別途、上記反応性アミノ酸残基を前記ペプチドに導入することが好ましい。 The number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
For example, when a peptide that is cyclized by introducing a cell membrane permeable molecule is used as the peptide, the number of the reactive amino acid residues in the peptide is preferably 2 or more. The upper limit of the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. The position is not stable and it may be difficult to compare the characteristics of the peptide derived from the amino acid sequence, so 10 or less is preferable.
For example, when the cysteine residue in the peptide is involved in the stabilization of the higher-order structure of the peptide by disulfide bond, the reactive amino acid residue may be separately introduced into the peptide. preferable.
前記反応性アミノ酸残基の前記ペプチドにおける位置としては、特に制限はなく、目的に応じて適宜選択することができる。
The position of the reactive amino acid residue in the peptide is not particularly limited and can be appropriately selected depending on the intended purpose.
例えば、前記ペプチドとして、ｍＲＮＡ分子、その翻訳物であるペプチド鎖（以下、「ポリペプチド鎖」と称することもある。）、及びリボソームを含むリボソームディスプレイ複合体（以下、「ＲＤ複合体」と称することがある。）を用いる場合には、例えば、リボソームの出口トンネル（ｅｘｉｔ ｔｕｎｎｅｌ）から外に出ている部分であり、具体的にはＮ末端から２番目～Ｃ末端から３０番目の位置（Ｎ末端から２番目の位置及びＣ末端から３０番目の位置を含む）の間とすることが、前記リンカー分子による修飾反応がリボソームにより立体的に阻害され難くなり得る点で、好ましい。
前記Ｃ末端からの位置としては、Ｃ末端から５０番目が好ましく、１００番目がより好ましい。
また、前記反応性アミノ酸残基の位置をＮ末端側から数えた場合、その位置は、ペプチドの鎖長に応じて適宜設定できるが、例えば、Ｎ末端から２～１，０００番目の位置であり、Ｎ末端から２～１００番目の位置が好ましく、Ｎ末端から２～５０番目の位置がより好ましい。 For example, as the peptide, an mRNA molecule, a peptide chain which is a translation thereof (hereinafter, also referred to as “polypeptide chain”), and a ribosome display complex containing ribosome (hereinafter, referred to as “RD complex”). When (may be used) is used, for example, it is a portion protruding from the exit tunnel (exit tunnel) of the ribosome, specifically, the second position from the N-terminal to the 30th position (N) from the C-terminal. It is preferable to use it between the position 2nd from the terminal and the position 30th from the C end) in that the modification reaction by the linker molecule can be less likely to be sterically inhibited by the ribosome.
As the position from the C-terminal, the 50th position from the C-terminal is preferable, and the 100th position is more preferable.
Further, when the position of the reactive amino acid residue is counted from the N-terminal side, the position can be appropriately set according to the chain length of the peptide, and is, for example, the 2nd to 1,000th position from the N-terminal. , The 2nd to 100th positions from the N-terminal are preferable, and the 2nd to 50th positions from the N-terminal are more preferable.
前記Ｃ末端からの位置としては、Ｃ末端から５０番目が好ましく、１００番目がより好ましい。
また、前記反応性アミノ酸残基の位置をＮ末端側から数えた場合、その位置は、ペプチドの鎖長に応じて適宜設定できるが、例えば、Ｎ末端から２～１，０００番目の位置であり、Ｎ末端から２～１００番目の位置が好ましく、Ｎ末端から２～５０番目の位置がより好ましい。 For example, as the peptide, an mRNA molecule, a peptide chain which is a translation thereof (hereinafter, also referred to as “polypeptide chain”), and a ribosome display complex containing ribosome (hereinafter, referred to as “RD complex”). When (may be used) is used, for example, it is a portion protruding from the exit tunnel (exit tunnel) of the ribosome, specifically, the second position from the N-terminal to the 30th position (N) from the C-terminal. It is preferable to use it between the position 2nd from the terminal and the position 30th from the C end) in that the modification reaction by the linker molecule can be less likely to be sterically inhibited by the ribosome.
As the position from the C-terminal, the 50th position from the C-terminal is preferable, and the 100th position is more preferable.
Further, when the position of the reactive amino acid residue is counted from the N-terminal side, the position can be appropriately set according to the chain length of the peptide, and is, for example, the 2nd to 1,000th position from the N-terminal. , The 2nd to 100th positions from the N-terminal are preferable, and the 2nd to 50th positions from the N-terminal are more preferable.
前記ＲＤ複合体の製造方法としては、特に制限はなく、公知の方法を適宜選択することができ、例えば、国際公開第２０１７／２１３１５８号に記載の方法などが挙げられる。また、市販のキットを利用して製造することもできる。
The method for producing the RD complex is not particularly limited, and a known method can be appropriately selected. Examples thereof include the method described in International Publication No. 2017/213158. It can also be manufactured using a commercially available kit.
前記ペプチドのアミノ酸配列としては、特に制限はなく、目的に応じて適宜選択することができるが、ペプチドライブラリとして有用であるように、特定の位置にランダム配列を含むものが好ましい。かかるランダム配列の中から、所定の目的に応じて有用なアミノ酸配列を特定し得る。
The amino acid sequence of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, but one containing a random sequence at a specific position is preferable so as to be useful as a peptide library. From such a random sequence, a useful amino acid sequence can be identified according to a predetermined purpose.
前記ランダム配列の前記ペプチドにおける位置としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記反応性アミノ酸残基の位置と同様に、ＲＤ複合体を用いる場合には、Ｎ末端から２番目～Ｃ末端から３０番目の位置（Ｎ末端から２番目の位置及びＣ末端から３０番目の位置を含む）の間とすることが好ましい。即ち、反応性アミノ酸残基は、ランダム配列内に含まれることが好ましい。従ってランダム配列の好ましい位置は、反応性アミノ酸残基の好ましい位置と同じ範囲から設定できる。
The position of the random sequence in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. For example, when the RD complex is used as in the position of the reactive amino acid residue, the position is not particularly limited. It is preferably between the 2nd position from the N-terminal to the 30th position from the C-terminal (including the 2nd position from the N-terminal and the 30th position from the C-terminal). That is, the reactive amino acid residue is preferably contained in a random sequence. Therefore, the preferred position of the random sequence can be set from the same range as the preferred position of the reactive amino acid residue.
前記ランダム配列の前記ペプチドにおける数は、１つであってもよく、２つ以上であってもよい。前記ランダム配列の数の上限としては、特に制限はなく、目的に応じて適宜選択することができるが、１０以下が好ましい。
前記ランダム配列１つあたりのアミノ酸残基数としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１以上、３０以下とすることができる。
１つのランダム配列が長くなるほど、またランダム配列の数が多くなるほど、ペプチドライブラリの多様性を高めることができる。 The number of the random sequences in the peptide may be one or two or more. The upper limit of the number of the random sequences is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 or less.
The number of amino acid residues per random sequence is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 1 or more and 30 or less.
The longer one random sequence and the larger the number of random sequences, the greater the diversity of the peptide library.
前記ランダム配列１つあたりのアミノ酸残基数としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１以上、３０以下とすることができる。
１つのランダム配列が長くなるほど、またランダム配列の数が多くなるほど、ペプチドライブラリの多様性を高めることができる。 The number of the random sequences in the peptide may be one or two or more. The upper limit of the number of the random sequences is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 or less.
The number of amino acid residues per random sequence is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 1 or more and 30 or less.
The longer one random sequence and the larger the number of random sequences, the greater the diversity of the peptide library.
前記ペプチドは、更に、ＦＬＡＧ（登録商標）配列やポリＨｉｓ配列等のポリペプチド鎖の精製のための配列、プロテアーゼなどにより選択的に切断される配列、スペーサー配列などを含んでいてもよい。
The peptide may further contain a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.
前記ペプチドのアミノ酸残基数としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１０以上、５，０００以下とすることができる。
前記ペプチドのアミノ酸残基数の下限値としては、１５０以上が好ましく、２００以上がより好ましい。また、ペプチドのアミノ酸残基数の上限値としては、８００以下が好ましく、６００以下がより好ましく、５００以下が特に好ましい。前記下限値と上限値とは、適宜組み合わせて選択することができる。 The number of amino acid residues of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 10 or more and 5,000 or less.
The lower limit of the number of amino acid residues of the peptide is preferably 150 or more, more preferably 200 or more. The upper limit of the number of amino acid residues of the peptide is preferably 800 or less, more preferably 600 or less, and particularly preferably 500 or less. The lower limit value and the upper limit value can be appropriately combined and selected.
前記ペプチドのアミノ酸残基数の下限値としては、１５０以上が好ましく、２００以上がより好ましい。また、ペプチドのアミノ酸残基数の上限値としては、８００以下が好ましく、６００以下がより好ましく、５００以下が特に好ましい。前記下限値と上限値とは、適宜組み合わせて選択することができる。 The number of amino acid residues of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 10 or more and 5,000 or less.
The lower limit of the number of amino acid residues of the peptide is preferably 150 or more, more preferably 200 or more. The upper limit of the number of amino acid residues of the peptide is preferably 800 or less, more preferably 600 or less, and particularly preferably 500 or less. The lower limit value and the upper limit value can be appropriately combined and selected.
前記ペプチドの合成方法としては、特に制限はなく、公知の方法を適宜選択することができる。
The method for synthesizing the peptide is not particularly limited, and a known method can be appropriately selected.
＜細胞膜透過性分子＞
前記細胞膜透過性分子は、上記した本発明の細胞膜透過性分子である。
前記細胞膜透過性分子は、ペプチドに導入される際に、前記ペプチドにおける反応性アミノ酸残基と反応する部位の構造が変化してもよい。 <Cell membrane permeable molecule>
The cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
When the cell membrane permeable molecule is introduced into a peptide, the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
前記細胞膜透過性分子は、上記した本発明の細胞膜透過性分子である。
前記細胞膜透過性分子は、ペプチドに導入される際に、前記ペプチドにおける反応性アミノ酸残基と反応する部位の構造が変化してもよい。 <Cell membrane permeable molecule>
The cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
When the cell membrane permeable molecule is introduced into a peptide, the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
＜その他の構成＞
前記ペプチド複合体におけるその他の構成としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、蛍光物質などの発光物質、色素、放射性物質、薬剤、毒素、核酸、アミノ酸、糖類、脂質、各種ポリマーなどが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。
前記蛍光物質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、フルオレセイン類、ローダミン類、クマリン類、ピレン類、シアニン類などの蛍光色素が挙げられる。
前記その他の構成は、例えば、上記したペプチドに、直接又は連結基などを介して結合させることができる。 <Other configurations>
The other constitution of the peptide complex is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the intended purpose. For example, a luminescent substance such as a fluorescent substance, a dye, a radioactive substance, and the like. Examples include drugs, toxins, nucleic acids, amino acids, sugars, lipids, various polymers and the like. These may be used alone or in combination of two or more.
The fluorescent substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene and cyanine.
The other constitution can be attached to the above-mentioned peptide, for example, directly or via a linking group or the like.
前記ペプチド複合体におけるその他の構成としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、蛍光物質などの発光物質、色素、放射性物質、薬剤、毒素、核酸、アミノ酸、糖類、脂質、各種ポリマーなどが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。
前記蛍光物質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、フルオレセイン類、ローダミン類、クマリン類、ピレン類、シアニン類などの蛍光色素が挙げられる。
前記その他の構成は、例えば、上記したペプチドに、直接又は連結基などを介して結合させることができる。 <Other configurations>
The other constitution of the peptide complex is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the intended purpose. For example, a luminescent substance such as a fluorescent substance, a dye, a radioactive substance, and the like. Examples include drugs, toxins, nucleic acids, amino acids, sugars, lipids, various polymers and the like. These may be used alone or in combination of two or more.
The fluorescent substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene and cyanine.
The other constitution can be attached to the above-mentioned peptide, for example, directly or via a linking group or the like.
本発明のペプチド複合体は、優れた細胞透過性を有しつつ、生体への毒性も低い。したがって、例えば、ランダム配列を含むペプチド複合体ライブラリとし、スクリーニングを行うことで、優れた細胞透過性を有しつつ、生体への毒性も低く、かつ対象物質への親和性が高い等の有用なアミノ酸配列を特定し得る。
The peptide complex of the present invention has excellent cell permeability and low toxicity to the living body. Therefore, for example, by preparing a peptide complex library containing a random sequence and performing screening, it is useful to have excellent cell permeability, low toxicity to a living body, and high affinity for a target substance. The amino acid sequence can be specified.
（ペプチド複合体の製造方法）
本発明のペプチド複合体の製造方法は、ペプチドに、本発明の細胞膜透過性分子を導入する導入工程を少なくとも含み、必要に応じて更にその他の工程を含む。 (Method for producing peptide complex)
The method for producing a peptide complex of the present invention includes at least an introduction step of introducing the cell membrane permeable molecule of the present invention into a peptide, and further includes other steps as necessary.
本発明のペプチド複合体の製造方法は、ペプチドに、本発明の細胞膜透過性分子を導入する導入工程を少なくとも含み、必要に応じて更にその他の工程を含む。 (Method for producing peptide complex)
The method for producing a peptide complex of the present invention includes at least an introduction step of introducing the cell membrane permeable molecule of the present invention into a peptide, and further includes other steps as necessary.
＜導入工程＞
前記導入工程は、ペプチドに、本発明の細胞膜透過性分子を導入する（以下、「結合する」、「挿入する」、「連結する」と称することもある。）工程である。
前記導入工程により、ペプチドへ細胞膜透過性を付与することができる。また、上記した環状化基を有する細胞膜透過性分子を導入する場合には、ペプチドの環状化と、ペプチドへの細胞膜透過性の付与とを同時に行うことができる。
前記導入工程では、反応物中における少なくとも１つのペプチドに細胞膜透過性分子が導入されればよいが、全てのペプチドに細胞膜透過性分子が導入されることが好ましい。 <Introduction process>
The introduction step is a step of introducing the cell membrane-permeable molecule of the present invention into a peptide (hereinafter, may be referred to as "binding", "inserting", or "linking").
By the introduction step, cell membrane permeability can be imparted to the peptide. Further, when the cell membrane permeable molecule having the above-mentioned cyclization group is introduced, the cyclization of the peptide and the impartation of the cell membrane permeability to the peptide can be performed at the same time.
In the introduction step, the cell membrane-permeable molecule may be introduced into at least one peptide in the reaction product, but it is preferable that the cell membrane-permeable molecule is introduced into all the peptides.
前記導入工程は、ペプチドに、本発明の細胞膜透過性分子を導入する（以下、「結合する」、「挿入する」、「連結する」と称することもある。）工程である。
前記導入工程により、ペプチドへ細胞膜透過性を付与することができる。また、上記した環状化基を有する細胞膜透過性分子を導入する場合には、ペプチドの環状化と、ペプチドへの細胞膜透過性の付与とを同時に行うことができる。
前記導入工程では、反応物中における少なくとも１つのペプチドに細胞膜透過性分子が導入されればよいが、全てのペプチドに細胞膜透過性分子が導入されることが好ましい。 <Introduction process>
The introduction step is a step of introducing the cell membrane-permeable molecule of the present invention into a peptide (hereinafter, may be referred to as "binding", "inserting", or "linking").
By the introduction step, cell membrane permeability can be imparted to the peptide. Further, when the cell membrane permeable molecule having the above-mentioned cyclization group is introduced, the cyclization of the peptide and the impartation of the cell membrane permeability to the peptide can be performed at the same time.
In the introduction step, the cell membrane-permeable molecule may be introduced into at least one peptide in the reaction product, but it is preferable that the cell membrane-permeable molecule is introduced into all the peptides.
－ペプチド－
前記ペプチドは、上記した（ペプチド複合体）の＜ペプチド＞の項目に記載したものと同様である。前記導入工程に用いるペプチドは、ペプチドライブラリの態様であってもよい。なお、このペプチドライブラリにおけるペプチドは本発明の細胞膜透過性分子が導入されていない状態のものである。 -peptide-
The peptide is the same as that described in the <Peptide> section of the above (peptide complex). The peptide used in the introduction step may be an embodiment of a peptide library. The peptide in this peptide library is in a state in which the cell membrane-permeable molecule of the present invention has not been introduced.
前記ペプチドは、上記した（ペプチド複合体）の＜ペプチド＞の項目に記載したものと同様である。前記導入工程に用いるペプチドは、ペプチドライブラリの態様であってもよい。なお、このペプチドライブラリにおけるペプチドは本発明の細胞膜透過性分子が導入されていない状態のものである。 -peptide-
The peptide is the same as that described in the <Peptide> section of the above (peptide complex). The peptide used in the introduction step may be an embodiment of a peptide library. The peptide in this peptide library is in a state in which the cell membrane-permeable molecule of the present invention has not been introduced.
－細胞膜透過性分子－
前記細胞膜透過性分子は、上記した本発明の細胞膜透過性分子である。 -Cell membrane permeable molecule-
The cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
前記細胞膜透過性分子は、上記した本発明の細胞膜透過性分子である。 -Cell membrane permeable molecule-
The cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
－導入－
前記導入の方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記細胞膜透過性分子におけるペプチド連結用基と、前記ペプチドにおける反応性アミノ酸残基とを反応させる方法などが挙げられる。例えば、還元剤の存在下で、前記細胞膜透過性分子と、前記ペプチドとを反応させる方法などが挙げられる。前記還元剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トリス（２－カルボキシエチル）ホスフィン塩酸塩などが挙げられる。
前記反応の温度、時間等の条件としては、特に制限はなく、目的に応じて適宜選択することができる。 -Introduction-
The method of introduction is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a method of reacting a peptide linking group in the cell membrane permeable molecule with a reactive amino acid residue in the peptide. And so on. For example, a method of reacting the cell membrane permeable molecule with the peptide in the presence of a reducing agent can be mentioned. The reducing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tris (2-carboxyethyl) phosphine hydrochloride and the like.
The conditions such as the temperature and time of the reaction are not particularly limited and may be appropriately selected depending on the intended purpose.
前記導入の方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記細胞膜透過性分子におけるペプチド連結用基と、前記ペプチドにおける反応性アミノ酸残基とを反応させる方法などが挙げられる。例えば、還元剤の存在下で、前記細胞膜透過性分子と、前記ペプチドとを反応させる方法などが挙げられる。前記還元剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トリス（２－カルボキシエチル）ホスフィン塩酸塩などが挙げられる。
前記反応の温度、時間等の条件としては、特に制限はなく、目的に応じて適宜選択することができる。 -Introduction-
The method of introduction is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a method of reacting a peptide linking group in the cell membrane permeable molecule with a reactive amino acid residue in the peptide. And so on. For example, a method of reacting the cell membrane permeable molecule with the peptide in the presence of a reducing agent can be mentioned. The reducing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tris (2-carboxyethyl) phosphine hydrochloride and the like.
The conditions such as the temperature and time of the reaction are not particularly limited and may be appropriately selected depending on the intended purpose.
前記ペプチド複合体を塩の形態とする方法としては、特に制限はなく、公知の化学合成の技術を適宜選択して行うことができ、例えば、上記した＜細胞膜透過性分子の製造方法＞の項目に記載した方法と同様の方法などが挙げられる。
The method for forming the peptide complex in the form of a salt is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, the above-mentioned item <Method for producing a cell membrane-permeable molecule>. The same method as the method described in 1) can be mentioned.
＜その他の工程＞
前記ペプチド複合体の製造方法におけるその他の工程としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができる。 <Other processes>
The other steps in the method for producing the peptide complex are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
前記ペプチド複合体の製造方法におけるその他の工程としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができる。 <Other processes>
The other steps in the method for producing the peptide complex are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
得られたペプチド複合体が所望の構造を有するか否かを確認する方法としては、特に制限はなく、公知の分析方法を適宜選択することができ、例えば、質量分析法、プロトン核磁気共鳴分光法、炭素１３核磁気共鳴分光法、紫外分光法、赤外分光法、液体クロマトグラフィーなどの分析方法が挙げられる。
The method for confirming whether or not the obtained peptide complex has a desired structure is not particularly limited, and a known analytical method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected. Analytical methods such as method, carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, and liquid chromatography can be mentioned.
（ペプチドライブラリ）
本発明のペプチドライブラリは、本発明のペプチド複合体を少なくとも含み、必要に応じて更にその他の構成を含む。即ち、本発明のペプチドライブラリは、本発明の細胞膜透過性分子が導入されたペプチド複合体を含むものである。
前記ペプチドライブラリは、本発明のペプチド複合体のみからなるものであってもよいし、前記細胞膜透過性分子が導入されていないペプチドが含まれていてもよい。 (Peptide library)
The peptide library of the present invention contains at least the peptide complex of the present invention, and further contains other configurations as required. That is, the peptide library of the present invention contains a peptide complex into which the cell membrane permeable molecule of the present invention has been introduced.
The peptide library may consist only of the peptide complex of the present invention, or may contain a peptide into which the cell membrane permeable molecule has not been introduced.
本発明のペプチドライブラリは、本発明のペプチド複合体を少なくとも含み、必要に応じて更にその他の構成を含む。即ち、本発明のペプチドライブラリは、本発明の細胞膜透過性分子が導入されたペプチド複合体を含むものである。
前記ペプチドライブラリは、本発明のペプチド複合体のみからなるものであってもよいし、前記細胞膜透過性分子が導入されていないペプチドが含まれていてもよい。 (Peptide library)
The peptide library of the present invention contains at least the peptide complex of the present invention, and further contains other configurations as required. That is, the peptide library of the present invention contains a peptide complex into which the cell membrane permeable molecule of the present invention has been introduced.
The peptide library may consist only of the peptide complex of the present invention, or may contain a peptide into which the cell membrane permeable molecule has not been introduced.
前記ペプチドライブラリは、上記した（ペプチド複合体の製造方法）と同様にして、製造することができる。
The peptide library can be produced in the same manner as described above (method for producing a peptide complex).
（機能性ペプチドのスクリーニング方法）
本発明の機能性ペプチドのスクリーニング方法は、本発明のペプチドライブラリを用いて機能性ペプチドをスクリーニングする工程を少なくとも含み、必要に応じて更にその他の工程を含む。
前記スクリーニングの方法としては、本発明のペプチドライブラリを用いる限り、特に制限はなく、公知の方法を適宜選択することができる。例えば、所望の対象物質と、前記ペプチドライブラリとを混合し、結合したペプチド複合体（例えば、ＲＤ複合体）を選択し、前記ＲＤ複合体からＲＮＡを解離させ、前記ＲＮＡからＤＮＡを調製し、増幅した後、ｍＲＮＡに転写し、再度ＲＤ複合体ライブラリを作製するという工程を繰り返し、前記対象物質に対する親和性を有する機能性ペプチドをスクリーニングする、リボソームディスプレイ法によるスクリーニング方法が挙げられる。また、ファージディスプレイ法、ｍＲＮＡディスプレイ法、ＤＮＡディスプレイ法、１ビーズ１化合物（ｏｎｅ－ｂｅａｄ ｏｎｅ－ｃｏｍｐｏｕｎｄ）法などを用いたスクリーニング方法なども挙げられる。
前記スクリーニング方法では、スクリーニング工程を繰返す過程などにおいて、選択されたペプチドに、本発明の細胞膜透過性分子を導入する導入工程を含んでいてもよい。前記導入工程は、上記した（ペプチド複合体の製造方法）における＜導入工程＞と同様にして行うことができる。 (Screening method for functional peptides)
The method for screening a functional peptide of the present invention includes at least a step of screening a functional peptide using the peptide library of the present invention, and further includes other steps as necessary.
The screening method is not particularly limited as long as the peptide library of the present invention is used, and a known method can be appropriately selected. For example, a desired target substance and the peptide library are mixed, a bound peptide complex (for example, an RD complex) is selected, RNA is dissociated from the RD complex, and DNA is prepared from the RNA. A screening method based on the ribosome display method, which repeats the steps of transcribing to mRNA and then producing an RD complex library again to screen for a functional peptide having an affinity for the target substance, can be mentioned. Further, a screening method using a phage display method, an mRNA display method, a DNA display method, a one-bead one-compound method, or the like can also be mentioned.
The screening method may include an introduction step of introducing the cell membrane-permeable molecule of the present invention into the selected peptide in a process of repeating the screening step or the like. The introduction step can be performed in the same manner as the <introduction step> in the above-mentioned (method for producing a peptide complex).
本発明の機能性ペプチドのスクリーニング方法は、本発明のペプチドライブラリを用いて機能性ペプチドをスクリーニングする工程を少なくとも含み、必要に応じて更にその他の工程を含む。
前記スクリーニングの方法としては、本発明のペプチドライブラリを用いる限り、特に制限はなく、公知の方法を適宜選択することができる。例えば、所望の対象物質と、前記ペプチドライブラリとを混合し、結合したペプチド複合体（例えば、ＲＤ複合体）を選択し、前記ＲＤ複合体からＲＮＡを解離させ、前記ＲＮＡからＤＮＡを調製し、増幅した後、ｍＲＮＡに転写し、再度ＲＤ複合体ライブラリを作製するという工程を繰り返し、前記対象物質に対する親和性を有する機能性ペプチドをスクリーニングする、リボソームディスプレイ法によるスクリーニング方法が挙げられる。また、ファージディスプレイ法、ｍＲＮＡディスプレイ法、ＤＮＡディスプレイ法、１ビーズ１化合物（ｏｎｅ－ｂｅａｄ ｏｎｅ－ｃｏｍｐｏｕｎｄ）法などを用いたスクリーニング方法なども挙げられる。
前記スクリーニング方法では、スクリーニング工程を繰返す過程などにおいて、選択されたペプチドに、本発明の細胞膜透過性分子を導入する導入工程を含んでいてもよい。前記導入工程は、上記した（ペプチド複合体の製造方法）における＜導入工程＞と同様にして行うことができる。 (Screening method for functional peptides)
The method for screening a functional peptide of the present invention includes at least a step of screening a functional peptide using the peptide library of the present invention, and further includes other steps as necessary.
The screening method is not particularly limited as long as the peptide library of the present invention is used, and a known method can be appropriately selected. For example, a desired target substance and the peptide library are mixed, a bound peptide complex (for example, an RD complex) is selected, RNA is dissociated from the RD complex, and DNA is prepared from the RNA. A screening method based on the ribosome display method, which repeats the steps of transcribing to mRNA and then producing an RD complex library again to screen for a functional peptide having an affinity for the target substance, can be mentioned. Further, a screening method using a phage display method, an mRNA display method, a DNA display method, a one-bead one-compound method, or the like can also be mentioned.
The screening method may include an introduction step of introducing the cell membrane-permeable molecule of the present invention into the selected peptide in a process of repeating the screening step or the like. The introduction step can be performed in the same manner as the <introduction step> in the above-mentioned (method for producing a peptide complex).
（細胞膜透過性分子の細胞膜透過性の向上方法）
本発明の細胞膜透過性分子の細胞膜透過性の向上方法（以下、「膜透過性向上方法」と称することがある。）は、塩形成工程を少なくとも含み、必要に応じて更にその他の工程を含む。 (Method for improving cell membrane permeability of cell membrane-permeable molecules)
The method for improving the cell membrane permeability of a cell membrane-permeable molecule of the present invention (hereinafter, may be referred to as "membrane permeability improving method") includes at least a salt forming step, and further includes other steps as necessary. ..
本発明の細胞膜透過性分子の細胞膜透過性の向上方法（以下、「膜透過性向上方法」と称することがある。）は、塩形成工程を少なくとも含み、必要に応じて更にその他の工程を含む。 (Method for improving cell membrane permeability of cell membrane-permeable molecules)
The method for improving the cell membrane permeability of a cell membrane-permeable molecule of the present invention (hereinafter, may be referred to as "membrane permeability improving method") includes at least a salt forming step, and further includes other steps as necessary. ..
＜塩形成工程＞
本発明の膜透過性向上方法における塩形成工程は、下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とする工程である。
（前記一般式（Ｉ）中、Ｒは結合手を表す。）。
<Salt formation process>
The salt forming step in the method for improving membrane permeability of the present invention is a step of converting a cell membrane-permeable molecule having a structure represented by the following general formula (I) into a salt with an acid.
(In the general formula (I), R represents a bond).
本発明の膜透過性向上方法における塩形成工程は、下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とする工程である。
The salt forming step in the method for improving membrane permeability of the present invention is a step of converting a cell membrane-permeable molecule having a structure represented by the following general formula (I) into a salt with an acid.
＜＜一般式（Ｉ）で表される構造＞＞
本発明の膜透過性向上方法における前記一般式（Ｉ）で表される構造は、上記した（細胞膜透過性分子）における＜一般式（Ｉ）で表される構造＞の項目に記載したものと同様である。 << Structure represented by the general formula (I) >>
The structure represented by the general formula (I) in the method for improving the membrane permeability of the present invention is the same as that described in the item of <Structure represented by the general formula (I)> in the above-mentioned (cell membrane permeability molecule). The same is true.
本発明の膜透過性向上方法における前記一般式（Ｉ）で表される構造は、上記した（細胞膜透過性分子）における＜一般式（Ｉ）で表される構造＞の項目に記載したものと同様である。 << Structure represented by the general formula (I) >>
The structure represented by the general formula (I) in the method for improving the membrane permeability of the present invention is the same as that described in the item of <Structure represented by the general formula (I)> in the above-mentioned (cell membrane permeability molecule). The same is true.
－一般式（Ｉ）で表される構造を有する細胞膜透過性分子の製造方法－
前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子の製造方法としては、特に制限はなく、公知の化学合成の技術を適宜選択して行うことができる。例えば、米国特許第７，８６２，８０７号明細書などに記載の方法を参考にして、公知の化学合成の技術を適宜選択することにより製造することができる。 -Method for producing a cell membrane permeable molecule having a structure represented by the general formula (I)-
The method for producing the cell membrane-permeable molecule having the structure represented by the general formula (I) is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807.
前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子の製造方法としては、特に制限はなく、公知の化学合成の技術を適宜選択して行うことができる。例えば、米国特許第７，８６２，８０７号明細書などに記載の方法を参考にして、公知の化学合成の技術を適宜選択することにより製造することができる。 -Method for producing a cell membrane permeable molecule having a structure represented by the general formula (I)-
The method for producing the cell membrane-permeable molecule having the structure represented by the general formula (I) is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807.
＜＜塩の形成＞＞
本発明の膜透過性向上方法において、前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とする方法としては、特に制限はなく、公知の化学合成の技術を適宜選択して行うことができる。例えば、塩の形態ではない細胞膜透過性分子の粗生成物を、目的の塩の種類に応じた移動相を用いたＨＰＬＣにより精製し、目的とする塩の形態の細胞膜透過性分子とする方法、塩の形態である細胞膜透過性分子を、対イオンを目的の塩のイオンへと置換した陰イオン交換樹脂に通じ、溶出することで目的とする塩の形態の細胞膜透過性分子とする方法などが挙げられる。 << Formation of salt >>
In the method for improving membrane permeability of the present invention, the method of using a cell membrane-permeable molecule having a structure represented by the general formula (I) as a salt with an acid is not particularly limited, and is a known chemical synthesis technique. Can be selected as appropriate. For example, a method of purifying a crude product of a cell membrane-permeable molecule that is not in the form of a salt by HPLC using a mobile phase according to the type of the target salt to obtain a cell membrane-permeable molecule in the form of the target salt. A method of passing a cell membrane-permeable molecule in the form of a salt through an anion exchange resin in which a counterion is replaced with an ion of the target salt and eluting the molecule to obtain a cell membrane-permeable molecule in the form of the target salt, etc. Can be mentioned.
本発明の膜透過性向上方法において、前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とする方法としては、特に制限はなく、公知の化学合成の技術を適宜選択して行うことができる。例えば、塩の形態ではない細胞膜透過性分子の粗生成物を、目的の塩の種類に応じた移動相を用いたＨＰＬＣにより精製し、目的とする塩の形態の細胞膜透過性分子とする方法、塩の形態である細胞膜透過性分子を、対イオンを目的の塩のイオンへと置換した陰イオン交換樹脂に通じ、溶出することで目的とする塩の形態の細胞膜透過性分子とする方法などが挙げられる。 << Formation of salt >>
In the method for improving membrane permeability of the present invention, the method of using a cell membrane-permeable molecule having a structure represented by the general formula (I) as a salt with an acid is not particularly limited, and is a known chemical synthesis technique. Can be selected as appropriate. For example, a method of purifying a crude product of a cell membrane-permeable molecule that is not in the form of a salt by HPLC using a mobile phase according to the type of the target salt to obtain a cell membrane-permeable molecule in the form of the target salt. A method of passing a cell membrane-permeable molecule in the form of a salt through an anion exchange resin in which a counterion is replaced with an ion of the target salt and eluting the molecule to obtain a cell membrane-permeable molecule in the form of the target salt, etc. Can be mentioned.
－酸－
本発明の膜透過性向上方法の塩形成工程で生成される細胞膜透過性分子は、酸との塩である。
前記酸の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トリフルオロ酢酸、塩酸、硫酸、硝酸、リン酸、メシル酸、トシル酸、酒石酸、クエン酸、酢酸、各種アミノ酸などが挙げられる。これらの中でも、酸性アミノ酸以外の酸が好ましく、ｐＫａ（酸解離定数）の値が－８．０以上４．７以下の酸がより好ましい。
本発明において、酸が複数のｐＫａの値を有している場合には、１段階目の解離におけるｐＫａの値であるｐＫａ１の値をｐＫａの値とする。
前記ｐＫａの値が－８．０以上４．７以下の酸としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トリフルオロ酢酸（－０．３）、塩酸（－８．０）、硫酸（－３．０）、硝酸（－１．３）、リン酸（２．１（ｐＫａ１））、メシル酸（－２．６）、トシル酸（－２．８）、酒石酸（３．２（ｐＫａ１））、クエン酸（３．１（ｐＫａ１））、酢酸（４．７）などが挙げられる。前記酸の名称の後のかっこ内の数値はｐＫａの値を表す。なお、ｐＫａ１の値の場合は、数値の後にｐＫａ１と記載した。 -acid-
The cell membrane-permeable molecule produced in the salt-forming step of the method for improving membrane permeability of the present invention is a salt with an acid.
The type of the acid is not particularly limited and may be appropriately selected depending on the intended purpose. For example, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartrate acid, citric acid, acetic acid. , Various amino acids and the like. Among these, acids other than acidic amino acids are preferable, and acids having a pKa (acid dissociation constant) value of −8.0 or more and 4.7 or less are more preferable.
In the present invention, when the acid has a plurality of pK a values, the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
The acid having a pK a value of −8.0 or more and 4.7 or less is not particularly limited and may be appropriately selected depending on the intended purpose. For example, trifluoroacetic acid (-0.3), hydrochloric acid ( -8.0), sulfuric acid (-3.0), nitric acid (-1.3), phosphoric acid (2.1 (pK a1 )), mesylic acid (-2.6), tosylic acid (-2.8) ), Tartrate acid (3.2 (pK a1 )), citric acid (3.1 (pK a1 )), acetic acid (4.7) and the like. The number in parentheses after the acid name represents the value of pK a . In the case of the value of pK a1 , it is described as pK a1 after the numerical value.
本発明の膜透過性向上方法の塩形成工程で生成される細胞膜透過性分子は、酸との塩である。
前記酸の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トリフルオロ酢酸、塩酸、硫酸、硝酸、リン酸、メシル酸、トシル酸、酒石酸、クエン酸、酢酸、各種アミノ酸などが挙げられる。これらの中でも、酸性アミノ酸以外の酸が好ましく、ｐＫａ（酸解離定数）の値が－８．０以上４．７以下の酸がより好ましい。
本発明において、酸が複数のｐＫａの値を有している場合には、１段階目の解離におけるｐＫａの値であるｐＫａ１の値をｐＫａの値とする。
前記ｐＫａの値が－８．０以上４．７以下の酸としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トリフルオロ酢酸（－０．３）、塩酸（－８．０）、硫酸（－３．０）、硝酸（－１．３）、リン酸（２．１（ｐＫａ１））、メシル酸（－２．６）、トシル酸（－２．８）、酒石酸（３．２（ｐＫａ１））、クエン酸（３．１（ｐＫａ１））、酢酸（４．７）などが挙げられる。前記酸の名称の後のかっこ内の数値はｐＫａの値を表す。なお、ｐＫａ１の値の場合は、数値の後にｐＫａ１と記載した。 -acid-
The cell membrane-permeable molecule produced in the salt-forming step of the method for improving membrane permeability of the present invention is a salt with an acid.
The type of the acid is not particularly limited and may be appropriately selected depending on the intended purpose. For example, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartrate acid, citric acid, acetic acid. , Various amino acids and the like. Among these, acids other than acidic amino acids are preferable, and acids having a pKa (acid dissociation constant) value of −8.0 or more and 4.7 or less are more preferable.
In the present invention, when the acid has a plurality of pK a values, the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
The acid having a pK a value of −8.0 or more and 4.7 or less is not particularly limited and may be appropriately selected depending on the intended purpose. For example, trifluoroacetic acid (-0.3), hydrochloric acid ( -8.0), sulfuric acid (-3.0), nitric acid (-1.3), phosphoric acid (2.1 (pK a1 )), mesylic acid (-2.6), tosylic acid (-2.8) ), Tartrate acid (3.2 (pK a1 )), citric acid (3.1 (pK a1 )), acetic acid (4.7) and the like. The number in parentheses after the acid name represents the value of pK a . In the case of the value of pK a1 , it is described as pK a1 after the numerical value.
＜その他の工程＞
本発明の膜透過性向上方法におけるその他の工程としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができる。 <Other processes>
The other steps in the method for improving membrane permeability of the present invention are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
本発明の膜透過性向上方法におけるその他の工程としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができる。 <Other processes>
The other steps in the method for improving membrane permeability of the present invention are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
本発明の膜透過性向上方法で生成される細胞膜透過性分子は、上記した一般式（Ｉ）で表される構造を有し、酸との塩であって、本発明の効果を損なわない限りにおいて、その他の構成を有していてもよい。
The cell membrane-permeable molecule produced by the method for improving membrane permeability of the present invention has a structure represented by the above-mentioned general formula (I) and is a salt with an acid as long as the effect of the present invention is not impaired. In, it may have other configurations.
前記細胞膜透過性分子が所望の構造を有するか否かを確認する方法としては、特に制限はなく、公知の分析方法を適宜選択することができ、例えば、質量分析法、プロトン核磁気共鳴分光法、炭素１３核磁気共鳴分光法、紫外分光法、赤外分光法、液体クロマトグラフィーなどの分析方法が挙げられる。
The method for confirming whether or not the cell membrane permeable molecule has a desired structure is not particularly limited, and a known analytical method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected. , Carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, liquid chromatography and other analytical methods.
本発明の膜透過性向上方法によれば、分子量が小さな細胞膜透過性分子の細胞膜透過性を向上することができる。そのため、前記細胞膜透過性分子をペプチド、核酸、タンパク質などの有効成分に導入することで、これらの細胞内への取込み効率を向上することができる。これらの中でも前記細胞膜透過性分子は、ペプチドに導入されたものであることが好ましい。
According to the method for improving membrane permeability of the present invention, it is possible to improve the cell membrane permeability of a cell membrane-permeable molecule having a small molecular weight. Therefore, by introducing the cell membrane-permeable molecule into an active ingredient such as a peptide, nucleic acid, or protein, the efficiency of incorporation into these cells can be improved. Among these, the cell membrane permeable molecule is preferably one introduced into a peptide.
したがって、本発明は、前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子が導入されたペプチド複合体を、酸との塩とすることを含むことを特徴とするペプチド複合体の細胞膜透過性の向上方法にも関する。
Therefore, the present invention is characterized in that the peptide complex into which a cell membrane permeable molecule having a structure represented by the general formula (I) is introduced is used as a salt with an acid. It is also related to the method of improving cell membrane permeability.
［ペプチド複合体の細胞膜透過性の向上方法］
前記ペプチド複合体の細胞膜透過性の向上方法は、塩形成工程を少なくとも含み、必要に応じて更にその他の工程を含む。 [Method for improving cell membrane permeability of peptide complex]
The method for improving the cell membrane permeability of the peptide complex includes at least a salt forming step, and further includes other steps as necessary.
前記ペプチド複合体の細胞膜透過性の向上方法は、塩形成工程を少なくとも含み、必要に応じて更にその他の工程を含む。 [Method for improving cell membrane permeability of peptide complex]
The method for improving the cell membrane permeability of the peptide complex includes at least a salt forming step, and further includes other steps as necessary.
＜塩形成工程＞
前記塩形成工程は、前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子が導入されたペプチド複合体を、酸との塩とする工程であり、前記細胞膜透過性分子がペプチドに導入されたものである点以外は、上記した細胞膜透過性分子の細胞膜透過性の向上方法における塩形成工程と同様にして行うことができる。 <Salt formation process>
The salt forming step is a step of converting a peptide complex into which a cell membrane-permeable molecule having a structure represented by the general formula (I) into a salt with an acid, and the cell membrane-permeable molecule becomes a peptide. Except for the fact that it has been introduced, it can be carried out in the same manner as the salt forming step in the method for improving the cell membrane permeability of the cell membrane permeable molecule described above.
前記塩形成工程は、前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子が導入されたペプチド複合体を、酸との塩とする工程であり、前記細胞膜透過性分子がペプチドに導入されたものである点以外は、上記した細胞膜透過性分子の細胞膜透過性の向上方法における塩形成工程と同様にして行うことができる。 <Salt formation process>
The salt forming step is a step of converting a peptide complex into which a cell membrane-permeable molecule having a structure represented by the general formula (I) into a salt with an acid, and the cell membrane-permeable molecule becomes a peptide. Except for the fact that it has been introduced, it can be carried out in the same manner as the salt forming step in the method for improving the cell membrane permeability of the cell membrane permeable molecule described above.
＜＜ペプチド複合体＞＞
前記ペプチド複合体は、ペプチドと、前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子とを少なくとも含み、必要に応じて更にその他の構成を含む。 << Peptide Complex >>
The peptide complex contains at least a peptide and a cell membrane-permeable molecule having a structure represented by the general formula (I), and further contains other configurations as necessary.
前記ペプチド複合体は、ペプチドと、前記一般式（Ｉ）で表される構造を有する細胞膜透過性分子とを少なくとも含み、必要に応じて更にその他の構成を含む。 << Peptide Complex >>
The peptide complex contains at least a peptide and a cell membrane-permeable molecule having a structure represented by the general formula (I), and further contains other configurations as necessary.
－ペプチド－
前記ペプチド複合体の細胞膜透過性の向上方法におけるペプチドは、上記した（ペプチド複合体）における＜ペプチド＞の項目に記載したものと同様である。 -peptide-
The peptide in the method for improving the cell membrane permeability of the peptide complex is the same as that described in the item of <Peptide> in the above-mentioned (peptide complex).
前記ペプチド複合体の細胞膜透過性の向上方法におけるペプチドは、上記した（ペプチド複合体）における＜ペプチド＞の項目に記載したものと同様である。 -peptide-
The peptide in the method for improving the cell membrane permeability of the peptide complex is the same as that described in the item of <Peptide> in the above-mentioned (peptide complex).
－細胞膜透過性分子－
前記ペプチド複合体の細胞膜透過性の向上方法における細胞膜透過性分子は、上記した膜透過性向上方法における一般式（Ｉ）で表される構造を有する細胞膜透過性分子である。
前記細胞膜透過性分子は、ペプチドに導入される際に、前記ペプチドにおける反応性アミノ酸残基と反応する部位の構造が変化してもよい。 -Cell membrane permeable molecule-
The cell membrane-permeable molecule in the method for improving the cell membrane permeability of the peptide complex is a cell membrane-permeable molecule having a structure represented by the general formula (I) in the above-mentioned method for improving the membrane permeability.
When the cell membrane permeable molecule is introduced into a peptide, the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
前記ペプチド複合体の細胞膜透過性の向上方法における細胞膜透過性分子は、上記した膜透過性向上方法における一般式（Ｉ）で表される構造を有する細胞膜透過性分子である。
前記細胞膜透過性分子は、ペプチドに導入される際に、前記ペプチドにおける反応性アミノ酸残基と反応する部位の構造が変化してもよい。 -Cell membrane permeable molecule-
The cell membrane-permeable molecule in the method for improving the cell membrane permeability of the peptide complex is a cell membrane-permeable molecule having a structure represented by the general formula (I) in the above-mentioned method for improving the membrane permeability.
When the cell membrane permeable molecule is introduced into a peptide, the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
－その他の構成－
前記ペプチド複合体の細胞膜透過性の向上方法におけるその他の構成は、上記した（ペプチド複合体）における＜その他の構成＞の項目に記載したものと同様である。 -Other configurations-
Other configurations in the method for improving the cell membrane permeability of the peptide complex are the same as those described in the item of <Other configurations> in the above-mentioned (peptide complex).
前記ペプチド複合体の細胞膜透過性の向上方法におけるその他の構成は、上記した（ペプチド複合体）における＜その他の構成＞の項目に記載したものと同様である。 -Other configurations-
Other configurations in the method for improving the cell membrane permeability of the peptide complex are the same as those described in the item of <Other configurations> in the above-mentioned (peptide complex).
前記ペプチドに、前記細胞膜透過性分子を導入する（以下、「結合する」、「挿入する」、「連結する」と称することもある。）方法は、上記した（ペプチド複合体の製造方法）における＜導入工程＞の項目に記載したものと同様である。
The method for introducing the cell membrane-permeable molecule into the peptide (hereinafter, also referred to as “binding”, “inserting”, or “linking”) is described in the above-mentioned (method for producing a peptide complex). It is the same as that described in the item of <Introduction process>.
前記ペプチド複合体に用いるペプチドは、ペプチドライブラリの態様であってもよい。即ち、上記した細胞膜透過性分子が導入される前のペプチドは、ペプチドライブラリの態様のものを用いることもできる。
The peptide used in the peptide complex may be in the form of a peptide library. That is, as the peptide before the introduction of the above-mentioned cell membrane permeable molecule, a peptide in the form of a peptide library can also be used.
＜その他の工程＞
前記ペプチド複合体の細胞膜透過性の向上方法におけるその他の工程としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができる。 <Other processes>
The other steps in the method for improving the cell membrane permeability of the peptide complex are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
前記ペプチド複合体の細胞膜透過性の向上方法におけるその他の工程としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができる。 <Other processes>
The other steps in the method for improving the cell membrane permeability of the peptide complex are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
以下に実施例等を挙げて本発明をより具体的に説明するが、本発明はこれらの実施例等に何ら限定されるものではない。
The present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited to these Examples and the like.
（実施例Ａ１：細胞膜透過性分子Ｇ３－ＤＨＸトリフルオロ酢酸塩の合成）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸトリフルオロ酢酸塩を下記のようにして合成した。 (Example A1: Synthesis of cell membrane permeable molecule G3-DHX trifluoroacetic acid salt)
For toxicity evaluation, G3-DHX trifluoroacetic acid salt, which is an example of a cell membrane permeable molecule, was synthesized as follows.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸトリフルオロ酢酸塩を下記のようにして合成した。 (Example A1: Synthesis of cell membrane permeable molecule G3-DHX trifluoroacetic acid salt)
For toxicity evaluation, G3-DHX trifluoroacetic acid salt, which is an example of a cell membrane permeable molecule, was synthesized as follows.
＜化合物Ｇ３－ＤＣＸの合成＞
－化合物Ｃ１の合成－
米国特許第７，８６２，８０７号明細書に記載の方法と同様の方法により、下記構造式で表される化合物Ｃ１を合成した。
なお、構造式中の「Ｂｏｃ」は、「ｔｅｒｔ－ブトキシカルボニル基」を表す。
<Synthesis of compound G3-DCX>
-Synthesis of compound C1-
Compound C1 represented by the following structural formula was synthesized by the same method as described in US Pat. No. 7,862,807.
In addition, "Boc" in the structural formula represents "tert-butoxycarbonyl group".
－化合物Ｃ１の合成－
米国特許第７，８６２，８０７号明細書に記載の方法と同様の方法により、下記構造式で表される化合物Ｃ１を合成した。
なお、構造式中の「Ｂｏｃ」は、「ｔｅｒｔ－ブトキシカルボニル基」を表す。
-Synthesis of compound C1-
Compound C1 represented by the following structural formula was synthesized by the same method as described in US Pat. No. 7,862,807.
In addition, "Boc" in the structural formula represents "tert-butoxycarbonyl group".
－化合物Ｃ３の合成－
-Synthesis of compound C3-
上記反応式のようにして、上記構造式で表される化合物Ｃ３を合成した。
具体的には、化合物Ｃ１（５００ｍｇ，０．４２ｍｍｏｌ）の塩化メチレン溶液（１５ｍＬ）を０℃に冷却し、化合物Ｃ２（東京化成工業株式会社製、製品番号Ａ２２９３）（１１７．５ｍｇ，０．５０４ｍｍｏｌ）と１－ヒドロキシベンゾトリアゾール（ＨＯＢＴ）（８５．１ｍｇ，０．６３０ｍｍｏｌ）と１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（ＥＤＣ／ＨＣｌ）（１２０ｍｇ，０．６３０ｍｍｏｌ）を加えて２５℃で１７時間撹拌した。ここにＨ２Ｏ（２０ｍＬ）を加え、塩化メチレンで抽出を行い（３０ｍＬで３回）、有機層は１Ｎ塩酸（２０ｍＬで２回）と飽和重曹水（２０ｍＬで２回）と水（２０ｍＬで２回）で洗浄した。Ｎａ２ＳＯ４で乾燥しろ過と濃縮を行い、得られた残渣をシリカゲルクロマトグラフィー精製（メタノール（ＭｅＯＨ）／ＣＨ２Ｃｌ２＝１／１０）することにより、化合物Ｃ３を白色固体として取得した（５３３ｍｇ，０．３７９ｍｍｏｌ，収率９０％）。
前記化合物Ｃ３の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ １１．４（ｓ， ３Ｈ）， ８．５８（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ３Ｈ）， ７．６９（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ３Ｈ）， ６．８４（ｓ， １Ｈ）， ３．８９（ｓ， ２Ｈ）， ３．７１－３．６５（ｍ， ２２Ｈ）， ３．５６－３．５３（ｍ， ６Ｈ）， ３．４２－３．３８（ｍ， ８Ｈ）， ２．４３（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４８（ｓ， ２７Ｈ） The compound C3 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, a methylene chloride solution (15 mL) of compound C1 (500 mg, 0.42 mmol) was cooled to 0 ° C., and compound C2 (manufactured by Tokyo Kasei Kogyo Co., Ltd., product number A2293) (117.5 mg, 0.504 mmol) was cooled. ), 1-Hydroxybenzotriazole (HOBT) (85.1 mg, 0.630 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC / HCl) (120 mg, 0.630 mmol). The mixture was stirred at 25 ° C. for 17 hours. Add H 2 O (20 mL) and extract with methylene chloride (30 mL 3 times), and the organic layer is 1N hydrochloric acid (20 mL twice), saturated sodium bicarbonate solution (20 mL twice) and water (20 mL). Washed twice). The compound C3 was obtained as a white solid by drying with Na 2 SO 4 , filtering and concentrating, and purifying the obtained residue by silica gel chromatography (methanol (MeOH) / CH 2 Cl 2 = 1/10) (methanol (MeOH) / CH 2 Cl 2 = 1/10). 533 mg, 0.379 mmol, 90% yield).
The identification data of the compound C3 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 11.4 (s, 3H), 8.58 (t, 3 J HH = 6.0 Hz, 3 H), 7.69 (t, 3 J HH = 5.0 Hz, 3 H) , 6.84 (s, 1H), 3.89 (s, 2H), 3.71-3.65 (m, 22H), 3.56-3.53 (m, 6H), 3.42-3 .38 (m, 8H), 2.43 (t, 3 JHH = 6.0Hz, 6H), 1.49 (s, 27H), 1.48 (s, 27H)
具体的には、化合物Ｃ１（５００ｍｇ，０．４２ｍｍｏｌ）の塩化メチレン溶液（１５ｍＬ）を０℃に冷却し、化合物Ｃ２（東京化成工業株式会社製、製品番号Ａ２２９３）（１１７．５ｍｇ，０．５０４ｍｍｏｌ）と１－ヒドロキシベンゾトリアゾール（ＨＯＢＴ）（８５．１ｍｇ，０．６３０ｍｍｏｌ）と１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（ＥＤＣ／ＨＣｌ）（１２０ｍｇ，０．６３０ｍｍｏｌ）を加えて２５℃で１７時間撹拌した。ここにＨ２Ｏ（２０ｍＬ）を加え、塩化メチレンで抽出を行い（３０ｍＬで３回）、有機層は１Ｎ塩酸（２０ｍＬで２回）と飽和重曹水（２０ｍＬで２回）と水（２０ｍＬで２回）で洗浄した。Ｎａ２ＳＯ４で乾燥しろ過と濃縮を行い、得られた残渣をシリカゲルクロマトグラフィー精製（メタノール（ＭｅＯＨ）／ＣＨ２Ｃｌ２＝１／１０）することにより、化合物Ｃ３を白色固体として取得した（５３３ｍｇ，０．３７９ｍｍｏｌ，収率９０％）。
前記化合物Ｃ３の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ １１．４（ｓ， ３Ｈ）， ８．５８（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ３Ｈ）， ７．６９（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ３Ｈ）， ６．８４（ｓ， １Ｈ）， ３．８９（ｓ， ２Ｈ）， ３．７１－３．６５（ｍ， ２２Ｈ）， ３．５６－３．５３（ｍ， ６Ｈ）， ３．４２－３．３８（ｍ， ８Ｈ）， ２．４３（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４８（ｓ， ２７Ｈ） The compound C3 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, a methylene chloride solution (15 mL) of compound C1 (500 mg, 0.42 mmol) was cooled to 0 ° C., and compound C2 (manufactured by Tokyo Kasei Kogyo Co., Ltd., product number A2293) (117.5 mg, 0.504 mmol) was cooled. ), 1-Hydroxybenzotriazole (HOBT) (85.1 mg, 0.630 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC / HCl) (120 mg, 0.630 mmol). The mixture was stirred at 25 ° C. for 17 hours. Add H 2 O (20 mL) and extract with methylene chloride (30 mL 3 times), and the organic layer is 1N hydrochloric acid (20 mL twice), saturated sodium bicarbonate solution (20 mL twice) and water (20 mL). Washed twice). The compound C3 was obtained as a white solid by drying with Na 2 SO 4 , filtering and concentrating, and purifying the obtained residue by silica gel chromatography (methanol (MeOH) / CH 2 Cl 2 = 1/10) (methanol (MeOH) / CH 2 Cl 2 = 1/10). 533 mg, 0.379 mmol, 90% yield).
The identification data of the compound C3 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 11.4 (s, 3H), 8.58 (t, 3 J HH = 6.0 Hz, 3 H), 7.69 (t, 3 J HH = 5.0 Hz, 3 H) , 6.84 (s, 1H), 3.89 (s, 2H), 3.71-3.65 (m, 22H), 3.56-3.53 (m, 6H), 3.42-3 .38 (m, 8H), 2.43 (t, 3 JHH = 6.0Hz, 6H), 1.49 (s, 27H), 1.48 (s, 27H)
－化合物ＤＢＸＡの合成－
水素化ナトリウム（１３２ｍｇ，３．０２ｍｍｏｌ）をテトラヒドロフラン（ＴＨＦ）（６ｍＬ）に懸濁させ０℃に冷却した。ここに２－プロピン－１－オール（０．１６５ｍＬ，２．８０ｍｍｏｌ）を加え０℃で３０分間撹拌した。１，３，５－トリス（ブロモメチル）ベンゼン（１．００ｇ，２．８０ｍｍｏｌ）を加え２５℃で２０時間撹拌した。酢酸エチル（１００ｍＬ）を加えてから水（１００ｍＬ）と飽和食塩水（１００ｍＬ）で洗浄し、有機層を硫酸マグネシウムで洗浄した。ろ過と濃縮を行い得られた残渣を分取薄層クロマトグラフィー（ＰＴＬＣ）で精製し（塩化メチレン／ヘキサン＝１／２）、下記構造式で表される化合物ＤＢＸＡ（４１６．６ｍｇ，１．２５ｍｍｏｌ，収率４５％）を淡黄色油状物として取得した。
前記化合物ＤＢＸＡの１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ ７．２９（ｓ， １Ｈ）， ７．２６（ｓ， ２Ｈ）， ４．５３（ｓ， ２Ｈ）， ４．４０（ｓ， ４Ｈ）， ４．１５（４ＪＨＨ＝２．５Ｈｚ， ２Ｈ）， ２．４３（４ＪＨＨ＝２．０Ｈｚ， １Ｈ）
-Synthesis of compound DBXA-
Sodium hydride (132 mg, 3.02 mmol) was suspended in tetrahydrofuran (THF) (6 mL) and cooled to 0 ° C. 2-Propin-1-ol (0.165 mL, 2.80 mmol) was added thereto, and the mixture was stirred at 0 ° C. for 30 minutes. 1,3,5-Tris (bromomethyl) benzene (1.00 g, 2.80 mmol) was added, and the mixture was stirred at 25 ° C. for 20 hours. Ethyl acetate (100 mL) was added and then washed with water (100 mL) and saturated brine (100 mL), and the organic layer was washed with magnesium sulfate. The residue obtained by filtration and concentration was purified by preparative thin layer chromatography (PTLC) (methylene chloride / hexane = 1/2), and the compound DBXA (416.6 mg, 1.25 mmol) represented by the following structural formula was used. , Yield 45%) was obtained as a pale yellow oil.
The identification data of the compound DBXA by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 7.29 (s, 1H), 7.26 (s, 2H), 4.53 (s, 2H), 4.40 (s, 4H), 4.15 ( 4 J) HH = 2.5Hz, 2H), 2.43 ( 4 J HH = 2.0Hz, 1H)
水素化ナトリウム（１３２ｍｇ，３．０２ｍｍｏｌ）をテトラヒドロフラン（ＴＨＦ）（６ｍＬ）に懸濁させ０℃に冷却した。ここに２－プロピン－１－オール（０．１６５ｍＬ，２．８０ｍｍｏｌ）を加え０℃で３０分間撹拌した。１，３，５－トリス（ブロモメチル）ベンゼン（１．００ｇ，２．８０ｍｍｏｌ）を加え２５℃で２０時間撹拌した。酢酸エチル（１００ｍＬ）を加えてから水（１００ｍＬ）と飽和食塩水（１００ｍＬ）で洗浄し、有機層を硫酸マグネシウムで洗浄した。ろ過と濃縮を行い得られた残渣を分取薄層クロマトグラフィー（ＰＴＬＣ）で精製し（塩化メチレン／ヘキサン＝１／２）、下記構造式で表される化合物ＤＢＸＡ（４１６．６ｍｇ，１．２５ｍｍｏｌ，収率４５％）を淡黄色油状物として取得した。
前記化合物ＤＢＸＡの１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ ７．２９（ｓ， １Ｈ）， ７．２６（ｓ， ２Ｈ）， ４．５３（ｓ， ２Ｈ）， ４．４０（ｓ， ４Ｈ）， ４．１５（４ＪＨＨ＝２．５Ｈｚ， ２Ｈ）， ２．４３（４ＪＨＨ＝２．０Ｈｚ， １Ｈ）
Sodium hydride (132 mg, 3.02 mmol) was suspended in tetrahydrofuran (THF) (6 mL) and cooled to 0 ° C. 2-Propin-1-ol (0.165 mL, 2.80 mmol) was added thereto, and the mixture was stirred at 0 ° C. for 30 minutes. 1,3,5-Tris (bromomethyl) benzene (1.00 g, 2.80 mmol) was added, and the mixture was stirred at 25 ° C. for 20 hours. Ethyl acetate (100 mL) was added and then washed with water (100 mL) and saturated brine (100 mL), and the organic layer was washed with magnesium sulfate. The residue obtained by filtration and concentration was purified by preparative thin layer chromatography (PTLC) (methylene chloride / hexane = 1/2), and the compound DBXA (416.6 mg, 1.25 mmol) represented by the following structural formula was used. , Yield 45%) was obtained as a pale yellow oil.
The identification data of the compound DBXA by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 7.29 (s, 1H), 7.26 (s, 2H), 4.53 (s, 2H), 4.40 (s, 4H), 4.15 ( 4 J) HH = 2.5Hz, 2H), 2.43 ( 4 J HH = 2.0Hz, 1H)
－化合物Ｃ４の合成－
-Synthesis of compound C4-
上記反応式のようにして、上記構造式で表される化合物Ｃ４を合成した。
具体的には、化合物Ｃ３（５３３ｍｇ， ０．３７９ｍｍｏｌ）と化合物ＤＢＸＡ（１８８ｍｇ， ０．５６８ｍｍｏｌ）とＴＨＦ（４０ｍＬ）からなる溶液に窒素ガスバブリングを行い、窒素雰囲気下とした。ここに、硫酸銅水溶液（２００ｍＭ； １．８９ｍＬ，０．３７９ｍｍｏｌ）とアスコルビン酸ナトリウム水溶液（１００ｍＭ； ７．５８ｍＬ，０．７５８ｍｍｏｌ）を加え、２５℃で３時間撹拌した。反応液に水を加え塩化メチレンで抽出を行い（２０ｍＬで３回）、有機層をＮａ２ＳＯ４で乾燥しろ過と濃縮を行い、得られた残渣をシリカゲルクロマトグラフィー精製（ＭｅＯＨ／ＣＨ２Ｃｌ２＝１／２０）することにより、化合物Ｃ４を白色固体として取得した（４９７ｍｇ，０．２８６ｍｍｏｌ，収率７６％）。
前記化合物Ｃ４の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ ８．５８（ｔ， ３ＪＨＨ＝５．５Ｈｚ， ３Ｈ）， ７．７７（ｓ， １Ｈ）， ７．７４（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ３Ｈ）， ７．３４（ｓ， １Ｈ）， ７．３２（ｓ， ２Ｈ）， ６．８２（ｓ， １Ｈ）， ４．６９（ｓ， ２Ｈ）， ４．５８（ｓ， ２Ｈ）， ４．５６（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ２Ｈ）， ４．４７（ｓ， ４Ｈ）， ３．８９（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ２Ｈ）， ３．８７（ｓ， ２Ｈ）， ３．６９（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， ３．６６（ｓ， ６Ｈ）， ３．６１（ｓ， ８Ｈ）， ３．５６－３．５２（ｍ， ６Ｈ）， ３．４１－３．３８（ｍ， ６Ｈ）， ２．４２（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４８（ｓ， ２７Ｈ） The compound C4 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, nitrogen gas bubbling was performed on a solution consisting of compound C3 (533 mg, 0.379 mmol), compound DBXA (188 mg, 0.568 mmol) and THF (40 mL) to create a nitrogen atmosphere. A copper sulfate aqueous solution (200 mM; 1.89 mL, 0.379 mmol) and an ascorbic acid sodium aqueous solution (100 mM; 7.58 mL, 0.758 mmol) were added thereto, and the mixture was stirred at 25 ° C. for 3 hours. Water was added to the reaction solution, extraction was performed with methylene chloride (20 mL three times), the organic layer was dried with Na 2 SO 4 , filtered and concentrated, and the obtained residue was purified by silica gel chromatography (MeOH / CH 2 Cl). 2 = 1/20) to obtain compound C4 as a white solid (497 mg, 0.286 mmol, yield 76%).
The identification data of the compound C4 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 8.58 (t, 3 J HH = 5.5 Hz, 3 H), 7.77 (s, 1 H), 7.74 (t, 3 J HH = 5.0 Hz, 3 H) , 7.34 (s, 1H), 7.32 (s, 2H), 6.82 (s, 1H), 4.69 (s, 2H), 4.58 (s, 2H), 4.56 ( t, 3 J HH = 5.0 Hz, 2H), 4.47 (s, 4H), 3.89 (t, 3 J HH = 5.0 Hz, 2H), 3.87 (s, 2H), 3. 69 (t, 3 J HH = 6.0 Hz, 6H), 3.66 (s, 6H), 3.61 (s, 8H), 3.56-3.52 (m, 6H), 3.41- 3.38 (m, 6H), 2.42 (t, 3J HH = 6.0Hz , 6H), 1.49 (s, 27H), 1.48 (s, 27H)
具体的には、化合物Ｃ３（５３３ｍｇ， ０．３７９ｍｍｏｌ）と化合物ＤＢＸＡ（１８８ｍｇ， ０．５６８ｍｍｏｌ）とＴＨＦ（４０ｍＬ）からなる溶液に窒素ガスバブリングを行い、窒素雰囲気下とした。ここに、硫酸銅水溶液（２００ｍＭ； １．８９ｍＬ，０．３７９ｍｍｏｌ）とアスコルビン酸ナトリウム水溶液（１００ｍＭ； ７．５８ｍＬ，０．７５８ｍｍｏｌ）を加え、２５℃で３時間撹拌した。反応液に水を加え塩化メチレンで抽出を行い（２０ｍＬで３回）、有機層をＮａ２ＳＯ４で乾燥しろ過と濃縮を行い、得られた残渣をシリカゲルクロマトグラフィー精製（ＭｅＯＨ／ＣＨ２Ｃｌ２＝１／２０）することにより、化合物Ｃ４を白色固体として取得した（４９７ｍｇ，０．２８６ｍｍｏｌ，収率７６％）。
前記化合物Ｃ４の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ ８．５８（ｔ， ３ＪＨＨ＝５．５Ｈｚ， ３Ｈ）， ７．７７（ｓ， １Ｈ）， ７．７４（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ３Ｈ）， ７．３４（ｓ， １Ｈ）， ７．３２（ｓ， ２Ｈ）， ６．８２（ｓ， １Ｈ）， ４．６９（ｓ， ２Ｈ）， ４．５８（ｓ， ２Ｈ）， ４．５６（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ２Ｈ）， ４．４７（ｓ， ４Ｈ）， ３．８９（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ２Ｈ）， ３．８７（ｓ， ２Ｈ）， ３．６９（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， ３．６６（ｓ， ６Ｈ）， ３．６１（ｓ， ８Ｈ）， ３．５６－３．５２（ｍ， ６Ｈ）， ３．４１－３．３８（ｍ， ６Ｈ）， ２．４２（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４８（ｓ， ２７Ｈ） The compound C4 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, nitrogen gas bubbling was performed on a solution consisting of compound C3 (533 mg, 0.379 mmol), compound DBXA (188 mg, 0.568 mmol) and THF (40 mL) to create a nitrogen atmosphere. A copper sulfate aqueous solution (200 mM; 1.89 mL, 0.379 mmol) and an ascorbic acid sodium aqueous solution (100 mM; 7.58 mL, 0.758 mmol) were added thereto, and the mixture was stirred at 25 ° C. for 3 hours. Water was added to the reaction solution, extraction was performed with methylene chloride (20 mL three times), the organic layer was dried with Na 2 SO 4 , filtered and concentrated, and the obtained residue was purified by silica gel chromatography (MeOH / CH 2 Cl). 2 = 1/20) to obtain compound C4 as a white solid (497 mg, 0.286 mmol, yield 76%).
The identification data of the compound C4 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 8.58 (t, 3 J HH = 5.5 Hz, 3 H), 7.77 (s, 1 H), 7.74 (t, 3 J HH = 5.0 Hz, 3 H) , 7.34 (s, 1H), 7.32 (s, 2H), 6.82 (s, 1H), 4.69 (s, 2H), 4.58 (s, 2H), 4.56 ( t, 3 J HH = 5.0 Hz, 2H), 4.47 (s, 4H), 3.89 (t, 3 J HH = 5.0 Hz, 2H), 3.87 (s, 2H), 3. 69 (t, 3 J HH = 6.0 Hz, 6H), 3.66 (s, 6H), 3.61 (s, 8H), 3.56-3.52 (m, 6H), 3.41- 3.38 (m, 6H), 2.42 (t, 3J HH = 6.0Hz , 6H), 1.49 (s, 27H), 1.48 (s, 27H)
－化合物Ｇ３－ＤＣＸの合成－
-Synthesis of compound G3-DCX-
上記反応式のようにして、上記構造式で表される化合物Ｇ３－ＤＣＸを合成した。
具体的には、化合物Ｃ４（４９７ｍｇ，０．２８６ｍｍｏｌ）と４Ｎ塩酸ジオキサン溶液（３０ｍＬ）を混合し、２５℃で４６時間撹拌した。反応後、白色固体が析出した。上清を除去し、化合物Ｇ３－ＤＣＸの粗生成物を取得した。分取ＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）で精製し、化合物Ｇ３－ＤＣＸを白色固体として取得した（１１６ｍｇ， ０．１１０ｍｍｏｌ， 収率５４％）。
前記化合物Ｇ３－ＤＣＸのマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ２４Ｈ５１Ｎ１４Ｏ８ 計算値（[Ｍ＋Ｈ]＋）１０４７．５０２、測定値１０４７．９５３ The compound G3-DCX represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound C4 (497 mg, 0.286 mmol) and a 4N dioxane hydrochloride solution (30 mL) were mixed, and the mixture was stirred at 25 ° C. for 46 hours. After the reaction, a white solid was precipitated. The supernatant was removed to obtain a crude product of compound G3-DCX. Purification by preparative HPLC (high performance liquid chromatography) gave compound G3-DCX as a white solid (116 mg, 0.110 mmol, yield 54%).
The identification data of the compound G3-DCX by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 24 H 51 N 14 O 8 Calculated value ([M + H] + ) 1047.502, measured value 1047.953
具体的には、化合物Ｃ４（４９７ｍｇ，０．２８６ｍｍｏｌ）と４Ｎ塩酸ジオキサン溶液（３０ｍＬ）を混合し、２５℃で４６時間撹拌した。反応後、白色固体が析出した。上清を除去し、化合物Ｇ３－ＤＣＸの粗生成物を取得した。分取ＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）で精製し、化合物Ｇ３－ＤＣＸを白色固体として取得した（１１６ｍｇ， ０．１１０ｍｍｏｌ， 収率５４％）。
前記化合物Ｇ３－ＤＣＸのマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ２４Ｈ５１Ｎ１４Ｏ８ 計算値（[Ｍ＋Ｈ]＋）１０４７．５０２、測定値１０４７．９５３ The compound G3-DCX represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound C4 (497 mg, 0.286 mmol) and a 4N dioxane hydrochloride solution (30 mL) were mixed, and the mixture was stirred at 25 ° C. for 46 hours. After the reaction, a white solid was precipitated. The supernatant was removed to obtain a crude product of compound G3-DCX. Purification by preparative HPLC (high performance liquid chromatography) gave compound G3-DCX as a white solid (116 mg, 0.110 mmol, yield 54%).
The identification data of the compound G3-DCX by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 24 H 51 N 14 O 8 Calculated value ([M + H] + ) 1047.502, measured value 1047.953
＜Ｇ３－ＤＨＸトリフルオロ酢酸塩の合成＞
<Synthesis of G3-DHX trifluoroacetic acid salt>
上記反応式のようにして、上記構造式で表される細胞膜透過性分子Ｇ３－ＤＨＸトリフルオロ酢酸塩を合成した。
具体的には、化合物Ｇ３－ＤＣＸ（５０６ｍｇ，０．４８０ｍｍｏｌ）とＨ２Ｏ（６ｍＬ）を混合し、８０℃で２時間撹拌した。反応液を濃縮し、化合物Ｇ３－ＤＨＸ粗生成物を取得した。トリフルオロ酢酸（ＴＦＡ）を含む移動相を用いたＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）で精製し、細胞膜透過性分子Ｇ３－ＤＨＸトリフルオロ酢酸塩を白色固体として取得した（２２４．６ｍｇ， ０．１６９ｍｍｏｌ， 収率３５％）。
前記Ｇ３－ＤＨＸトリフルオロ酢酸塩のマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ４２Ｈ７４Ｎ１６Ｏ１３ 計算値（[Ｍ＋Ｈ]＋）１０１１．１５３、測定値１０１１．５５０。 The cell membrane permeable molecule G3-DHX trifluoroacetate represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound G3-DCX (506 mg, 0.480 mmol) and H2O (6 mL) were mixed and stirred at 80 ° C. for 2 hours. The reaction solution was concentrated to obtain a crude product of compound G3-DHX. Purification by HPLC (high performance liquid chromatography) using a mobile phase containing trifluoroacetic acid (TFA) gave the cell membrane permeable molecule G3-DHX trifluoroacetate as a white solid (224.6 mg, 0.169 mmol, Yield 35%).
The identification data of the G3-DHX trifluoroacetic acid salt by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 42 H 74 N 16 O 13 Calculated value ([M + H] + ) 1011.153, measured value 1011.550.
具体的には、化合物Ｇ３－ＤＣＸ（５０６ｍｇ，０．４８０ｍｍｏｌ）とＨ２Ｏ（６ｍＬ）を混合し、８０℃で２時間撹拌した。反応液を濃縮し、化合物Ｇ３－ＤＨＸ粗生成物を取得した。トリフルオロ酢酸（ＴＦＡ）を含む移動相を用いたＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）で精製し、細胞膜透過性分子Ｇ３－ＤＨＸトリフルオロ酢酸塩を白色固体として取得した（２２４．６ｍｇ， ０．１６９ｍｍｏｌ， 収率３５％）。
前記Ｇ３－ＤＨＸトリフルオロ酢酸塩のマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ４２Ｈ７４Ｎ１６Ｏ１３ 計算値（[Ｍ＋Ｈ]＋）１０１１．１５３、測定値１０１１．５５０。 The cell membrane permeable molecule G3-DHX trifluoroacetate represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound G3-DCX (506 mg, 0.480 mmol) and H2O (6 mL) were mixed and stirred at 80 ° C. for 2 hours. The reaction solution was concentrated to obtain a crude product of compound G3-DHX. Purification by HPLC (high performance liquid chromatography) using a mobile phase containing trifluoroacetic acid (TFA) gave the cell membrane permeable molecule G3-DHX trifluoroacetate as a white solid (224.6 mg, 0.169 mmol, Yield 35%).
The identification data of the G3-DHX trifluoroacetic acid salt by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 42 H 74 N 16 O 13 Calculated value ([M + H] + ) 1011.153, measured value 1011.550.
（実施例Ａ２：細胞膜透過性分子Ｇ３－ＤＨＸ塩酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ塩酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１９．７ｍｇ）を、超純水に溶解し（５ｍｇ／ｍＬ）、対イオンを塩化物イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ塩酸塩（１７．８ｍｇ）を得た。 (Example A2: Preparation of cell membrane permeable molecule G3-DHX hydrochloride)
For toxicity evaluation, G3-DHX hydrochloride, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (19.7 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (5 mg / mL) and counterions were replaced with chloride ions (19.7 mg). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX hydrochloride (17.8 mg) as a white to off-white powder.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ塩酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１９．７ｍｇ）を、超純水に溶解し（５ｍｇ／ｍＬ）、対イオンを塩化物イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ塩酸塩（１７．８ｍｇ）を得た。 (Example A2: Preparation of cell membrane permeable molecule G3-DHX hydrochloride)
For toxicity evaluation, G3-DHX hydrochloride, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (19.7 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (5 mg / mL) and counterions were replaced with chloride ions (19.7 mg). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX hydrochloride (17.8 mg) as a white to off-white powder.
（実施例Ａ３：細胞膜透過性分子Ｇ３－ＤＨＸ硝酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ硝酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１．５ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンを硝酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ硝酸塩（０．９ｍｇ）を得た。 (Example A3: Preparation of cell membrane permeable molecule G3-DHX nitrate)
For toxicity evaluation, G3-DHX nitrate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with nitrate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX nitrate (0.9 mg) as a white to off-white powder.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ硝酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１．５ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンを硝酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ硝酸塩（０．９ｍｇ）を得た。 (Example A3: Preparation of cell membrane permeable molecule G3-DHX nitrate)
For toxicity evaluation, G3-DHX nitrate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with nitrate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX nitrate (0.9 mg) as a white to off-white powder.
（実施例Ａ４：細胞膜透過性分子Ｇ３－ＤＨＸ硫酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ硫酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１．５ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンを硫酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ硫酸塩（１．０ｍｇ）を得た。 (Example A4: Preparation of cell membrane permeable molecule G3-DHX sulfate)
For toxicity evaluation, G3-DHX sulfate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with sulfate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX sulfate (1.0 mg) as a white to off-white powder.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ硫酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１．５ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンを硫酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ硫酸塩（１．０ｍｇ）を得た。 (Example A4: Preparation of cell membrane permeable molecule G3-DHX sulfate)
For toxicity evaluation, G3-DHX sulfate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with sulfate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX sulfate (1.0 mg) as a white to off-white powder.
（実施例Ａ５：細胞膜透過性分子Ｇ３－ＤＨＸリン酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸリン酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１．５ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをリン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸリン酸塩（１．４ｍｇ）を得た。 (Example A5: Preparation of cell membrane permeable molecule G3-DHX phosphate)
For toxicity evaluation, G3-DHX phosphate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and counterions were replaced with phosphate ions (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX phosphate (1.4 mg) as a white to off-white powder.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸリン酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（１．５ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをリン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸリン酸塩（１．４ｍｇ）を得た。 (Example A5: Preparation of cell membrane permeable molecule G3-DHX phosphate)
For toxicity evaluation, G3-DHX phosphate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and counterions were replaced with phosphate ions (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX phosphate (1.4 mg) as a white to off-white powder.
（実施例Ａ６：細胞膜透過性分子Ｇ３－ＤＨＸメシル酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸメシル酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをメシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、無色液体としてＧ３－ＤＨＸメシル酸塩（２．２ｍｇ）を得た。 (Example A6: Preparation of cell membrane permeable molecule G3-DHX mesylate)
For toxicity evaluation, G3-DHX mesylate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with mesylate ion (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX mesylate (2.2 mg) as a colorless liquid.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸメシル酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをメシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、無色液体としてＧ３－ＤＨＸメシル酸塩（２．２ｍｇ）を得た。 (Example A6: Preparation of cell membrane permeable molecule G3-DHX mesylate)
For toxicity evaluation, G3-DHX mesylate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with mesylate ion (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX mesylate (2.2 mg) as a colorless liquid.
（実施例Ａ７：細胞膜透過性分子Ｇ３－ＤＨＸトシル酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸトシル酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをトシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色粉末としてＧ３－ＤＨＸトシル酸塩（２．４ｍｇ）を得た。 (Example A7: Preparation of cell membrane permeable molecule G3-DHX tosylate)
For toxicity evaluation, G3-DHX tosylate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with tosylate ion (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX tosylate (2.4 mg) as a white powder.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸトシル酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをトシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色粉末としてＧ３－ＤＨＸトシル酸塩（２．４ｍｇ）を得た。 (Example A7: Preparation of cell membrane permeable molecule G3-DHX tosylate)
For toxicity evaluation, G3-DHX tosylate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with tosylate ion (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX tosylate (2.4 mg) as a white powder.
（実施例Ａ８：細胞膜透過性分子Ｇ３－ＤＨＸ酒石酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ酒石酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンを酒石酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてＧ３－ＤＨＸ酒石酸塩（１．８ｍｇ）を得た。 (Example A8: Preparation of cell membrane permeable molecule G3-DHX tartrate)
For toxicity evaluation, G3-DHX tartrate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin (diamond) in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with tartrate ion. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX tartrate (1.8 mg) as a pale yellow powder.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸ酒石酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンを酒石酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてＧ３－ＤＨＸ酒石酸塩（１．８ｍｇ）を得た。 (Example A8: Preparation of cell membrane permeable molecule G3-DHX tartrate)
For toxicity evaluation, G3-DHX tartrate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin (diamond) in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with tartrate ion. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX tartrate (1.8 mg) as a pale yellow powder.
（実施例Ａ９：細胞膜透過性分子Ｇ３－ＤＨＸクエン酸塩の調製）
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸクエン酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをクエン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてＧ３－ＤＨＸクエン酸塩（１．６ｍｇ）を得た。 (Example A9: Preparation of cell membrane permeable molecule G3-DHX citrate)
For toxicity evaluation, G3-DHX citrate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with citrate ion (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX citrate (1.6 mg) as a pale yellow powder.
毒性評価用として、細胞膜透過性分子の一例であるＧ３－ＤＨＸクエン酸塩を下記のようにして調製した。
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（２．２ｍｇ）を、超純水に溶解し（２ｍｇ／ｍＬ）、対イオンをクエン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてＧ３－ＤＨＸクエン酸塩（１．６ｍｇ）を得た。 (Example A9: Preparation of cell membrane permeable molecule G3-DHX citrate)
For toxicity evaluation, G3-DHX citrate, which is an example of a cell membrane permeable molecule, was prepared as follows.
An anion exchange resin in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with citrate ion (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX citrate (1.6 mg) as a pale yellow powder.
（参考例Ａ１：細胞膜透過性分子Ｇ３－ＤＨＸ酢酸塩の調製）
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（４３ｍｇ）を、超純水に溶解し（５ｍｇ／ｍＬ）、対イオンを酢酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ酢酸塩（３６ｍｇ）を得た。 (Reference Example A1: Preparation of cell membrane permeable molecule G3-DHX acetate)
An anion exchange resin (Diaion PA306S) in which G3-DHX trifluoroacetate (43 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (5 mg / mL) and counterions were replaced with acetate ions. ). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX acetate (36 mg) as a white to off-white powder.
実施例Ａ１と同様にして合成したＧ３－ＤＨＸトリフルオロ酢酸塩（４３ｍｇ）を、超純水に溶解し（５ｍｇ／ｍＬ）、対イオンを酢酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＨＸ酢酸塩（３６ｍｇ）を得た。 (Reference Example A1: Preparation of cell membrane permeable molecule G3-DHX acetate)
An anion exchange resin (Diaion PA306S) in which G3-DHX trifluoroacetate (43 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (5 mg / mL) and counterions were replaced with acetate ions. ). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX acetate (36 mg) as a white to off-white powder.
（実施例Ａ１０：ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩の合成）
細胞膜透過性分子とペプチドとの複合体の一例であるペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を下記のようにして合成した。 (Example A10: Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt)
A peptide complex G3-DCX-P trifluoroacetate, which is an example of a complex of a cell membrane permeable molecule and a peptide, was synthesized as follows.
細胞膜透過性分子とペプチドとの複合体の一例であるペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を下記のようにして合成した。 (Example A10: Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt)
A peptide complex G3-DCX-P trifluoroacetate, which is an example of a complex of a cell membrane permeable molecule and a peptide, was synthesized as follows.
＜ペプチドの合成＞
マイクロウェーブを用いた固相合成法により、リンクアミド（Ｒｉｎｋ Ａｍｉｄｅ）樹脂（０．２ｍｍｏｌ／ｇ）上で、以下の配列を有するペプチドを合成した。
ＦＩＴＣ－Ａｈｘ－Ｃｙｓ－Ｇｌｙ－Ｓｅｒ－Ｇｌｙ－Ｌｅｕ－Ａｌａ－Ｓｅｒ－Ｐｒｏ－Ａｓｎ－Ｇｌｙ－Ｔｙｒ－Ｃｙｓ－ＮＨ２
（上記配列中、「ＦＩＴＣ」はフルオレセインイソチオシアネートを表し、「Ａｈｘ」は６－アミノヘキサン酸を表す。） <Peptide synthesis>
A peptide having the following sequence was synthesized on a link amide resin (0.2 mmol / g) by a solid-phase synthesis method using microwaves.
FITC-Ahx-Cys-Gly-Ser-Gly-Leu-Ala-Ser-Pro-Asn-Gly-Tyr-Cys-NH 2
(In the above sequence, "FITC" represents fluorescein isothiocyanate and "Ahx" represents 6-aminocaproic acid.)
マイクロウェーブを用いた固相合成法により、リンクアミド（Ｒｉｎｋ Ａｍｉｄｅ）樹脂（０．２ｍｍｏｌ／ｇ）上で、以下の配列を有するペプチドを合成した。
ＦＩＴＣ－Ａｈｘ－Ｃｙｓ－Ｇｌｙ－Ｓｅｒ－Ｇｌｙ－Ｌｅｕ－Ａｌａ－Ｓｅｒ－Ｐｒｏ－Ａｓｎ－Ｇｌｙ－Ｔｙｒ－Ｃｙｓ－ＮＨ２
（上記配列中、「ＦＩＴＣ」はフルオレセインイソチオシアネートを表し、「Ａｈｘ」は６－アミノヘキサン酸を表す。） <Peptide synthesis>
A peptide having the following sequence was synthesized on a link amide resin (0.2 mmol / g) by a solid-phase synthesis method using microwaves.
FITC-Ahx-Cys-Gly-Ser-Gly-Leu-Ala-Ser-Pro-Asn-Gly-Tyr-Cys-NH 2
(In the above sequence, "FITC" represents fluorescein isothiocyanate and "Ahx" represents 6-aminocaproic acid.)
前記ペプチドを形成した樹脂を、ＴＦＡ／水／トリイソプロピルシラン／３，６－ジオキサ－１，８－オクタンジチオール（９２．５／２．５／２．５／２．５（容量比））に３時間浸漬し、前記ペプチドを樹脂から切り出した。得られたペプチドをＨＰＬＣで精製し凍結乾燥することにより、上記配列を有するペプチド（以下、「Ｐ」と表すことがある。下記構造式参照。）を取得した。前記ペプチドＰのエレクトロスプレーイオン化質量分析（ＥＳＩ－ＭＳ）による同定データは、以下のとおりであった。
ＥＳＩ－ＭＳ Ｃ７２Ｈ９２Ｎ１６Ｏ２２Ｏ３ 計算値（[Ｍ＋２Ｈ]２＋）８１５．２９５、測定値８１４．６７
The resin on which the peptide was formed was converted to TFA / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (volume ratio)). After soaking for 3 hours, the peptide was excised from the resin. The obtained peptide was purified by HPLC and freeze-dried to obtain a peptide having the above sequence (hereinafter, may be referred to as "P"; see the following structural formula). The identification data of the peptide P by electrospray ionization mass spectrometry (ESI-MS) were as follows.
ESI-MS C 72 H 92 N 16 O 22 O 3 Calculated value ([M + 2H] 2+ ) 815.295, measured value 814.67
ＥＳＩ－ＭＳ Ｃ７２Ｈ９２Ｎ１６Ｏ２２Ｏ３ 計算値（[Ｍ＋２Ｈ]２＋）８１５．２９５、測定値８１４．６７
ESI-MS C 72 H 92 N 16 O 22 O 3 Calculated value ([M + 2H] 2+ ) 815.295, measured value 814.67
＜ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩の合成＞
<Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt>
上記反応式のようにして、上記構造式で表されるペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を合成した。
具体的には、上記で合成したペプチドＰ（１１．０ｍｇ，６．７５μｍｏｌ）を２０ｍＭ重炭酸アンモニウム緩衝液（１１．８ｍＬ）とアセトニトリル（ＭｅＣＮ）（１．７ｍＬ）の混合溶液に溶解させた。この溶液に、トリス（２－カルボキシエチル）ホスフィン（ＴＣＥＰ）（５００ｍＭ ｉｎ Ｈ２Ｏ；１４．８μＬ，７．４μｍｏｌ）を添加し、２５℃下で１５分間撹拌した。続いて、上記で合成したＧ３－ＤＣＸ（１０．６ｍｇ，１０．１μｍｏｌ）と２５℃下で２４時間撹拌した。その後、反応液を、ＴＦＡを含む移動相を用いたＨＰＬＣで精製することにより、環状ペプチド（ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩）を取得した（３．２５ｍｇ，１．１０μｍｏｌ，収率１６％）。
前記ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩のＭＡＬＤＩ－ＴＯＦ ＭＳによる同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ１１４Ｈ１６３Ｎ３２Ｏ３３Ｓ３ 計算値（[Ｍ＋Ｈ]＋）２６０４．９２、測定値２６０４．３３０ The peptide complex G3-DCX-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula.
Specifically, the peptide P (11.0 mg, 6.75 μmol) synthesized above was dissolved in a mixed solution of 20 mM ammonium bicarbonate buffer (11.8 mL) and acetonitrile (MeCN) (1.7 mL). To this solution was added tris (2-carboxyethyl) phosphine (TCEP) (500 mM in H2O ; 14.8 μL, 7.4 μmol) and stirred at 25 ° C. for 15 minutes. Subsequently, the mixture was stirred with G3-DCX (10.6 mg, 10.1 μmol) synthesized above at 25 ° C. for 24 hours. Then, the reaction solution was purified by HPLC using a mobile phase containing TFA to obtain a cyclic peptide (peptide complex G3-DCX-P trifluoroacetate) (3.25 mg, 1.10 μmol, yield). Rate 16%).
The identification data of the peptide complex G3-DCX-P trifluoroacetic acid salt by MALDI-TOF MS were as follows.
MALDI-TOF MS C 114 H 163 N 32 O 33 S 3 Calculated value ([M + H] + ) 2604.92, measured value 2604.330
具体的には、上記で合成したペプチドＰ（１１．０ｍｇ，６．７５μｍｏｌ）を２０ｍＭ重炭酸アンモニウム緩衝液（１１．８ｍＬ）とアセトニトリル（ＭｅＣＮ）（１．７ｍＬ）の混合溶液に溶解させた。この溶液に、トリス（２－カルボキシエチル）ホスフィン（ＴＣＥＰ）（５００ｍＭ ｉｎ Ｈ２Ｏ；１４．８μＬ，７．４μｍｏｌ）を添加し、２５℃下で１５分間撹拌した。続いて、上記で合成したＧ３－ＤＣＸ（１０．６ｍｇ，１０．１μｍｏｌ）と２５℃下で２４時間撹拌した。その後、反応液を、ＴＦＡを含む移動相を用いたＨＰＬＣで精製することにより、環状ペプチド（ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩）を取得した（３．２５ｍｇ，１．１０μｍｏｌ，収率１６％）。
前記ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩のＭＡＬＤＩ－ＴＯＦ ＭＳによる同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ１１４Ｈ１６３Ｎ３２Ｏ３３Ｓ３ 計算値（[Ｍ＋Ｈ]＋）２６０４．９２、測定値２６０４．３３０ The peptide complex G3-DCX-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula.
Specifically, the peptide P (11.0 mg, 6.75 μmol) synthesized above was dissolved in a mixed solution of 20 mM ammonium bicarbonate buffer (11.8 mL) and acetonitrile (MeCN) (1.7 mL). To this solution was added tris (2-carboxyethyl) phosphine (TCEP) (500 mM in H2O ; 14.8 μL, 7.4 μmol) and stirred at 25 ° C. for 15 minutes. Subsequently, the mixture was stirred with G3-DCX (10.6 mg, 10.1 μmol) synthesized above at 25 ° C. for 24 hours. Then, the reaction solution was purified by HPLC using a mobile phase containing TFA to obtain a cyclic peptide (peptide complex G3-DCX-P trifluoroacetate) (3.25 mg, 1.10 μmol, yield). Rate 16%).
The identification data of the peptide complex G3-DCX-P trifluoroacetic acid salt by MALDI-TOF MS were as follows.
MALDI-TOF MS C 114 H 163 N 32 O 33 S 3 Calculated value ([M + H] + ) 2604.92, measured value 2604.330
（実施例Ａ１１：ペプチド複合体Ｇ３－ＤＣＸ－Ｐ塩酸塩の調製）
実施例Ａ１０と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩（１．１ｍｇ）を、超純水に溶解し（０．５ｍｇ／ｍＬ）、対イオンを塩化物イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）を添加した。１時間振とうしたのち、陰イオン交換樹脂を濾別し、超純水で溶出した。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＣＸ－Ｐ塩酸塩（０．６ｍｇ）を得た。 (Example A11: Preparation of peptide complex G3-DCX-P hydrochloride)
The peptide complex G3-DCX-P trifluoroacetate (1.1 mg) synthesized in the same manner as in Example A10 was dissolved in ultrapure water (0.5 mg / mL), and the counterion was converted to chloride ion. A substituted anion exchange resin (Diaion PA306S) was added. After shaking for 1 hour, the anion exchange resin was filtered off and eluted with ultrapure water. The obtained solution was freeze-dried to obtain G3-DCX-P hydrochloride (0.6 mg) as a white to off-white powder.
実施例Ａ１０と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩（１．１ｍｇ）を、超純水に溶解し（０．５ｍｇ／ｍＬ）、対イオンを塩化物イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）を添加した。１時間振とうしたのち、陰イオン交換樹脂を濾別し、超純水で溶出した。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＣＸ－Ｐ塩酸塩（０．６ｍｇ）を得た。 (Example A11: Preparation of peptide complex G3-DCX-P hydrochloride)
The peptide complex G3-DCX-P trifluoroacetate (1.1 mg) synthesized in the same manner as in Example A10 was dissolved in ultrapure water (0.5 mg / mL), and the counterion was converted to chloride ion. A substituted anion exchange resin (Diaion PA306S) was added. After shaking for 1 hour, the anion exchange resin was filtered off and eluted with ultrapure water. The obtained solution was freeze-dried to obtain G3-DCX-P hydrochloride (0.6 mg) as a white to off-white powder.
（参考例Ａ２：ペプチド複合体Ｇ３－ＤＣＸ－Ｐ酢酸塩の調製）
実施例Ａ１０と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩（１．０ｍｇ）を、超純水に溶解し（０．５ｍｇ／ｍＬ）、対イオンを酢酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）を添加した。１時間振とうしたのち、陰イオン交換樹脂を濾別し、超純水で溶出した。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＣＸ－Ｐ酢酸塩（０．９ｍｇ）を得た。 (Reference Example A2: Preparation of peptide complex G3-DCX-P acetate)
The peptide complex G3-DCX-P trifluoroacetate (1.0 mg) synthesized in the same manner as in Example A10 was dissolved in ultrapure water (0.5 mg / mL), and the counterion was replaced with acetate ion. The anion exchange resin (Diaion PA306S) was added. After shaking for 1 hour, the anion exchange resin was filtered off and eluted with ultrapure water. The obtained solution was freeze-dried to obtain G3-DCX-P acetate (0.9 mg) as a white to off-white powder.
実施例Ａ１０と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩（１．０ｍｇ）を、超純水に溶解し（０．５ｍｇ／ｍＬ）、対イオンを酢酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）を添加した。１時間振とうしたのち、陰イオン交換樹脂を濾別し、超純水で溶出した。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてＧ３－ＤＣＸ－Ｐ酢酸塩（０．９ｍｇ）を得た。 (Reference Example A2: Preparation of peptide complex G3-DCX-P acetate)
The peptide complex G3-DCX-P trifluoroacetate (1.0 mg) synthesized in the same manner as in Example A10 was dissolved in ultrapure water (0.5 mg / mL), and the counterion was replaced with acetate ion. The anion exchange resin (Diaion PA306S) was added. After shaking for 1 hour, the anion exchange resin was filtered off and eluted with ultrapure water. The obtained solution was freeze-dried to obtain G3-DCX-P acetate (0.9 mg) as a white to off-white powder.
（試験例Ａ１：細胞膜透過性分子の毒性評価）
＜マウスでのｉｎ ｖｉｖｏ毒性評価＞
供与動物は、７週齢のマウス（雌性、１５～２５ｇ、日本チャールズ・リバー社）を購入し、約７日間、環境馴化させたものを用いた。
上記した実施例及び参考例で合成した細胞膜透過性分子Ｇ３－ＤＨＸの各種酸塩（トリフルオロ酢酸塩、塩酸塩、硫酸塩、リン酸塩、メシル酸塩、トシル酸塩、酒石酸塩、クエン酸塩、酢酸塩）は、０．１％（ｖ／ｖ）ＤＭＳＯ ＰＢＳに１～１０ｍｇ／ｍＬで溶解後、シリンジフィルターユニット（酢酸セルロース製、０．２２μｍ）でろ過した。
マウスに、下記の表１に記載の量となるように調製した細胞膜透過性分子Ｇ３－ＤＨＸの各種酸塩又はプラセボ緩衝液対照（０．１％（ｖ／ｖ）ＤＭＳＯ ＰＢＳ）を尾静脈に単回投与し、投与後３０分間については一般症状及び生死等について経時的に観察し、その後７日間については１回／日のペースで継続的に一般症状及び生死等について観察した。細胞膜透過性分子Ｇ３－ＤＨＸの各種酸塩及びプラセボ緩衝液対照は、各群３匹のマウスに投与し、観察を実施した。また、投与日及び投与後１、２、７日後には体重測定を行い、体重の著しい増減を調査した。死亡例がみられた場合には、その都度解剖を実施した。また、生存例では、観察最終日（投与後７日目）に、全例についてイソフルラン吸入麻酔下で腹大動脈及び後大静脈より放血させ、安楽死させたうえで解剖し、肉眼的観察を行った。表１には投与５分後の一般症状の観察結果を示す。 (Test Example A1: Toxicity evaluation of cell membrane permeable molecule)
<In vivo toxicity evaluation in mice>
As the donor animal, a 7-week-old mouse (female, 15 to 25 g, Charles River Laboratories, Japan) was purchased and conditioned for about 7 days.
Various acid salts of the cell membrane permeable molecule G3-DHX synthesized in the above Examples and Reference Examples (trifluoroacetate, hydrochloride, sulfate, phosphate, mesylate, tosilate, tartrate, citric acid) The salt (salt, acetate) was dissolved in 0.1% (v / v) DMSO PBS at 1-10 mg / mL and then filtered through a syringe filter unit (cellulose acetate, 0.22 μm).
Mice were fed with various acid salts of the cell membrane permeable molecule G3-DHX or placebo buffer control (0.1% (v / v) DMSO PBS) prepared to the amounts shown in Table 1 below in the tail vein. A single dose was administered, and general symptoms and life / death were observed over time for 30 minutes after administration, and then general symptoms and life / death were continuously observed at a pace of once / day for 7 days. Various acid salts of the cell membrane permeable molecule G3-DHX and placebo buffer control were administered to 3 mice in each group and observed. In addition, body weight was measured on the day of administration and 1, 2, and 7 days after administration to investigate a significant increase or decrease in body weight. Whenever a fatal case was found, an autopsy was performed. In surviving cases, on the final day of observation (7th day after administration), all cases were euthanized by exhaling blood from the abdominal aorta and posterior vena cava under isoflurane inhalation anesthesia, and then dissected and visually observed. rice field. Table 1 shows the observation results of general symptoms 5 minutes after administration.
＜マウスでのｉｎ ｖｉｖｏ毒性評価＞
供与動物は、７週齢のマウス（雌性、１５～２５ｇ、日本チャールズ・リバー社）を購入し、約７日間、環境馴化させたものを用いた。
上記した実施例及び参考例で合成した細胞膜透過性分子Ｇ３－ＤＨＸの各種酸塩（トリフルオロ酢酸塩、塩酸塩、硫酸塩、リン酸塩、メシル酸塩、トシル酸塩、酒石酸塩、クエン酸塩、酢酸塩）は、０．１％（ｖ／ｖ）ＤＭＳＯ ＰＢＳに１～１０ｍｇ／ｍＬで溶解後、シリンジフィルターユニット（酢酸セルロース製、０．２２μｍ）でろ過した。
マウスに、下記の表１に記載の量となるように調製した細胞膜透過性分子Ｇ３－ＤＨＸの各種酸塩又はプラセボ緩衝液対照（０．１％（ｖ／ｖ）ＤＭＳＯ ＰＢＳ）を尾静脈に単回投与し、投与後３０分間については一般症状及び生死等について経時的に観察し、その後７日間については１回／日のペースで継続的に一般症状及び生死等について観察した。細胞膜透過性分子Ｇ３－ＤＨＸの各種酸塩及びプラセボ緩衝液対照は、各群３匹のマウスに投与し、観察を実施した。また、投与日及び投与後１、２、７日後には体重測定を行い、体重の著しい増減を調査した。死亡例がみられた場合には、その都度解剖を実施した。また、生存例では、観察最終日（投与後７日目）に、全例についてイソフルラン吸入麻酔下で腹大動脈及び後大静脈より放血させ、安楽死させたうえで解剖し、肉眼的観察を行った。表１には投与５分後の一般症状の観察結果を示す。 (Test Example A1: Toxicity evaluation of cell membrane permeable molecule)
<In vivo toxicity evaluation in mice>
As the donor animal, a 7-week-old mouse (female, 15 to 25 g, Charles River Laboratories, Japan) was purchased and conditioned for about 7 days.
Various acid salts of the cell membrane permeable molecule G3-DHX synthesized in the above Examples and Reference Examples (trifluoroacetate, hydrochloride, sulfate, phosphate, mesylate, tosilate, tartrate, citric acid) The salt (salt, acetate) was dissolved in 0.1% (v / v) DMSO PBS at 1-10 mg / mL and then filtered through a syringe filter unit (cellulose acetate, 0.22 μm).
Mice were fed with various acid salts of the cell membrane permeable molecule G3-DHX or placebo buffer control (0.1% (v / v) DMSO PBS) prepared to the amounts shown in Table 1 below in the tail vein. A single dose was administered, and general symptoms and life / death were observed over time for 30 minutes after administration, and then general symptoms and life / death were continuously observed at a pace of once / day for 7 days. Various acid salts of the cell membrane permeable molecule G3-DHX and placebo buffer control were administered to 3 mice in each group and observed. In addition, body weight was measured on the day of administration and 1, 2, and 7 days after administration to investigate a significant increase or decrease in body weight. Whenever a fatal case was found, an autopsy was performed. In surviving cases, on the final day of observation (7th day after administration), all cases were euthanized by exhaling blood from the abdominal aorta and posterior vena cava under isoflurane inhalation anesthesia, and then dissected and visually observed. rice field. Table 1 shows the observation results of general symptoms 5 minutes after administration.
表１に示されるとおり、Ｇ３－ＤＨＸトシル酸塩、Ｇ３－ＤＨＸメシル酸塩、Ｇ３－ＤＨＸリン酸塩、Ｇ３－ＤＨＸクエン酸塩、又はＧ３－ＤＨＸ酒石酸塩を１０ｍｇ／ｋｇの用量で投与した群では、いずれの個体においても歩行障害や呼吸不整等の症状や体重の著しい増減は認められなかった。また投与後７日間にわたって生存を確認できた。
また、Ｇ３－ＤＨＸトリフルオロ酢酸塩については、１０ｍｇ／ｋｇ又は２０ｍｇ／ｋｇのいずれの用量で投与しても、歩行障害や呼吸不整等症状や体重の著しい増減は認められなかった。
また、Ｇ３－ＤＨＸ塩酸塩を投与した個体は、投与直後には歩行障害が僅かに認められたが３分後には回復した。その後７日間にわたって症状は認められず、体重の著しい増減もなかった。Ｇ３－ＤＨＸ硫酸塩を投与した個体は、投与直後には呼吸不整が認められたが３分後には回復した。その後７日間にわたって異常症状は認められ体重の著しい増減もなかった。
一方、Ｇ３－ＤＨＸ酢酸塩を１０ｍｇ／ｋｇの用量で投与した個体は、投与直後から振戦がみられ、投与から２分後に死亡した。 As shown in Table 1, G3-DHX tosylate, G3-DHX mesylate, G3-DHX phosphate, G3-DHX citrate, or G3-DHX tartrate was administered at a dose of 10 mg / kg. In the group, no symptoms such as walking disorders or respiratory irregularities or significant increase or decrease in body weight were observed in any of the individuals. In addition, survival was confirmed for 7 days after administration.
In addition, regarding G3-DHX trifluoroacetic acid salt, no significant increase or decrease in body weight was observed, such as gait disturbance and respiratory irregularity, regardless of the dose of 10 mg / kg or 20 mg / kg.
In addition, the individual to which G3-DHX hydrochloride was administered had a slight gait disorder immediately after the administration, but recovered after 3 minutes. No symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight. Individuals treated with G3-DHX sulfate showed respiratory irregularities immediately after administration, but recovered 3 minutes later. Abnormal symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight.
On the other hand, the individual to which G3-DHX acetate was administered at a dose of 10 mg / kg showed tremor immediately after the administration and died 2 minutes after the administration.
また、Ｇ３－ＤＨＸトリフルオロ酢酸塩については、１０ｍｇ／ｋｇ又は２０ｍｇ／ｋｇのいずれの用量で投与しても、歩行障害や呼吸不整等症状や体重の著しい増減は認められなかった。
また、Ｇ３－ＤＨＸ塩酸塩を投与した個体は、投与直後には歩行障害が僅かに認められたが３分後には回復した。その後７日間にわたって症状は認められず、体重の著しい増減もなかった。Ｇ３－ＤＨＸ硫酸塩を投与した個体は、投与直後には呼吸不整が認められたが３分後には回復した。その後７日間にわたって異常症状は認められ体重の著しい増減もなかった。
一方、Ｇ３－ＤＨＸ酢酸塩を１０ｍｇ／ｋｇの用量で投与した個体は、投与直後から振戦がみられ、投与から２分後に死亡した。 As shown in Table 1, G3-DHX tosylate, G3-DHX mesylate, G3-DHX phosphate, G3-DHX citrate, or G3-DHX tartrate was administered at a dose of 10 mg / kg. In the group, no symptoms such as walking disorders or respiratory irregularities or significant increase or decrease in body weight were observed in any of the individuals. In addition, survival was confirmed for 7 days after administration.
In addition, regarding G3-DHX trifluoroacetic acid salt, no significant increase or decrease in body weight was observed, such as gait disturbance and respiratory irregularity, regardless of the dose of 10 mg / kg or 20 mg / kg.
In addition, the individual to which G3-DHX hydrochloride was administered had a slight gait disorder immediately after the administration, but recovered after 3 minutes. No symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight. Individuals treated with G3-DHX sulfate showed respiratory irregularities immediately after administration, but recovered 3 minutes later. Abnormal symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight.
On the other hand, the individual to which G3-DHX acetate was administered at a dose of 10 mg / kg showed tremor immediately after the administration and died 2 minutes after the administration.
以上より、マウス単回静脈投与時の毒性は、Ｇ３－ＤＨＸ酢酸塩が最も高く、オクタアルギニン（構造式は下記参照）と同程度である（Ｍａｒｃｅｌ Ｇｒｏｇｇら， Ｃｅｌｌ Ｐｅｎｅｔｒａｔｉｏｎ， Ｈｅｒｂｉｃｉｄａｌ Ａｃｔｉｖｉｔｙ， ａｎｄ ｉｎ‐ｖｉｖｏ‐Ｔｏｘｉｃｉｔｙ ｏｆ Ｏｌｉｇｏ‐Ａｒｇｉｎｉｎｅ Ｄｅｒｉｖａｔｉｖｅｓ ａｎｄ ｏｆ Ｎｏｖｅｌ Ｇｕａｎｉｄｉｎｉｕｍ‐Ｒｉｃｈ Ｃｏｍｐｏｕｎｄｓ Ｄｅｒｉｖｅｄ ｆｒｏｍ ｔｈｅ Ｂｉｏｐｏｌｙｍｅｒ Ｃｙａｎｏｐｈｙｃｉｎ， Ｈｅｌｖ Ｃｈｉｍ Ａｃｔａ． ２０１８ Ｏｃｔｏｂｅｒ ； １０１（１０），ｅ１８００１１２）ことが分かった。
これに対し、Ｇ３－ＤＨＸトリフルオロ酢酸塩、Ｇ３－ＤＨＸ塩酸塩、Ｇ３－ＤＨＸ硫酸塩、Ｇ３－ＤＨＸリン酸塩、Ｇ３－ＤＨＸメシル酸塩、Ｇ３－ＤＨＸトシル酸塩、Ｇ３－ＤＨＸ酒石酸塩、及びＧ３－ＤＨＸクエン酸塩は、Ｇ３－ＤＨＸ酢酸塩よりも低毒性であることが分かった。各酸のｐＫａの値と各酸塩の毒性の結果から、ｐＫａの値が４．７の酢酸が最も毒性が高く、ｐＫａの値が酢酸より小さい酸については毒性が低いことが分かる。すなわち、細胞膜透過性分子は、ｐＫａの値が４．７よりも小さい酸との塩とすることで低毒性となることが示唆された。また、Ｇ３－ＤＨＸ塩酸塩とＧ３－ＤＨＸ硫酸塩については、投与直後に僅からながらも症状が認められており、低毒性という観点からは、ｐＫａ－８．０超の酸との塩が好ましく、またｐＫａ－３．０超の酸との塩がより好ましいことが明らかとなった。
Based on the above, G3-DHX acetate has the highest toxicity after single intravenous administration to mice and is comparable to octaarginine (see below for the structural formula) (Marcel Grogg et al., Cell Penetration, Herbicidal Activity, and in- vivo-Toxicity of Oligo-Arginine Radivatives and of Novel Guandinium-Rich Compounds Dived from the Biopolymer Cyanophycin, Heil.
In contrast, G3-DHX trifluoroacetate, G3-DHX hydrochloride, G3-DHX sulfate, G3-DHX phosphate, G3-DHX mesylate, G3-DHX tosylate, G3-DHX tartrate. , And G3-DHX citrate were found to be less toxic than G3-DHX acetate. From the pK a value of each acid and the toxicity result of each acid salt, it can be seen that acetic acid having a pK a value of 4.7 is the most toxic, and an acid having a pK a value smaller than acetic acid is less toxic. .. That is, it was suggested that the cell membrane permeable molecule becomes less toxic by using a salt with an acid having a pKa value of less than 4.7. In addition, with regard to G3-DHX hydrochloride and G3-DHX sulfate, slight symptoms were observed immediately after administration, and from the viewpoint of low toxicity, salts with acids exceeding pK a -8.0 were observed. It has been found to be preferable, and salts with acids above pK a -3.0 are more preferred.
これに対し、Ｇ３－ＤＨＸトリフルオロ酢酸塩、Ｇ３－ＤＨＸ塩酸塩、Ｇ３－ＤＨＸ硫酸塩、Ｇ３－ＤＨＸリン酸塩、Ｇ３－ＤＨＸメシル酸塩、Ｇ３－ＤＨＸトシル酸塩、Ｇ３－ＤＨＸ酒石酸塩、及びＧ３－ＤＨＸクエン酸塩は、Ｇ３－ＤＨＸ酢酸塩よりも低毒性であることが分かった。各酸のｐＫａの値と各酸塩の毒性の結果から、ｐＫａの値が４．７の酢酸が最も毒性が高く、ｐＫａの値が酢酸より小さい酸については毒性が低いことが分かる。すなわち、細胞膜透過性分子は、ｐＫａの値が４．７よりも小さい酸との塩とすることで低毒性となることが示唆された。また、Ｇ３－ＤＨＸ塩酸塩とＧ３－ＤＨＸ硫酸塩については、投与直後に僅からながらも症状が認められており、低毒性という観点からは、ｐＫａ－８．０超の酸との塩が好ましく、またｐＫａ－３．０超の酸との塩がより好ましいことが明らかとなった。
In contrast, G3-DHX trifluoroacetate, G3-DHX hydrochloride, G3-DHX sulfate, G3-DHX phosphate, G3-DHX mesylate, G3-DHX tosylate, G3-DHX tartrate. , And G3-DHX citrate were found to be less toxic than G3-DHX acetate. From the pK a value of each acid and the toxicity result of each acid salt, it can be seen that acetic acid having a pK a value of 4.7 is the most toxic, and an acid having a pK a value smaller than acetic acid is less toxic. .. That is, it was suggested that the cell membrane permeable molecule becomes less toxic by using a salt with an acid having a pKa value of less than 4.7. In addition, with regard to G3-DHX hydrochloride and G3-DHX sulfate, slight symptoms were observed immediately after administration, and from the viewpoint of low toxicity, salts with acids exceeding pK a -8.0 were observed. It has been found to be preferable, and salts with acids above pK a -3.0 are more preferred.
（試験例Ａ２：ペプチド複合体の細胞膜透過能評価）
実施例Ａ１０で合成したペプチドＰ及び実施例Ａ１０で合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩のいずれかを２μＭ含む細胞培養液中、５％（ｖ／ｖ）ＣＯ２、３７℃の条件下で、ＨｅＬａ細胞（Ｈｕｍａｎ ｃｅｒｖｉｘ ａｄｅｎｏｃａｒｃｉｎｏｍａ ｃｅｌｌ）を２時間培養した。ＨｅＬａ細胞の培養には、ＦｌｕｏｒｏＢｒｉｔｅ Ｄ－ＭＥＭ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）（１０％（ｖ／ｖ）ＦＣＳ（ウシ胎児血清）、２％（ｖ／ｖ）ＧｌｕｔａＭａｘ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）添加）を用いた。
次いで、Ｄ－ＰＢＳ（－）（ヘパリン（２０ｕｎｉｔｓ／ｍＬ）添加）で細胞表面を洗浄後、細胞を回収し、Ｄ－ＰＢＳ（－）（０．５％（ｖ／ｖ）ＢＳＡ（ウシ血清アルブミン）、２００ｍＭ ＥＤＴＡ（エチレンジアミン四酢酸）、０．２％（ｖ／ｖ）ヨウ化プロピジウム（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）添加）に懸濁させ、フローサイトメーター（ＢＤ ＦＡＣＳ ＡｒｉａＩＩＩ）を用いて蛍光強度を測定した。フローサイトメトリー解析時には、ヨウ化プロピジウム陽性の死細胞を除いた生細胞集団に対し、蛍光強度最頻値を算出した。結果を表２に示す。 (Test Example A2: Evaluation of cell membrane permeability of peptide complex)
5% (v / v) CO 2 , 37 ° C. in a cell culture medium containing 2 μM of either peptide P synthesized in Example A10 or peptide complex G3-DCX-P trifluoroacetic acid salt synthesized in Example A10. HeLa cells (Human cervix adenocarcinoma cell) were cultured for 2 hours under the above conditions. For the culture of HeLa cells, FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used.
Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected. ), 200 mM EDTA (ethylenediaminetetraacetic acid), 0.2% (v / v) propidium iodide (manufactured by Sigma-Aldrich) was suspended), and the fluorescence intensity was measured using a flow cytometer (BD FACS Maria III). It was measured. At the time of flow cytometry analysis, the mode of fluorescence intensity was calculated for the living cell population excluding the dead cells positive for propidium iodide. The results are shown in Table 2.
実施例Ａ１０で合成したペプチドＰ及び実施例Ａ１０で合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩のいずれかを２μＭ含む細胞培養液中、５％（ｖ／ｖ）ＣＯ２、３７℃の条件下で、ＨｅＬａ細胞（Ｈｕｍａｎ ｃｅｒｖｉｘ ａｄｅｎｏｃａｒｃｉｎｏｍａ ｃｅｌｌ）を２時間培養した。ＨｅＬａ細胞の培養には、ＦｌｕｏｒｏＢｒｉｔｅ Ｄ－ＭＥＭ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）（１０％（ｖ／ｖ）ＦＣＳ（ウシ胎児血清）、２％（ｖ／ｖ）ＧｌｕｔａＭａｘ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）添加）を用いた。
次いで、Ｄ－ＰＢＳ（－）（ヘパリン（２０ｕｎｉｔｓ／ｍＬ）添加）で細胞表面を洗浄後、細胞を回収し、Ｄ－ＰＢＳ（－）（０．５％（ｖ／ｖ）ＢＳＡ（ウシ血清アルブミン）、２００ｍＭ ＥＤＴＡ（エチレンジアミン四酢酸）、０．２％（ｖ／ｖ）ヨウ化プロピジウム（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）添加）に懸濁させ、フローサイトメーター（ＢＤ ＦＡＣＳ ＡｒｉａＩＩＩ）を用いて蛍光強度を測定した。フローサイトメトリー解析時には、ヨウ化プロピジウム陽性の死細胞を除いた生細胞集団に対し、蛍光強度最頻値を算出した。結果を表２に示す。 (Test Example A2: Evaluation of cell membrane permeability of peptide complex)
5% (v / v) CO 2 , 37 ° C. in a cell culture medium containing 2 μM of either peptide P synthesized in Example A10 or peptide complex G3-DCX-P trifluoroacetic acid salt synthesized in Example A10. HeLa cells (Human cervix adenocarcinoma cell) were cultured for 2 hours under the above conditions. For the culture of HeLa cells, FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used.
Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected. ), 200 mM EDTA (ethylenediaminetetraacetic acid), 0.2% (v / v) propidium iodide (manufactured by Sigma-Aldrich) was suspended), and the fluorescence intensity was measured using a flow cytometer (BD FACS Maria III). It was measured. At the time of flow cytometry analysis, the mode of fluorescence intensity was calculated for the living cell population excluding the dead cells positive for propidium iodide. The results are shown in Table 2.
表２に示されるとおり、培養開始２時間後において、実施例Ａ１０で合成したペプチドＰはほぼ膜透過しないのに対し、実施例Ａ１０で合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩は膜透過していた。このことから、本発明の細胞膜透過性分子をペプチドに導入することにより、細胞膜透過性を獲得したことが実証された。
As shown in Table 2, the peptide P synthesized in Example A10 hardly permeates the membrane 2 hours after the start of culture, whereas the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 has no membrane permeation. It was permeated through the membrane. From this, it was demonstrated that the cell membrane permeability was obtained by introducing the cell membrane permeability molecule of the present invention into the peptide.
（試験例Ａ３：ペプチド複合体塩酸塩の細胞膜透過能評価）
実施例Ａ１０で合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩及び実施例Ａ１１で合成した合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐ塩酸塩のいずれかを２μＭ含む細胞培養液中、５％（ｖ／ｖ）ＣＯ２、３７℃の条件下で、ＨｅＬａ細胞（Ｈｕｍａｎ ｃｅｒｖｉｘ ａｄｅｎｏｃａｒｃｉｎｏｍａ ｃｅｌｌ）を８時間培養した。ＨｅＬａ細胞の培養には、ＦｌｕｏｒｏＢｒｉｔｅ Ｄ－ＭＥＭ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）（１０％（ｖ／ｖ）ＦＣＳ（ウシ胎児血清）、２％（ｖ／ｖ）ＧｌｕｔａＭａｘ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）添加）を用いた。
次いで、Ｄ－ＰＢＳ（－）（ヘパリン（２０ｕｎｉｔｓ／ｍＬ）添加）で細胞表面を洗浄後、細胞を回収し、Ｄ－ＰＢＳ（－）（０．５％（ｖ／ｖ）ＢＳＡ（ウシ血清アルブミン）、２００ｍＭ ＥＤＴＡ（エチレンジアミン四酢酸）、０．２％（ｖ／ｖ）ヨウ化プロピジウム（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）添加）に懸濁させ、フローサイトメーター（ＢＤ ＦＡＣＳ ＡｒｉａＩＩＩ）を用いて蛍光強度を測定した。フローサイトメトリー解析時には、ヨウ化プロピジウム陽性の死細胞を除いた生細胞集団に対し、蛍光強度最頻値を算出した。結果を表３に示す。 (Test Example A3: Evaluation of cell membrane permeability of peptide complex hydrochloride)
5% in a cell culture medium containing 2 μM of either the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 or the peptide complex G3-DCX-P hydrochloride synthesized in Example A11. HeLa cells (Human cervix adenocarcinoma cell) were cultured for 8 hours under the condition of (v / v) CO 2 , 37 ° C. For the culture of HeLa cells, FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used.
Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected. ), 200 mM EDTA (ethylenediaminetetraacetic acid), 0.2% (v / v) propidium iodide (manufactured by Sigma-Aldrich) was suspended), and the fluorescence intensity was measured using a flow cytometer (BD FACS Maria III). It was measured. At the time of flow cytometry analysis, the mode of fluorescence intensity was calculated for the living cell population excluding the dead cells positive for propidium iodide. The results are shown in Table 3.
実施例Ａ１０で合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩及び実施例Ａ１１で合成した合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐ塩酸塩のいずれかを２μＭ含む細胞培養液中、５％（ｖ／ｖ）ＣＯ２、３７℃の条件下で、ＨｅＬａ細胞（Ｈｕｍａｎ ｃｅｒｖｉｘ ａｄｅｎｏｃａｒｃｉｎｏｍａ ｃｅｌｌ）を８時間培養した。ＨｅＬａ細胞の培養には、ＦｌｕｏｒｏＢｒｉｔｅ Ｄ－ＭＥＭ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）（１０％（ｖ／ｖ）ＦＣＳ（ウシ胎児血清）、２％（ｖ／ｖ）ＧｌｕｔａＭａｘ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）添加）を用いた。
次いで、Ｄ－ＰＢＳ（－）（ヘパリン（２０ｕｎｉｔｓ／ｍＬ）添加）で細胞表面を洗浄後、細胞を回収し、Ｄ－ＰＢＳ（－）（０．５％（ｖ／ｖ）ＢＳＡ（ウシ血清アルブミン）、２００ｍＭ ＥＤＴＡ（エチレンジアミン四酢酸）、０．２％（ｖ／ｖ）ヨウ化プロピジウム（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）添加）に懸濁させ、フローサイトメーター（ＢＤ ＦＡＣＳ ＡｒｉａＩＩＩ）を用いて蛍光強度を測定した。フローサイトメトリー解析時には、ヨウ化プロピジウム陽性の死細胞を除いた生細胞集団に対し、蛍光強度最頻値を算出した。結果を表３に示す。 (Test Example A3: Evaluation of cell membrane permeability of peptide complex hydrochloride)
5% in a cell culture medium containing 2 μM of either the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 or the peptide complex G3-DCX-P hydrochloride synthesized in Example A11. HeLa cells (Human cervix adenocarcinoma cell) were cultured for 8 hours under the condition of (v / v) CO 2 , 37 ° C. For the culture of HeLa cells, FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used.
Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected. ), 200 mM EDTA (ethylenediaminetetraacetic acid), 0.2% (v / v) propidium iodide (manufactured by Sigma-Aldrich) was suspended), and the fluorescence intensity was measured using a flow cytometer (BD FACS Maria III). It was measured. At the time of flow cytometry analysis, the mode of fluorescence intensity was calculated for the living cell population excluding the dead cells positive for propidium iodide. The results are shown in Table 3.
表３に示されるとおり、培養開始４時間後及び８時間後において、実施例Ａ１０で合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩と実施例Ａ１１で合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐ塩酸塩は同程度膜透過していた。このことから、本発明の細胞膜透過性分子を導入したペプチド複合体の塩形態を変更しても、同程度の細胞膜透過性を示すことが実証された。
As shown in Table 3, the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 and the peptide complex G3-DCX- synthesized in Example A11 4 hours and 8 hours after the start of culture. The P hydrochloride was permeated to the same extent. From this, it was demonstrated that even if the salt morphology of the peptide complex into which the cell membrane-permeable molecule of the present invention was introduced was changed, the same degree of cell membrane permeability was exhibited.
（実施例Ｂ１：細胞膜透過性分子Ｇ３－ＤＣＸトリフルオロ酢酸塩の合成）
＜化合物Ｇ３－ＤＣＸの合成＞
－化合物Ｃ１の合成－
上記した実施例Ａ１における－化合物Ｃ１の合成－と同様にして、上記した構造式で表される化合物Ｃ１を合成した。 (Example B1: Synthesis of cell membrane permeable molecule G3-DCX trifluoroacetic acid salt)
<Synthesis of compound G3-DCX>
-Synthesis of compound C1-
In the same manner as in-Synthesis of compound C1 in Example A1 described above, compound C1 represented by the above-mentioned structural formula was synthesized.
＜化合物Ｇ３－ＤＣＸの合成＞
－化合物Ｃ１の合成－
上記した実施例Ａ１における－化合物Ｃ１の合成－と同様にして、上記した構造式で表される化合物Ｃ１を合成した。 (Example B1: Synthesis of cell membrane permeable molecule G3-DCX trifluoroacetic acid salt)
<Synthesis of compound G3-DCX>
-Synthesis of compound C1-
In the same manner as in-Synthesis of compound C1 in Example A1 described above, compound C1 represented by the above-mentioned structural formula was synthesized.
－化合物Ｃ３の合成－
上記した実施例Ａ１における－化合物Ｃ３の合成－と同様にして、上記した構造式で表される化合物Ｃ３を合成した。 -Synthesis of compound C3-
In the same manner as in-Synthesis of compound C3 in Example A1 described above, compound C3 represented by the above-mentioned structural formula was synthesized.
上記した実施例Ａ１における－化合物Ｃ３の合成－と同様にして、上記した構造式で表される化合物Ｃ３を合成した。 -Synthesis of compound C3-
In the same manner as in-Synthesis of compound C3 in Example A1 described above, compound C3 represented by the above-mentioned structural formula was synthesized.
－化合物ＤＢＸＡの合成－
上記した実施例Ａ１における－化合物ＤＢＸＡの合成－と同様にして、上記した構造式で表される化合物ＤＢＸＡを合成した。 -Synthesis of compound DBXA-
In the same manner as in-Synthesis of compound DBXA-in Example A1 described above, compound DBXA represented by the above structural formula was synthesized.
上記した実施例Ａ１における－化合物ＤＢＸＡの合成－と同様にして、上記した構造式で表される化合物ＤＢＸＡを合成した。 -Synthesis of compound DBXA-
In the same manner as in-Synthesis of compound DBXA-in Example A1 described above, compound DBXA represented by the above structural formula was synthesized.
－化合物Ｃ４の合成－
上記した実施例Ａ１における－化合物Ｃ４の合成－と同様にして、上記した構造式で表される化合物Ｃ４を合成した。 -Synthesis of compound C4-
In the same manner as in-Synthesis of compound C4 in Example A1 described above, compound C4 represented by the above-mentioned structural formula was synthesized.
上記した実施例Ａ１における－化合物Ｃ４の合成－と同様にして、上記した構造式で表される化合物Ｃ４を合成した。 -Synthesis of compound C4-
In the same manner as in-Synthesis of compound C4 in Example A1 described above, compound C4 represented by the above-mentioned structural formula was synthesized.
－化合物Ｇ３－ＤＣＸの合成－
上記した実施例Ａ１における－化合物Ｇ３－ＤＣＸの合成－と同様にして、上記した構造式で表される化合物Ｇ３－ＤＣＸの粗生成物を合成した。 -Synthesis of compound G3-DCX-
In the same manner as in-Synthesis of compound G3-DCX-in Example A1 described above, a crude product of compound G3-DCX represented by the above structural formula was synthesized.
上記した実施例Ａ１における－化合物Ｇ３－ＤＣＸの合成－と同様にして、上記した構造式で表される化合物Ｇ３－ＤＣＸの粗生成物を合成した。 -Synthesis of compound G3-DCX-
In the same manner as in-Synthesis of compound G3-DCX-in Example A1 described above, a crude product of compound G3-DCX represented by the above structural formula was synthesized.
＜細胞膜透過性分子Ｇ３－ＤＣＸトリフルオロ酢酸塩の合成＞
上述の方法により得られたＧ３－ＤＣＸの粗生成物（１１６ｍｇ）を水に溶解し、トリフルオロ酢酸（ＴＦＡ）を含む移動相を用いたＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）により精製し、下記構造式で表される細胞膜透過性分子Ｇ３－ＤＣＸトリフルオロ酢酸塩（５３．１ｍｇ， ３８．２μｍｏｌ）を取得した。
<Synthesis of cell membrane permeable molecule G3-DCX trifluoroacetic acid salt>
The crude product (116 mg) of G3-DCX obtained by the above method is dissolved in water and purified by HPLC (high performance liquid chromatography) using a mobile phase containing trifluoroacetic acid (TFA), and has the following structural formula. The cell membrane permeable molecule G3-DCX trifluoroacetic acid salt (53.1 mg, 38.2 μmol) represented by the above was obtained.
上述の方法により得られたＧ３－ＤＣＸの粗生成物（１１６ｍｇ）を水に溶解し、トリフルオロ酢酸（ＴＦＡ）を含む移動相を用いたＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）により精製し、下記構造式で表される細胞膜透過性分子Ｇ３－ＤＣＸトリフルオロ酢酸塩（５３．１ｍｇ， ３８．２μｍｏｌ）を取得した。
The crude product (116 mg) of G3-DCX obtained by the above method is dissolved in water and purified by HPLC (high performance liquid chromatography) using a mobile phase containing trifluoroacetic acid (TFA), and has the following structural formula. The cell membrane permeable molecule G3-DCX trifluoroacetic acid salt (53.1 mg, 38.2 μmol) represented by the above was obtained.
（実施例Ｂ２：ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩の合成）
上記した実施例Ａ１０と同様にして、ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を合成した。 (Example B2: Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt)
The peptide complex G3-DCX-P trifluoroacetic acid salt was synthesized in the same manner as in Example A10 described above.
上記した実施例Ａ１０と同様にして、ペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を合成した。 (Example B2: Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt)
The peptide complex G3-DCX-P trifluoroacetic acid salt was synthesized in the same manner as in Example A10 described above.
（実施例Ｂ３：ペプチド複合体Ｇ３－ＤＣＸ－Ｐ塩酸塩の調製）
上記した実施例Ａ１１と同様にして、ペプチド複合体Ｇ３－ＤＣＸ－Ｐ塩酸塩を合成した。 (Example B3: Preparation of peptide complex G3-DCX-P hydrochloride)
The peptide complex G3-DCX-P hydrochloride was synthesized in the same manner as in Example A11 described above.
上記した実施例Ａ１１と同様にして、ペプチド複合体Ｇ３－ＤＣＸ－Ｐ塩酸塩を合成した。 (Example B3: Preparation of peptide complex G3-DCX-P hydrochloride)
The peptide complex G3-DCX-P hydrochloride was synthesized in the same manner as in Example A11 described above.
（実施例Ｂ４：ペプチド複合体Ｇ３－ＤＣＸ－Ｐ酢酸塩の調製）
上記した参考例Ａ２と同様にして、ペプチド複合体Ｇ３－ＤＣＸ－Ｐ酢酸塩を合成した。 (Example B4: Preparation of peptide complex G3-DCX-P acetate)
The peptide complex G3-DCX-P acetate was synthesized in the same manner as in Reference Example A2 described above.
上記した参考例Ａ２と同様にして、ペプチド複合体Ｇ３－ＤＣＸ－Ｐ酢酸塩を合成した。 (Example B4: Preparation of peptide complex G3-DCX-P acetate)
The peptide complex G3-DCX-P acetate was synthesized in the same manner as in Reference Example A2 described above.
（実施例Ｂ５：ペプチド複合体Ｇ３－ＤＣＸ－Ｐ硝酸塩の調製）
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンを硝酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐ硝酸塩を得た。 (Example B5: Preparation of peptide complex G3-DCX-P nitrate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with nitrate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P nitrate as a white to off-white powder.
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンを硝酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐ硝酸塩を得た。 (Example B5: Preparation of peptide complex G3-DCX-P nitrate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with nitrate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P nitrate as a white to off-white powder.
（実施例Ｂ６：ペプチド複合体Ｇ３－ＤＣＸ－Ｐ硫酸塩の調製）
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンを硫酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐ硫酸塩を得た。 (Example B6: Preparation of peptide complex G3-DCX-P sulfate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with sulfate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P sulfate as a white to off-white powder.
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンを硫酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐ硫酸塩を得た。 (Example B6: Preparation of peptide complex G3-DCX-P sulfate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with sulfate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P sulfate as a white to off-white powder.
（実施例Ｂ７：ペプチド複合体Ｇ３－ＤＣＸ－Ｐリン酸塩の調製）
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをリン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐリン酸塩を得た。 (Example B7: Preparation of peptide complex G3-DCX-P phosphate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which counterions were replaced with phosphate ions. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P phosphate as a white to off-white color powder.
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをリン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色乃至オフホワイト色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐリン酸塩を得た。 (Example B7: Preparation of peptide complex G3-DCX-P phosphate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which counterions were replaced with phosphate ions. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P phosphate as a white to off-white color powder.
（実施例Ｂ８：ペプチド複合体Ｇ３－ＤＣＸ－Ｐメシル酸塩の調製）
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをメシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、無色液体としてペプチド複合体Ｇ３－ＤＣＸ－Ｐメシル酸塩を得た。 (Example B8: Preparation of peptide complex G3-DCX-P mesylate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with a mesylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P mesylate as a colorless liquid.
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをメシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、無色液体としてペプチド複合体Ｇ３－ＤＣＸ－Ｐメシル酸塩を得た。 (Example B8: Preparation of peptide complex G3-DCX-P mesylate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with a mesylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P mesylate as a colorless liquid.
（実施例Ｂ９：ペプチド複合体Ｇ３－ＤＣＸ－Ｐトシル酸塩の調製）
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをトシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐトシル酸塩を得た。 (Example B9: Preparation of peptide complex G3-DCX-P tosylate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with tosylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tosylate as a white powder.
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをトシル酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、白色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐトシル酸塩を得た。 (Example B9: Preparation of peptide complex G3-DCX-P tosylate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with tosylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tosylate as a white powder.
（実施例Ｂ１０：ペプチド複合体Ｇ３－ＤＣＸ－Ｐ酒石酸塩の調製）
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンを酒石酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐ酒石酸塩を得た。 (Example B10: Preparation of peptide complex G3-DCX-P tartrate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which the counterion was replaced with tartrate ion. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tartrate as a pale yellow powder.
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンを酒石酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐ酒石酸塩を得た。 (Example B10: Preparation of peptide complex G3-DCX-P tartrate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which the counterion was replaced with tartrate ion. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tartrate as a pale yellow powder.
（実施例Ｂ１１：ペプチド複合体Ｇ３－ＤＣＸ－Ｐクエン酸塩の調製）
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをクエン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐクエン酸塩を得た。 (Example B11: Preparation of peptide complex G3-DCX-P citrate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with citrate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P citrate as a pale yellow powder.
実施例Ｂ２と同様にして合成したペプチド複合体Ｇ３－ＤＣＸ－Ｐトリフルオロ酢酸塩を、超純水に溶解し、対イオンをクエン酸イオンへと置換した陰イオン交換樹脂（ダイヤイオン ＰＡ３０６Ｓ）に通じた。溶出には超純水を用いた。得られた溶液を凍結乾燥して、淡黄色粉末としてペプチド複合体Ｇ３－ＤＣＸ－Ｐクエン酸塩を得た。 (Example B11: Preparation of peptide complex G3-DCX-P citrate)
The peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with citrate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P citrate as a pale yellow powder.
（実施例Ｂ１２：ペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩の合成）
＜化合物Ｆｍｏｃ－Ｇ３－Ｐの合成＞
－化合物Ｋ１の合成－
Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｏｒｇａｎｏｍｅｔａｌｌｉｃ Ｃｈｅｍｉｓｔｒｙ ２０１３，１７－２４に記載の方法に従い、下記構造式で表される化合物Ｋ１を合成した。なお、化合物の構造式中の「Ｆｍｏｃ」は「９－フルオレニルメチルオキシカルボニル基」を表す。
(Example B12: Synthesis of peptide complex G3-P trifluoroacetate)
<Synthesis of compound Fmoc-G3-P>
-Synthesis of compound K1-
The compound K1 represented by the following structural formula was synthesized according to the method described in The Journal of Organometallic Chemistry 2013, 17-24. In addition, "Fmoc" in the structural formula of a compound represents "9-fluorenylmethyloxycarbonyl group".
＜化合物Ｆｍｏｃ－Ｇ３－Ｐの合成＞
－化合物Ｋ１の合成－
Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｏｒｇａｎｏｍｅｔａｌｌｉｃ Ｃｈｅｍｉｓｔｒｙ ２０１３，１７－２４に記載の方法に従い、下記構造式で表される化合物Ｋ１を合成した。なお、化合物の構造式中の「Ｆｍｏｃ」は「９－フルオレニルメチルオキシカルボニル基」を表す。
<Synthesis of compound Fmoc-G3-P>
-Synthesis of compound K1-
The compound K1 represented by the following structural formula was synthesized according to the method described in The Journal of Organometallic Chemistry 2013, 17-24. In addition, "Fmoc" in the structural formula of a compound represents "9-fluorenylmethyloxycarbonyl group".
－化合物Ｋ３の合成－
-Synthesis of compound K3-
上記反応式のようにして、上記構造式で表される化合物Ｋ３を合成した。
具体的には、化合物Ｋ１（５０ｍｇ，０．１５９ｍｍｏｌ）を塩化メチレン（５ｍＬ）に溶解し、０℃に冷却した。ここに、米国特許第７，８６２，８０７号明細書に記載の方法と同様の方法により合成した化合物Ｋ２（２８４ｍｇ，０．２３９ｍｍｏｌ）と１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（ＥＤＣ／ＨＣｌ）（４５．８ｍｇ，０．２３９ｍｍｏｌ）と１－ヒドロキシベンゾトリアゾール（ＨＯＢＴ）（３２．２ｍｇ，０．２３９ｍｍｏｌ）を加え０℃で１２時間撹拌した。飽和食塩水（２０ｍＬ）を加え、塩化メチレンで抽出を行い（２０ｍＬで３回）、有機相を硫酸マグネシウムで乾燥し、濾過後、濃縮した。残渣を分取薄層クロマトグラフィー（ＰＴＬＣ）で精製し（塩化メチレン／メタノール（ＭｅＯＨ）＝１０／１；Ｒｆ＝０．７）、化合物Ｋ３（２１０ｍｇ，０．１４１ｍｍｏｌ，収率８９％）を無色油状物として取得した。
なお、化合物の構造式中の「Ｆｍｏｃ」は「９－フルオレニルメチルオキシカルボニル基」、「Ｂｏｃ」は「ｔｅｒｔ－ブトキシカルボニル基」を表す。
前記化合物Ｋ３の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ ８．５８（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ３Ｈ）， ７．７４（ｄ， ３ＪＨＨ＝８．０Ｈｚ， ２Ｈ）， ７．５８－７．５７（ｍ， ４Ｈ）， ７．３８（ｔ， ３ＪＨＨ＝７．５Ｈｚ， ２Ｈ）， ７．２８（ｔ， ３ＪＨＨ＝７．０Ｈｚ， ２Ｈ）， ４．４７（ｄ， ３ＪＨＨ＝７．０Ｈｚ， ２Ｈ）， ４．２６（ｓ， ６Ｈ）， ３．６８（ｔ， ３ＪＨＨ＝５．５Ｈｚ， ６Ｈ）， ３．４９－３．４６（ｍ， ６Ｈ）， ３．３７－３．３３（ｍ， ６Ｈ）， ２．４３（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４６（ｓ， ２７Ｈ） The compound K3 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound K1 (50 mg, 0.159 mmol) was dissolved in methylene chloride (5 mL) and cooled to 0 ° C. Here, compounds K2 (284 mg, 0.239 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride synthesized by the same method as described in US Pat. No. 7,862,807 are used. Salt (EDC / HCl) (45.8 mg, 0.239 mmol) and 1-hydroxybenzotriazole (HOBT) (32.2 mg, 0.239 mmol) were added and stirred at 0 ° C. for 12 hours. Saturated saline (20 mL) was added, extraction was performed with methylene chloride (20 mL 3 times), the organic phase was dried over magnesium sulfate, filtered, and concentrated. The residue was purified by preparative thin layer chromatography (PTLC) (methylene chloride / methanol (MeOH) = 10/1; Rf = 0.7), and compound K3 (210 mg, 0.141 mmol, yield 89%) was colorless. Obtained as an oil.
In the structural formula of the compound, "Fmoc" represents "9-fluorenylmethyloxycarbonyl group" and "Boc" represents "tert-butoxycarbonyl group".
The identification data of the compound K3 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 8.58 (t, 3 J HH = 6.0 Hz, 3 H), 7.74 (d, 3 J HH = 8.0 Hz, 2 H), 7.58-7.57 ( m, 4H), 7.38 (t, 3 J HH = 7.5 Hz, 2 H), 7.28 (t, 3 J HH = 7.0 Hz, 2 H), 4.47 (d, 3 J HH = 7). .0Hz, 2H), 4.26 (s, 6H), 3.68 (t, 3JHH = 5.5Hz , 6H), 3.49-3.46 (m, 6H), 3.37-3 .33 (m, 6H), 2.43 (t, 3 JHH = 6.0Hz, 6H), 1.49 (s, 27H), 1.46 (s, 27H)
具体的には、化合物Ｋ１（５０ｍｇ，０．１５９ｍｍｏｌ）を塩化メチレン（５ｍＬ）に溶解し、０℃に冷却した。ここに、米国特許第７，８６２，８０７号明細書に記載の方法と同様の方法により合成した化合物Ｋ２（２８４ｍｇ，０．２３９ｍｍｏｌ）と１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（ＥＤＣ／ＨＣｌ）（４５．８ｍｇ，０．２３９ｍｍｏｌ）と１－ヒドロキシベンゾトリアゾール（ＨＯＢＴ）（３２．２ｍｇ，０．２３９ｍｍｏｌ）を加え０℃で１２時間撹拌した。飽和食塩水（２０ｍＬ）を加え、塩化メチレンで抽出を行い（２０ｍＬで３回）、有機相を硫酸マグネシウムで乾燥し、濾過後、濃縮した。残渣を分取薄層クロマトグラフィー（ＰＴＬＣ）で精製し（塩化メチレン／メタノール（ＭｅＯＨ）＝１０／１；Ｒｆ＝０．７）、化合物Ｋ３（２１０ｍｇ，０．１４１ｍｍｏｌ，収率８９％）を無色油状物として取得した。
なお、化合物の構造式中の「Ｆｍｏｃ」は「９－フルオレニルメチルオキシカルボニル基」、「Ｂｏｃ」は「ｔｅｒｔ－ブトキシカルボニル基」を表す。
前記化合物Ｋ３の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ ８．５８（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ３Ｈ）， ７．７４（ｄ， ３ＪＨＨ＝８．０Ｈｚ， ２Ｈ）， ７．５８－７．５７（ｍ， ４Ｈ）， ７．３８（ｔ， ３ＪＨＨ＝７．５Ｈｚ， ２Ｈ）， ７．２８（ｔ， ３ＪＨＨ＝７．０Ｈｚ， ２Ｈ）， ４．４７（ｄ， ３ＪＨＨ＝７．０Ｈｚ， ２Ｈ）， ４．２６（ｓ， ６Ｈ）， ３．６８（ｔ， ３ＪＨＨ＝５．５Ｈｚ， ６Ｈ）， ３．４９－３．４６（ｍ， ６Ｈ）， ３．３７－３．３３（ｍ， ６Ｈ）， ２．４３（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４６（ｓ， ２７Ｈ） The compound K3 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound K1 (50 mg, 0.159 mmol) was dissolved in methylene chloride (5 mL) and cooled to 0 ° C. Here, compounds K2 (284 mg, 0.239 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride synthesized by the same method as described in US Pat. No. 7,862,807 are used. Salt (EDC / HCl) (45.8 mg, 0.239 mmol) and 1-hydroxybenzotriazole (HOBT) (32.2 mg, 0.239 mmol) were added and stirred at 0 ° C. for 12 hours. Saturated saline (20 mL) was added, extraction was performed with methylene chloride (20 mL 3 times), the organic phase was dried over magnesium sulfate, filtered, and concentrated. The residue was purified by preparative thin layer chromatography (PTLC) (methylene chloride / methanol (MeOH) = 10/1; Rf = 0.7), and compound K3 (210 mg, 0.141 mmol, yield 89%) was colorless. Obtained as an oil.
In the structural formula of the compound, "Fmoc" represents "9-fluorenylmethyloxycarbonyl group" and "Boc" represents "tert-butoxycarbonyl group".
The identification data of the compound K3 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 8.58 (t, 3 J HH = 6.0 Hz, 3 H), 7.74 (d, 3 J HH = 8.0 Hz, 2 H), 7.58-7.57 ( m, 4H), 7.38 (t, 3 J HH = 7.5 Hz, 2 H), 7.28 (t, 3 J HH = 7.0 Hz, 2 H), 4.47 (d, 3 J HH = 7). .0Hz, 2H), 4.26 (s, 6H), 3.68 (t, 3JHH = 5.5Hz , 6H), 3.49-3.46 (m, 6H), 3.37-3 .33 (m, 6H), 2.43 (t, 3 JHH = 6.0Hz, 6H), 1.49 (s, 27H), 1.46 (s, 27H)
－化合物Ｋ４の合成－
-Synthesis of compound K4-
上記反応式のようにして、上記構造式で表される化合物Ｋ４を合成した。
具体的には、化合物Ｋ３（２１０ｍｇ，０．１４１ｍｍｏｌ）と２０％ピペリジン／Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）溶液（１．０ｍＬ）を混合し、２５℃で１時間撹拌した。反応液に窒素ガスを吹き付けて溶媒を揮発させた。得られた残渣をＰＴＬＣで精製し（塩化メチレン／ＭｅＯＨ＝１０／１）、化合物Ｋ４（１６８ｍｇ，０．１３３ｍｍｏｌ，収率９４％）を無色透明油状物として取得した。
前記化合物Ｋ４の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ １１．４（ｓ， ３Ｈ）， ８．５９（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ３Ｈ）， ７．７１（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ３Ｈ）， ６．７９（ｓ， １Ｈ）， ４．０３（ｓ， ２Ｈ）， ３．７１－３．６７（ｍ， １２Ｈ）， ３．５５－３．５２（ｍ， ６Ｈ）， ３．４２－３．３９（ｍ， ６Ｈ）， ２．４３（ｔ， ３ＪＨＨ＝５．５Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４８（ｓ， ２７Ｈ） The compound K4 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound K3 (210 mg, 0.141 mmol) and a 20% piperidine / N, N-dimethylformamide (DMF) solution (1.0 mL) were mixed and stirred at 25 ° C. for 1 hour. Nitrogen gas was blown onto the reaction solution to volatilize the solvent. The obtained residue was purified by PTLC (methylene chloride / MeOH = 10/1) to obtain compound K4 (168 mg, 0.133 mmol, yield 94%) as a colorless transparent oil.
The identification data of the compound K4 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 11.4 (s, 3H), 8.59 (t, 3 J HH = 6.0 Hz, 3 H), 7.71 (t, 3 J HH = 5.0 Hz, 3 H) , 6.79 (s, 1H), 4.03 (s, 2H), 3.71-3.67 (m, 12H), 3.55-3.52 (m, 6H), 3.42-3 .39 (m, 6H), 2.43 (t, 3 JHH = 5.5Hz, 6H), 1.49 (s, 27H), 1.48 (s, 27H)
具体的には、化合物Ｋ３（２１０ｍｇ，０．１４１ｍｍｏｌ）と２０％ピペリジン／Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）溶液（１．０ｍＬ）を混合し、２５℃で１時間撹拌した。反応液に窒素ガスを吹き付けて溶媒を揮発させた。得られた残渣をＰＴＬＣで精製し（塩化メチレン／ＭｅＯＨ＝１０／１）、化合物Ｋ４（１６８ｍｇ，０．１３３ｍｍｏｌ，収率９４％）を無色透明油状物として取得した。
前記化合物Ｋ４の１Ｈ ＮＭＲによる同定データは、以下のとおりであった。
１Ｈ ＮＭＲ（ＣＤＣｌ３）： δ １１．４（ｓ， ３Ｈ）， ８．５９（ｔ， ３ＪＨＨ＝６．０Ｈｚ， ３Ｈ）， ７．７１（ｔ， ３ＪＨＨ＝５．０Ｈｚ， ３Ｈ）， ６．７９（ｓ， １Ｈ）， ４．０３（ｓ， ２Ｈ）， ３．７１－３．６７（ｍ， １２Ｈ）， ３．５５－３．５２（ｍ， ６Ｈ）， ３．４２－３．３９（ｍ， ６Ｈ）， ２．４３（ｔ， ３ＪＨＨ＝５．５Ｈｚ， ６Ｈ）， １．４９（ｓ， ２７Ｈ）， １．４８（ｓ， ２７Ｈ） The compound K4 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound K3 (210 mg, 0.141 mmol) and a 20% piperidine / N, N-dimethylformamide (DMF) solution (1.0 mL) were mixed and stirred at 25 ° C. for 1 hour. Nitrogen gas was blown onto the reaction solution to volatilize the solvent. The obtained residue was purified by PTLC (methylene chloride / MeOH = 10/1) to obtain compound K4 (168 mg, 0.133 mmol, yield 94%) as a colorless transparent oil.
The identification data of the compound K4 by 1H NMR were as follows.
1H NMR (CDCl 3 ): δ 11.4 (s, 3H), 8.59 (t, 3 J HH = 6.0 Hz, 3 H), 7.71 (t, 3 J HH = 5.0 Hz, 3 H) , 6.79 (s, 1H), 4.03 (s, 2H), 3.71-3.67 (m, 12H), 3.55-3.52 (m, 6H), 3.42-3 .39 (m, 6H), 2.43 (t, 3 JHH = 5.5Hz, 6H), 1.49 (s, 27H), 1.48 (s, 27H)
－細胞膜透過性分子Ｇ３の合成－
-Synthesis of cell membrane permeable molecule G3-
上記反応式のようにして、上記構造式で表される細胞膜透過性分子Ｇ３を合成した。
具体的には、化合物Ｋ４に塩酸ジオキサン溶液（４Ｎ； １．０ｍＬ）を加えて溶液とし、２５℃で２時間撹拌した。析出した白色固体を遠心分離により取得し、さらにジエチルエーテルで洗浄することにより（３ｍＬで３回）、細胞膜透過性分子Ｇ３（９０ｍｇ，定量的収率）を取得した。
前記細胞膜透過性分子Ｇ３のマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ２４Ｈ５０Ｎ１４Ｏ８ 計算値（［Ｍ＋Ｈ］＋）６６３．４０１、測定値６６３．４０２ The cell membrane permeable molecule G3 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, a dioxane hydrochloride solution (4N; 1.0 mL) was added to compound K4 to prepare a solution, and the mixture was stirred at 25 ° C. for 2 hours. The precipitated white solid was obtained by centrifugation and further washed with diethyl ether (3 times at 3 mL) to obtain a cell membrane permeable molecule G3 (90 mg, quantitative yield).
The identification data of the cell membrane-permeable molecule G3 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 24 H 50 N 14 O 8 Calculated value ([M + H] + ) 663.401, measured value 663.402
具体的には、化合物Ｋ４に塩酸ジオキサン溶液（４Ｎ； １．０ｍＬ）を加えて溶液とし、２５℃で２時間撹拌した。析出した白色固体を遠心分離により取得し、さらにジエチルエーテルで洗浄することにより（３ｍＬで３回）、細胞膜透過性分子Ｇ３（９０ｍｇ，定量的収率）を取得した。
前記細胞膜透過性分子Ｇ３のマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ２４Ｈ５０Ｎ１４Ｏ８ 計算値（［Ｍ＋Ｈ］＋）６６３．４０１、測定値６６３．４０２ The cell membrane permeable molecule G3 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, a dioxane hydrochloride solution (4N; 1.0 mL) was added to compound K4 to prepare a solution, and the mixture was stirred at 25 ° C. for 2 hours. The precipitated white solid was obtained by centrifugation and further washed with diethyl ether (3 times at 3 mL) to obtain a cell membrane permeable molecule G3 (90 mg, quantitative yield).
The identification data of the cell membrane-permeable molecule G3 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 24 H 50 N 14 O 8 Calculated value ([M + H] + ) 663.401, measured value 663.402
－化合物Ｋ５の合成－
マイクロウェーブを用いた固相合成法により、リンクアミド（Ｒｉｎｋ Ａｍｉｄｅ）樹脂（０．２ｍｍｏｌ／ｇ）上で、以下の配列を有するペプチドを合成した。
Ｆｍｏｃ－Ａｈｘ－Ｃｙｓ－Ｍｅｔ－Ｌｅｕ－Ｔｙｒ－Ｉｌｅ－Ｖａｌ－Ｐｒｏ－Ｔｙｒ－Ｐｈｅ－Ｓｅｒ－Ｖａｌ－Ｇｌｙ－Ｃｙｓ－ＮＨ２
［上記配列中、「Ｆｍｏｃ」は９－フルオレニルメチルオキシカルボニル基を表し、「Ａｈｘ」は６－アミノヘキサン酸を表す。］
前記ペプチドを形成した樹脂を、トリフルオロ酢酸（ＴＦＡ）／水／トリイソプロピルシラン／３，６－ジオキサ－１，８－オクタンジチオール（９２．５／２．５／２．５／２．５（容量比））に３時間浸漬し、前記ペプチドを樹脂から切り出した。
得られたペプチド（５５．０ｍｇ，３０．０μｍｏｌ）をＤＭＦ（２．５ｍＬ）に溶解し、１，３－ジブロモ－２－プロパノン（２０ｍＭ ＤＭＦ溶液；１．５ｍＬ，３０μｍｏｌ）及びＮ－メチルモルホリン（１０ｍＭ ＤＭＦ溶液；６．０ｍＬ，６０μｍｏｌ）を加えて２５℃で１時間撹拌した。ここにジエチルエーテル（１００ｍＬ）を加えて上清を除去し、残渣をジエチルエーテル（１００ｍＬ）で洗浄することにより化合物Ｋ５を白色固体として取得した（５４．６ｍｇ，２８．９μｍｏｌ，収率９７％）。
なお、化合物Ｋ５の構造式中の「ＣＭＬＹＩＶＰＹＦＳＶＧＣ」は、ペプチドのアミノ酸配列を表す。
前記化合物Ｋ５のマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ９４Ｈ１２８Ｎ１５Ｏ２０Ｓ３ ＋ 計算値（［Ｍ＋Ｈ］＋）１８８２．８６２、測定値１８８３．１４０
-Synthesis of compound K5-
A peptide having the following sequence was synthesized on a link amide resin (0.2 mmol / g) by a solid-phase synthesis method using microwaves.
Fmoc-Ahx-Cys-Met-Leu-Tyr-Ile-Val-Pro-Tyr-Phe-Ser-Val-Gly-Cys-NH 2
[In the above sequence, "Fmoc" represents a 9-fluorenylmethyloxycarbonyl group and "Ahx" represents 6-aminohexanoic acid. ]
The resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5). The peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
The obtained peptide (55.0 mg, 30.0 μmol) was dissolved in DMF (2.5 mL), and 1,3-dibromo-2-propanol (20 mM DMF solution; 1.5 mL, 30 μmol) and N-methylmorpholine ( 10 mM DMF solution; 6.0 mL, 60 μmol) was added and stirred at 25 ° C. for 1 hour. Diethyl ether (100 mL) was added thereto to remove the supernatant, and the residue was washed with diethyl ether (100 mL) to obtain compound K5 as a white solid (54.6 mg, 28.9 μmol, yield 97%). ..
In addition, "CMLYIVPYFSVGC" in the structural formula of compound K5 represents the amino acid sequence of the peptide.
The identification data of the compound K5 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 94 H 128 N 15 O 20 S 3 + Calculated value ([M + H] + ) 1882.862, Measured value 1883.140
マイクロウェーブを用いた固相合成法により、リンクアミド（Ｒｉｎｋ Ａｍｉｄｅ）樹脂（０．２ｍｍｏｌ／ｇ）上で、以下の配列を有するペプチドを合成した。
Ｆｍｏｃ－Ａｈｘ－Ｃｙｓ－Ｍｅｔ－Ｌｅｕ－Ｔｙｒ－Ｉｌｅ－Ｖａｌ－Ｐｒｏ－Ｔｙｒ－Ｐｈｅ－Ｓｅｒ－Ｖａｌ－Ｇｌｙ－Ｃｙｓ－ＮＨ２
［上記配列中、「Ｆｍｏｃ」は９－フルオレニルメチルオキシカルボニル基を表し、「Ａｈｘ」は６－アミノヘキサン酸を表す。］
前記ペプチドを形成した樹脂を、トリフルオロ酢酸（ＴＦＡ）／水／トリイソプロピルシラン／３，６－ジオキサ－１，８－オクタンジチオール（９２．５／２．５／２．５／２．５（容量比））に３時間浸漬し、前記ペプチドを樹脂から切り出した。
得られたペプチド（５５．０ｍｇ，３０．０μｍｏｌ）をＤＭＦ（２．５ｍＬ）に溶解し、１，３－ジブロモ－２－プロパノン（２０ｍＭ ＤＭＦ溶液；１．５ｍＬ，３０μｍｏｌ）及びＮ－メチルモルホリン（１０ｍＭ ＤＭＦ溶液；６．０ｍＬ，６０μｍｏｌ）を加えて２５℃で１時間撹拌した。ここにジエチルエーテル（１００ｍＬ）を加えて上清を除去し、残渣をジエチルエーテル（１００ｍＬ）で洗浄することにより化合物Ｋ５を白色固体として取得した（５４．６ｍｇ，２８．９μｍｏｌ，収率９７％）。
なお、化合物Ｋ５の構造式中の「ＣＭＬＹＩＶＰＹＦＳＶＧＣ」は、ペプチドのアミノ酸配列を表す。
前記化合物Ｋ５のマトリックス支援レーザー脱離イオン化飛行時間質量分析（ＭＡＬＤＩ－ＴＯＦ ＭＳ）による同定データは、以下のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ９４Ｈ１２８Ｎ１５Ｏ２０Ｓ３ ＋ 計算値（［Ｍ＋Ｈ］＋）１８８２．８６２、測定値１８８３．１４０
A peptide having the following sequence was synthesized on a link amide resin (0.2 mmol / g) by a solid-phase synthesis method using microwaves.
Fmoc-Ahx-Cys-Met-Leu-Tyr-Ile-Val-Pro-Tyr-Phe-Ser-Val-Gly-Cys-NH 2
[In the above sequence, "Fmoc" represents a 9-fluorenylmethyloxycarbonyl group and "Ahx" represents 6-aminohexanoic acid. ]
The resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5). The peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
The obtained peptide (55.0 mg, 30.0 μmol) was dissolved in DMF (2.5 mL), and 1,3-dibromo-2-propanol (20 mM DMF solution; 1.5 mL, 30 μmol) and N-methylmorpholine ( 10 mM DMF solution; 6.0 mL, 60 μmol) was added and stirred at 25 ° C. for 1 hour. Diethyl ether (100 mL) was added thereto to remove the supernatant, and the residue was washed with diethyl ether (100 mL) to obtain compound K5 as a white solid (54.6 mg, 28.9 μmol, yield 97%). ..
In addition, "CMLYIVPYFSVGC" in the structural formula of compound K5 represents the amino acid sequence of the peptide.
The identification data of the compound K5 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C 94 H 128 N 15 O 20 S 3 + Calculated value ([M + H] + ) 1882.862, Measured value 1883.140
－化合物Ｆｍｏｃ－Ｇ３－Ｐの合成－
-Synthesis of compound Fmoc-G3-P-
上記反応式のようにして、上記構造式で表される化合物Ｆｍｏｃ－Ｇ３－Ｐを合成した。
具体的には、化合物Ｋ５（３０ｍｇ，０．０１５９ｍｍｏｌ）と細胞膜透過性分子Ｇ３（５２．７ｍｇ，０．０７９６ｍｍｏｌ）とＤＭＦ（０．５ｍＬ）と水（０．０５ｍＬ）とを混合し、２５℃で２４時間撹拌した。ジエチルエーテル（５ｍＬ）を加えて遠心分離を行い、上澄を除去した。残渣をジエチルエーテルで洗浄（３ｍＬで３回）することにより化合物Ｆｍｏｃ－Ｇ３－Ｐの粗生成物を取得した。逆相ＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）により精製し凍結乾燥することにより、化合物Ｆｍｏｃ－Ｇ３－Ｐ（３．８ｍｇ，０．００１５０ｍｍｏｌ，収率９％）を取得した。 The compound Fmoc-G3-P represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound K5 (30 mg, 0.0159 mmol), cell membrane permeable molecule G3 (52.7 mg, 0.0796 mmol), DMF (0.5 mL) and water (0.05 mL) were mixed and 25 ° C. Was stirred for 24 hours. Diethyl ether (5 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (3 times with 3 mL) to obtain a crude product of compound Fmoc-G3-P. The compound Fmoc-G3-P (3.8 mg, 0.00150 mmol, yield 9%) was obtained by purification by reverse phase HPLC (high performance liquid chromatography) and freeze-drying.
具体的には、化合物Ｋ５（３０ｍｇ，０．０１５９ｍｍｏｌ）と細胞膜透過性分子Ｇ３（５２．７ｍｇ，０．０７９６ｍｍｏｌ）とＤＭＦ（０．５ｍＬ）と水（０．０５ｍＬ）とを混合し、２５℃で２４時間撹拌した。ジエチルエーテル（５ｍＬ）を加えて遠心分離を行い、上澄を除去した。残渣をジエチルエーテルで洗浄（３ｍＬで３回）することにより化合物Ｆｍｏｃ－Ｇ３－Ｐの粗生成物を取得した。逆相ＨＰＬＣ（ｈｉｇｈ ｐｅｒｆｏｒｍａｎｃｅ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒａｐｈｙ）により精製し凍結乾燥することにより、化合物Ｆｍｏｃ－Ｇ３－Ｐ（３．８ｍｇ，０．００１５０ｍｍｏｌ，収率９％）を取得した。 The compound Fmoc-G3-P represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound K5 (30 mg, 0.0159 mmol), cell membrane permeable molecule G3 (52.7 mg, 0.0796 mmol), DMF (0.5 mL) and water (0.05 mL) were mixed and 25 ° C. Was stirred for 24 hours. Diethyl ether (5 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (3 times with 3 mL) to obtain a crude product of compound Fmoc-G3-P. The compound Fmoc-G3-P (3.8 mg, 0.00150 mmol, yield 9%) was obtained by purification by reverse phase HPLC (high performance liquid chromatography) and freeze-drying.
＜ペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩の合成＞
<Synthesis of peptide complex G3-P trifluoroacetic acid salt>
上記反応式のようにして、上記構造式で表されるペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩を合成した。
具体的には、上記で合成した化合物Ｆｍｏｃ－Ｇ３－Ｐと２０％ピペリジン／ＤＭＦ溶液（１．０ｍＬ）を混合し、２５℃で２時間ボルテックスミキサーを用いて撹拌した。窒素ガスを吹き付けて溶媒を揮発させた。この残渣をジエチルエーテルで洗浄した（１ｍＬで３回）。ここに、フルオレセインイソチオシアネート（ＦＩＴＣ）（０．８ｍｇ，０．００２ｍｍｏｌ）とジイソプロピルエチルアミン（ＤＩＰＥＡ）（０．０２２ｍＬ，０．０１２４ｍｍｏｌ）とＤＭＦ（０．５ｍＬ）を混合し、２５℃で２０時間撹拌した。ジエチルエーテル（１５ｍＬ）を加えて遠心分離を行い、上澄みを除去した。残渣をジエチルエーテルで洗浄し（５ｍＬで２回）、ペプチド複合体Ｇ３－Ｐ粗生成物を取得した。ＴＦＡを含む移動相を用いたＨＰＬＣで精製し凍結乾燥することにより、ペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩（１．６ｍｇ，０．０００５９ｍｍｏｌ，収率４０％）を白色固体として取得した。
前記ペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩のＭＡＬＤＩ－ＴＯＦ ＭＳによる同定データは、次のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ１２４Ｈ１７７Ｎ３０Ｏ３０Ｓ４ 計算値（［Ｍ＋Ｈ］＋）２６９４．２１２、測定値２６９４．２０８ The peptide complex G3-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula.
Specifically, the compound Fmoc-G3-P synthesized above and a 20% piperidine / DMF solution (1.0 mL) were mixed and stirred at 25 ° C. for 2 hours using a vortex mixer. The solvent was volatilized by spraying nitrogen gas. The residue was washed with diethyl ether (1 mL 3 times). Here, fluorescein isothiocyanate (FITC) (0.8 mg, 0.002 mmol), diisopropylethylamine (DIPEA) (0.022 mL, 0.0124 mmol) and DMF (0.5 mL) are mixed and stirred at 25 ° C. for 20 hours. did. Diethyl ether (15 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (twice at 5 mL) to give the peptide complex G3-P crude product. The peptide complex G3-P trifluoroacetic acid salt (1.6 mg, 0.00059 mmol, yield 40%) was obtained as a white solid by purification by HPLC using a mobile phase containing TFA and freeze-drying.
The identification data of the peptide complex G3-P trifluoroacetic acid salt by MALDI-TOF MS were as follows.
MALDI-TOF MS C 124 H 177 N 30 O 30 S 4 Calculated value ([M + H] + ) 2694.212, measured value 2694.208
具体的には、上記で合成した化合物Ｆｍｏｃ－Ｇ３－Ｐと２０％ピペリジン／ＤＭＦ溶液（１．０ｍＬ）を混合し、２５℃で２時間ボルテックスミキサーを用いて撹拌した。窒素ガスを吹き付けて溶媒を揮発させた。この残渣をジエチルエーテルで洗浄した（１ｍＬで３回）。ここに、フルオレセインイソチオシアネート（ＦＩＴＣ）（０．８ｍｇ，０．００２ｍｍｏｌ）とジイソプロピルエチルアミン（ＤＩＰＥＡ）（０．０２２ｍＬ，０．０１２４ｍｍｏｌ）とＤＭＦ（０．５ｍＬ）を混合し、２５℃で２０時間撹拌した。ジエチルエーテル（１５ｍＬ）を加えて遠心分離を行い、上澄みを除去した。残渣をジエチルエーテルで洗浄し（５ｍＬで２回）、ペプチド複合体Ｇ３－Ｐ粗生成物を取得した。ＴＦＡを含む移動相を用いたＨＰＬＣで精製し凍結乾燥することにより、ペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩（１．６ｍｇ，０．０００５９ｍｍｏｌ，収率４０％）を白色固体として取得した。
前記ペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩のＭＡＬＤＩ－ＴＯＦ ＭＳによる同定データは、次のとおりであった。
ＭＡＬＤＩ－ＴＯＦ ＭＳ Ｃ１２４Ｈ１７７Ｎ３０Ｏ３０Ｓ４ 計算値（［Ｍ＋Ｈ］＋）２６９４．２１２、測定値２６９４．２０８ The peptide complex G3-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula.
Specifically, the compound Fmoc-G3-P synthesized above and a 20% piperidine / DMF solution (1.0 mL) were mixed and stirred at 25 ° C. for 2 hours using a vortex mixer. The solvent was volatilized by spraying nitrogen gas. The residue was washed with diethyl ether (1 mL 3 times). Here, fluorescein isothiocyanate (FITC) (0.8 mg, 0.002 mmol), diisopropylethylamine (DIPEA) (0.022 mL, 0.0124 mmol) and DMF (0.5 mL) are mixed and stirred at 25 ° C. for 20 hours. did. Diethyl ether (15 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (twice at 5 mL) to give the peptide complex G3-P crude product. The peptide complex G3-P trifluoroacetic acid salt (1.6 mg, 0.00059 mmol, yield 40%) was obtained as a white solid by purification by HPLC using a mobile phase containing TFA and freeze-drying.
The identification data of the peptide complex G3-P trifluoroacetic acid salt by MALDI-TOF MS were as follows.
MALDI-TOF MS C 124 H 177 N 30 O 30 S 4 Calculated value ([M + H] + ) 2694.212, measured value 2694.208
（比較例Ｂ１：ペプチド複合体Ｇ６－Ｐの合成）
国際出願番号：ＰＣＴ／ＪＰ２０２０／００５７６２に記載の実施例１の方法により、下記構造式で表されるペプチド複合体Ｇ６－Ｐを合成した。なお、下記構造式中の「ＣＭＬＹＩＶＰＹＦＳＶＧＣ」は、ペプチドのアミノ酸配列を表す。
(Comparative Example B1: Synthesis of peptide complex G6-P)
The peptide complex G6-P represented by the following structural formula was synthesized by the method of Example 1 described in International Application No .: PCT / JP2020 / 005762. In addition, "CMLYIVPYFSVGC" in the following structural formula represents the amino acid sequence of the peptide.
国際出願番号：ＰＣＴ／ＪＰ２０２０／００５７６２に記載の実施例１の方法により、下記構造式で表されるペプチド複合体Ｇ６－Ｐを合成した。なお、下記構造式中の「ＣＭＬＹＩＶＰＹＦＳＶＧＣ」は、ペプチドのアミノ酸配列を表す。
The peptide complex G6-P represented by the following structural formula was synthesized by the method of Example 1 described in International Application No .: PCT / JP2020 / 005762. In addition, "CMLYIVPYFSVGC" in the following structural formula represents the amino acid sequence of the peptide.
（比較例Ｂ２：ペプチド複合体Ｇ９－Ｐの合成）
国際出願番号：ＰＣＴ／ＪＰ２０２０／００５７６２に記載の実施例２の方法により、下記構造式で表されるペプチド複合体Ｇ９－Ｐを合成した。なお、下記構造式中の「ＣＭＬＹＩＶＰＹＦＳＶＧＣ」は、ペプチドのアミノ酸配列を表す。
(Comparative Example B2: Synthesis of peptide complex G9-P)
The peptide complex G9-P represented by the following structural formula was synthesized by the method of Example 2 described in International Application No .: PCT / JP2020 / 005762. In addition, "CMLYIVPYFSVGC" in the following structural formula represents the amino acid sequence of the peptide.
国際出願番号：ＰＣＴ／ＪＰ２０２０／００５７６２に記載の実施例２の方法により、下記構造式で表されるペプチド複合体Ｇ９－Ｐを合成した。なお、下記構造式中の「ＣＭＬＹＩＶＰＹＦＳＶＧＣ」は、ペプチドのアミノ酸配列を表す。
The peptide complex G9-P represented by the following structural formula was synthesized by the method of Example 2 described in International Application No .: PCT / JP2020 / 005762. In addition, "CMLYIVPYFSVGC" in the following structural formula represents the amino acid sequence of the peptide.
（試験例Ｂ１：ペプチド複合体の細胞膜透過能評価）
実施例Ｂ１２で合成したペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩、比較例Ｂ１で合成したペプチド複合体Ｇ６－Ｐ、及び比較例Ｂ２で合成したペプチド複合体Ｇ９－Ｐのいずれかを２μＭを含む細胞培養液中、５％（ｖ／ｖ）ＣＯ２、３７℃の条件下で、ＨｅＬａ細胞（Ｈｕｍａｎ ｃｅｒｖｉｘ ａｄｅｎｏｃａｒｃｉｎｏｍａ ｃｅｌｌ）を４時間培養した。ＨｅＬａ細胞の培養には、ＦｌｕｏｒｏＢｒｉｔｅ Ｄ－ＭＥＭ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）（１０％（ｖ／ｖ）ＦＣＳ（ウシ胎児血清）、１０％（ｖ／ｖ）ＧｌｕｔａＭａｘ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）添加）を用いた。
次いで、Ｄ－ＰＢＳ（－）（ヘパリン（２０ｕｎｉｔｓ／ｍＬ）添加）で細胞表面を洗浄後、細胞を回収し、Ｄ－ＰＢＳ（－）（０．５％（ｖ／ｖ）ＢＳＡ（ウシ血清アルブミン）、２００ｍＭ ＥＤＴＡ（エチレンジアミン四酢酸）、０．２％（ｖ／ｖ）ヨウ化プロピジウム（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）添加）に懸濁させ、フローサイトメーター（ＢＤ ＦＡＣＳ ＡｒｉａＩＩＩ）を用いて蛍光強度を測定した。フローサイトメトリー解析時には、ヨウ化プロピジウム陽性の死細胞を除いた生細胞集団に対し、蛍光強度最頻値を算出した。結果を表４に示す。 (Test Example B1: Evaluation of cell membrane permeability of peptide complex)
Contains 2 μM of any one of the peptide complex G3-P trifluoroacetate synthesized in Example B12, the peptide complex G6-P synthesized in Comparative Example B1, and the peptide complex G9-P synthesized in Comparative Example B2. HeLa cells (Human cervix adenocarcinoma cell) were cultured for 4 hours in a cell culture medium under the conditions of 5% (v / v) CO 2 and 37 ° C. For the culture of HeLa cells, FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum) added) and 10% (v / v) GlutaMax (manufactured by Thermo Fisher) was added).
Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected. ), 200 mM EDTA (ethylenediaminetetraacetic acid), 0.2% (v / v) propidium iodide (manufactured by Sigma-Aldrich) was suspended), and the fluorescence intensity was measured using a flow cytometer (BD FACS Maria III). It was measured. At the time of flow cytometry analysis, the mode of fluorescence intensity was calculated for the living cell population excluding the dead cells positive for propidium iodide. The results are shown in Table 4.
実施例Ｂ１２で合成したペプチド複合体Ｇ３－Ｐトリフルオロ酢酸塩、比較例Ｂ１で合成したペプチド複合体Ｇ６－Ｐ、及び比較例Ｂ２で合成したペプチド複合体Ｇ９－Ｐのいずれかを２μＭを含む細胞培養液中、５％（ｖ／ｖ）ＣＯ２、３７℃の条件下で、ＨｅＬａ細胞（Ｈｕｍａｎ ｃｅｒｖｉｘ ａｄｅｎｏｃａｒｃｉｎｏｍａ ｃｅｌｌ）を４時間培養した。ＨｅＬａ細胞の培養には、ＦｌｕｏｒｏＢｒｉｔｅ Ｄ－ＭＥＭ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）（１０％（ｖ／ｖ）ＦＣＳ（ウシ胎児血清）、１０％（ｖ／ｖ）ＧｌｕｔａＭａｘ（ＴｈｅｒｍｏＦｉｓｈｅｒ社製）添加）を用いた。
次いで、Ｄ－ＰＢＳ（－）（ヘパリン（２０ｕｎｉｔｓ／ｍＬ）添加）で細胞表面を洗浄後、細胞を回収し、Ｄ－ＰＢＳ（－）（０．５％（ｖ／ｖ）ＢＳＡ（ウシ血清アルブミン）、２００ｍＭ ＥＤＴＡ（エチレンジアミン四酢酸）、０．２％（ｖ／ｖ）ヨウ化プロピジウム（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）添加）に懸濁させ、フローサイトメーター（ＢＤ ＦＡＣＳ ＡｒｉａＩＩＩ）を用いて蛍光強度を測定した。フローサイトメトリー解析時には、ヨウ化プロピジウム陽性の死細胞を除いた生細胞集団に対し、蛍光強度最頻値を算出した。結果を表４に示す。 (Test Example B1: Evaluation of cell membrane permeability of peptide complex)
Contains 2 μM of any one of the peptide complex G3-P trifluoroacetate synthesized in Example B12, the peptide complex G6-P synthesized in Comparative Example B1, and the peptide complex G9-P synthesized in Comparative Example B2. HeLa cells (Human cervix adenocarcinoma cell) were cultured for 4 hours in a cell culture medium under the conditions of 5% (v / v) CO 2 and 37 ° C. For the culture of HeLa cells, FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum) added) and 10% (v / v) GlutaMax (manufactured by Thermo Fisher) was added).
Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected. ), 200 mM EDTA (ethylenediaminetetraacetic acid), 0.2% (v / v) propidium iodide (manufactured by Sigma-Aldrich) was suspended), and the fluorescence intensity was measured using a flow cytometer (BD FACS Maria III). It was measured. At the time of flow cytometry analysis, the mode of fluorescence intensity was calculated for the living cell population excluding the dead cells positive for propidium iodide. The results are shown in Table 4.
国際出願番号：ＰＣＴ／ＪＰ２０２０／００５７６２においては、ペプチド複合体の細胞膜透過能は、Ｇ３－Ｐ、Ｇ６－Ｐ及びＧ９－Ｐの順に高くなり、グアニジル基を有する樹状骨格の数が多いほど膜透過性が大きかった。一方、表４に示すように、培養開始４時間後におけるＧ３－Ｐトリフルオロ酢酸塩の細胞膜透過能は、Ｇ６－ＰやＧ９－Ｐと比較して顕著に高かった。このことから、酸との塩の形態をとることでＧ３－Ｐは、Ｇ６－Ｐ及びＧ９－Ｐと同等以上の細胞膜透過性を示すことが明らかとなった。
分子量４，０００Ｄａを超える分子は免疫原性を引き起こす可能性があり、分子量が１，０００を超えるペプチドと結合させて用いる細胞膜透過性分子は、分子量はより小さいものが望ましい。そのため、医薬品製造においては、Ｇ６－ＰやＧ９－Ｐよりも分子量の小さいＧ３－Ｐを使用するのが好ましい。また、上述したように、Ｇ３－Ｐを酸との塩の形態にすることで、膜透過能を顕著に向上させることが可能となり、Ｇ３－Ｐが課題としていた細胞膜透過能の不足についても克服できたといえる。つまり本発明の細胞膜透過性分子の細胞膜透過性の向上方法は、医薬品としてのペプチドを含むペプチド複合体の細胞膜透過性能を向上することができる有用な方法であるといえる。 In international application number: PCT / JP2020 / 005762, the cell membrane permeability of the peptide complex increases in the order of G3-P, G6-P and G9-P, and the larger the number of dendritic skeletons having guanidyl groups, the more the membrane. The permeability was high. On the other hand, as shown in Table 4, the cell membrane permeability of G3-P trifluoroacetic acid salt 4 hours after the start of culture was significantly higher than that of G6-P and G9-P. From this, it was clarified that G3-P exhibits cell membrane permeability equal to or higher than that of G6-P and G9-P by taking the form of a salt with an acid.
Molecules with a molecular weight of more than 4,000 Da may cause immunogenicity, and it is desirable that the cell membrane-permeable molecule used in combination with a peptide having a molecular weight of more than 1,000 has a smaller molecular weight. Therefore, in the manufacture of pharmaceutical products, it is preferable to use G3-P having a smaller molecular weight than G6-P or G9-P. Further, as described above, by forming G3-P in the form of a salt with an acid, it is possible to remarkably improve the membrane permeability, and overcome the problem of lack of cell membrane permeability that G3-P has been a problem. It can be said that it was possible. That is, it can be said that the method for improving the cell membrane permeability of the cell membrane-permeable molecule of the present invention is a useful method capable of improving the cell membrane permeability of the peptide complex containing the peptide as a pharmaceutical product.
分子量４，０００Ｄａを超える分子は免疫原性を引き起こす可能性があり、分子量が１，０００を超えるペプチドと結合させて用いる細胞膜透過性分子は、分子量はより小さいものが望ましい。そのため、医薬品製造においては、Ｇ６－ＰやＧ９－Ｐよりも分子量の小さいＧ３－Ｐを使用するのが好ましい。また、上述したように、Ｇ３－Ｐを酸との塩の形態にすることで、膜透過能を顕著に向上させることが可能となり、Ｇ３－Ｐが課題としていた細胞膜透過能の不足についても克服できたといえる。つまり本発明の細胞膜透過性分子の細胞膜透過性の向上方法は、医薬品としてのペプチドを含むペプチド複合体の細胞膜透過性能を向上することができる有用な方法であるといえる。 In international application number: PCT / JP2020 / 005762, the cell membrane permeability of the peptide complex increases in the order of G3-P, G6-P and G9-P, and the larger the number of dendritic skeletons having guanidyl groups, the more the membrane. The permeability was high. On the other hand, as shown in Table 4, the cell membrane permeability of G3-P trifluoroacetic acid salt 4 hours after the start of culture was significantly higher than that of G6-P and G9-P. From this, it was clarified that G3-P exhibits cell membrane permeability equal to or higher than that of G6-P and G9-P by taking the form of a salt with an acid.
Molecules with a molecular weight of more than 4,000 Da may cause immunogenicity, and it is desirable that the cell membrane-permeable molecule used in combination with a peptide having a molecular weight of more than 1,000 has a smaller molecular weight. Therefore, in the manufacture of pharmaceutical products, it is preferable to use G3-P having a smaller molecular weight than G6-P or G9-P. Further, as described above, by forming G3-P in the form of a salt with an acid, it is possible to remarkably improve the membrane permeability, and overcome the problem of lack of cell membrane permeability that G3-P has been a problem. It can be said that it was possible. That is, it can be said that the method for improving the cell membrane permeability of the cell membrane-permeable molecule of the present invention is a useful method capable of improving the cell membrane permeability of the peptide complex containing the peptide as a pharmaceutical product.
本発明の態様としては、例えば、以下のものなどが挙げられる。
＜１＞ 下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩であることを特徴とする細胞膜透過性分子である。
（前記一般式（Ｉ）中、Ｒは結合手を表す。）。
＜２＞ 前記一般式（Ｉ）で表される構造の前記Ｒの部分が、ヘテロ原子含有基と結合している前記＜１＞に記載の細胞膜透過性分子である。
＜３＞ 前記ヘテロ原子含有基におけるヘテロ原子の含有割合が、１０％以上である前記＜２＞に記載の細胞膜透過性分子である。
＜４＞ 前記ヘテロ原子含有基におけるヘテロ原子が、酸素原子、窒素原子、硫黄原子及びリン原子からなる群から選択される少なくとも１種である前記＜２＞から＜３＞のいずれかに記載の細胞膜透過性分子である。
＜５＞ 前記ヘテロ原子含有基が、繰返し単位を含む前記＜２＞から＜４＞のいずれかに記載の細胞膜透過性分子である。
＜６＞ 前記繰返し単位が、アルキレンオキサイドである前記＜５＞に記載の細胞膜透過性分子である。
＜７＞ 前記アルキレンオキサイドが、メチレンオキサイド、エチレンオキサイド及びプロピレンオキサイドからなる群から選択される少なくとも１種である前記＜６＞に記載の細胞膜透過性分子である。
＜８＞ 前記ｐＫａの値が４．７未満の酸が、トリフルオロ酢酸、塩酸、硫酸、硝酸、リン酸、メシル酸、トシル酸、酒石酸及びクエン酸からなる群から選択されるいずれかである前記＜１＞から＜７＞のいずれかに記載の細胞膜透過性分子である。
＜９＞ ペプチドと、前記＜１＞から＜８＞のいずれかに記載の細胞膜透過性分子とを含むことを特徴とするペプチド複合体である。
＜１０＞ 前記＜９＞に記載のペプチド複合体を含むことを特徴とするペプチドライブラリである。
＜１１＞ ペプチドに、前記＜１＞から＜８＞のいずれかに記載の細胞膜透過性分子を導入することを含むことを特徴とするペプチド複合体の製造方法である。
＜１２＞ 前記ペプチドが、ペプチドライブラリに含まれるペプチドである前記＜１１＞に記載のペプチド複合体の製造方法である。
＜１３＞ 前記＜１０＞に記載のペプチドライブラリを用いて機能性ペプチドをスクリーニングすることを含むことを特徴とする機能性ペプチドのスクリーニング方法である。
＜１４＞ ペプチドに、前記＜１＞から＜８＞のいずれかに記載の細胞膜透過性分子を導入することを含む前記＜１３＞に記載の機能性ペプチドのスクリーニング方法である。
＜１５＞ 下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とすることを含むことを特徴とする細胞膜透過性分子の細胞膜透過性の向上方法である。
（前記一般式（Ｉ）中、Ｒは結合手を表す。）。
＜１６＞ 前記一般式（Ｉ）で表される構造の前記Ｒの部分が、ヘテロ原子含有基と結合している前記＜１５＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜１７＞ 前記ヘテロ原子含有基におけるヘテロ原子の含有割合が、１０％以上である前記＜１６＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜１８＞ 前記ヘテロ原子含有基におけるヘテロ原子が、酸素原子、窒素原子、硫黄原子及びリン原子からなる群から選択される少なくとも１種である前記＜１６＞から＜１７＞のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜１９＞ 前記ヘテロ原子含有基が、繰返し単位を含む前記＜１６＞から＜１８＞のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜２０＞ 前記繰返し単位が、アルキレンオキサイドである前記＜１９＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜２１＞ 前記アルキレンオキサイドが、メチレンオキサイド、エチレンオキサイド及びプロピレンオキサイドからなる群から選択される少なくとも１種である前記＜２０＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜２２＞ 前記細胞膜透過性分子がペプチドに導入されたものである前記＜１５＞から＜２１＞のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法である。 Examples of aspects of the present invention include the following.
<1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid.
(In the general formula (I), R represents a bond).
<2> The cell membrane-permeable molecule according to <1>, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group.
<3> The cell membrane-permeable molecule according to <2>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
<4> The above-mentioned <2> to <3>, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a cell membrane permeable molecule.
<5> The cell membrane-permeable molecule according to any one of <2> to <4>, wherein the heteroatom-containing group contains a repeating unit.
<6> The cell membrane permeable molecule according to <5>, wherein the repeating unit is an alkylene oxide.
<7> The cell membrane-permeable molecule according to <6>, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide, and propylene oxide.
<8> The acid having a pKa value of less than 4.7 is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartaric acid and citric acid. The cell membrane permeable molecule according to any one of <1> to <7>.
<9> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to any one of <1> to <8>.
<10> A peptide library comprising the peptide complex according to <9>.
<11> A method for producing a peptide complex, which comprises introducing the cell membrane-permeable molecule according to any one of <1> to <8> into a peptide.
<12> The method for producing a peptide complex according to <11>, wherein the peptide is a peptide contained in a peptide library.
<13> A method for screening a functional peptide, which comprises screening a functional peptide using the peptide library according to <10>.
<14> The method for screening a functional peptide according to <13>, which comprises introducing the cell membrane permeable molecule according to any one of <1> to <8> into the peptide.
<15> A method for improving the cell membrane permeability of a cell membrane-permeable molecule, which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid.
(In the general formula (I), R represents a bond).
<16> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <15>, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group. ..
<17> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <16>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
<18> The above-mentioned <16> to <17>, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a method for improving the cell membrane permeability of a cell membrane permeability molecule.
<19> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of <16> to <18>, wherein the heteroatom-containing group contains a repeating unit.
<20> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <19>, wherein the repeating unit is an alkylene oxide.
<21> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <20>, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide, and propylene oxide.
<22> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of <15> to <21>, wherein the cell membrane-permeable molecule is introduced into a peptide.
＜１＞ 下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩であることを特徴とする細胞膜透過性分子である。
＜２＞ 前記一般式（Ｉ）で表される構造の前記Ｒの部分が、ヘテロ原子含有基と結合している前記＜１＞に記載の細胞膜透過性分子である。
＜３＞ 前記ヘテロ原子含有基におけるヘテロ原子の含有割合が、１０％以上である前記＜２＞に記載の細胞膜透過性分子である。
＜４＞ 前記ヘテロ原子含有基におけるヘテロ原子が、酸素原子、窒素原子、硫黄原子及びリン原子からなる群から選択される少なくとも１種である前記＜２＞から＜３＞のいずれかに記載の細胞膜透過性分子である。
＜５＞ 前記ヘテロ原子含有基が、繰返し単位を含む前記＜２＞から＜４＞のいずれかに記載の細胞膜透過性分子である。
＜６＞ 前記繰返し単位が、アルキレンオキサイドである前記＜５＞に記載の細胞膜透過性分子である。
＜７＞ 前記アルキレンオキサイドが、メチレンオキサイド、エチレンオキサイド及びプロピレンオキサイドからなる群から選択される少なくとも１種である前記＜６＞に記載の細胞膜透過性分子である。
＜８＞ 前記ｐＫａの値が４．７未満の酸が、トリフルオロ酢酸、塩酸、硫酸、硝酸、リン酸、メシル酸、トシル酸、酒石酸及びクエン酸からなる群から選択されるいずれかである前記＜１＞から＜７＞のいずれかに記載の細胞膜透過性分子である。
＜９＞ ペプチドと、前記＜１＞から＜８＞のいずれかに記載の細胞膜透過性分子とを含むことを特徴とするペプチド複合体である。
＜１０＞ 前記＜９＞に記載のペプチド複合体を含むことを特徴とするペプチドライブラリである。
＜１１＞ ペプチドに、前記＜１＞から＜８＞のいずれかに記載の細胞膜透過性分子を導入することを含むことを特徴とするペプチド複合体の製造方法である。
＜１２＞ 前記ペプチドが、ペプチドライブラリに含まれるペプチドである前記＜１１＞に記載のペプチド複合体の製造方法である。
＜１３＞ 前記＜１０＞に記載のペプチドライブラリを用いて機能性ペプチドをスクリーニングすることを含むことを特徴とする機能性ペプチドのスクリーニング方法である。
＜１４＞ ペプチドに、前記＜１＞から＜８＞のいずれかに記載の細胞膜透過性分子を導入することを含む前記＜１３＞に記載の機能性ペプチドのスクリーニング方法である。
＜１５＞ 下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とすることを含むことを特徴とする細胞膜透過性分子の細胞膜透過性の向上方法である。
＜１６＞ 前記一般式（Ｉ）で表される構造の前記Ｒの部分が、ヘテロ原子含有基と結合している前記＜１５＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜１７＞ 前記ヘテロ原子含有基におけるヘテロ原子の含有割合が、１０％以上である前記＜１６＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜１８＞ 前記ヘテロ原子含有基におけるヘテロ原子が、酸素原子、窒素原子、硫黄原子及びリン原子からなる群から選択される少なくとも１種である前記＜１６＞から＜１７＞のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜１９＞ 前記ヘテロ原子含有基が、繰返し単位を含む前記＜１６＞から＜１８＞のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜２０＞ 前記繰返し単位が、アルキレンオキサイドである前記＜１９＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜２１＞ 前記アルキレンオキサイドが、メチレンオキサイド、エチレンオキサイド及びプロピレンオキサイドからなる群から選択される少なくとも１種である前記＜２０＞に記載の細胞膜透過性分子の細胞膜透過性の向上方法である。
＜２２＞ 前記細胞膜透過性分子がペプチドに導入されたものである前記＜１５＞から＜２１＞のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法である。 Examples of aspects of the present invention include the following.
<1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid.
<2> The cell membrane-permeable molecule according to <1>, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group.
<3> The cell membrane-permeable molecule according to <2>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
<4> The above-mentioned <2> to <3>, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a cell membrane permeable molecule.
<5> The cell membrane-permeable molecule according to any one of <2> to <4>, wherein the heteroatom-containing group contains a repeating unit.
<6> The cell membrane permeable molecule according to <5>, wherein the repeating unit is an alkylene oxide.
<7> The cell membrane-permeable molecule according to <6>, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide, and propylene oxide.
<8> The acid having a pKa value of less than 4.7 is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartaric acid and citric acid. The cell membrane permeable molecule according to any one of <1> to <7>.
<9> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to any one of <1> to <8>.
<10> A peptide library comprising the peptide complex according to <9>.
<11> A method for producing a peptide complex, which comprises introducing the cell membrane-permeable molecule according to any one of <1> to <8> into a peptide.
<12> The method for producing a peptide complex according to <11>, wherein the peptide is a peptide contained in a peptide library.
<13> A method for screening a functional peptide, which comprises screening a functional peptide using the peptide library according to <10>.
<14> The method for screening a functional peptide according to <13>, which comprises introducing the cell membrane permeable molecule according to any one of <1> to <8> into the peptide.
<15> A method for improving the cell membrane permeability of a cell membrane-permeable molecule, which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid.
<16> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <15>, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group. ..
<17> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <16>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
<18> The above-mentioned <16> to <17>, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a method for improving the cell membrane permeability of a cell membrane permeability molecule.
<19> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of <16> to <18>, wherein the heteroatom-containing group contains a repeating unit.
<20> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <19>, wherein the repeating unit is an alkylene oxide.
<21> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to <20>, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide, and propylene oxide.
<22> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of <15> to <21>, wherein the cell membrane-permeable molecule is introduced into a peptide.
Claims (22)
- 下記一般式（Ｉ）で表される構造を有し、ｐＫａの値が４．７未満の酸との塩であることを特徴とする細胞膜透過性分子：
- 前記一般式（Ｉ）で表される構造の前記Ｒの部分が、ヘテロ原子含有基と結合している請求項１に記載の細胞膜透過性分子。 The cell membrane-permeable molecule according to claim 1, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group.
- 前記ヘテロ原子含有基におけるヘテロ原子の含有割合が、１０％以上である請求項２に記載の細胞膜透過性分子。 The cell membrane-permeable molecule according to claim 2, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
- 前記ヘテロ原子含有基におけるヘテロ原子が、酸素原子、窒素原子、硫黄原子及びリン原子からなる群から選択される少なくとも１種である請求項２から３のいずれかに記載の細胞膜透過性分子。 The cell membrane permeable molecule according to any one of claims 2 to 3, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom.
- 前記ヘテロ原子含有基が、繰返し単位を含む請求項２から４のいずれかに記載の細胞膜透過性分子。 The cell membrane-permeable molecule according to any one of claims 2 to 4, wherein the heteroatom-containing group contains a repeating unit.
- 前記繰返し単位が、アルキレンオキサイドである請求項５に記載の細胞膜透過性分子。 The cell membrane-permeable molecule according to claim 5, wherein the repeating unit is an alkylene oxide.
- 前記アルキレンオキサイドが、メチレンオキサイド、エチレンオキサイド及びプロピレンオキサイドからなる群から選択される少なくとも１種である請求項６に記載の細胞膜透過性分子。 The cell membrane permeable molecule according to claim 6, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide and propylene oxide.
- 前記ｐＫａの値が４．７未満の酸が、トリフルオロ酢酸、塩酸、硫酸、硝酸、リン酸、メシル酸、トシル酸、酒石酸及びクエン酸からなる群から選択されるいずれかである請求項１から７のいずれかに記載の細胞膜透過性分子。 Claim that the acid having a pK a value of less than 4.7 is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartaric acid and citric acid. The cell membrane permeable molecule according to any one of 1 to 7.
- ペプチドと、請求項１から８のいずれかに記載の細胞膜透過性分子とを含むことを特徴とするペプチド複合体。 A peptide complex comprising a peptide and the cell membrane-permeable molecule according to any one of claims 1 to 8.
- 請求項９に記載のペプチド複合体を含むことを特徴とするペプチドライブラリ。 A peptide library comprising the peptide complex according to claim 9.
- ペプチドに、請求項１から８のいずれかに記載の細胞膜透過性分子を導入することを含むことを特徴とするペプチド複合体の製造方法。 A method for producing a peptide complex, which comprises introducing the cell membrane permeable molecule according to any one of claims 1 to 8 into a peptide.
- 前記ペプチドが、ペプチドライブラリに含まれるペプチドである請求項１１に記載のペプチド複合体の製造方法。 The method for producing a peptide complex according to claim 11, wherein the peptide is a peptide contained in a peptide library.
- 請求項１０に記載のペプチドライブラリを用いて機能性ペプチドをスクリーニングすることを含むことを特徴とする機能性ペプチドのスクリーニング方法。 A method for screening a functional peptide, which comprises screening a functional peptide using the peptide library according to claim 10.
- ペプチドに、請求項１から８のいずれかに記載の細胞膜透過性分子を導入することを含む請求項１３に記載の機能性ペプチドのスクリーニング方法。 The method for screening a functional peptide according to claim 13, which comprises introducing the cell membrane-permeable molecule according to any one of claims 1 to 8 into the peptide.
- 下記一般式（Ｉ）で表される構造を有する細胞膜透過性分子を、酸との塩とすることを含むことを特徴とする細胞膜透過性分子の細胞膜透過性の向上方法：
- 前記一般式（Ｉ）で表される構造の前記Ｒの部分が、ヘテロ原子含有基と結合している請求項１５に記載の細胞膜透過性分子の細胞膜透過性の向上方法。 The method for improving the cell membrane permeability of a cell membrane-permeable molecule according to claim 15, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group.
- 前記ヘテロ原子含有基におけるヘテロ原子の含有割合が、１０％以上である請求項１６に記載の細胞膜透過性分子の細胞膜透過性の向上方法。 The method for improving the cell membrane permeability of a cell membrane-permeable molecule according to claim 16, wherein the heteroatom content ratio in the heteroatom-containing group is 10% or more.
- 前記ヘテロ原子含有基におけるヘテロ原子が、酸素原子、窒素原子、硫黄原子及びリン原子からなる群から選択される少なくとも１種である請求項１６から１７のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法。 The cell membrane of the cell membrane permeable molecule according to any one of claims 16 to 17, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. How to improve transparency.
- 前記ヘテロ原子含有基が、繰返し単位を含む請求項１６から１８のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法。 The method for improving the cell membrane permeability of a cell membrane-permeable molecule according to any one of claims 16 to 18, wherein the heteroatom-containing group contains a repeating unit.
- 前記繰返し単位が、アルキレンオキサイドである請求項１９に記載の細胞膜透過性分子の細胞膜透過性の向上方法。 The method for improving the cell membrane permeability of a cell membrane-permeable molecule according to claim 19, wherein the repeating unit is an alkylene oxide.
- 前記アルキレンオキサイドが、メチレンオキサイド、エチレンオキサイド及びプロピレンオキサイドからなる群から選択される少なくとも１種である請求項２０に記載の細胞膜透過性分子の細胞膜透過性の向上方法。 The method for improving the cell membrane permeability of a cell membrane-permeable molecule according to claim 20, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide and propylene oxide.
- 前記細胞膜透過性分子がペプチドに導入されたものである請求項１５から２１のいずれかに記載の細胞膜透過性分子の細胞膜透過性の向上方法。
The method for improving cell membrane permeability of a cell membrane-permeable molecule according to any one of claims 15 to 21, wherein the cell membrane-permeable molecule is introduced into a peptide.
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