WO2016148125A1

WO2016148125A1 - Novel fluorescently labeled sphingomyelin and use thereof

Info

Publication number: WO2016148125A1
Application number: PCT/JP2016/058079
Authority: WO
Inventors: 道雄村田; 信明松森; 祥尚木下; 明弘楠見; 鈴木　健一
Original assignee: 国立大学法人大阪大学; 国立大学法人京都大学
Priority date: 2015-03-16
Filing date: 2016-03-15
Publication date: 2016-09-22
Also published as: JPWO2016148125A1; JP6398055B2

Abstract

A compound represented by formula (1) (wherein A represents a residue of a water-soluble fluorescence-emitting compound; B and E represent linkers which are the same as or different from each other; R₁ to R₄ independently represent a hydrogen atom or an alkyl group which may be substituted; X represents 3 to 50; m and n independently represent 10 to 30; and the group represented by formula (2) represents a single bond or a double bond) is novel fluorescently labeled sphingomyelin that exhibits the same behavior as that of native sphingomyelin. The use of the fluorescently labeled sphingomyelin makes it possible to provide: a lipid raft visualizing agent, a diagnosis marker for a lipid raft-related disease, a reagent for detecting or quantifying a lipid raft, and a method for visualizing a lipid raft.

Description

Novel fluorescently labeled sphingomyelin and its use

The present invention relates to a novel labeled fluorescent sphingomyelin and use thereof.

Since the discovery of sphingomyelin (hereinafter also referred to as “SM”) in brain tissue by Thudicum in the 1880s, the behavior of SM (distribution, mobility, intermolecular) was aimed at elucidating the role of SM in lipid membranes. Interaction) has been studied. According to several studies using model membranes, the most basic characteristic of SM is that it has a higher hydrogen bond-forming ability than glycerophospholipid, thereby causing the formation of SM clusters in the presence of cholesterol. Such microdomains containing a large amount of SM (and cholesterol) are called lipid rafts, and are important in biology such as protein localization and signal transduction as well as viral infections and serious diseases. It has attracted the attention of scientists because it is involved in the phenomena. Raft-related virus infections and diseases are summarized in past literature (for example, Non-Patent Literature 1). However, although SM is an essential component of lipid rafts, direct information regarding SM behavior has not yet been obtained, particularly in biological membranes.

Recent advances in microscopy have made it possible to directly visualize lipid distribution in artificial membranes. For example, CARS (coherent anti-Stokes Raman scattering), which is an image method using Raman spectroscopy, is composed of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and DOPC (dioleoylphosphatidylcholine). In the L _d / L _β phase separation membrane, the distribution of DSPC, which is one of the typical similar substances of SM, with a Raman tag is revealed. Secondary ion mass spectrometry is used to determine the distribution of the isotope-labeled lipid in the absence and presence of G _M1 in a double layer with a composition of SM / DOPC / chol, lipid distribution and phase separation was elucidated to be strongly modulated by the presence of G _M1. However, these advanced techniques are not simple due to technical problems such as preparation and freezing of fixed samples, observation under ultra-high vacuum, and sample destruction. Thus, some membrane scientists are skeptical of this discovery and no information has been available from these techniques beyond the lipid distribution so far.

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. That is, preferentially binds to a non-raft-like L _d phase rather than the raft-like L _o phase in the phase separation membrane (Non-Patent Documents 2 and 3). 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. This is because lysenin binds to the SM cluster and the endogenous tryptophan residue functions as a fluorophore (Non-patent Document 7). However, as these authors are concerned, 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). After all, in order to directly observe the SM behavior, it is necessary to find a new fluorescent SM exhibiting the same behavior as the original SM.

In general, lipid rafts (less than 200 to 300 nm) of biological membranes are smaller than lipid rafts (over 1 μm) 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)

(In the 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, and 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)

Is a single bond or a double bond. ) A compound represented by
[2] In the formula (1), A represents 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 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.
[3] In formula (1), B represents the following formula (4)

(Wherein 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, and B3 is —CONR ₁₀ —, -COOCO -, - COO -, - NHCOO -, - O -, - OCO -, - O -, - C≡C -, - CH = CH -, - CH 2 -CH 2 -, - S -, - SO —, —SO ₂ —, —NR ₁₁ —, —NR ₁₂ CO—, —NR ₁₃ CONR ₁₄ —, the following formula (5)

A group represented by formula (6):

A group represented by formula (7):

A group represented by formula (8):

A group represented by formula (9):

Or a group represented by the following formula (10)

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). The compound according to [1] or [2] above.
[4] In formula (1), E represents 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):

A group represented by formula (8):

A group represented by formula (9):

Or a group represented by the following formula (10)

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, and 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].
[8] Use of the compound according to any one of [1] to [4] for producing a lipid raft visualization agent.
[9] 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.

[Compound of the present invention]
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.

In the formula (1), 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. It is particularly preferred that 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. . Specifically, 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.
In the formula (3), 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). Among these, it is preferable that 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.
In R ₅ to R ₈ , examples of the substituent include an aliphatic group and an aromatic group. Examples of the aliphatic group include an alkyl group and a cycloalkyl group (for example, a cyclohexyl group). Examples of the alkyl group 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.
The aliphatic group and the aromatic group further include a group substituted with a substituent. 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).
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.

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)

A group represented by formula (6):

A group represented by formula (7):

A group represented by formula (8):

A group represented by formula (9):

Or a group represented by the following formula (10)

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).
In B _1b , B _2b and B3, 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. _1-20 alkylene groups, preferably _{C 1 ~ 16} alkylene group, more preferably the like _{C 2 ~ 10} alkylene group. The alkylene group may be linear or branched, and may be particularly linear.
In R ₉ to R ₁₄ , 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. Representative _{_{_{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.
When 1f is 2 or 3, B _1b , B _1c , 1d, and 1e may be different from each other. When 2f is 2 or 3, B _2b , B _2c , 2d and 2e may be different from each other. 2f is preferably 0. Usually, when 1e is 1, it is often 1d.

Specific examples of B are shown in Table 1.

In the above table, 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 —. In this case, 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. Examples of the substituent 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.
m 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):

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):

Or a group represented by the following formula (10)

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, and 1i and 2i are each independently Or an integer of 0 to 3). When 1i is 2 or 3, E _1b , E _1c , 1g and 1h may be different from each other. When 2i is 2 or 3, E _2b , E _2c , 2g and 2h may be different from each other. In order to maintain the basicity of N in the sphingomyelin part, 2g and 2i are preferably 1.

Specific examples of E are shown in Table 2.

In the above table, 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. In particular, 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.

〔Production method〕
Although 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.

That is, in the example of the above formula, 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. For example, 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 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.)

(C) obtained in Reaction Formula 1 is reacted with Compound (d) containing a sphingomyelin moiety as shown in Reaction Formula 2 below. 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 You may make it react with the water-soluble fluorescent coloring compound (I) which has a group. That is, compound (ii) may be reacted after reacting compounds (ii) and (b), or compound (ii) may be reacted after reacting compounds (b) and (d). Compounds (a), (b) and (d) may be reacted at the same time. A solvent etc. can be suitably selected by those skilled in the art.

That is, in the example of the above formula, and 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. For example, 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. For example, 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. For example, 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 μL 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.

[Lipid raft visualization agent]
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. When 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. Note that FRET is 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. In addition, since 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.

[Diagnosis marker for lipid raft-related diseases]
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. When 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.

In the present specification, 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, hemorrhagic joint, non Connective fracture, rheumatoid arthritis (eg, malignant rheumatoid arthritis), osteoarthritis, follicular cyst, ovarian hypertrophy syndrome, polycystic ovary, age-related macular degeneration, diabetic retinopathy, Live blood vessel glaucoma, trachoma, emphysema, chronic bronchitis, obesity, periodontal disease, angiogenesis of corneal graft, aneurysm, etc.), lifestyle diseases (diabetes, hypertension, hyperlipidemia, etc.), viral infection, infection Symptoms and the like. For example, since systemic lupus erythematosus is known to have an increased lipid raft region associated with exacerbation of the disease, 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, including the use of the present invention and the method 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. 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. For example, 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 Furthermore, 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.

[Reagent for lipid raft detection or quantification]
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.

〔use〕
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).

[Lipid raft visualization method]
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).

In the method of the present invention, 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.

In the method of the present invention, the step of mixing the compound of the present invention with cells containing lipid rafts is not particularly limited. For example, 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.

In the method of the present invention, the step of irradiating the cells containing lipid rafts with light to detect fluorescence is not particularly limited. For example, 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. For example, it is possible to detect the fluorescence of the water-soluble fluorescent coloring compound of the compound of the present invention which has been irradiated to emit light. 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. When 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. For example, 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. Moreover, it is possible to obtain a preferable excitation wavelength or detect only fluorescence using various optical filters.

In the method of the present invention, for example, 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. 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.

EXAMPLES Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples at all, and many variations are within the technical idea of the present invention. This is possible by those with ordinary knowledge.

[Production Example 1] Synthesis of Compound 5 First, a reaction intermediate (compound 3) was obtained through the following steps.

6-amino-3- (14-azaniridin) -9- (2-((2-((2,5-dioxopyrrolidin-1-yl) oxy) which is ATTO-488NHS-ester (ATTO-TEC GmbH) -2-Oxoethyl) (methyl) carbamoyl) phenyl) -3H-xanthene-4,5-disulfonic acid (Compound 1) 1.45 μmol was dissolved in 450 μL of DMF (N, N-dimethylformamide). To this, 3.71 μL (18.3 μmol) of triethylamine was added, followed by 32-azido-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontan-1-amine ( 1.45 μmol of compound 2) (O- (2-Aminoethyl) -O ′-(2-azidoethyl) nonaethylene glycol, purchased from Aldrich) was added and stirred at room temperature for 7 hours. The solvent was distilled off, and the residue was subjected to silica gel chromatography (mobile phase; mixed solution of chloroform, methanol and water (CHCl ₃ : CH ₃ OH: H ₂ O = 13: 9: 2 (volume ratio)) and gel permeation chromatography. Purified by (JAIGEL-GS, solvent CH ₃ OH) and purified by 6-amino-3- (14-azanilysine) -9- (2-((35-azido-2-oxo-6,9,12,15,18, 21,24,27,30,33-Decaoxa-3-azapentatriacontyl) (methyl) carbamoyl) phenyl) -3H-xanthene-4,5-disulfonic acid (Compound 3) was obtained.

Next, Compound 5 was obtained through the following steps.

The obtained compound 3 (1.0 μmol) is dissolved in t-BuOH / H ₂ O (4/1, 400 μL), copper sulfate (0.5 μmol) and sodium ascorbate (1.5 uμmol) are added, and then 2- (ethynyldimethylammonio) ethyl ((2S, 3R, E) -3-hydroxy-2-stearamidooctadec-4-en-1-yl) phosphate (compound 4) dissolved in 100 μL of methanol ) (Manufactured by the method described in Sarah A. Goretta, Masanao Kinoshita, Shoko Mori, Hiroshi Tsuchikawa, Nobuaki Matsumori, Michio Murata Bioorg. Med. Chem. 20, 4012-4019 (2012)) and stirred at room temperature for 1 day . The solvent was distilled off, and the residue was separated and purified by thin layer chromatography (developing solvent CHCl ₃ / MeOH / H ₂ O = 13/9/1) and gel permeation chromatography (JAIGEL-GS, solvent CH ₃ OH), 2-(((1- (1- (2- (6-Amino-3- (14-azaniridin) -4,5-disulfo-3H-xanthen-9-yl) phenyl) -2-methyl-1,4 -Dioxo-8,11,14,17,20,23,26,29,32,35-decaoxa-2,5-diazapentatriacontan-37-yl) -1H-1,2,3-triazole- 4-yl) methyl) dimethylammonio) ethyl ((2S, 3R, E) -3-hydroxy-2-stearamidooctadec-4-en-1-yl) (compound 5) (0.29 μmol (total) Yield 1 %) Was obtained.

Regarding the NMR of Compound 5, the results of one-dimensional data processing and two-dimensional data processing using an NMR data processing program (Delta 5.0.0) are shown in FIGS. 29 and 30, respectively. The HRMS chart (Xcalibur) of Compound 5 is shown in FIG.

(Compound 5)
NMR (400 MHz, CD3OD) δ 0.90 (t, J = 6.64 Hz, 6H), 1.07-1.50 (m, 50H), 1.52-1.69 (m, 2H), 1.94 -2.08 (m, 2H), 2.11-2.24 (m, 2H), 2.87 (s, 3H), 3.17 (s, 6H), 3.44-3.76 (m , 40H, PEG), 3.82-4.16 (m, 4H), 4.22-4.41 (m, 2H), 4.58-4.70 (m, 1H), 5.40-5 .51 (m, 1H), 5.61-5.78 (m, 1H), 6.94-7.03 (m, 2H, aromatic), 7.23-7.28 (m, 2H, aromatic) , 7.33-7.81 (m, 4H, aromatic), 8.36-8.60 (m, 1H).
HRMS calcd for C ₉₀ H ₁₅₁ N ₉ O ₂₅ PS ₂ ⁺ [M] ⁺ 1852.9995, found: 1852.997.

[Production Example 2] Synthesis of Compound 6 The same method as in Production Example 1 except that ATTO-594N-hydroxysuccinimide derivative (ATTO-TEC GmbH) was used instead of ATTO-488N-hydroxysuccinimide derivative. To obtain 0.34 μmol of compound 6 (total yield 22%).

Regarding the NMR of Compound 6, the results of one-dimensional data processing and two-dimensional data processing using an NMR data processing program (Delta 5.0.0) are shown in FIGS. 32 and 33, respectively. An HRMS chart (Xcalibur) of Compound 5 is shown in FIG.

(Compound 6)
NMR (500 MHz, CD3OD) δ 0.90 (t, J = 6.73 Hz, 6H), 1.20-1.45 (m, 50H), 1.51-1.66 (m, 6H), 1.78 (T, J = 5.80 Hz, 1H), 1.97-2.09 (m, 2H), 2.13-2.23 (m, 2H), 2.64 (s, 1H), 2.72 (S, 1H), 3.19 (s, 6H), 3.43-3.52 (m, 2H), 3.54-3.70 (m, 40H, PEG), 3.70-3.78 (M, 2H), 3.85 (d, J = 15.75 Hz, 1H), 3.90-4.01 (m, 2H), 4.03-4.09 (m, 1H), 4.10 -4.18 (m, 1H), 4.33-4.39 (m, 1H), 4.58 (s, 12H), 4.64-4.68 (m, 1H), 4.76 (s , 2H), .41-5.48 (m, 1H), 5.66-5.74 (m, 1H), 5.89 (s, 1H), 6.79 (s, 1H), 7.38 (d, J = 15.32 Hz, 1H), 7.46-7.53 (m, 1H), 7.57-7.64 (m, 1H), 7.68-7.76 (m, 2H), 8.41. (S, 1H).
HRMS found: [M + 2Na] ²⁺ 1056.5724.

[Production Example 3] Production of artificial membrane liposome (GUV) Sphingomyelin (SM) and DOPC were purchased from Avanti Polar Lipid (Alabaster AL). Cholesterol was purchased from Sigma Aldrich (St. Louis, LO). A ternary giant lipid membrane liposome (GUV) composed of SM, DOPC and cholesterol is disclosed in Dimitrov, D. et al. S. Liposome Electro formation. It was prepared using the electro-formation method according to 303-311 (1986). Specifically, 5 to 10 μL of SM / DOPC / chol solution (1 mg / mL) to which an appropriate amount of fluorescent probe was added was spread on the surface of the electrode (platinum wire, diameter = 100 μm). The electrode was dried under vacuum for 24 hours and then coated with a thin lipid membrane. Next, 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 μL 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.). (Sine wave function, 10 Vpp, 10 Hz) was applied. After electroformation, the GUV was cooled to 25 ° C for subsequent observation, equilibrated for 15 minutes, and then heated to 28.5 ° C. The temperature of the bulk solution was separately measured using a K-type thermometer (AD-5602a, Sansho Co., Ltd., Tokyo, Japan), and the temperature difference between the sample stage and the sample was calibrated.

[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.

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. Similarly, 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).

[Example 2] Ratio of distribution of compounds 5 and 6 to raft-like and non-raft-like phases (confocal laser microscope observation)
The SM distribution in the ordered phase (Lo, raft-like phase) and disordered phase (Ld, non-raft-like phase) in GUV consisting of SM / DOPC / chol (1/1/1 mol / mol / mol) is 0.2 mol%. It investigated by adding the compound 5 or the compound 6. For each excitation, confocal lasers (λex = 473 ± 2 nm and 559 ± 2 nm) were used. Each emission was examined at 485 nm-585 nm and 570 nm-670 nm. The laser power is 9 mW / cm ^{2 and} 6 mW / cm ² , and the scanning speed is 2 μs / pix and 4 μs / pix for GUV containing Compound 5 and GUV containing Compound 6, respectively, and the confocal image is 1024 pix × 1024 pix. Got in.

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. FRET images (1024 pix × 1024 pix) obtained at laser powers 71 mW / cm ² and 89 mW / cm ² , scan speeds 4 μs / pix and 8 μs / pix are shown in FIGS. 12 and 14 (scale bar is 10 μm). The white part of FIG.12 and FIG.14 shows a red fluorescent part.

FIG. 13 shows a FRET intensity profile obtained by selecting 21 GUVs of the same size and detecting radiation only from Compound 6 along the GUV surface (see FIG. 12) at a wavelength (λem = 610-630 nm). Indicated. As a result, 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 μL of the obtained erythrocytes were suspended in 990 mL of PBS. To this, 5 μL 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. Lipid raft detecting in membranes of live erythrocytes. Biochim. Biophys. Acta 1808, 1930-9 (2011)). It was helpful. Immediately thereafter, the cells were washed 5 times with 10 times the amount of PBS to completely remove compounds 5 and 6 outside the erythrocyte membrane. Red blood cells into which compounds 5 and 6 were inserted were diluted with PBS to 1% (v / v) or less. This diluted solution was placed on a cover glass (width 24 mm × 60 mm, thickness 0.12-0.17 mm) and observed with a fluorescence microscope and a confocal microscope.

FIG. 15 shows the results of fluorescence microscope observation in which compound 6 was excited with a laser of λex = 559 ± 2 nm (178 mW / cm ² ) and emission of λem = 610-630 nm was detected. Compound 5 was excited with a laser of λex = 473 ± 2 nm (178 mW / cm ² ), and emission from compound 6 (λem = 610-630 nm) was detected at a lateral scanning speed of 8 μs / pix (1024 pix. The result of × 1024 pix.) Is shown in FIG. The white part of FIG.15 and FIG.16 shows a red fluorescent part. In this region, it was confirmed that no crosstalk (duplication of emission wavelength) between compound 5 and compound 6 occurred. A confocal microscope image (1024 pix. × 1024 pix.) Was obtained at a lateral scanning speed of 100 μs / pix. Compared to a normal fluorescent microscope image, the FRET method requires a long exposure and thus suffers a relatively large background (digital noise). Therefore, FIG. 16 shows a photograph with the background subtracted.

In FIGS. 15 and 16, the brightness is normalized by the brightness of the SM-rich region so that the contrast can be easily compared (arrowhead). In the latter, 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. On the other hand, the SM-rich region (˜1 μm) obtained by these fluorescence observations was confirmed to be larger than the commonly known raft (<200 nm).

Thus, the SM aggregation obtained here was considered to be involved in the formation of protrusions on erythrocytes, and the incorporation of compounds 5 and 6 into the protrusions on erythrocytes was examined. 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 μm). 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.

In addition, research to date suggests that the GPI-anchored protein, which is a major component of rafts, is localized in the processes on the erythrocytes, and that proteins are exchanged between the cells via the processes on the erythrocytes. 15-19, it is suggested that not only GPI-anchored protein but also SM is involved in protein exchange in erythrocytes. Considering that protein selection and exchange is one of the typical functions of rafts in biological membranes, it is an interesting result.

[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.

Therefore, in order to quantitatively evaluate the colocalization of Compound 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 μL 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. This operation was repeated three times, and the obtained 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.

10 μL of the obtained erythrocyte ghost membrane solution was suspended in 1 mL of PBS buffer (pH 7.4). Subsequently, compounds 5 and 6 and ATTO-DOPE were added to this by the method described in Example 4. This was placed on a glass coated with an appropriate amount (about 3 μL) of poly-Lycine.
200 μL of 1% TX-100 (surfactant Triton X-100) was added to an erythrocyte ghost membrane fixed on a cover glass and incubated for 15 minutes under ice cooling. Thereafter, the plate was washed twice with PBS buffer to remove the surfactant.

FIG. 23 shows a fluorescence micrograph obtained by adding Compound 6 to the erythrocyte ghost membrane, and 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, and 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, and 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.

From the above results, it was confirmed that Compound 5 and Compound 6 selectively label the lipid raft region.

[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.

At the Osaka University clinic, blood was collected by injection by a nurse. Red blood cells were extracted by washing 500 μL 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 μL of buffer solution. Next, 10 μL 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. Cell, Biol, 177, 717-730 (2007)) (150 μg / mL) In addition to the solution, GPI-anchored CD59 was labeled by incubating at 37 ° C. for 1 hour. Next, 200 μL of PBS buffer containing 1 μg of Compound 5 was added and left at room temperature for 7.5 minutes. Thereafter, the sample was washed 5 times with a PBS buffer solution to remove fluorescent substances not incorporated in the membrane. Finally, PBS buffer was added to erythrocytes and diluted to ˜1% (v / v). The sample was placed on a cover glass (24 mm × 60 mm, thickness 0.12 mm to 0.17 mm) and observed with a confocal microscope. The collected blood was used within 3 days. Labeling was performed immediately before observation.

The distribution of 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 μW / cm ² ) laser with a 473 ± 2 nm (8.9 μW / 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 μs / pix. In order to clarify each distribution, the brightness and contrast were adjusted with Adobe Photoshop.

Even when the echinosite was ready, co-localization of Compound 5 and Cy3-GPI-anchored CD59 could be observed. This suggests that a slight change in curvature triggers GPI-anchored CD59 and SM to form a raft-like region of ˜1 μM size.

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. In addition, the present invention can visualize a raft region on erythrocytes and is industrially useful. Furthermore, 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).

Claims

Following formula (1)

(In the 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, and 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)

Is a single bond or a double bond. ) A compound represented by
In formula (1), A represents 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 The compound of claim 1, wherein at least one of G 1 to G 6 and four G 7 is a water-soluble functional group.
In formula (1), B represents the following formula (4)

(Wherein 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, and B3 is —CONR 10 —, -COOCO -, - COO -, - NHCOO -, - O -, - OCO -, - C≡C -, - CH = CH -, - CH 2 -CH 2 -, - S -, - SO -, - SO 2 -, - NR 11 -, - NR 12 CO -, - NR 13 CONR 14 -, the following formula (5)

A group represented by formula (6):

A group represented by formula (7):

A group represented by formula (8):

A group represented by formula (9):

Or a group represented by the following formula (10)

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 The compound according to claim 1 or 2, wherein the compound is an integer of 0 to 3.
In formula (1), E represents 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)

Group shown, the following formula (6)

A group represented by formula (7):

A group represented by formula (8):

A group represented by formula (9):

Or a group represented by the following formula (10)

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, and 1i and 2i are each independently The compound according to any one of claims 1 to 3, which is an integer of 0 to 3).
A lipid raft visualization agent comprising the compound according to any one of claims 1 to 4.
A marker for diagnosis of a lipid raft-related disease comprising the compound according to any one of claims 1 to 4 or the agent according to claim 5.
A lipid raft detection or quantification reagent comprising the compound according to any one of claims 1 to 4 or the agent according to claim 5.
Use of the compound according to any one of claims 1 to 4 for producing a lipid raft visualization agent.
A lipid raft visualization method comprising the steps of mixing the compound according to any one of claims 1 to 4 and a cell containing lipid raft, and detecting fluorescence by irradiating the cell with light.