WO2016148125A1 - Nouveau composé de sphingomyéline marqué par fluorescence et utilisation de celui-ci - Google Patents

Nouveau composé de sphingomyéline marqué par fluorescence et utilisation de celui-ci Download PDF

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WO2016148125A1
WO2016148125A1 PCT/JP2016/058079 JP2016058079W WO2016148125A1 WO 2016148125 A1 WO2016148125 A1 WO 2016148125A1 JP 2016058079 W JP2016058079 W JP 2016058079W WO 2016148125 A1 WO2016148125 A1 WO 2016148125A1
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compound
group
formula
independently
raft
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Japanese (ja)
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道雄 村田
信明 松森
祥尚 木下
明弘 楠見
鈴木　健一
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国立大学法人大阪大学
国立大学法人京都大学
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Publication of WO2016148125A1 publication Critical patent/WO2016148125A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/335—Polymers modified by chemical after-treatment with organic compounds containing phosphorus
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to a novel labeled fluorescent sphingomyelin and use thereof.
  • SM sphingomyelin
  • Non-Patent Literature 1 SM is an essential component of lipid rafts, direct information regarding SM behavior has not yet been obtained, particularly in biological membranes.
  • CARS coherent anti-Stokes Raman scattering
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DOPC dioleoylphosphatidylcholine
  • Fluorescence microscopy is a useful tool for observing lipid distribution and domain formation. Furthermore, recent advances in SFMT (fluorescence single molecule tracking method) have made it possible to obtain detailed information on behavior and interactions as well as the distribution of biomolecules at the nanoscale.
  • the simplest approach for examining the behavior of SM in such microscopic observation is to use a fluorescently labeled SM that is confirmed to behave similarly to the original SM. Many fluorescently-labeled SMs have already been synthesized and sold in part, but these fluorescently-labeled SMs hardly reflect the behavior of the original SM.
  • Non-Patent Documents 2 and 3 preferentially binds to a non-raft-like L d phase rather than the raft-like L o phase in the phase separation membrane. It is conceivable that a large fluorescent substance (fluorophore) bound to SM causes steric hindrance, which may impair lipid packing or be excluded from the domain of the raft-like structure (Non-patent Document 4). Inevitably, the apparent properties of rafts vary greatly, especially in living cells (Non-Patent Documents 5 and 6). Instead, lysenin is used as a fluorescent probe for SM.
  • fluorophore fluorophore
  • Non-patent Document 7 lysenin binds to the SM cluster and the endogenous tryptophan residue functions as a fluorophore.
  • Non-patent Document 8 one potential problem with using proteins as lipid probes is the difference in size between lipids and proteins.
  • the molecular weight of lysenin is 30 times the molecular weight of general phospholipids, and may change the properties of the membrane (Non-patent Document 8).
  • lipid rafts (less than 200 to 300 nm) of biological membranes are smaller than lipid rafts (over 1 ⁇ m) of artificial membranes, so that they are hardly visible with ordinary fluorescence microscopy. Due to such problems in imaging, it was not possible to obtain direct information on SM behavior and the role of SM in rafting.
  • An object of the present invention is to provide a new fluorescently labeled sphingomyelin that exhibits the same behavior as the original sphingomyelin.
  • An object of the present invention is to provide a lipid raft visualization agent, a diagnostic marker for lipid raft-related diseases, a reagent for detection or quantification of lipid rafts, and a lipid raft visualization method.
  • the present invention relates to the following inventions. [1] The following formula (1)
  • A represents a residue of a water-soluble fluorescent coloring compound
  • B and E represent the same or different linkers
  • R 1 to R 4 each independently represents a hydrogen atom or a substituent
  • X represents An integer of 3 to 50
  • m and n are each independently an integer of 10 to 30, and the following formula (2)
  • G 1 to G 7 are each independently a hydrogen atom or a water-soluble functional group
  • p is 4
  • R 5 to R 8 are each independently a hydrogen atom or a substituent, provided that The compound according to [1] above, wherein at least one of G 1 to G 6 and four G 7 is a water-soluble functional group.
  • B represents the following formula (4)
  • B 1c and B 2c are each independently —CONR 9 — or —COO—
  • B 1b and B 2b are each independently a C 1-20 alkylene group
  • R 9 to R 14 each independently represents a hydrogen atom or a substituent, 1d, 1e, 2d and 2e are each independently 0 or 1, and 1f and 2f are each independently Or an integer of 0 to 3).
  • E represents the following formula (11)
  • E 1c and E 2c are each independently —CONR 15 — or —COO—
  • E 1b and E 2b are each independently an alkylene group
  • E3 is —CONR 16 —, —COOCO—, —COO—, —NHCOO—, —O—, —OCO—, —C ⁇ C—, —CH ⁇ CH—, an alkylene group, —S—, —SO—, —SO 2 —, —NR 17 —, — NR 18 CO—, —NR 19 CONR 20 —, the following formula (5)
  • R 15 to R 20 each independently represents a hydrogen atom or a substituent
  • 1g, 1h, 2g and 2h are each independently 0 or 1
  • 1i and 2i are each independently The compound according to any one of the above [1] to [3], which is a group represented by [5]
  • a lipid raft visualization agent containing the compound according to any one of [1] to [4].
  • [6] A lipid raft-related disease diagnostic marker containing the compound according to any one of [1] to [4] or the agent according to [5].
  • [7] A lipid raft detection or quantification reagent containing the compound according to any one of [1] to [4] or the agent according to [5].
  • a lipid raft comprising a step of mixing the compound according to any one of [1] to [4] above with a cell containing a lipid raft, and a step of detecting fluorescence by irradiating the cell with light. Visualization method.
  • the present invention can provide a new fluorescently labeled sphingomyelin that exhibits the same behavior as the original sphingomyelin.
  • the present invention can provide a lipid raft visualization agent, a diagnostic marker for lipid raft-related diseases, a reagent for detection or quantification of lipid rafts, and a lipid raft visualization method.
  • FIG. 1 shows a fluorescence micrograph of GUV containing compound 5.
  • FIG. 2 shows a GUV fluorescence micrograph containing TX-red DHPE.
  • FIG. 3 shows a photograph in which FIGS. 1 and 2 are superimposed.
  • FIG. 4 shows a fluorescence micrograph of GUV containing compound 6.
  • FIG. 5 shows a fluorescence micrograph of GUV containing Bodipy-PC.
  • FIG. 6 shows a photograph in which FIGS. 4 and 5 are superimposed.
  • FIG. 7 shows a confocal laser micrograph of a cross section of GUV containing compound 5.
  • FIG. 8 shows the fluorescence intensity distribution of the cross section of GUV containing Compound 5.
  • FIG. 9 shows a confocal laser micrograph of a cross section of GUV containing compound 6.
  • FIG. 10 shows the fluorescence intensity distribution of the cross section of GUV containing Compound 6.
  • FIG. 11 shows the ratio of the fluorescence intensity of the raft-like phase / the fluorescence intensity of the non-raft-like phase in the GUV cross section.
  • FIG. 12 shows a confocal laser micrograph (FRET method) of a cross section of one GUV containing compounds 5 and 6.
  • FIG. 13 shows the fluorescence intensity distribution (FRET method) of the cross section of GUV containing compounds 5 and 6.
  • FIG. 14 shows a confocal laser scanning micrograph (FRET method) of a cross section of GUV containing a plurality of compounds 5 and 6.
  • FIG. 15 shows a fluorescence micrograph when compound 6 is added to one erythrocyte.
  • FIG. 16 shows a fluorescence micrograph (FRET method) when compounds 5 and 6 are added to one red blood cell.
  • FIG. 17 shows a differential interference microscopic photograph of an artificially formed echinosite obtained by adding compound 6 to a plurality of red blood cells.
  • FIG. 18 shows a fluorescence micrograph of an echinocyte artificially formed by adding Compound 6 to a plurality of red blood cells.
  • FIG. 19 shows a photograph in which FIGS. 17 and 18 are superimposed.
  • FIG. 20 shows the time course of diffusion movement of two molecules of compound 6 in CHO-K1 cells.
  • FIG. 21 shows the results of quantifying the colocalization of compound 6 in the raft region.
  • FIG. 22 shows the result of quantifying the colocalization of ATTO594-DOPE in the raft region.
  • FIG. 23 shows a fluorescence micrograph of compound 6 added to an erythrocyte ghost membrane.
  • FIG. 24 shows a fluorescence micrograph after treatment of the erythrocyte ghost membrane of FIG. 23 with a surfactant TX-100.
  • FIG. 25 shows a fluorescence micrograph of compound 6 added to a CHO—K1 ghost film.
  • FIG. 26 shows a fluorescence micrograph after the CHO-K1 ghost film of FIG. 25 has been treated with a surfactant TX-100.
  • FIG. 27 shows a fluorescence micrograph of compound 6 added to an ECV304 ghost membrane.
  • FIG. 28 shows a fluorescence micrograph after the ECV304 ghost membrane of FIG. 27 has been treated with a surfactant TX-100.
  • FIG. 29 shows an NMR chart of Compound 5 subjected to one-dimensional data processing.
  • FIG. 30 shows an NMR chart of Compound 5 that has been subjected to two-dimensional data processing.
  • FIG. 31 shows the HRMS chart of Compound 5.
  • FIG. 32 shows an NMR chart of Compound 6 subjected to one-dimensional data processing.
  • FIG. 33 shows an NMR chart of Compound 6 that has been subjected to two-dimensional data processing.
  • FIG. 34 shows the HRMS chart of Compound 6.
  • FIG. 35 shows a differential interference micrograph of echinosite.
  • FIG. 36 shows the distribution of CD59 labeled with the fluorescent antibody Cy3-IgG (confocal laser micrograph).
  • FIG. 37 shows a confocal laser micrograph in which compound 5 was added to the echinosite film.
  • FIG. 38 shows a photograph in which FIGS. 36 and 37 are superimposed.
  • FIG. 39 shows the distribution (confocal fluorescence micrograph) of compound 5 in the early stage of echinosite.
  • FIG. 40 shows the distribution (confocal fluorescence microscope image) of GPI-anchored CD59 labeled with Cy3-IgG at the early stage of echinosite.
  • FIG. 41 shows a differential interference micrograph of the initial echinosite.
  • FIG. 42 shows a photograph in which FIGS. 39 to 41 are superimposed.
  • the present invention includes a compound represented by the following formula (1) (hereinafter also referred to as the compound of the present invention).
  • the group represented by the following formula (2) is a single bond or a double bond.
  • A represents a residue of the water-soluble fluorescent coloring compound.
  • the water-soluble fluorescent coloring compound is, for example, a compound in which an electron excitation source emits light by an electromagnetic wave having a wavelength shorter than that of visible light.
  • the water-soluble fluorescent coloring compound is not particularly limited, and examples thereof include a fluorescent coloring compound having a water-soluble functional group such as a sulfonic acid group (sulfo group), a carboxyl group, and a quaternary amine. It is preferably a group (sulfo group).
  • the number of water-soluble functional groups possessed by the water-soluble fluorescent coloring compound is not particularly limited, but is preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3.
  • Examples of the water-soluble fluorescent coloring compound represented by A include compounds having a rhodamine skeleton, an acridine skeleton, a cyanine skeleton, a fluorescein skeleton, an oxazine skeleton, a phenanthridine skeleton, etc., among which a compound having a rhodamine skeleton is preferable.
  • the water-soluble fluorescent coloring compound is preferably ATTO-488 (trade name) or ATTO-594 (trade name) sold by ATTO-TEC GmbH.
  • Examples of the residue of the water-soluble fluorescent coloring compound represented by A include the following formula (3): (In the formula, G 1 to G 7 are each independently a hydrogen atom or a water-soluble functional group, p is 4, and R 5 to R 8 are each independently a hydrogen atom or a substituent, provided that It is preferable that at least one of G 1 to G 6 and four G 7 is a water-soluble functional group.
  • examples of the water-soluble functional group include the groups exemplified above such as a sulfonic acid group.
  • the number of water-soluble functional groups can also be selected from the same range as described above (for example, about 1 to 10, preferably about 1 to 4).
  • G 3 and G 4 are sulfonic acid groups (sulfo groups), and G 1 , G 2 , G 5 , G 6 and G 7 are hydrogen atoms.
  • substituents include an aliphatic group and an aromatic group.
  • the aliphatic group include an alkyl group and a cycloalkyl group (for example, a cyclohexyl group).
  • alkyl group examples include C 1-20 alkyl groups such as methyl group, ethyl group, propyl group, isopropyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, preferably C 1-10 alkyl group. More preferably, a C 1-4 alkyl group and the like can be mentioned.
  • the aromatic group for example, an aromatic hydrocarbon group [for example, a phenyl group, a tolyl group, a xylyl group, an aryl group such as a naphthyl group (e.g., C 6 ⁇ 20 aryl group, preferably a C 6 ⁇ 12 aryl group) Etc.], and aromatic heterocyclic groups.
  • aromatic hydrocarbon group for example, a phenyl group, a tolyl group, a xylyl group, an aryl group such as a naphthyl group (e.g., C 6 ⁇ 20 aryl group, preferably a C 6 ⁇ 12 aryl group) Etc.
  • aromatic heterocyclic groups for example, an aromatic hydrocarbon group [for example, a phenyl group, a tolyl group, a xylyl group, an aryl group such as a naphthyl group (e.g., C 6 ⁇ 20 aryl group, preferably a
  • Examples of the aliphatic group having a substituent include, for example, an alkyl group substituted with an aromatic group [for example, an aralkyl group (for example, an arylalkyl group such as benzyl group, phenethyl group), a haloalkyl group (for example, a chloromethyl group, a bromoethyl group).
  • An alkyl group having a substituent such as a group etc.
  • the aromatic group having a substituent for example, haloaryl group (e.g., chlorophenyl group, halo C 6 ⁇ 20 aryl group such as a bromophenyl group, preferably halo C 6 ⁇ 12 aryl group).
  • R 5 ⁇ R 8 Preferred hydrogen atom as R 5 ⁇ R 8, an alkyl group (C 1 ⁇ 4 alkyl group such as a methyl group, an ethyl group), an aralkyl group (e.g., C 6 ⁇ 10 aryl C 1 ⁇ 4 such as a benzyl group, a phenethyl group An alkyl group), and it is more preferable that all of R 5 to R 8 are hydrogen atoms.
  • R 5 ⁇ R 8 an alkyl group (C 1 ⁇ 4 alkyl group such as a methyl group, an ethyl group), an aralkyl group (e.g., C 6 ⁇ 10 aryl C 1 ⁇ 4 such as a benzyl group, a phenethyl group An alkyl group), and it is more preferable that all of R 5 to R 8 are hydrogen atoms.
  • B and E represent the same or different linkers.
  • B is not particularly limited as long as it is a linker for binding A and the polyethylene glycol moiety.
  • the polyethylene glycol moiety refers to a moiety represented by the structural formula — (CH 2 CH 2 O) x— in formula (1).
  • X is an integer of 3 to 50, preferably 5 to 40, more preferably 7 to 30, still more preferably 8 to 20, and particularly preferably 8 to 12.
  • B is typically represented by the following formula (4): (Wherein B 1c and B 2c each independently represent —CONR 9 — or —COO—, B 1b and B 2b each independently represent an alkylene group, and B3 represents —CONR 10 —, —COOCO—, —COO—, —NHCOO—, —O—, —OCO—, —C ⁇ C—, —CH ⁇ CH—, an alkylene group, —S—, —SO—, —SO 2 —, —NR 11 —, — NR 12 CO—, —NR 13 CONR 14 —, the following formula (5)
  • R 9 to R 14 each independently represents a hydrogen atom or a substituent, 1d, 1e, 2d and 2e are each independently 0 or 1, and 1f and 2f are each independently Or an integer of 0 to 3).
  • examples of the alkylene group include a methylene group, an ethylene group, a trimethylene group, a propylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group.
  • the alkylene group may be linear or branched, and may be particularly linear.
  • the substituent is the same as that of R 5 to R 8 [eg, alkyl group (methyl group, ethyl group, etc.), aryl group, haloalkyl group, haloaryl group, aralkyl group, etc.] Is mentioned.
  • R 9 ⁇ R 14, C 6 ⁇ 10 such as a hydrogen atom, an alkyl group (e.g., C 1 ⁇ 4 alkyl group such as a methyl group, an ethyl group), an aralkyl group (e.g., benzyl group, phenethyl group Aryl C 1-4 alkyl group) and the like.
  • an alkyl group e.g., C 1 ⁇ 4 alkyl group such as a methyl group, an ethyl group
  • an aralkyl group e.g., benzyl group, phenethyl group Aryl C 1-4 alkyl group
  • 2f is 2 or 3
  • B 2b , B 2c , 2d and 2e may be different from each other.
  • 2f is preferably 0.
  • B is shown in Table 1.
  • p 1 , p 2 and p 3 represent an integer of 1 or more, preferably 1 to 20, more preferably 1 to 12, particularly 2 to 8.
  • B is particularly preferably —CONR 9 — (CH 2 ) p 3 CONR 10 —.
  • R 9 , p 3 and R 10 are each independently described as above.
  • E is not particularly limited as long as it is a linker for binding the polyethylene glycol moiety and the sphingomyelin moiety represented by the following formula (12).
  • R 1 to R 4 each independently represent a hydrogen atom or a substituent.
  • substituents include the same substituents as R 5 to R 8 [eg, alkyl group (methyl group, ethyl group, etc.), aryl group, haloalkyl group, haloaryl group, aralkyl group, etc.].
  • Representative R 1 ⁇ R 4, C 6 ⁇ 10 such as a hydrogen atom, an alkyl group (e.g., C 1 ⁇ 4 alkyl group such as a methyl group, an ethyl group), an aralkyl group (e.g., benzyl group, phenethyl group Aryl C 1-4 alkyl group) and the like.
  • n is not particularly limited as long as it is within the range of 10 to 30, but is preferably 10 to 25, more preferably 10 to 20, further preferably 11 to 15, and preferably 13. Particularly preferred.
  • n is not particularly limited as long as it is within the range of 10 to 30, but it is preferably 14 to 25, more preferably 15 to 20, further preferably 16 to 18, and preferably 17. Particularly preferred.
  • E is typically represented by the following formula (11): Wherein E 1c and E 2c are each independently —CONR 15 — or —COO—, E 1b and E 2b are each independently an alkylene group, E3 is —CONR 16 —, —COOCO—, —COO—, —NHCOO—, —O—, —OCO—, —C ⁇ C—, —CH ⁇ CH—, an alkylene group, —S—, —SO—, —SO 2 —, —NR 17 —, — NR 18 CO—, —NR 19 CONR 20 —, the following formula (5) A group represented by formula (6): A group represented by formula (7): Or a group represented by the following formula (8) A group represented by formula (9):
  • R 15 to R 20 each independently represents a hydrogen atom or a substituent
  • 1g, 1h, 2g and 2h are each independently 0 or 1
  • 1i and 2i are each independently Or an integer of 0 to 3).
  • E 1b , E 1c , 1g and 1h may be different from each other.
  • E 2b , E 2c , 2g and 2h may be different from each other.
  • 2g and 2i are preferably 1.
  • E is shown in Table 2.
  • q represents an integer of 1 or more, and q is preferably 1 to 20, more preferably 1 to 12, and particularly 1 to 8.
  • E is preferably a combination of E-1 in Table 2.
  • the compound of the present invention can specifically migrate to the lipid raft region.
  • the compounds of the present invention are capable of visualizing lipid raft regions and are very useful, for example, in diagnosing lipid raft-related diseases.
  • the compound of this invention is not specifically limited, For example, it manufactures by referring the below-mentioned Example.
  • the compound of the present invention can be obtained, for example, as follows.
  • a water-soluble fluorescent coloring compound (a) having a functional group and a polyethylene glycol moiety derivative (b) are reacted as shown in the following reaction formula 1.
  • B 3a is formed by the reaction of B 1a of compound (I) and B 2a of compound (b).
  • Such functional groups B 1a and B 2a can be appropriately selected depending on the type of B3.
  • Examples include —N-succinimide group, maleimide group, tosyloxy group (—OTs), trifluoromethanesulfonyloxy group, cyano group and the like.
  • Table 3 shows representative combinations of B 1a , B 2a and B3. (In the table, X represents halogen.)
  • Reaction Formula 2 Reaction of reaction formula 2 is carried out using the derivative (b) of the polyethylene glycol moiety instead of (c) in reaction formula 2, and the resulting compound is used in place of compound (b) in reaction formula 1
  • E 1a compounds (c), E3 by the E 2a is the reaction of the compound (II) is formed.
  • Such functional groups E 1a and E 2a can be appropriately selected according to the type of E3.
  • hydroxyl group (—OH), alkylamino group, carboxyl group (—COOH), —NCO group, thiol group ( —SH), azide group (—N 3 ), acetylene group (—C ⁇ CH), vinyl group (—CH ⁇ CH 2 ), halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), —COO -N-succinimide group, maleimide group, tosyloxy group, trifluoromethanesulfonyloxy group and the like can be mentioned.
  • Table 4 shows typical combinations of E 1a , E 2a and E3.
  • Solvents and the like can be appropriately selected by those skilled in the art.
  • the compound (c) is not particularly limited.
  • the compound (b) is dissolved in DMF (N, N-dimethylformamide), and the compound (b) containing triethylamine and a polyethylene glycol part is added thereto in order, For 7 hours, the solvent is distilled off and the residue is purified by silica gel chromatography and gel permeation chromatography.
  • the compound of the present invention is not particularly limited.
  • a sphingomyelin portion in which compound (c) is dissolved in t-BuOH / H 2 O, copper sulfate and sodium ascorbate are added, and then dissolved in 100 ⁇ L of methanol is used.
  • the compound (d) containing the mixture is added, the solvent stirred at room temperature for 1 day is distilled off, and the residue is separated and purified by thin layer chromatography and gel permeation chromatography.
  • the present invention includes a lipid raft visualization agent (hereinafter also referred to as the agent of the present invention) containing the compound of the present invention.
  • the agent of this invention should just contain the compound of this invention, and may contain the other component, as long as there exists the effect of this invention.
  • the agent of the present invention can be appropriately selected by those skilled in the art according to the absorption wavelength of the water-soluble fluorescent coloring compound of A in Formula (1) representing the compound of the present invention, the type of excitation light, and the like.
  • the water-soluble fluorescent coloring compound is ATTO-488, ATTO-594, or a combination thereof, it is 450 nm or more, preferably 480 to 800 nm, particularly preferably 480 to irradiating with excitation light having a wavelength of 400 to 600 nm. It emits fluorescence with a wavelength of 670 nm. Since the agent of the present invention specifically migrates to the lipid raft region, the lipid raft region can be visualized. The agent of the present invention can enhance the contrast between the fluorescent portion and the non-fluorescent portion by using the FRET (Forster Resonance Energy Transfer) method.
  • FRET Fluorescence resonance energy transfer, which is a phenomenon in which excitation energy moves directly between two adjacent dye molecules not by electromagnetic waves but by electron resonance.
  • the usage mode of the agent of the present invention is not limited to those applied to a living body, and can be applied to cells, tissues, specimens and the like extracted outside the living body.
  • the agent of the present invention is useful because it does not cause damage to living tissue or DNA.
  • the agent of the present invention specifically migrates to the lipid raft region, the lipid raft region can be visualized, and is very useful for diagnosing lipid raft-related diseases, for example.
  • the agent of the present invention can be administered by intravenous injection, oral administration or the like, and can diagnose a disease by specific binding to a target biological substance in vivo.
  • the present invention includes a lipid raft-related disease diagnostic marker (hereinafter referred to as the diagnostic marker of the present invention) containing the compound of the present invention or the agent of the present invention.
  • the diagnostic marker of the present invention only needs to contain the compound of the present invention, and may contain other components as long as the effects of the present invention are exhibited.
  • the diagnostic marker of the present invention may contain one or more agents of the present invention.
  • the diagnostic marker of the present invention can be appropriately selected by those skilled in the art depending on the absorption wavelength of the water-soluble fluorescent coloring compound of A in formula (1) representing the compound of the present invention, the type of excitation light, and the like.
  • the water-soluble fluorescent coloring compound is ATTO-488, ATTO-594, or a combination thereof, it is 450 nm or more, preferably 480 to 800 nm, particularly preferably 480 to irradiating with excitation light having a wavelength of 400 to 600 nm. It emits fluorescence with a wavelength of 670 nm. Since the diagnostic marker of the present invention specifically moves to the lipid raft region, the lipid raft region can be visualized and is very useful for diagnosing a lipid raft-related disease.
  • lipid raft-related disease is a disease in which increase or decrease in lipid raft region is known to be related to exacerbation of the disease, and increase or decrease in lipid raft region may be related to exacerbation of the disease Includes diseases and the like.
  • Lipid raft-related diseases include, for example, systemic lupus erythematosus, central diseases (Alzheimer's disease, Parkinson's disease, etc.), inflammatory diseases (autoimmune arthritis, atopic dermatitis, asthma, emphysema, Behcet's disease, multiple sclerosis) , Spinocerebellar degeneration, uveitis, Guillain-Barre syndrome, Fisher syndrome, chronic inflammatory demyelinating polyneuritis, polymyositis, scleroderma, autoimmune hepatitis, sarcoidosis, chronic pancreatitis, inflammatory bowel disease, clone Disease, solid cancer, multiple myeloma, angiofibroma, atherosclerosis, arteriovenous malformation, granulation, hemangioma, hypertrophic scar, keloid, premature aging, psoriasis, febrile granuloma, hemorrhoid, hemor
  • the present invention (the compound of the present invention, the agent of the present invention, the diagnostic marker of the present invention, described later)
  • the reagent of the present invention is very useful for the diagnosis and prevention of lipid-related diseases such as systemic lupus erythematosus.
  • the diagnostic marker of the present invention is, for example, a solution prepared by dissolving the compound of the present invention or the agent of the present invention in an aqueous medium such as physiological saline or phosphate buffer, a solid agent such as fine particle powder, lyophilized powder, etc. Offered as.
  • aqueous medium such as physiological saline or phosphate buffer
  • solid agent such as fine particle powder, lyophilized powder, etc. Offered as.
  • the form of the diagnostic marker of the present invention is not particularly limited, and can be appropriately selected by those skilled in the art according to the purpose of use and the like.
  • Pharmacologically and pharmaceutically acceptable additives can be added to the diagnostic marker of the present invention.
  • excipients such as glucose, lactose, D-mannitol, starch, crystalline cellulose; disintegrating agents or disintegrating aids such as carboxymethylcellulose, starch, carboxymethylcellulose calcium; petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin Bases such as purified water and hard fat; isotonic agents such as glucose, sodium chloride, D-mannitol, glycerin; pH regulators such as inorganic acids, organic acids, inorganic bases, organic bases; vitamin A, vitamin E
  • pharmaceutical additives such as drugs that can contribute to stabilization such as coenzyme Q may be added.
  • the diagnostic marker of the present invention can be applied to, for example, a tissue, a specimen, etc. extracted outside the living body.
  • the diagnostic marker of the present invention can be used for, for example, inspection using a confocal laser microscope. It is possible to detect a foci by detecting fluorescence by irradiating near infrared rays or far infrared rays after bringing the reagent of the present invention into contact with tissues, cells, etc. suspected of having a foci of lipid raft-related diseases and washing them appropriately. it can.
  • the present invention includes a reagent for lipid raft detection or quantification (hereinafter also referred to as the reagent of the present invention) containing the compound of the present invention or the lipid raft visualization agent of the present invention.
  • the reagent of the present invention can be used, for example, for inspection using a confocal laser microscope. It is possible to detect a foci by detecting fluorescence by irradiating near infrared rays or far infrared rays after bringing the reagent of the present invention into contact with tissues, cells, etc. suspected of having a foci of lipid raft-related diseases and washing them appropriately. It is possible to know the presence / absence of disease, the degree of progression, the severity, etc. by quantifying the fluorescence.
  • the present invention includes the use of a compound of the present invention for producing a lipid raft visualization agent (hereinafter also referred to as use of the present invention).
  • the present invention relates to a lipid raft visualization method (hereinafter also referred to as the method of the present invention) comprising a step of mixing the compound of the present invention and a cell containing lipid raft, and a step of irradiating the cell with light to detect fluorescence. Included).
  • cells containing lipid rafts include cells known to contain lipid rafts, cells unknown whether lipid rafts are contained, and the like.
  • Cells containing lipid rafts are not particularly limited, and examples include red blood cells, white blood cells, and human bladder cancer cells. Examples of red blood cells include sea urchin red blood cells (echinocytes).
  • the method of the present invention is very useful in that a process on a sea urchin erythrocyte can be visualized and detected.
  • the step of mixing the compound of the present invention with cells containing lipid rafts is not particularly limited.
  • a solvent or the like is appropriately used so that the compound of the present invention and cells containing lipid rafts come into contact with each other. Can be selected and used.
  • the step of irradiating the cells containing lipid rafts with light to detect fluorescence is not particularly limited.
  • the cells containing lipid rafts are irradiated with visible light or near infrared light
  • Light from an excitation light source is applied to cells containing lipid rafts using infrared light observation with a CCD, fluorescence microscope, fluorescence endoscope, multiphoton excitation fluorescence microscope, confocal microscope, confocal endoscope, etc.
  • the detection method is not particularly limited, and examples thereof include Western blotting, protein array, flow cytometry, fluorescent ELISA, fluorescent immunostaining, FRET, and in vivo imaging.
  • the wavelength for excitation used in the present invention is not particularly limited as long as it does not affect the cell containing the compound of the present invention and lipid raft, but varies depending on the water-soluble fluorescent coloring compound of the compound of the present invention to be used. There is no particular limitation as long as the water-soluble fluorescent coloring compound of the compound of the present invention emits fluorescence efficiently.
  • the water-soluble fluorescent coloring compound is ATTO-488, ATTO-594, or a combination thereof, it is 450 nm or more, preferably 480 to 800 nm, particularly preferably 480 to irradiating with excitation light having a wavelength of 400 to 600 nm. It emits fluorescence with a wavelength of 670 nm.
  • the light source used in the method of the present invention is not particularly limited as long as it does not affect the cells containing the compound of the present invention and lipid rafts.
  • a dye laser, a semiconductor laser, an ion laser, a fiber laser, a halogen lamp, A xenon lamp, an evanescent wave, a tungsten lamp, etc. are mentioned.
  • a cell containing lipid rafts is irradiated with light to cause the water-soluble fluorescent coloring compound of the compound of the present invention to emit light in the lipid raft region of the cells containing lipid rafts.
  • the water-soluble fluorescent coloring compound of the compound of the present invention By imaging, a lipid raft region that emits light can be easily detected, and the state, localization, change, and the like of the lipid raft can be captured as an image.
  • the present invention includes embodiments in which the above configurations are combined in various ways within the technical scope of the present invention as long as the effects of the present invention are exhibited.
  • the electrode was dried under vacuum for 24 hours and then coated with a thin lipid membrane.
  • the electrodes arranged in parallel were sandwiched between two cover glasses (24 mm ⁇ 60 mm, thickness 0.12 to 0.17 mm) using a rubber spacer (thickness 1 mm) in the shape of a square frame. Placed in about 400 ⁇ L of MilliQ water. This chamber was fixed on a temperature-controlled sample stage (Thermoplate, Tokai Hit, Shizuoka, Japan). Samples were then incubated for 60 minutes at 50 ° C., a temperature well above the Tm of pure SM bilayers, and low frequency alternating current (AC) using a function generator (20 MHz, Agilent, Santa Clara, Calif.).
  • AC low frequency alternating current
  • Example 1 Transition to a raft-like phase in a ternary giant lipid membrane liposome (GUV) containing compound 5 and compound 6 (observation with a fluorescence microscope) Confocal GUV formed with SM / DOPC / chol (1/1/1 mol / mol / mol) containing 0.2% mol of Compound 5 or 0.2% mol of TX-red DHPE at 28.5 ° C. The sample was observed with a fluorescence microscope. GUV is known to phase-separate into a raft-like ordered phase and a non-raft-like disordered phase under this condition. In addition, the lipid composition is different in each phase, and it has been reported that SM, which is a major component of raft, is distributed abundantly in the ordered phase.
  • SM which is a major component of raft
  • FIGS. 1 and 5 Fluorescence microscope observation results are shown in FIGS.
  • the white portions in FIGS. 1 and 5 are blue fluorescent portions, and the white portions in FIGS. 2 and 4 are red fluorescent portions.
  • 3 and 6 are photographs in which FIGS. 1 and 2 and FIGS. 4 and 5 are superimposed. It was found that Compound 5 exhibited a distribution opposite to TX-red DHPE (Invitrogen, Eugene, OR, USA), which is known to bind to a disordered phase (non-raft-like phase) (FIGS. 1 to 3). This indicates that compound 5 is preferentially incorporated into the ordered phase (raft-like phase) as in normal SM.
  • TX-red DHPE Invitrogen, Eugene, OR, USA
  • compound 6 is preferentially distributed in the ordered phase (raft-like phase) because it exhibits the opposite distribution to Boipy-PC (Invitrogen, Eugene, OR, USA) that binds to the disordered phase (non-raft-like phase). (Figs. 4-6).
  • the laser power is 9 mW / cm 2 and 6 mW / cm 2
  • the scanning speed is 2 ⁇ s / pix and 4 ⁇ s / pix for GUV containing Compound 5 and GUV containing Compound 6, respectively
  • the confocal image is 1024 pix ⁇ 1024 pix.
  • the distribution of the ordered and non-ordered phases of Compound 5 and Compound 6 was examined by fluorescence intensity.
  • a cross section of the GUV is shown in FIGS.
  • the white portion in FIG. 7 indicates a blue fluorescent portion, and the white portion in FIG. 9 indicates a red fluorescent portion.
  • Twenty spherical GUVs were selected, and the intensity around the cross section was plotted (FIGS. 8 and 10). Both the fluorescence intensity of the ordered phase of Compound 5 and Compound 6 is 4 times the fluorescence intensity of the non-ordered phase (FIG. 11), indicating that Compound 5 and Compound 6 are highly distributed in the ordered phase.
  • Example 3 Detection of ordered phase Lo transition region of compound 6 by FRET method 0.2 mol% Compound 5 or compound 6 was added to a mixture of SM / DOPC / chol (1: 1: 0.5 mol / mol / mol). A GUV was created. The GUV containing compound 5 was irradiated with a laser having a wavelength of 473 ⁇ 2 nm. After energy transfer from compound 5 to compound 6, radiation from compound 6 was detected at a wavelength of 610-630 nm.
  • the white part of FIG.12 and FIG.14 shows a red fluorescent part.
  • the average intensity ratio of the ordered phase (Lo, raft-like phase) and the non-ordered phase (Ld, non-raft-like phase) is 5.9 ⁇ 1.1, confirming that the contrast is strengthened by the FRET phenomenon. It was done.
  • Example 4 Labeling of SM cluster in the protrusion structure of erythrocytes Blood was collected by injection at a clinic in Osaka University by a nurse. 1 mL of blood was suspended in 4 mL phosphate buffer solution (PBS, pH 7.4) and centrifuged. This operation was repeated twice, and 10 ⁇ L of the obtained erythrocytes were suspended in 990 mL of PBS. To this, 5 ⁇ L of an ethanol solution (0.5 mM) of compounds 5 and 6 was added and incubated at room temperature for 7 minutes. The method of adding compounds 5 and 6 to the erythrocyte membrane is a known method (Mikhalyov, I. & Samsonov, A.
  • the white part of FIG.15 and FIG.16 shows a red fluorescent part.
  • FIG. 16 shows a photograph with the background subtracted.
  • the brightness is normalized by the brightness of the SM-rich region so that the contrast can be easily compared (arrowhead).
  • a new domain that was not seen in the former appears (arrow in FIG. 16), and it can be seen that the contrast between the SM-rich / SM-poor regions is enhanced by using FRET.
  • the SM-rich region ( ⁇ 1 ⁇ m) obtained by these fluorescence observations was confirmed to be larger than the commonly known raft ( ⁇ 200 nm).
  • FIGS. 17 and 18 show differential interference microscopic images of protrusions on erythrocytes artificially induced by adding a higher concentration of compound 6 and fluorescent microscopic images of compound 6, respectively.
  • the white part of FIG. 18 is a red fluorescent part.
  • a photograph in which FIGS. 17 and 18 are superimposed is shown in FIG. 19 (scale bar is 5 ⁇ m). From these results, it was found that Compound 6 was localized in the protrusion on the erythrocyte. This result is the first example to directly visualize that SM is localized in a process on an erythrocyte. From this, it is considered that the change in the film curvature caused by the lipid embedding caused the aggregation of the compound 6.
  • Example 5 Time-dependent change in diffusion movement of Compound 6 in CHO-K1 cells
  • the time-dependent change in diffusion movement of Compound 6 in a Chinese hamster ovary-derived cultured cell line (CHO-K1 cell line) was measured using the SFMT method (1 fluorescent molecule). The pursuit method).
  • the result of 4 ms / frame focusing on the bimolecular compound 6 is shown in the upper column of FIG. 20, and the locus of the diffusion movement of the bimolecular compound 6 is shown in the lower column of FIG. From the results shown in FIG. 20, two molecules of Compound 6 that originally performed Brownian motion co-localize in the 9th frame, and continue co-diffusion until 29th frame, but the co-localization is resolved in the 30th frame. I found out.
  • the co-localization mentioned here means that the compound 6 is close to a distance below the resolution of the microscope ( ⁇ 240 nm). Moreover, this repeated process of free diffusion, co-diffusion, and free diffusion was also observed in other compounds 6.
  • the number of colocalizations was plotted as a function of the colocalization time (FIG. 21). Furthermore, in order to clarify the difference in colocalization time between different molecules, the apparent dimer lifetime (relaxation time) was estimated by fitting the obtained histogram with a first-order exponential function (line in FIG. 21). . As a result, the apparent dimer lifetime of Compound 6 was 50 ms, and a result showing that it was significantly longer than that of ATTO594-DOPE (34 ms) (FIG. 22), which is known to have no affinity for rafts, was obtained. It was. Presumably, the colocalization is stabilized by incorporating a plurality of compounds 6 into the raft region. This result is the first example of visualizing the behavior of SM in a biological membrane at a single molecule level.
  • Example 6 Confirmation of specific distribution of compound 6 to raft phase of erythrocyte ghost membrane
  • the erythrocyte ghost membrane is a non-patent document (Tsuji, a, Kawasaki, K., Ohnishi, S., Merkle, H. & Kusumi, a. Regulation of band 3 mobilities in erythrocyte ghost membranes by protein association and cytoskeletal meshwork. Biochemistry 27, 7447-52 (1988)). That is, 50 ⁇ L of blood collected by a nurse at a clinic in Osaka University was suspended in 450 mL phosphate buffer solution (PBS, pH 7.4) and centrifuged.
  • PBS phosphate buffer solution
  • erythrocytes were suspended in 1 mL of 5P8 buffer solution (140 mM NaCl, 5 mM Na 3 PO 4 / Na 2 HPO 4, and 20 mM phenylmethylsulfurfluoride (pH 8.0)) and incubated under ice-cooling for 20 minutes. did. Next, it was suspended in 5P8 buffer and centrifuged. This operation was performed 4 times.
  • 5P8 buffer solution 140 mM NaCl, 5 mM Na 3 PO 4 / Na 2 HPO 4, and 20 mM phenylmethylsulfurfluoride (pH 8.0)
  • FIG. 23 shows a fluorescence micrograph obtained by adding Compound 6 to the erythrocyte ghost membrane
  • FIG. 24 shows a fluorescence micrograph after the erythrocyte ghost membrane of FIG. 23 is treated with the surfactant TX-100. Since compound 6 remained in the erythrocyte ghost membrane after treatment with surfactant TX-100, it was confirmed that compound 6 selectively transferred to the raft phase of the erythrocyte ghost membrane.
  • Example 7 Confirmation of specific distribution of compound 6 to raft phase of CHO-K1 ghost membrane or ECV304 ghost membrane instead of erythrocytes CHO-K1 (Chinese hamster ovary cells) or ECV304 (human bladder cancer-derived cells) A ghost film was produced by the same method as in Example 6 except that (1) was used. The distribution of compound 6 was examined in the same manner as in Example 6.
  • FIG. 25 shows a fluorescence micrograph obtained by adding Compound 6 to the CHO-K1 ghost film
  • FIG. 26 shows a fluorescence micrograph after the CHO-K1 ghost film of FIG. 25 is treated with the surfactant TX-100. Since compound 6 remained in the ghost film after treatment with surfactant TX-100, it was confirmed that compound 6 selectively transferred to the raft phase of the CHO-K1 ghost film.
  • FIG. 27 shows a fluorescence micrograph obtained by adding Compound 6 to the ECV304 ghost film
  • FIG. 28 shows a fluorescence micrograph after the ECV304 ghost film of FIG. 27 is treated with the surfactant TX-100. Since compound 6 remained in the ECV304 ghost membrane after treatment with surfactant TX-100, it was confirmed that compound 6 selectively transferred to the raft phase of the ECV304 ghost membrane.
  • Example 8 Detection of red blood membrane protein (GPI-anchored CD59) existing region Spines of echinocytes (spinous erythrocytes) known to aggregate the raft-specific protein GPI-anchored CD59 (Fig. 37 white area), compound 5 was co-localized with GPI-anchored CD59 using a confocal laser microscope.
  • GPI-anchored CD59 red blood membrane protein
  • Red blood cells were extracted by washing 500 ⁇ L of blood twice with 10 times the amount of PBS buffer (pH 7.4).
  • the extracted erythrocytes were diluted 10-fold by suspending them in 450 ⁇ L of buffer solution.
  • 10 ⁇ L of Cy3-IgG (Suzuki, K. G. N., Fujiwara, T. K., Sanematsu, F., Iino, R., Edidin, M., Kusumi, A.
  • recruited red blood cell suspension prepared by the method described in recruitment, recruitment, Lyn, and Galpha, fortemporary, cluster, immobilization, and Lyn, activation: single-molecule, tracking, study, 1. J.
  • CD59 labeled with the fluorescent antibody Cy3-IgG is shown in FIG. From FIG. 36, a result was obtained showing that Cy3 aggregates in the echinocytic spines. Interestingly, compound 5 was also aggregated in the spines (FIG. 37) and its distribution was found to overlap with CD59 (FIG. 38).
  • FIGS. 39 and 40 show confocal fluorescence microscope images showing the distribution of GPI-anchored CD59 labeled with compound 5 and Cy3-IgG at the early stage of erythrocyte echinocytosis, respectively.
  • the differential interference microscope image is shown in FIG. 41, and the overlapping of FIGS. 39 to 41 is shown in FIG. Cy3 was excited with an ATTO488, 559 ⁇ 2 nm (120 ⁇ W / cm 2 ) laser with a 473 ⁇ 2 nm (8.9 ⁇ W / cm 2 ) laser. Each emission was detected at 485-515 nm and 590-690 nm. In such a spectral region, ATTO488 and Cy3 crosstalk (excitation wavelength overlap) is sufficiently small.
  • An image of 1024 pix ⁇ 1024 pix was obtained by scanning the laser at 10 ⁇ s / pix. In order to clarify each distribution, the brightness and contrast were adjusted with Adobe Photoshop.
  • the compound of the present invention is industrially useful because it can provide diagnostic markers and diagnostic compositions for lipid raft-related diseases, lipid raft detection or quantification methods, and lipid raft detection or quantification reagents.
  • the present invention can visualize a raft region on erythrocytes and is industrially useful.
  • the present invention is industrially useful because it can visualize the behavior and temporal capture of SM in a raft using SFMT (one fluorescent molecular tracking method).

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Abstract

L'invention concerne un composé représenté par la formule (1) (dans laquelle A représente un résidu d'un composé soluble dans l'eau émetteur de fluorescence; B et E représentent des lieurs qui sont identiques ou différents l'un de l'autre; R1 à R4 représentent indépendamment un atome d'hydrogène ou un groupe alkyle pouvant être substitué; X représente un nombre compris entre 3 à 50; m et n représentent indépendamment un nombre compris entre 10 et 30; et le groupe représenté par la formule (2) représente une liaison simple ou une liaison double), qui constitue un nouveau composé de sphingomyéline marqué par fluorescence dont le comportement est identique à celui de la sphingomyéline native. L'utilisation de la sphingomyéline marquée par fluorescence permet de fournir : un agent de visualisation de radeau lipidique, un marqueur diagnostique pour une maladie associée aux radeaux lipidiques, un réactif pour détecter ou quantifier un radeau lipidique et un procédé de visualisation d'un radeau lipidique.
PCT/JP2016/058079 2015-03-16 2016-03-15 Nouveau composé de sphingomyéline marqué par fluorescence et utilisation de celui-ci WO2016148125A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003344419A (ja) * 2002-05-31 2003-12-03 Inst Of Physical & Chemical Res コレステロール検出試薬
US20050026235A1 (en) * 2003-06-30 2005-02-03 Graham Ronald J. Fluorescent phospholipase assays and compositions
WO2007102396A1 (fr) * 2006-03-02 2007-09-13 Kyushu University Procede de production de sphingolipide marque
US20120028290A1 (en) * 2009-02-04 2012-02-02 President And Fellows Of Harvard College Compositions and Methods for Labeling and Imaging Phospholipids

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JP5271912B2 (ja) * 2006-10-24 2013-08-21 ケレオス インコーポレーティッド 標的化リガンドを固定化するための改良型リンカー

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003344419A (ja) * 2002-05-31 2003-12-03 Inst Of Physical & Chemical Res コレステロール検出試薬
US20050026235A1 (en) * 2003-06-30 2005-02-03 Graham Ronald J. Fluorescent phospholipase assays and compositions
WO2007102396A1 (fr) * 2006-03-02 2007-09-13 Kyushu University Procede de production de sphingolipide marque
US20120028290A1 (en) * 2009-02-04 2012-02-02 President And Fellows Of Harvard College Compositions and Methods for Labeling and Imaging Phospholipids

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ISHITSUKA, REIKO ET AL.: "Imaging Lipid Rafts", J. BIOCHEM., vol. 137, 2005, pages 249 - 254, XP055311887 *
MUKHERJEE, SOUMI ET AL.: "Organization and dynamics of N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)-labeled lipids: a fluorescence approach", CHEMISTRY AND PHYSICS OF LIPIDS, vol. 127, 2004, pages 91 - 101, XP055311889 *

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