Fluorescence intensity enhancer, method for enhancing fluorescence intensity of fluorescently-labeled target biological substance, and kit for fluorescence detection

Abstract

A fluorescence intensity enhancer containing a water-soluble compound having a melting point of 50 DEG C or higher, a method for enhancing the fluorescence intensity of a fluorescently labeled target biological substance using the fluorescence intensity enhancer, and a fluorescence detection kit containing the fluorescence intensity enhancer.

Description

Technical Field

[0001] The present invention relates to a fluorescence intensity enhancer, a method for enhancing the fluorescence intensity of a fluorescently labeled target biological substance, and a kit for fluorescence detection. Background Art

[0002] There is known a bioimaging technique for analyzing the dynamics or functions of substances constituting a living organism, such as biomolecules, cells, or tissues. For the purpose of observing or analyzing the dynamics, interactions, concentration changes, or functional changes of substances such as proteins, nucleic acids, or low-molecular-weight substances or their complexes, fluorescence imaging is widely used. Regarding fluorescence imaging, a fluorescent dye-labeled biomolecule (fluorescent dye-labeled antibody, etc.) obtained by labeling a biomolecule (antibody, etc.) having a bonding property to a substance to be detected with a fluorescent dye is used, and information is obtained by visualizing it through fluorescence labeling. For example, in immunoblotting (Western blotting) in which a sample derived from a living organism is transferred onto a membrane and a specific protein is detected from a protein mixture, and cell staining for observing the dynamics of a target substance in a cell in a cell state, fluorescence imaging using the above-described fluorescent dye-labeled biomolecule is also utilized.

[0003] In the above fluorescence imaging, organic fluorescent dye molecules are usually used. Generally, by using a fluorescent dye-labeled biomolecule obtained by bonding a plurality of organic fluorescent dye molecules to one molecule of a biomolecule, the brightness (fluorescence intensity) is increased to a desired level. However, since most of the organic dyes showing fluorescence, such as cyanine dyes and rhodamine dyes, contain aromatic chromophores having high planarity, intermolecular interactions between the dyes are likely to occur. As a result, a decrease in fluorescence intensity due to intermolecular interactions such as self-association between the dyes is likely to occur. Moreover, as the number of fluorescent dye molecules per molecule of the biomolecule (fluorescence labeling rate: DOL) increases, the fluorescence intensity tends to further decrease due to self-association or the like.

[0004] Furthermore, there is known a technique for performing fluorescence imaging by coexisting a fluorescent dye-labeled biomolecule with other molecules. For example, Patent Document 1 describes the use of a conjugate compound of chlorotoxin peptide and a cyanine dye for tumor imaging purposes, and a composition containing mannitol in a preparation (tablet) for the conjugate compound. Patent Document 2 describes administering to a subject 89A method for labeling an antigen-binding construct with Zr is used for tumor imaging, and it is described that sucrose is included in the composition of the preparation for the antigen-binding construct. In Patent Document 3, there is described a dried mammalian cell which is obtained by fixing the cell with a fixative, reducing it with a Schiff base reducing agent, and drying it in the presence of trehalose as a membrane stabilizing compound, and this dried mammalian cell is useful as a control or standard in immunoassay.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2016-534147

[0008] Patent Document 2: Japanese Patent Application Laid-Open No. 2022-501402

[0009] Patent Document 3: Japanese Patent Application Laid-Open No. 05-133957 Summary of the Invention

[0010] Technical Problem to be Solved by the Invention

[0011] As a technique for suppressing self-association between fluorescent dyes in a fluorescent dye-labeled biomolecule, the present inventors have repeatedly studied a technique for suppressing self-association by adjusting the chemical structure of an organic fluorescent dye molecule introduced into the fluorescent dye-labeled biomolecule. However, in this technique, although self-association can be suppressed in an organic fluorescent dye molecule having a specific chemical structure, it lacks generality. Therefore, in fluorescence imaging, as a more general technique, there is a need to develop a technique that acts on a target biomolecule fluorescently labeled with a fluorescent dye-labeled biomolecule to suppress self-association and enhance fluorescence intensity.

[0012] That is, an object of the present invention is to provide a fluorescence intensity enhancer that can enhance the fluorescence intensity of a fluorescently labeled target biomolecule by acting on the fluorescently labeled target biomolecule. Another object of the present invention is to provide a method for enhancing the fluorescence intensity of a fluorescently labeled target biomolecule using the fluorescence intensity enhancer and a fluorescence detection kit containing the fluorescence intensity enhancer.

[0013] Means for Solving the Technical Problem

[0014] The inventors of the present invention have conducted repeated studies and found that by allowing a water-soluble compound having a melting point of 50 °C or higher, such as mannitol, sucrose, or trehalose, to act on a fluorescently labeled target biological substance, the fluorescence intensity of the fluorescently labeled target biological substance can be enhanced.

[0015] That is, the above problems of the present invention have been solved by the following method.

[0016] 〔1〕

[0017] A fluorescence intensity enhancer containing a water-soluble compound having a melting point of 50 °C or higher.

[0018] 〔2〕

[0019] The fluorescence intensity enhancer according to 〔1〕, wherein

[0020] the content of the above water-soluble compound is 4.5% by mass or more.

[0021] 〔3〕

[0022] The fluorescence intensity enhancer according to 〔1〕 or 〔2〕, wherein

[0023] the above water-soluble compound does not contain a salt structure.

[0024] 〔4〕

[0025] The fluorescence intensity enhancer according to any one of 〔1〕 to 〔3〕, wherein

[0026] the above water-soluble compound is a polyol compound or a betaine compound.

[0027] 〔5〕

[0028] The fluorescence intensity enhancer according to any one of 〔1〕 to 〔4〕, wherein

[0029] the content of the above water-soluble compound is 20% by mass or more.

[0030] 〔6〕

[0031] The fluorescence intensity enhancer according to any one of 〔1〕 to 〔5〕, wherein

[0032] the above melting point is 80 °C or higher.

[0033] 〔7〕

[0034] The fluorescence intensity enhancer according to any one of 〔1〕 to 〔6〕, which is in a solution state.

[0035] 〔8〕

[0036] The fluorescence intensity enhancer according to any one of [1] to [7], which is used for fluorescence imaging.

[0037] [9]

[0038] A method for enhancing the fluorescence intensity of a fluorescently labeled target biomolecule, which includes the step of allowing the fluorescence intensity enhancer according to any one of [1] to [8] to act on the fluorescently labeled target biomolecule.

[0039]

[10]

[0040] A method for enhancing the fluorescence intensity of a fluorescently labeled target biomolecule, which includes the step of allowing the fluorescence intensity enhancer according to any one of [1] to [8] to act on the fluorescently labeled target biomolecule to enhance the fluorescence intensity emitted from the fluorescent label.

[0041]

[11]

[0042] A kit for fluorescence detection, which contains the following (a) and (b),

[0043] (a) A fluorescence intensity enhancer containing a water-soluble compound having a melting point of 50 °C or higher,

[0044] (b) A reagent for producing a fluorescent dye-labeled biomolecule or a fluorescent dye-labeled biomolecule containing a fluorescent dye.

[0045] In the present invention, when there are multiple substituents, linking groups, structural units, etc. (hereinafter referred to as substituents, etc.) represented by specific symbols or formulas, or when multiple substituents, etc. are defined simultaneously, unless otherwise specified, each of the substituents, etc. may be the same or different from each other. This also applies to the definition of the number of substituents, etc. And when multiple substituents, etc. are close (especially adjacent), unless otherwise specified, they may be connected to each other to form a ring. And unless otherwise specified, rings, such as alicyclic rings, aromatic rings, and heterocyclic rings, may be further fused to form a fused ring.

[0046] For example, in the present invention, the structure represented by the following general formula (I) means that n (n is an integer of 2 or more) structures represented by the following general formula (i) are continuous. In this case, the n structures represented by the general formula (i) may be the same or different from each other. In addition, X in the following general formula (i) 1 ~X 3 has the same meaning as X in the following general formula (I) 1 ~X 3 . This also applies to the structures enclosed by ()s, the structures enclosed by () t , the structures enclosed by () u , the structures enclosed by () m , the structures enclosed by () n1Enclosed structure, using () na Enclosed structure and using () nb The enclosed structures are also the same. There are s structures that can be the same or different from each other, t structures that can be the same or different from each other, u structures that can be the same or different from each other, m structures that can be the same or different from each other, n1 structures that can be the same or different from each other, na structures that can be the same or different from each other, and nb structures that can be the same or different from each other.

[0047] [Chemical formula 1]

[0048]

[0049] In the present invention, regarding the double bond, unless otherwise specified, when both E-type and Z-type exist within the molecule, it can be either one of them, and it can also be a mixture thereof. And, unless otherwise specified, when there are asymmetric carbon atoms or asymmetric centers in the compound, the configuration can be either R or S in the R and S representations of the configuration, and it can also be a mixture thereof.

[0050] In the present invention, regarding the representation of compounds and substituents, it is used in the meaning that includes its salts and its ions in addition to the compound itself and the substituent itself. For example, dissociative anionic groups such as carboxyl group, sulfo group, and phosphono group (-P(=O)(OH)2) can dissociate through hydrogen ions to take an ionic structure, or can take a salt structure. That is, in the present invention, "carboxyl group" is used in the meaning of a group including carboxylate ion or its salt, "sulfo group" is used in the meaning of a group including sulfonate ion or its salt, and "phosphono group" is used in the meaning of a group including phosphonate ion or its salt. As the monovalent or polyvalent cations when forming the above salt structure, there is no particular limitation, and examples include inorganic cations, organic cations, etc. Specifically, examples include Na + , Li + and K + and other cations of alkali metals, Mg 2+ , Ca 2+ and Ba 2+ and other cations of alkaline earth metals, and organic ammonium cations such as trialkylammonium cations and tetraalkylammonium cations.

[0051] In the case of a salt structure, the type of the salt can be one kind, or two or more kinds can be mixed and present, or a salt-type group and a free acid structure group can be mixed and present in the compound, and also a compound with a salt structure and a compound with a free acid structure can be mixed and present.

[0052] The compounds of the present invention and those described in this specification are all neutral compounds. In the present invention and this specification, a compound being neutral means electrically neutral. Specifically, through charged groups or counterions within the compound, the overall charge of the compound is adjusted to 0. For example, in the cyanine pigment represented by the general formula (α), which is listed as an example of the pigment constituting the phosphor part, the formal charge of the nitrogen atom to which R 42 is bonded is +1, and in a manner that counteracts this formal charge, dissociable groups such as sulfonic groups in other structures in the cyanine pigment or the fluorescent pigment (F) have ionic structures such as sulfonate ions, whereby the fluorescent pigment (F) becomes a compound with an overall charge of 0 for the compound.

[0053] In each general formula related to the cyanine pigments defined in the present invention, for convenience, the positive charge possessed by the compound is represented by determining the structure possessed by a specific nitrogen atom. However, since the cyanine pigments defined in the present invention have a conjugated system, in fact, other atoms other than the above nitrogen atom may sometimes carry a positive charge, and as long as it is a cyanine pigment that can adopt the structure represented by each general formula as one of its chemical structures, it is included in the cyanine pigments represented by each general formula. The same applies to negative charges.

[0054] And it means compounds obtained by changing a part of the structure within the scope that does not impair the effects of the present invention. In addition, regarding compounds for which substitution or non-substitution is not clearly described, it means that they can have any substituent within the scope that does not impair the effects of the present invention. The same applies to substituents (for example, groups expressed as "alkyl", "methyl group", "methyl", etc.) and linking groups (for example, groups expressed as "alkylene", "methylene group", "methylene", etc.). Among such arbitrary substituents, the substituents preferred in the present invention are substituents selected from the substituent group T described later.

[0055] In the present invention, when defining the number of carbon atoms of a certain group, unless otherwise specified in the present invention or this specification, the number of carbon atoms refers to the total number of carbon atoms of the group as a whole. That is, when the group has substituents in a further manner, it refers to the total number of carbon atoms including the substituents.

[0056] And, in the present invention, the numerical range represented by "~" means a range that includes the numerical values described before and after "~" as the lower limit value and the upper limit value.

[0057] Advantages of the Invention

[0058] The fluorescence intensity enhancer of the present invention can enhance the fluorescence intensity of the above-mentioned fluorescently labeled target biological substance by acting on the fluorescently labeled target biological substance.

[0059] Moreover, the method for enhancing the fluorescence intensity of a fluorescently labeled target biological substance according to the present invention can enhance the fluorescence intensity of the fluorescently labeled target biological substance.

[0060] Moreover, the kit for fluorescence detection according to the present invention can enhance the fluorescence intensity of a fluorescently labeled target biological substance. Detailed Embodiments

[0061] <<Fluorescence Intensity Enhancer>>

[0062] The fluorescence intensity enhancer of the present invention contains a water-soluble compound having a melting point of 50°C or higher. Hereinafter, the water-soluble compound having a melting point of 50°C or higher is also referred to as "water-soluble compound (W)".

[0063] In the present invention, the "water-soluble compound" means a compound having a solubility in water of 1 g / 100 mL-H2O or more at 25°C, that is, a compound that dissolves 1 g or more in 100 mL of water at 25°C. Here, "dissolve" means that the liquid becomes uniformly transparent.

[0064] The solubility of the above water-soluble compound (W) in water at 25°C is preferably 5 to 500 g / 100 mL-H2O, more preferably 10 to 400 g / 100 mL-H2O, and still more preferably 30 to 300 g / 100 mL-H2O.

[0065] In the present invention, the melting point of the compound is the value obtained by using a thermogravimetry-differential thermal simultaneous measurement device and by the method described in the following examples. In addition, as long as the melting point of the compound can be measured, the decomposition of the water-soluble compound (W) can occur at a lower temperature than the melting of the water-soluble compound (W).

[0066] The melting point of the above water-soluble compound (W) is 50°C or higher, and from the viewpoint of further enhancing the fluorescence intensity, it is preferably 80°C or higher, more preferably 90°C or higher. As long as the effects of the present invention are exhibited, there is no particular limitation on the upper limit value. For example, it can be set to 600°C or lower, preferably 500°C or lower, and more preferably 400°C or lower. That is, the melting point of the water-soluble compound (W) is preferably 50 to 600°C, more preferably 80 to 500°C, and still more preferably 90 to 400°C.

[0067] In addition, when the fluorescence intensity enhancer of the present invention contains two or more water-soluble compounds (W), the melting point of the above water-soluble compound (W) is set to the lowest value among the melting points of the respective water-soluble compounds (W) contained in the fluorescence intensity enhancer of the present invention.

[0068] By allowing the fluorescence intensity enhancer of the present invention to act on a fluorescently labeled target biological substance, a water-soluble compound having a melting point of 50°C or higher acts as an active ingredient, thereby enabling enhancement of the fluorescence intensity of the above-mentioned fluorescently labeled target biological substance. Although the reason is not clear, it is considered that the water-soluble compound having a melting point of 50°C or higher contained in the fluorescence intensity enhancer of the present invention is interposed between the fluorochrome-labeled biomolecules and / or between the fluorochromes in the fluorescently labeled target biological substance, inhibiting the interaction between the fluorochromes, thereby being able to inhibit the quenching caused by the association of the fluorochromes and enhance the fluorescence intensity. In contrast, a water-soluble compound having a melting point lower than 50°C cannot intervene in the form of a film to inhibit the interaction between the fluorochromes, and thus cannot obtain an enhancement effect on the fluorescence intensity. Also, compounds that are not water-soluble compounds, such as oil-soluble compounds, will undergo phase separation when acting on a fluorescently labeled target biological substance and cannot intervene between the fluorochromes to inhibit the interaction between the fluorochromes, and thus cannot obtain an enhancement effect on the fluorescence intensity.

[0069] The above-mentioned water-soluble compound (W) preferably contains a water-soluble group. For example, it preferably contains at least one of monovalent water-soluble groups such as a hydroxyl group, a carboxyl group, a sulfo group, a phosphonyl group, a phosphonyloxy group, an amino group, an ammonium group, and a phosphonio group, and divalent water-soluble groups such as an ether bond and an amide bond.

[0070] As described above, the carboxyl group, sulfo group, phosphonyl group, and phosphonyloxy group can be dissociated by hydrogen ions to adopt an ionic structure or can adopt a salt structure.

[0071] As the above-mentioned water-soluble compound (W), a compound having a betaine structure containing at least one anionic group selected from a carboxyl group, a sulfo group, a phosphonyl group, and a phosphonyloxy group and at least one cationic group selected from an ammonium group and a phosphonium group or a compound containing a hydroxyl group is more preferable.

[0072] The above-mentioned water-soluble compound (W) can be a low-molecular compound or a high-molecular compound. The molecular weight of the above-mentioned water-soluble compound (W) is preferably 100,000 to 300,000, more preferably 200,000 to 200,000, and further preferably 300,000 to 100,000. In addition, when the water-soluble compound is a compound having a repeating structure such as a high-molecular compound, the above-mentioned molecular weight refers to the weight-average molecular weight.

[0073] From the viewpoint of further enhancing the fluorescence intensity, it is preferable that the above-mentioned water-soluble compound (W) does not contain a salt structure. The salt structure refers to a compound composed of an anionic group and a cationic group, and a structure in which the anionic group and the cationic group exist as different molecules. Specifically, the description related to the salt structure at the beginning can be applied.

[0074] In addition, the betaine structure having an anionic group and a cationic group in the same molecule does not correspond to the above-mentioned salt structure.

[0075] By using the fluorescence intensity enhancer of the present invention, the fluorescence intensity of a fluorescently labeled target biological substance can be enhanced, and sometimes the fluorescence intensity in a region other than the fluorescence region shown by the fluorescently labeled target biological substance may also increase. From the viewpoint of suppressing the increase in the fluorescence intensity in a region other than the fluorescence region shown by the fluorescently labeled target biological substance, the above-mentioned water-soluble compound (W) is preferably a polyol compound or a betaine compound.

[0076] A polyol compound refers to a compound having two or more hydroxyl groups in the molecule. Examples of the polyol compound include glucose, sucrose, maltose, lactose, trehalose, ribitol, sorbitol, mannitol, maltitol, lactitol, xylitol, fructose, 1-kestose, nystose trihydrate, fucose, dulcitol, galactooligosaccharide, 4'-galactosyllactose, isomaltooligosaccharide, lactulose, palatinitol, isomaltose monohydrate, raffinose pentahydrate, arabinose, dihydroxyacetone dimer, galactose, glyceraldehyde dimer, mannose, ribose, xylose, lactosylfructoside, erythritol, Daucosteride, isosteviol, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, rubusoside, stevioside, 1-deoxynojirimycin, fucoidan, rhamnose, threose, lyxose, allose, altrose, gulose, idose, talose, 1,3-dihydroxyacetone, erythrose, xylulose, ribulose, psicose, sorbose, tagatose, polysucrose, fructooligosaccharide and other sugar compounds, polyoxyalkylene glycol. In addition, the sugar compound may be any one of monosaccharides, polysaccharides formed by bonding two or more sugars, sugar alcohols, chemically modified sugars obtained by copolymerizing epichlorohydrin or the like with sugars, and the like.

[0077] A betaine compound refers to a compound having a betaine structure, and preferably includes a betaine-structured compound containing at least one anionic group selected from a carboxyl group, a sulfo group, a phosphonyl group, and a phosphonyloxy group and at least one cationic group selected from an ammonium group and a phosphonium group, and more preferably a betaine-structured compound containing a carboxyl group as the anionic group and an ammonium group as the cationic group.

[0078] As the betaine compound, for example, trimethylglycine can be mentioned.

[0079] And, as the above-mentioned water-soluble compound (W), in addition to the above-mentioned polyol compound and betaine compound, water-soluble polymers such as polyvinylpyrrolidone can be preferably mentioned.

[0080] Regarding the content of the above-mentioned water-soluble compound (W), as long as the effects of the present invention are exhibited, there is no particular limitation. For example, it can be set to 1% by mass or more, preferably 2.5% by mass or more. From the viewpoint of further enhancing the fluorescence intensity, it is more preferably 4.5% by mass or more, further preferably 9% by mass or more, further preferably 12% by mass or more, further preferably 15% by mass or more, particularly preferably 20% by mass or more, and most preferably 25% by mass or more. The upper limit value is not particularly limited and can be set to 100% by mass or less.

[0081] In addition, the content of the water-soluble compound (W) refers to the content of the water-soluble compound (W) in the fluorescence intensity enhancer of the present invention. In addition, when the fluorescence intensity enhancer of the present invention in a solution state acts on a fluorescently labeled target biomolecule, from the viewpoint of further enhancing the fluorescence intensity, it is preferred that the content of the water-soluble compound (W) in the fluorescence intensity enhancer (solution state) of the present invention satisfies the content of the above-mentioned water-soluble compound (W).

[0082] And, the water-soluble compound (W) used in the present invention is preferably a colorless compound, and more preferably a compound that does not absorb the excitation light of the fluorescent dye.

[0083] The fluorescence intensity enhancer of the present invention can be either in a solid state or in a solution state.

[0084] As an example of the fluorescence intensity enhancer of the present invention being in a solid state, particulate powders and freeze-dried powders of the fluorescence intensity enhancer of the present invention can be mentioned.

[0085] When the fluorescence intensity enhancer of the present invention is in a solid state, components such as buffering agents and surfactants that constitute the buffer solution described later can be further contained in a solid state. In addition, a part of the fluorescence intensity enhancer of the present invention can exist in an aqueous medium such as the buffer solution described later in a solution state and the remaining part in a solid state.

[0086] As an example of the fluorescence intensity enhancer of the present invention being in a solution state, for example, a solution obtained by dissolving the fluorescence intensity enhancer of the present invention in an aqueous medium can be mentioned.

[0087] The aqueous medium may contain an organic solvent in addition to water within the range where the effects of the present invention can be exhibited. Examples of the organic solvent that can be contained include organic solvents miscible with water such as methanol, ethanol, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, acetonitrile, and acetic acid.

[0088] Moreover, the aqueous medium may contain an additive within the range where the effects of the present invention can be exhibited. Examples of the additive that can be contained include buffers such as TBS (Tris-buffered saline), PBS (phosphate-buffered saline), TAE (Tris-acetate EDTA), TBE (Tris-borate EDTA), TE (Tris EDTA), and Good's buffer (for example, available from NIPPON GENE CO., LTD., DOJINDO LABORATORIES), surfactants such as sodium dodecyl sulfate, Triton X (product name, Polyoxyethylene(10)octylphenyl ether, for example, available from Sigma-Aldrich), Tween-20 (product name, Polyoxyethylene Sorbitan Monolaurate, for example, available from FUJIFILM Wako Pure Chemical Corporation), and compounds contained in the buffer solution. And a blocker such as BSA (bovine serum albumin) may be included. That is, when the fluorescence intensity enhancer of the present invention is in a solution state, examples include buffer solutions such as TBS, PBS, TBS-T (TBS containing Tween-20), PBS-T (PBS containing Tween-20), TAE, TBE, TE, or Good's buffer containing the above-mentioned water-soluble compound (W).

[0089] As the form of the fluorescence intensity enhancer of the present invention, a solution state is preferred, and a buffer solution form containing the above-mentioned water-soluble compound (W) is more preferred.

[0090] The fluorescence intensity enhancer of the present invention is preferably used for fluorescence imaging applications.

[0091] Fluorescence imaging refers to biological imaging that visualizes and observes a biological substance as a target (also referred to as "target biological substance" in the present invention) by performing fluorescence labeling using a fluorescent dye to label a biomolecule. Specifically, the fluorescence of the target biological substance (also referred to as "fluorescently labeled target biological substance" in the present invention) labeled with a biomolecule labeled with a fluorescent dye is observed. Details of the fluorescent dye used in the biomolecule labeled with a fluorescent dye, the biomolecule labeled with a fluorescent dye, and the fluorescence detection performed using the biomolecule labeled with a fluorescent dye are described later.

[0092] As for the use of fluorescence imaging, there is no particular limitation, and it can be applied to commonly used fluorescence imaging applications. For example, it can be used for immunoblotting (hereinafter also simply referred to as WB), multicolor WB, cell staining, dot blotting, and other applications.

[0093] In particular, in WB, after separating the target biological substance (protein) according to the molecular mass difference by SDS-PAGE method (sodium dodecyl sulfate-polyacrylamide gel electrophoresis method), the protein is transferred onto a membrane, and a fluorescent dye-labeled antibody is specifically bonded to the protein transferred onto the membrane to obtain a fluorescently labeled target biological substance, and then evaluation is performed. Thereby, protein bands can be detected. In WB, after obtaining the above-mentioned fluorescently labeled target biological substance, the membrane is dried and evaluated, whereby the fluorescence intensity can be further increased. Therefore, even in the case of a smaller amount of protein, protein bands can be detected.

[0094] In WB, when the fluorescence intensity enhancer of the present invention acts on the fluorescently labeled target biological substance and the membrane is dried, the quenching effect caused by the interaction between the fluorescent dyes in the fluorescently labeled target biological substance can be suppressed, and the fluorescence intensity can be increased. Therefore, it is considered that compared with ordinary WB, bands of lower protein mass can be detected with high sensitivity.

[0095] In addition, the fluorescence intensity enhancement effect of the fluorescence intensity enhancer of the present invention is not limited to the case where the membrane is dried, and the fluorescence intensity enhancement effect can also be obtained when observing without drying the membrane.

[0096] <<Method for enhancing the fluorescence intensity of a fluorescently labeled target biological substance>>

[0097] The method for enhancing the fluorescence intensity of the fluorescently labeled target biological substance of the present invention includes the step of allowing the fluorescence intensity enhancer of the present invention to act on the fluorescently labeled target biological substance.

[0098] Specifically, it is preferred that the fluorescence intensity enhancer of the present invention acts on the fluorescently labeled target biological substance, that is, the fluorescence intensity enhancer of the present invention acts on the fluorescently labeled target biological substance to enhance the fluorescence intensity emitted from the fluorescent label.

[0099] In addition, when the fluorescence intensity enhancer of the present invention acts on the fluorescently labeled target biological substance, it preferably acts as the above solution.

[0100] In order to make the fluorescence intensity enhancer of the present invention act on the fluorescently labeled target biological substance to enhance the fluorescence intensity, it is only necessary to make the fluorescence intensity enhancer of the present invention act during the period from obtaining the fluorescently labeled target biological substance to performing fluorescence detection, so as to exhibit the function of inhibiting the interaction between the fluorescent dyes in the fluorescently labeled target biological substance. For example, it is preferred that the fluorescence intensity enhancer of the present invention acts in the washing step of the fluorescently labeled target biological substance, and it is more preferred to wash the fluorescently labeled target biological substance with a buffer solution containing the above water-soluble compound (W).

[0101] For example, in WB, in the step of washing the fluorescently labeled target biological substance with a washing buffer solution after the fluorescent dye-labeled antibody is bonded to the protein transferred to the membrane through an antibody reaction to obtain the fluorescently labeled target biological substance, it is preferred to wash with a buffer solution containing the above water-soluble compound (W).

[0102] As the above washing buffer solution, a washing buffer solution commonly used in WB can be used without particular limitation. For example, washing buffer solutions such as TBS, PBS, TBS-T, PBS-T, TAE, TBE, TE, or Good's buffer solution can be cited.

[0103] In addition, as the buffer solution containing the above water-soluble compound (W), buffer solutions such as TBS, PBS, TBS-T, PBS-T, TAE, TBE, TE, or Good's buffer solution containing the above water-soluble compound (W) can be cited.

[0104] In addition, regarding the washing of the fluorescently labeled target biological substance, usually when washing is performed by changing the washing buffer solution multiple times, the washing with the buffer solution containing the above water-soluble compound (W) is used as at least the last washing, thereby enhancing the fluorescence intensity emitted from the fluorescent label.

[0105] Regarding the washing time (shaking time) in the washing process using the buffer solution containing the above-mentioned water-soluble compound (W), the content of the water-soluble compound (W) in the buffer solution, and other washing conditions, as long as an enhancement effect of fluorescence intensity can be obtained, there are no particular limitations. For example, regarding the washing time (shaking time), it can be set to 5 to 15 minutes, and regarding the content of the water-soluble compound (W) in the buffer solution, the description of the content of the water-soluble compound (W) in the fluorescence intensity enhancer of the present invention described above can be applied.

[0106] <<Fluorescence Detection Kit>>

[0107] The fluorescence detection kit of the present invention includes the following (a) and (b).

[0108] (a) The fluorescence intensity enhancer of the present invention.

[0109] (b) A reagent for producing a fluorescent dye-labeled biomolecule containing a fluorescent dye or a fluorescent dye-labeled biomolecule.

[0110] Regarding the fluorescence intensity enhancer of the present invention in the above (a), it can be either of the above solid state or solution state.

[0111] When in the solid state, in addition to the above (a), it may contain a buffer solution such as TBS, PBS, TBS-T, PBS-T, TAE, TBE, TE, or Good's buffer solution, and may also contain components other than the aqueous medium constituting the buffer solution such as TBS, PBS, TBS-T, PBS-T, TAE, TBE, TE, or Good's buffer solution (buffer agents, surfactants, etc.). When containing components other than the aqueous medium constituting the buffer solution such as TBS, PBS, TBS-T, PBS-T, TAE, TBE, TE, Good's buffer solution (buffer agents, surfactants, etc.), when acting on the fluorescently labeled target biomolecule, it is preferably made to act in a solution state by adding an aqueous medium.

[0112] When in the solution state, it is preferably a buffer solution such as TBS, PBS, TBS-T, PBS-T, TAE, TBE, TE, or Good's buffer solution, and Tween-20 may also be added.

[0113] When the above (b) is a reagent for producing a fluorescent dye-labeled biomolecule containing a fluorescent dye, in addition to the fluorescent dye, it may contain other components for producing a fluorescent dye-labeled biomolecule. For example, a biomolecule (preferably an antibody) that binds to the fluorescent dye to produce a fluorescent dye-labeled biomolecule can be cited.

[0114] When the above (b) is a fluorescent dye-labeled biomolecule, the fluorescent dye-labeled biomolecule can act as either the first antibody or the second antibody to act on the applicable target biomolecule. In addition, when the fluorescent dye-labeled biomolecule acts as the second antibody, it may further contain the first antibody against the applicable target biomolecule.

[0115] The kit for fluorescence detection of the present invention may be any kit for fluorescence detection of a fluorescently labeled target biomolecule, and is preferably used for fluorescence detection in fluorescence imaging applications.

[0116] The kit for fluorescence detection of the present invention may, in addition to the above (a) and (b), further contain protein extraction and treatment solutions, gels, electrode solutions, blotting reagents, blocking agents, treatment solutions, cleaning solutions, buffer solutions, etc. that are commonly used for preparing fluorescently labeled target biomolecules.

[0117] Hereinafter, the fluorescent dyes for fluorescent dye-labeled biomolecules, the fluorescent dye-labeled biomolecules, and the fluorescence detection using the fluorescent dye-labeled biomolecules will be described in detail.

[0118] <Fluorescent Dyes>

[0119] The fluorescent dye-labeled biomolecule on which the fluorescence intensity enhancer of the present invention acts may be any biomolecule labeled with a fluorescent dye.

[0120] As the above fluorescent dyes, fluorescent dyes commonly used in the labeling of biomolecules can be used without particular limitation. For example, xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, squarylium dyes, phthalocyanine dyes, porphyrin dyes, pyridyl oxazole dyes, and pyrromethene dyes can be cited.

[0121] Specifically, for example, fluorescent compounds (compounds that exhibit fluorescence) described in International Publication No. 2019 / 230963, International Publication No. 2021 / 100814, International Publication No. 2021 / 125295, International Publication No. 2021 / 215514, International Publication No. 2022 / 025210, International Publication No. 2022 / 191123, Japanese Patent Application Laid-Open No. 2019-172826, Japanese Re-Publication No. 2019 / 230963, Japanese Patent Application Laid-Open No. 2022-131357, etc. can be cited.

[0122] Furthermore, commercially available fluorescent dyes can be used without particular limitation. For example, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, DyLight 350, DyLight 405, DyLight 425Q, DyLight 488, DyLight 510-LS, DyLight 515-LS, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 730-B1, DyLight 747-B4, DyLight 800, DyLight 650-4xPEG, DyLight 800-4xPEG, etc. (all are product names manufactured by Thermo Fisher Scientific Inc.), ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO RHO6G, ATTO 540Q, ATTO 550, ATTO 565, ATTO RHO11, ATTO 580Q, ATTO 590, ATTO 594, ATTO 610, ATTO 612Q, ATTO 620, ATTO 633, ATTO RHO14, ATTO 647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, etc. (all are product names manufactured by ATTO-TEC GmbH), Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc. (all are product names of Global Life Sciences Technologies Japan K.K.Product names manufactured), CF 350, CF 405S, CF 405M, CF 405L, CF 430, CF 440, CF 450, CF 488A, CF 503R, CF514, CF 532, CF 535ST, CF 543, CF 550R, CF 555, CF568, CF 570, CF 583, CF 583R, CF 594, CF 594ST, CF 597R, CF 620R, CF 633, CF 640R, CF647, CF 660C, CF 660R, CF 680, CF 680R, CF 700, CF 750, CF 770, CF 790, CF 800, CF 820, CF 850, CF 870, etc. (all are product names manufactured by Biotium).

[0123] The fluorescence intensity enhancer of the present invention can more effectively enhance the fluorescence intensity of a biological substance labeled with a fluorescent dye having two or more fluorophore moieties in one molecule. As the fluorescent dye having two or more fluorophore moieties in one molecule, for example, the following fluorescent dye (F) can be preferably cited.

[0124] <Fluorescent dye (F)>

[0125] The fluorescent dye (F) is a compound having two or more fluorophore moieties with equivalent light absorption characteristics, and is a compound in which adjacent fluorophore moieties are connected via a group containing a structure represented by the following general formula (I). The structure represented by the general formula (I) has a repeating unit (the number of repetitions is 2 or more) of a nitrogen-containing saturated 5-membered ring such as a proline-derived ring structure, and functions as a rigid linker for two fluorophore moieties connected via a group containing the structure represented by the general formula (I), and thus can effectively suppress quenching caused by the association of fluorophore moieties intermolecularly or intramolecularly. Therefore, it is considered that a fluorescent dye-labeled biomolecule obtained using the fluorescent dye (F) can exhibit excellent fluorescence intensity.

[0126] The fluorescent dye (F) may be a compound classified as a polymer or an oligomer.

[0127] In the present invention, "fluorophore moieties with equivalent light absorption characteristics" means that the difference between the maximum absorption wavelengths in the absorption spectra of the respective fluorophore moieties satisfies a relationship within 15 nm.

[0128] In the present invention, all the fluorophore moieties of the fluorescent dye (F) are preferably compounds that satisfy the relationship that the difference between the maximum absorption wavelength on the lowest wavelength side and the maximum absorption wavelength on the highest wavelength side in the absorption spectra of the respective fluorophore moieties is within 15 nm.

[0129] As a compound having two phosphor moieties in a fluorescent dye, a compound that exhibits the FRET phenomenon (Fluorescence Resonance Energy Transfer) is known. In this compound, the difference in the maximum absorption wavelengths in the absorption spectra of the phosphor moiety I (energy donor) excited by the excitation light and another phosphor moiety II (energy acceptor) that emits light or quenches upon receiving energy from the phosphor moiety I generally exceeds 15 nm. In such a compound, although fluorescence is emitted from the phosphor moiety I, the energy is received by the phosphor moiety II, resulting in a lower fluorescence intensity of the compound.

[0130] In contrast, as described above, since the fluorescent dye (F) has phosphor moieties with equivalent light absorption characteristics, the FRET phenomenon does not occur, and thus the fluorescence intensity proportional to the number of phosphor moieties can be exhibited.

[0131] The chemical structure of the phosphor moieties with equivalent light absorption characteristics as described above is not particularly limited as long as the difference in the maximum absorption wavelengths described above is satisfied, and it is preferred that the structures of the main skeletons of the phosphor moieties are the same. Among them, the configuration and chain length of the substituents can be different, and when there are anionic or cationic groups, the counterions can also be different. The difference in the maximum absorption wavelengths is preferably within 10 nm, more preferably within 5 nm.

[0132] In addition, the absorption spectrum of the phosphor moiety refers to the spectrum obtained by measuring the phosphor monomer that constitutes the phosphor moiety diluted with PBS buffer using a spectrophotometer.

[0133] Moreover, the "compound in which adjacent phosphor moieties are connected via a group containing a structure represented by the following general formula (I)" is preferably a compound in which two structures having a phosphor moiety as a substituent are connected via a group containing a structure represented by the following general formula (I).

[0134] In addition, as long as adjacent phosphor moieties are connected via a group containing a structure represented by the following general formula (I), the structure having the above phosphor moiety as a substituent is not particularly limited. For example, as described in detail in the following general formula (II), by the combination of L 1 and L 2 , the combination of L 1 and L 3 , the combination of L 4 and L 5 , or the combination of L 4 and L 6 , a structure in which carbon atoms, nitrogen atoms, and X 1 to X 3As a 5-membered ring that forms a ring atom, the 5-membered ring may have a group containing a phosphor portion as a substituent. That is, as a structure having a phosphor portion as a substituent, as long as each adjacent phosphor portion is connected via a group containing a structure represented by the following general formula (I), the carbon atom, nitrogen atom, and X described in the structure represented by the following general formula (I) 1 ~X 3 The 5-membered ring that forms a ring atom may be a structure having a group containing a phosphor portion as a substituent. As such a compound, for example, compounds represented by the following general formula (VI) or (VII) can be cited.

[0135] In the fluorescent pigment (F), the number of the above-described phosphor portions is 2 or more. The upper limit is not particularly limited, and for example, it can be set to 30 or less, preferably 20 or less, more preferably 15 or less.

[0136] Regarding the phosphor portion of the fluorescent pigment (F), in addition to the above, the description and specific examples of the phosphor portion in the following general formula (II) can also be applied.

[0137] (Structure represented by general formula (I))

[0138] [Chemical formula 2]

[0139]

[0140] In the formula, X 1 ~X 3 represents -O-, -S-, >NR 1 or >CR 2 R 3 .

[0141] R 1 ~R 3 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, -NR 8 R 9 , -OR 10 or an anionic group.

[0142] R 8 ~R 10 represents a hydrogen atom, an alkyl group, an alkenyl group, an acyl group, an aryl group, a heteroaryl group or an anionic group.

[0143] n is an integer of 2 or more.

[0144] * represents a linking bond.

[0145] When considering stereoisomers, the structure represented by the above general formula (I) is preferably a structure represented by any one of the following general formula (IA) or (IB). In addition, X in the following general formula1 to X 3 and n have the same meanings as X in the above general formula (I). 1 to X 3 and n have the same meanings.

[0146] [Chemical Formula 3]

[0147]

[0148] X 1 to X 3 represents -O-, -S-, >NR 1 or >CR 2 R 3 and R 1 to R 3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group, -NR 8 R 9 , -OR 10 or an anionic group.

[0149] The alkyl group, alkenyl group, alkynyl group, acyl group, aryl group, heteroaryl group and anionic group that can be used as R 1 to R 3 have the same meanings as the alkyl group, alkenyl group, alkynyl group, acyl group, aryl group, heteroaryl group and anionic group in the substituent group T described later, and the preferred ranges are also the same.

[0150] The alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group that can be used as the above R 1 to R 3 can be unsubstituted or can have substituents.

[0151] As the substituents that the alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group in R 1 to R 3 can have, the substituents of the substituent group T described later can be listed. For example, a halogen atom or an anionic group is preferred.

[0152] In -NR 1 to R 3 that can be used as R 8 R 9 and -OR 10 in, R 8 to R 10 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an aryl group, a heteroaryl group or an anionic group.

[0153] The alkyl group, alkenyl group, alkynyl group, acyl group, aryl group and heteroaryl group that can be used as R 8 to R 10The meanings of the alkyl, alkenyl, alkynyl, acyl, aryl, heteroaryl, and anionic groups are the same as those of the alkyl, alkenyl, alkynyl, acyl, aryl, heteroaryl, and anionic groups in the substituent group T described below, and the preferred ranges are also the same.

[0154] Can be used as R 8 ~R 10 The alkyl, alkenyl, alkynyl, acyl, aryl, and heteroaryl groups of can be unsubstituted or can have substituents.

[0155] As R 8 ~R 10 The substituents that the alkyl, alkenyl, alkynyl, acyl, aryl, and heteroaryl groups in can have include the substituents in the substituent group T described below. For example, a halogen atom, a carbamoyl group, an acylamino group, an alkoxy group (preferably an alkoxy group having an anionic group), or an anionic group is preferred, and a carbamoyl group, an acylamino group, an alkoxy group (preferably an alkoxy group having an anionic group), or an anionic group is more preferred.

[0156] As R 1 , a hydrogen atom, an alkyl group, -NR 8 R 9 , -OR 10 or an anionic group is preferred.

[0157] As R 2 and R 3 , a hydrogen atom, -NR 8 R 9 , -OR 10 or an anionic group is preferred, and it is more preferred that R 2 is a hydrogen atom, -NR 8 R 9 , -OR 10 or an anionic group and R 3 is a hydrogen atom.

[0158] As R 8 and R 10 , a hydrogen atom, an alkyl group, or an acyl group is preferred. The alkyl group can be an alkyl group having an anionic group, and the acyl group can be an acyl group containing an anionic group (preferably an acyl group having an anionic group or an acyl group substituted with an alkoxy group having an anionic group). The above alkyl and acyl groups can also have substituents other than anionic groups, and the substituents in the substituent group T described below can be cited. For example, a carbamoyl group or an acylamino group is preferred.

[0159] As the above X 1 ~X 3 , it is preferred that at least any one is >CR 2 R 3 , and it is more preferred that at least two are >CR 2R 3 and the remaining one is -O-, -S- or >CR 2 R 3 .

[0160] From the viewpoint of making the structure represented by the general formula (I) a more rigid structure, the structure represented by the general formula (I) preferably contains X 1 ~X 3 being >CR 2 R 3 . "The structure represented by the general formula (I) contains X 1 ~X 3 being >CR 2 R 3 " means that in at least one of the consecutive n structures represented by the foregoing general formula (i), X 1 ~X 3 are all >CR 2 R 3 .

[0161] In the structure represented by the general formula (I), the proportion of the number of the above X 1 ~X 3 being >CR 2 R 3 is preferably 30% or more, more preferably 60% or more, and further preferably 80% or more. The upper limit value is not particularly limited and can be set to 100% or less. In addition, it is also preferred that all the structures represented by the general formula (I) are the above X 1 ~X 3 being >CR 2 R 3 structures.

[0162] When at least one R 1 ~X 3 in the structure of >CR 2 R 3 exists between molecules and there are multiple, from the viewpoint of suppressing the interaction between the structures represented by the general formula (I) within the molecule, the at least one R 2 is preferably -NR 2 R 8 R 9 , -OR 10 or an anionic group, more preferably -NR 8 R 9 , -OR 10 or an anionic group and at least one of R 8 ~R 10 is a group containing an anionic group.

[0163] In addition, the above "X1 to X 3 is >CR 2 R 3 at least one R in the structure of 2 is -NR 8 R 9 , -OR 10 or an anionic group and R 8 to R 10 at least one of which is a group containing an anionic group" means that when R 2 is -NR 8 R 9 , at least one of R 8 and R 9 is a group containing an anionic group, and when R 2 is -OR 10 , R 10 is a group containing an anionic group.

[0164] And, R 10 being a group containing an anionic group means that R 10 is an anionic group or a group having an anionic group as a substituent. This is the same in the case where at least one of R 8 and R 9 is a group containing an anionic group.

[0165] n is an integer of 2 or more. Regarding the fluorescent dye (F), by n being an integer of 2 or more, it is possible to impart rigidity effective for suppressing the association of the structure represented by the general formula (I) to the pigment (fluorescent body part), and it is possible to suppress a decrease in fluorescence intensity.

[0166] In the present invention, when the number of the above-mentioned fluorescent body parts is 2, from the viewpoint of further increasing the fluorescence intensity, the lower limit value of n is preferably an integer of 3 or more, more preferably an integer of 7 or more, further preferably an integer of 9 or more, and particularly preferably an integer of 12 or more. The upper limit value is not particularly limited. For example, it can be set to an integer of 72 or less, preferably an integer of 36 or less, more preferably an integer of 24 or less, and further preferably an integer of 18 or less. That is, n is preferably an integer of 3 to 72, more preferably an integer of 7 to 36, further preferably an integer of 9 to 24, and particularly preferably an integer of 12 to 18.

[0167] On the other hand, when the number of the phosphor portions is 3 or more, compared with the case where the number of the phosphor portions is 2, the number of phosphor portions (pigment number) per 1 molecule is larger. Therefore, a certain amount of pigment association can be tolerated. Moreover, when it is an integer of 3 or more, the fluorescence intensity can be further increased, so it is preferred. Among them, when n is an integer of 6, although it has a certain association inhibition effect but it is not sufficient. Therefore, n is more preferably an integer of 7 or more. When the number of the phosphor portions is 3 or more, the upper limit value of n is preferably an integer of 72 or less, more preferably an integer of 36 or less, and still more preferably an integer of 24 or less. That is, when the number of the phosphor portions is 3 or more, n is preferably an integer of 3 to 72, more preferably an integer of 7 to 36, and still more preferably an integer of 7 to 24.

[0168] <Compound represented by general formula (II)>

[0169] The fluorescent pigment (F) is preferably represented by the following general formula (II).

[0170] [Chemical formula 4]

[0171]

[0172] In the formula, R 4 and R 5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxyl group, an alkoxy group, a thioalkyl group, an aryl group or a heteroaryl group.

[0173] R 6 and R 7 represent a hydrogen atom, a hydroxyl group, a thioalkyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, an amino group, an acyl group, a heteroaryl group, an anionic group, a cationic group or Q.

[0174] Q represents a carboxyl group, a substituent capable of bonding to a biological substance or a substituent capable of bonding to a solid support.

[0175] L 1 ~L 7 represent a single bond or a divalent linking group.

[0176] M represents a phosphor portion, a physiologically active substance portion, a prodrug portion or a radioactive isotope-containing portion.

[0177] Y represents a structure represented by the above general formula (I).

[0178] m is an integer of 1 or more.

[0179] Among them, at least two of M represent phosphor portions having equivalent light absorption characteristics to each other. In other words, it means that at least two of M are phosphor portions and each adjacent phosphor portion has equivalent light absorption characteristics to each other.

[0180] R 4 and R 5 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxyl group, an alkoxy group, a thioalkyl group, an aryl group or a heteroaryl group.

[0181] The alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group and heteroaryl group that can be used as R 4 or R 5 have the same meanings as the alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group and heteroaryl group in the substituent group T described below, and the preferred ranges are also the same.

[0182] The alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group and heteroaryl group that can be used as R 4 or R 5 can be unsubstituted or can have substituents. As the substituents that can be had, the substituents in the substituent group T described below can be enumerated. For example, a halogen atom is preferred.

[0183] As R 4 and R 5 , from the viewpoint of ease of synthesis, a hydrogen atom is preferred. When an amino acid is used as a raw material, R 4 and R 5 are mostly hydrogen atoms. However, since the substituents in R 4 and R 5 do not contribute much to the excellent fluorescence intensity of the fluorescent dye (F), R 4 and R 5 can be other substituents (alkyl group, alkenyl group, alkynyl group, acyl group, amino group, hydroxyl group, alkoxy group, thioalkyl group, aryl group or heteroaryl group) other than a hydrogen atom.

[0184] R 6 and R 7 represent a hydrogen atom, a hydroxyl group, a thioalkyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, an amino group, an acyl group, a heteroaryl group, an anionic group, a cationic group or Q.

[0185] The alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group, heteroaryl group, anionic group and cationic group that can be used as R 6 or R 7 have the same meanings as the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group, heteroaryl group, anionic group and cationic group in the substituent group T described below, and the preferred ranges are also the same.

[0186] The alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group, heteroaryl group, anionic group and cationic group that can be used as R 6 or R 7The alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group and heteroaryl group may be unsubstituted or may have substituents.

[0187] As R 6 or R 7 The substituents that the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, amino group, acyl group and heteroaryl group in may have include the substituents in the following-described substituent group T. Preferred are an alkyl group, an acyl group, an alkoxy group, an amino group, an anionic group, a cationic group, -(L-O) g R E or Q, or a substituent formed by combining two or more of these, and more preferably an alkyl group, an acyl group, an alkoxy group, an amino group, -(L-O) g R E or Q, or a substituent formed by combining two or more of these substituents.

[0188] Q that can be used as R 6 or R 7 represents a carboxyl group, a substituent capable of bonding to a biological substance or a substituent capable of bonding to a solid support.

[0189] As the substituent capable of bonding to a biological substance, the description of the substituent capable of bonding to the following-described biological substance can be applied, and as the substituent capable of bonding to a solid support, the description of the substituent capable of bonding to the following-described solid support can be applied.

[0190] As R 6 , the description of the substituent represented by the following -L 9 R 6A or -L 13 R 6A can be preferably cited, and as R 7 , the description of the substituent represented by the following -L 8 R 7A or -L 12 R 7A can be preferably cited. Specifically, the description of the substituent represented by -L 9 R 6A refers to a substituent obtained by combining a group that can be used as L 9 and a group that can be used as R 6A . This is the same for -L 13 R 6A , -L 8 R 7A and -L 12 R 7A .

[0191] In the present invention, from the viewpoint of being able to have appropriate hydrophilicity and an appropriate excluded volume effect and being able to obtain excellent fluorescence intensity, R is preferred6 and R 7 any one of them contains a structure represented by -(L-O) g R E more preferably, R 7 contains a structure represented by -(L-O) g R E represents the structure

[0192] L 2 ~L 5 and L 7 represent a single bond or a divalent linking group, preferably a single bond, or an alkylene, alkenylene, alkynylene, arylene, heteroarylene, -O-, -S-, >C=O, >NR A , >S=O, >S(=O)2 and >P(=O)OR B in one kind or a linking group formed by combining two or more kinds. R A and R B has the same meaning as R A and R B described later, and represents a hydrogen atom or a substituent

[0193] L 1 and L 6 represent a single bond or a divalent linking group, preferably a single bond, alkylene, alkenylene, alkynylene, arylene, heteroarylene, -O-, -S-, >C=O, >NR A , >S=O, >S(=O)2 or >P(=O)OR B . R A and R B has the same meaning as R A and R B described later, and represents a hydrogen atom or a substituent

[0194] The alkylene that can form L 1 ~L 7 has the same meaning as the group obtained by further removing one hydrogen atom from the alkyl groups selected from the substituent group T described later, and the preferred groups are also the same

[0195] The alkenylene that can form L 1 ~L 7 has the same meaning as the group obtained by further removing one hydrogen atom from the alkenyl groups selected from the substituent group T described later, and the preferred groups are also the same

[0196] The alkynylene that can form L 1 ~L 7 has the same meaning as the group obtained by further removing one hydrogen atom from the alkynyl groups selected from the substituent group T described later, and the preferred groups are also the same

[0197] can form L1 ~L 7 The arylene group of ~L has the same meaning as the group obtained by further removing one hydrogen atom from the aryl group selected from the substituent group T described later, and the preferred groups are also the same.

[0198] can form L 1 ~L 7 The heteroarylene group of ~L has the same meaning as the group obtained by further removing one hydrogen atom from the heteroaryl group selected from the substituent group T described later, and the preferred groups are also the same.

[0199] can form L 1 ~L 7 The alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group of ~L can be unsubstituted groups or groups having substituents.

[0200] As the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group that can form L 1 ~L 7 The substituents that the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group can have are not particularly limited, and are preferably selected from the substituent group T described later, and more preferably halogen atom, alkyl group, acylamino group or carbamoyl group can be listed. And, the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group that can form L 1 ~L 7 The substituents that the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group can have can be further substituted by substituents selected from the substituent group T described later. For example, an amino group is preferred.

[0201] And, regarding the number of substituents that the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group that can form L 1 ~L 7 can have, as long as it is a structure that can be adopted, it is not particularly limited, and can be set to at least 1 or more. As the upper limit value, it is not particularly limited. For example, all hydrogen atoms in the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group can be substituted by substituents.

[0202] As the >NR 1 ~L 7 that can be adopted as a structure that can form L A in R A and >P(=O)OR B in R B The substituents are not particularly limited and are preferably selected from the substituent group T described later. As R A , a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or an anionic group is preferred, a hydrogen atom or an alkyl group is more preferred, and a hydrogen atom is further preferred. As R B , a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group is preferred, a hydrogen atom or an alkyl group is more preferred, and a hydrogen atom is further preferred.

[0203] In addition, R A and R B of the above-mentioned alkyl, alkenyl, alkynyl, aryl and heteroaryl groups may be unsubstituted groups or groups having substituents.

[0204] Among the linking groups formed by combining two or more of the alkylene, alkenylene, alkynylene, arylene, heteroarylene, -O-, -S-, >C=O, >NR 2 ~L 5 and L 7 >, >S=O, >S(=O)2 and >P(=O)OR A as long as they form a proper chemical structure, the types of the combined groups are not particularly limited. For example, 2 to 6 types are preferred, and 2 to 4 types are more preferred. In addition, the alkylene, alkenylene, alkynylene, arylene, heteroarylene, -O-, -S-, >C=O, >NR B >, >S=O, >S(=O)2 and >P(=O)OR A are each counted as one type, and there are at most 12 types. B

[0205] In addition, among the linking groups formed by combining two or more of the alkylene, alkenylene, alkynylene, arylene, heteroarylene, -O-, -S-, >C=O, >NR 2 ~L 5 and L 7 >, >S=O, >S(=O)2 and >P(=O)OR A as long as they form a proper chemical structure, the number of the combined groups is not particularly limited. For example, 2 to 10 can be preferably listed, 2 to 6 are more preferred, and 2 to 4 are further preferred. B

[0206] (i) L 1 , L 6

[0207] L 1 More preferably, they are >C=O, >NR A , arylene, alkylene, -O- or -S-, and further preferably >C=O, >NR A , arylene or alkylene, and particularly preferably >C=O or >NR A .

[0208] L 6 More preferably, they are a single bond, >C=O, >NR A or arylene, and further preferably a single bond, >C=O or >NR A .

[0209] (ii) L 3 ​​, L 4 , L 7

[0210] L 3 , L 4 and L 7 More preferably, it is a single bond, >C=O, >NR A , alkylene, alkenylene, alkynylene, arylene or heteroarylene, or at least one of alkylene, alkenylene, alkynylene, arylene and heteroarylene and >C=O and >NR A The combination is further preferably a single bond, >C=O, >NR A , alkylene, alkenylene, alkynylene, arylene or heteroarylene, or -C(=O)NR A - and >C=O or -NR A C(=O)- and >NR A The group connected by at least one of alkylene, alkenylene, alkynylene, arylene and heteroarylene between them.

[0211] (iii) L 2 , L 5

[0212] L 2 and L 5 More preferably, it is one of alkylene, alkenylene, alkynylene, arylene, heteroarylene, -O-, -S-, >C=O and >NR A Or a group formed by combining two or more of them, and further preferably a group represented by *-L x -L y -**.

[0213] L x is a single bond, or one of alkylene, alkenylene, alkynylene, arylene and heteroarylene or a group formed by combining two or more of them, L y is a single bond, -O-, -S-, >C=O or >NR A . * represents the carbon atom bonded to L 1 and L 3 or the carbon atom bonded to L 4 and L 6 The connecting bond of the carbon atom, ** represents the connecting bond with M. Among them, when L y is a single bond, L x is heteroarylene, or a group composed of one of alkylene, alkenylene, alkynylene and arylene on the * side or a group formed by combining two or more of them and heteroarylene on the ** side.

[0214] Regarding L 2 and L 5 , in the above-mentioned *-Lx -L y Among the groups represented by -**, L is also preferred. y is a single bond, -S-, >C=O or >NR A group, and L is more preferred y is >C=O or >NR A group, and L is further preferred x is an alkylene group and L y is >C=O or >NR A group.

[0215] In addition, in the compound represented by the above general formula (II), M and L 1 and L 3 the carbon atom to which it is bonded or L 4 and L 6 the connecting chain (including the connecting chain containing L 2 the connecting chain and the connecting chain containing L 5 each connecting chain) connecting the carbon atoms to which it is bonded, for example, the shortest number of atoms can be 1 to 60, preferably 1 to 40. When M is a phosphor part, the above shortest number of atoms refers to the shortest chain of atoms in the connecting chain that forms the conjugated structure part for fluorescence display in the phosphor part M and connects the carbon atoms to which L 1 and L 3 are bonded or the carbon atoms to which L 4 and L 6 are bonded.

[0216] In addition, in the compound represented by the above general formula (II), it is also preferred that in the structure represented by the conjugated structure part of M - the connecting group ZZZ - L 2 - any part of the connecting chain represented by "- connecting group ZZZ - L 2 -", and in the structure represented by the conjugated structure part of M - the connecting group ZZZ - L 5 - any part of the connecting chain represented by "- connecting group ZZZ - L 5 -" has the structure represented by -(CH2 - CH2 - O) b - (b is also as described later).

[0217] In the compound represented by the general formula (II), adjacent groups can bond to each other to form a ring. As a combination of adjacent groups that can bond to each other to form a ring, for example, the combination of L 1 and L 2 , the combination of L 1 and L 3 , the combination of L 2 and R 4 , the combination of L 4 and L5 in combination with L 4 and L 6 in combination or L 5 and R 5 in combination.

[0218] The ring formed by the above adjacent groups bonding to each other can be any one of an aromatic ring and an aliphatic ring, and can also be any one of a hydrocarbon ring and a heterocyclic ring, preferably a 5- or 6-membered ring.

[0219] As the above aliphatic ring, a cyclopentane ring, a cyclohexane ring or a 5-membered ring having carbon atoms, nitrogen atoms and X as ring-constituting atoms as described in the structure represented by the above general formula (I) can be preferably exemplified, 1 ~X 3 more preferably a 5-membered ring having carbon atoms, nitrogen atoms and X as ring-constituting atoms as described in the structure represented by the above general formula (I), 1 ~X 3 as ring-constituting atoms.

[0220] As the above aromatic ring, a benzene ring or a nitrogen-containing aromatic heterocyclic ring is preferred, a benzene ring or a nitrogen-containing aromatic heterocyclic ring having ring-constituting atoms of carbon atoms and nitrogen atoms is more preferred, and a benzene ring or a pyridine ring is further preferred.

[0221] These rings can have substituents, and there is no particular limitation on the substituents that can be had, and they are selected from the substituent group T.

[0222] As the ring formed by the combination of L 2 and R 4 in combination or L 5 and R 5 in combination, it can be any one of the above aliphatic rings and aromatic rings, preferably the above aliphatic ring.

[0223] As the ring formed by the combination of L 1 and L 2 in combination, the combination of L 1 and L 3 in combination, the combination of L 4 and L 5 in combination or the combination of L 4 and L 6 in combination, it can be any one of the above aliphatic rings and aromatic rings, preferably a 5-membered ring having carbon atoms, nitrogen atoms and X as ring-constituting atoms as described in the structure represented by the above general formula (I) or a benzene ring. 1 ~X 3 as ring-constituting atoms.

[0224] For example, the following structure enclosed by a dotted line in the general formula (II) can be set as,

[0225] [Chemical formula 5]

[0226]

[0227] A structure including the following structures. In the following structures, * represents a connecting portion.

[0228] [Chemical formula 6]

[0229]

[0230] m is an integer of 1 or more. There is no particular limitation on the upper limit value. For example, it can be set as an integer of 30 or less, preferably 20 or less, more preferably 15 or less, and further preferably 10 or less. That is, m can be set as an integer from 1 to 30, preferably from 1 to 20, more preferably from 1 to 15, and further preferably from 1 to 10.

[0231] M represents a phosphor part, a physiologically active substance part, a prodrug part, or a radioactive isotope-containing part. Among them, at least two of Ms represent phosphor parts having equivalent light absorption characteristics.

[0232] As the phosphor part that can be adopted as M (hereinafter, also referred to as phosphor part M), as long as it is a structural part composed of an organic compound that exhibits fluorescence, it can be used without particular limitation. And the phosphor part M can be a structural part in which a structural part composed of an organic compound that exhibits fluorescence further has a linking group. As such a linking group, there is no particular limitation, and for example, the linking group ZZZ described later can be cited. For example, in the compound represented by the general formula (II), it is preferable to cite a compound in which the phosphor part M is bonded to L 2 or L 5 by this linking group ZZZ.

[0233] As the phosphor part M, for example, a structural part composed of at least one pigment selected from xanthene pigments, rhodamine pigments, coumarin pigments, cyanine pigments, pyrene pigments, oxazine pigments, squarylium pigments, pyridyl oxazole pigments, and pyrromethene pigments can be cited.

[0234] As the above-mentioned xanthene pigments, rhodamine pigments, coumarin pigments, cyanine pigments, pyrene pigments, oxazine pigments, squarylium pigments, pyridyl oxazole pigments, and pyrromethene pigments, pigments generally known as these pigments can be used without particular limitation.

[0235] As one mode, the phosphor part M is preferably a structural part composed of a pyrromethene pigment. As the pyrromethene pigment, a dipyrromethene boron complex can be cited. As the dipyrromethene boron complex, a fluorescent compound (dipyrromethene boron complex) represented by the general formula (1) or (4) described in International Publication No. 2019 / 230963 and a compound (dipyrromethene boron complex) represented by the general formula (1) described in International Publication No. 2021 / 100814 can be used, and these descriptions can be cited and incorporated into this specification.

[0236] In addition, with respect to the pigment constituting the phosphor part M, it is incorporated in such a manner that it does not have a substituent capable of bonding to a biological substance.

[0237] As another mode, the phosphor part M is preferably a structural part composed of a cyanine pigment, and more preferably a structural part composed of a cyanine pigment represented by the following general formula (α).

[0238] [Chemical formula 7]

[0239]

[0240] In the formula, R 1 ~R 4 represents an alkyl group or -(CH2-CH2-O) b -R 21 . b is 1 to 50, and R 21 represents an alkyl group.

[0241] R 11 ~R 13 represents a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group, or a halogen atom, and adjacent groups may bond to each other to form a 5-membered ring or a 6-membered ring.

[0242] R 22 ~R 25 and R 32 ~R 35 represent a hydrogen atom, an alkyl group, an alkoxy group, an aryl group, a sulfo group, a sulfamoyl group, a carboxyl group, an alkoxycarbonyl group, an aryloxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a nitro group, or a halogen atom.

[0243] R 41 and R 42 represent an alkyl group or -(CH2-CH2-O) b -R 21 . R 21 and b have the same meanings as R 21 and b described above. R 41 and R 42 may bond to each other to form a ring.

[0244] a is an integer from 1 to 3.

[0245] By removing one hydrogen atom from any one of the above R 1 ~R 4 、R 11 ~R 13 、R 22 ~R 25 、R 32 ~R 35 、R 41 or R 42 to form a monovalent structural moiety.

[0246] Among them, the cyanine pigment represented by the formula (α) is neutral.

[0247] The cyanine pigment represented by the above general formula (α) depends on the length of the methylene chain with a repeat number of 2a + 3 connected by conjugated double bonds. When a = 1, it has an excitation absorption wavelength in the wavelength range of 520 - 600 nm (near 585 nm); when a = 2, it has an excitation absorption wavelength in the wavelength range of 620 - 700 nm (near 685 nm); when a = 3, it has an excitation absorption wavelength in the wavelength range of 740 - 830 nm (near 785 nm). Therefore, the fluorescent pigment (F) having a structural moiety composed of the cyanine pigment represented by the general formula (α) as the phosphor moiety M can be used as a compound that exhibits excellent fluorescence intensity in a fluorescence labeling using a light source with an arbitrary wavelength (e.g., near 600 nm, 700 nm, 800 nm) in the wavelength range of approximately 500 - 800 nm corresponding to the absorption excitation wavelength of the compound as the excitation light source.

[0248] In multicolor WB, multiple emission colors are detected in the range from the visible region to the near-infrared region. Therefore, it is necessary to select in such a way that the absorption waveforms and emission waveforms of multiple pigments have an appropriate wavelength relationship to avoid crosstalk caused by interference when exciting the pigments to emit light. Ideally, it is adjusted so that only one pigment emits light under a certain excitation light, and other pigments do not emit light. From this point of view, for example, two excitation light sources with wavelengths separated by a certain degree, such as near 700 nm and near 800 nm, are used for the emission in the near-infrared region of multicolor WB.

[0249] Fluorescence detection using near-infrared light excitation can suppress the autofluorescence of the membrane, that is, background fluorescence, compared with detection using visible light excitation. Therefore, it is easy to improve the signal-to-noise ratio (S / N ratio) and detect the target protein with high sensitivity. Therefore, in recent years, the necessity of fluorescence detection WB using emission in the near-infrared region has increased in the analysis and research of trace proteins.

[0250] However, in the near-infrared region, the fluorescence quantum yield of fluorescent dyes is generally low, and it is not easy to obtain a high signal amount. Among fluorescent dyes (F) having a structural part composed of cyanine pigments represented by the general formula (α) as the phosphor part M, a compound in which a = 2 or 3 can also be used as a compound showing excellent fluorescence intensity in multicolor WB having the above two light sources near 700 nm and near 800 nm. In particular, for the requirement of observing and detecting proteins with higher sensitivity, compared with the fluorescence labeling using conventional cyanine pigments, it can show more excellent fluorescence intensity.

[0251] (i)R 1 ~R 4

[0252] R 1 ~R 4 represents an alkyl group or -(CH2-CH2-O) b -R 21 。

[0253] It can be adopted that the alkyl group for R 1 ~R 4 has the same meaning as the alkyl group in the substituent group T described later.

[0254] The number of carbon atoms of the unsubstituted alkyl group is preferably 1 to 6, more preferably 1 to 4, and further preferably 1 to 2.

[0255] When the alkyl group has a substituent, as the number of carbon atoms of the alkyl part of the alkyl group having a substituent, it is preferably 1 to 10, more preferably 1 to 8, further preferably 2 to 6, and particularly preferably 2 to 5. And, as the number of atoms of the longest chain constituting the alkyl group having a substituent, it is preferably 3 to 35, more preferably 3 to 25, further preferably 3 to 15, and particularly preferably 3 to 11.

[0256] In the present invention, "the number of carbon atoms of the alkyl part of the alkyl group having a substituent" means the number of carbon atoms other than the substituent part of the alkyl group.

[0257] In the present invention, "the number of atoms of the longest chain constituting the alkyl group having a substituent" means the number of atoms including the substituent part (that is, the number of atoms obtained by subtracting the number of atoms of the molecular chain that does not constitute the longest chain from the total number of atoms). In addition, in the case where a substituent having a dissociable hydrogen atom such as a sulfo group or a carboxyl group constitutes the longest chain, the hydrogen atom is counted regardless of dissociation. And, the number of atoms in the substituent part capable of bonding to a biological substance described later is not included.

[0258] As the alkyl group that can be adopted for R 1 ~R 4The alkyl group may have substituents, examples of which include alkoxy, carboxyl, alkoxycarbonyl, acyloxy, carbamoyl, acylamino, sulfo, phosphonyl, and -(CH2-CH2-O) b -R 21 , and groups formed by combinations of these substituents.

[0259] As the alkyl group that can be adopted as R 1 ~R 4 The alkyl group with substituents has no particular limitation as long as it is an alkyl group with the above substituents.

[0260] As the alkyl group that can be adopted as R 1 ~R 4 The alkyl group is preferably an unsubstituted alkyl group.

[0261] (-(CH2-CH2-O) b -R 21 )

[0262] The -(CH2-CH2-O) 1 ~R 4 can be adopted as R b -R 21 In this case, b is 1 to 50, and R 21 represents an alkyl group.

[0263] b refers to the average number of repetitions (also simply referred to as the number of repetitions). It is preferably 1 to 24, more preferably 1 to 12, further preferably 1 to 10, particularly preferably 4 to 10, and most preferably 4 to 8.

[0264] Regarding the above average number of repetitions, the compound is subjected to 1 1H-NMR measurement and calculated by the average integral value. The average number of repetitions defined in the present invention refers to the number obtained by rounding the first decimal place of the average number of repetitions calculated by the above method.

[0265] R 21 The alkyl group in can apply to the description of the alkyl group that can be adopted as the above R 1 ~R 4 .

[0266] As the -(CH2-CH2-O) 1 ~R 4 that can be adopted as R b -R 21 and the -(CH2-CH2-O) 1 ~R 4 that the alkyl group can have in the form of a substituent b -R 21 , preferably -(CH2-CH2-O) b -unsubstituted alkyl group.

[0267] In terms of the viewpoint of improving the fluorescence intensity of the fluorescent dye itself, R is preferred. 1 ~R 4 At least one of them contains a structure represented by -(CH2-CH2-O) b - is more preferably that at least one of R 1 and R 2 contains a structure represented by -(CH2-CH2-O) 3 and at least one of R 4 and R b contains a structure represented by -(CH2-CH2-O)

[0268] More preferably, all the fluorophore moieties M in the fluorescent dye (F) are structural moieties composed of cyanine pigments represented by the general formula (α) and at least one of R 1 and R 2 contains a structure represented by -(CH2-CH2-O) 3 and at least one of R 4 contains a structure represented by -(CH2-CH2-O) b -.

[0269] The structure represented by the above -(CH2-CH2-O) b - is preferably introduced by adopting -(CH2-CH2-O) b -R 21 as R 1 ~R 4 in this way.

[0270] The b in the above -(CH2-CH2-O) b - has the same meaning as the b in the above -(CH2-CH2-O) b -R 21 -.

[0271] R 1 ~R 4 The substituents of are extended in a direction perpendicular to the cyanine pigment skeleton (plane). Therefore, it is speculated that by including a structure represented by -(CH2-CH2-O) b - as the substituent, the π-π interaction in the condensed ring part is not likely to occur (the association inhibition effect is enhanced), thereby suppressing the decrease in fluorescence intensity caused by association.

[0272] (ii)R 11 ~R 13

[0273] R 11 ~R 13 Each independently represents a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group, or a halogen atom. Adjacent groups may bond to each other to form a 5-membered ring or a 6-membered ring.

[0274] It can be R 11 ~R 13 The alkyl, alkoxy, aryloxy, alkylthio, arylthio, amino and halogen atoms of are the same as the alkyl, alkoxy, aryloxy, alkylthio, arylthio, amino and halogen atoms in the substituent group T described later, and the preferred ranges are also the same.

[0275] As R 11 ~R 13 The substituents that the alkyl, alkoxy, aryloxy, alkylthio, arylthio and amino in can have include the substituents in the substituent group T described later.

[0276] In R 11 ~R 13 The 5-membered or 6-membered ring formed by bonding adjacent groups to each other can be either aromatic or aliphatic, preferably aliphatic. And, it is preferably a 6-membered ring. The number of the above-mentioned 5-membered or 6-membered rings in the compound is not particularly limited, preferably 1 or 2, more preferably 1.

[0277] For example, taking the case of a = 3 as an example, as having R 11 ~R 13 The ring structure formed by bonding adjacent groups to each other in can preferably include the following structures. In addition, the following examples describe the structure where R 11 ~R 13 is a hydrogen atom and the ring structure has no substituents, but it is not limited to these. And, the structure at the wavy line end is omitted in the following.

[0278] [Chemical formula 8]

[0279]

[0280] R 11 and the R 13 possessed by the carbon atom bonded to the indolenine ring are preferably hydrogen atoms.

[0281] R 12 and the R other than the above 13 are preferably hydrogen atoms or alkyl groups.

[0282] In R 11 ~R 13 Among them, R 11 and the R 13 possessed by the carbon atom bonded to the indolenine ring 12 ~R 13 Among the adjacent groups to each other (that is, the R other than the R 13 possessed by the carbon atom bonded to the indolenine ring13 and R 12 The adjacent groups in are preferably bonded to each other to form a 5- or 6-membered ring, more preferably a 6-membered ring. Further, it is preferred to form the above-mentioned 5- or 6-membered ring at the central portion of the bond connecting the indoline ring and the pseudoindole ring. The ring formed at the central portion of the bond connecting the indoline ring and the pseudoindole ring means a ring having as ring-constituting atoms carbon atoms with an equal number of bonding atoms from the indoline ring and the pseudoindole ring.

[0283] The phosphor portion M becomes a monovalent structural portion by removing one hydrogen atom from any one of the following: R 1 ~R 4 、R 11 ~R 13 、R 22 ~R 25 、R 32 ~R 35 、R 41 or R 42

[0284] Specifically, it becomes a monovalent structural portion by removing one hydrogen atom from the substituents that can be used as R 1 ~R 4 、R 11 ~R 13 、R 22 ~R 25 、R 32 ~R 35 、R 41 or R 42 ; or it becomes a monovalent structural portion having a linking bond on the carbon atom to which R 11 ~R 13 、R 22 ~R 25 or R 32 ~R 35 is bonded by removing the hydrogen atom of R 11 ~R 13 、R 22 ~R 25 or R 32 ~R 35

[0285] Among them, the phosphor portion M preferably becomes a monovalent structural portion by removing one hydrogen atom from the ring formed by the bonding of the above R 41 and R 42 ; or it becomes a monovalent structural portion by removing one hydrogen atom from the substituents that can be used as R 12 (preferably R 12 ) on the carbon atoms with an equal number of bonding atoms from the indoline ring and the pseudoindole ring.

[0286] (iii)R22 ~R 25 and R 32 ~R 35

[0287] R 22 ~R 25 and R 32 ~R 35 represent a hydrogen atom, an alkyl group, an alkoxy group, an aryl group, a sulfo group, a sulfamoyl group, a carboxyl group, an alkoxycarbonyl group, an aryloxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a nitro group or a halogen atom. Regarding these Rs 22 ~R 25 and R 32 ~R 35 , adjacent groups may be bonded to each other to form a fused ring.

[0288] The alkyl group, alkoxy group, aryl group, sulfo group, sulfamoyl group, carboxyl group, alkoxycarbonyl group, aryloxycarbonyl group, acyloxy group, carbamoyl group, acylamino group, nitro group and halogen atom that can be used for R 22 ~R 25 and R 32 ~R 35 have the same meanings as the alkyl group, alkoxy group, aryl group, sulfo group, sulfamoyl group, carboxyl group, alkoxycarbonyl group, aryloxycarbonyl group, acyloxy group, carbamoyl group, acylamino group, nitro group and halogen atom in the substituent group T described later, respectively.

[0289] As the fused ring formed by bonding adjacent groups to each other in R 22 ~R 25 and R 32 ~R 35 there is no particular limitation. For example, a naphthalene ring can be cited (a benzene ring formed by bonding adjacent groups to each other, and the benzene ring bonded to R 22 ~R 25 or the benzene ring bonded to R 32 ~R 35 form a naphthalene ring together). In addition, from the viewpoint of suppressing association, it is preferable that R 22 ~R 25 and R 32 ~R 35 do not bond to each other and do not form a fused ring.

[0290] From the viewpoints of improving water solubility and suppressing association, it is preferable that at least one of R 22 ~R 25 and at least one of R 32 ~R 35 have a hydrophilic group. More preferably, the ring bonded to R 22 ~R 25 and R 32 ~R35 Each of the bonded rings has at least 1 hydrophilic group. For example, R 22 ~R 25 and R 32 ~R 35 When adjacent groups in ~R 22 ~R 25 are bonded to each other to form a naphthalene ring in the form of a fused ring respectively, it means that the number of rings bonded by R 32 ~R 35 is 2 and the number of rings bonded by R 22 ~R 25 is 2. More preferably, at least 2 of R 32 ~R 35 and at least 2 of R

[0291] have hydrophilic groups. Regarding the upper limit value, as long as it is a feasible structure, there is no particular limitation, and it can be appropriately adjusted according to the number of hydrophilic groups in the compound as described later.

[0292] R 22 ~R 25 and R 32 ~R 35 are preferably a hydrogen atom, an alkyl group, a sulfo group, a nitro group or a halogen atom, more preferably a hydrogen atom, an alkyl group, a sulfo group or a halogen atom, and still more preferably a hydrogen atom, an alkyl group or a sulfo group.

[0293] (iv)R 41 and R 42

[0294] R 41 and R 42 represent an alkyl group or -(CH2-CH2-O) b -R 21 。R 21 and b have the same meanings as R 21 and b above.

[0295] As the substituents that the alkyl group in R 41 and R 42 can have, there may be mentioned an alkoxy group, a carboxyl group, an alkoxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a sulfo group and a phosphonyl group, and groups composed of combinations of these substituents.

[0296] The alkyl group used for R 41 and R 42 has the same meaning as the alkyl group in the substituent group T described later.

[0297] The number of carbon atoms of the unsubstituted alkyl group is preferably 1 to 6, more preferably 1 to 4, and still more preferably 1 to 3.

[0298] The number of carbon atoms of the alkyl moiety of the alkyl group having a substituent is preferably 1 to 10, more preferably 1 to 8, still more preferably 1 to 7, still more preferably 1 to 6, and still more preferably 1 to 5. Further, the number of atoms of the longest chain constituting the alkyl group having a substituent is preferably 3 to 14, more preferably 3 to 12, and still more preferably 3 to 10.

[0299] As the alkyl group having a substituent that can be used as R 41 and R 42 from the viewpoint of further improving water solubility, an alkyl group having at least one of an alkoxy group, a carboxyl group, a sulfo group, and a phosphonyl group as a substituent is preferred, and an alkyl group having at least one of a carboxyl group and a sulfo group as a substituent is more preferred. In addition, it may also be an alkyl group having a substituent composed of a combination of the above-mentioned preferred substituents (alkoxy group, carboxyl group, sulfo group, and phosphonyl group) and a group other than these substituents.

[0300] Further, the mode of the alkyl group having a substituent that can be used as the above R 1 to R 4 can be preferably applied.

[0301] Regarding -(CH2-CH2-O) 41 that can be used as R 42 and R b -R 21 , the description of -(CH2-CH2-O) 1 to R 4 in the above R b -R 21 can be preferably applied.

[0302] R 41 and R 42 may bond to each other to form a ring.

[0303] Among the cyanidin pigments represented by the above general formula (α), as the structure in which R 41 and R 42 bond to each other to form a ring, a cyanidin pigment represented by the following general formula (β) can be preferably cited.

[0304] [Chemical formula 9]

[0305]

[0306] In the formula, L x and L y represent an alkylene group or -(CH2-CH2-O) b -alkylene-*. * represents the bonding position to U.

[0307] The linking group U represents a divalent linking group having 1 to 100 atoms.

[0308] R 1 ~R 4 、R 11 ~R 13 、R 22 ~R 25 、R 32 ~R 35 、b and a have the same meanings as R 1 ~R 4 、R 11 ~R 13 、R 22 ~R 25 、R 32 ~R 35 、b and a have the same preferred ranges, unless otherwise specified.

[0309] R 1 ~R 4 、L x 、L y and at least one of U contains a structure represented by -(CH2-CH2-O) b -. b has the same meaning as b above.

[0310] Among them, the cyanidin represented by formula (β) is neutral.

[0311] It can be used as L x and L y The alkylene group is equivalent to the alkylene group obtained by removing one hydrogen atom or substituent from the substituted alkyl group that can be used as R 41 and R 42 .

[0312] It can be used as L x and L y The number of carbon atoms in the alkylene part of the alkylene group that can be used as L 41 and R 42 The description of the number of carbon atoms in the alkyl part of the substituted alkyl group can preferably be applied.

[0313] It can be used as L x and L y The -(CH2-CH2-O) b -alkylene -* is equivalent to the -(CH2-CH2-O) 41 and R 42 -R b (R 21 (R 21 represents a substituted alkyl group.) as R21 -(CH2-CH2-O) obtained by removing one hydrogen atom or substituent from the alkyl group of b -alkylene.

[0314] Among those that can be used as L x and L y of -(CH2-CH2-O) b -alkylene-*, b is preferably 1 to 10, more preferably 1 to 8, and the number of carbon atoms in the alkylene moiety can preferably be applied to the description of the number of carbon atoms in the alkyl moiety of the alkyl group having a substituent in R 41 and R 42 in.

[0315] From the viewpoint of further increasing the fluorescence intensity, it is preferable that L x and L y both contain a structure represented by -(CH2-CH2-O) b -.

[0316] The total number of atoms constituting the linking group U is 1 to 100, preferably 10 to 90, more preferably 20 to 90, and further preferably 30 to 80.

[0317] The linking group U is preferably a divalent linking group formed by bonding three or more selected from alkylene, -O-, -NR 50 -, -COO-, -CONR 50 -, and -SO2NR 50 -. R 50 represents a hydrogen atom or an alkyl group.

[0318] The number of carbon atoms in the alkylene moiety of the alkylene that can be used as the linking group U is preferably 1 to 10, more preferably 1 to 8, further preferably 1 to 7, particularly preferably 1 to 6, and most preferably 1 to 5.

[0319] In the present invention, "the number of carbon atoms in the alkylene moiety of the alkylene" refers to the number of carbon atoms other than the substituent moiety of the alkylene.

[0320] The alkyl group that can be used as R 50 can preferably be applied to the description of the alkyl group in the above R 1 to R 4 .

[0321] As R 50 , a hydrogen atom is preferred.

[0322] The above-mentioned alkylene, -O-, -NR 50 -, -COO-, -CONR 50 -, and -SO2NR 50The number of [-] is preferably 3 to 11, more preferably 3 to 7, still more preferably 3 to 5, and particularly preferably 3.

[0323] Regarding the linking group U, with L x and L y The linking part is preferably -O-, -NR 50 -, -COO-, -CONR 50 - or -SO2NR 50 -. That is, the linking group U is preferably bonded to L 50 and L 50 through -O-, -NR 50 -, -COO-, -CONR x - or -SO2NR y which constitute the linking group U. The linking group U is more preferably a divalent linking group in which the linking part with L x and L y is -O-, -NR 50 -, -COO-, -CONR 50 - or -SO2NR 50 - and the above linking parts are connected to each other through an alkylene group.

[0324] The linking group U preferably becomes a monovalent structural part, i.e., the phosphor part M, by removing one hydrogen atom. In the linking group U, as the site for removing one hydrogen atom, an alkylene group or an alkyl group as R 50 can be mentioned, and an alkylene group is preferred.

[0325] In the linking group U, when removing one hydrogen atom from the alkylene group or the alkyl group as R 50 , one hydrogen atom can be directly removed from the alkylene group or the alkyl group as R 50 , or the linking group ZZZ can be bonded to the alkylene group or the alkyl group as R 50 and the linking group ZZZ becomes a linking bond.

[0326] As the above linking group ZZZ, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, -O-, -S-, >C=O, >NR 60 , >S=O, >S(=O)2, >P(=O)OR 70 , -COO-, -CONR 60 - and -(CH2-CH2-O) p - and groups composed of combinations of these substituents can be mentioned. The number of combinations is not particularly limited. For example, it can be set to 2 to 20, preferably 2 to 7, and more preferably 2 to 5.

[0327] R 60 and R 70is a hydrogen atom or an alkyl group, preferably a hydrogen atom. As R that can be adopted 60 and R 70 for the alkyl group, the description of the alkyl group in the above R 50 can be preferably applied.

[0328] p represents the number of repetitions, preferably 1 to 10, more preferably 1 to 8, and further preferably 1 to 4.

[0329] (v) a

[0330] a is an integer of 1 to 3, preferably an integer of 2 or 3.

[0331] Among the cyanine pigments represented by the above general formula (α), preferably at least one of R 1 to R 4 , R 41 and R 42 contains a structure represented by -(CH2-CH2-O) b . b has the same meaning as b above. Thus, the fluorescent pigment (F) having a structural part composed of the cyanine pigment represented by the general formula (α) can have appropriate hydrophilicity and an appropriate excluded volume effect, and it is considered that the obtained fluorescent pigment-labeled biomolecule can exhibit excellent fluorescence intensity.

[0332] Moreover, regarding the cyanine pigment represented by the above general formula (α), from the viewpoint of imparting sufficient hydrophilicity as the fluorescent pigment (F), the number of hydrophilic groups per molecule of the cyanine pigment represented by the general formula (α) is preferably 2 or more, more preferably 2 to 8, further preferably 2 to 6, and particularly preferably 3 to 6.

[0333] As the hydrophilic group, the above-described R 22 to R 25 and R 32 to R 35 can be applied for the description of the adoptable hydrophilic groups.

[0334] Regarding the position of the hydrophilic group, unless otherwise specified, there is no particular limitation. As the group having the above hydrophilic group, for example, R 11 to R 13 , R 22 to R 25 , R 32 to R 35 , R 41 or R 42 can be preferably listed.

[0335] In addition, in the structure represented by the above general formula (α) or (β), one hydrogen atom can be removed from any substituent to form a monovalent structural part (phosphor part M). For example, it is preferably to remove one hydrogen atom from the linking group U to form a monovalent structural part, or by removing one hydrogen atom from a substituent that can be used as R 12 (preferably from a carbon atom with an equal number of bonding atoms from the indoline ring and the pseudoindoline ring of R 12 ) to form a monovalent structural part.

[0336] In addition, unless otherwise specified, the description of the substituents in the general formulas (α) and (β) is assumed to be only for the substituents applicable in the general formulas (α) and (β).

[0337] As the physiologically active substance part that can be used as M, as long as it is a structural part composed of a physiologically active substance, it can be used without particular limitation. As the physiologically active substance, for example, vitamins, coenzymes, hormones, antibiotics, neurotransmitters, cytokines, etc. can be cited. More specifically, calicheamicin, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, cisplatin or its derivatives, auristatin or its derivatives, maytansine or its derivatives, taxol or its derivatives, camptothecin or its derivatives, etc. described in paragraph

[0095] of JP-A-2021-020956 can be used, and the descriptions in paragraphs

[0095] to

[0099] of JP-A-2021-020956 can be applied.

[0338] As the prodrug part that can be used as M, as long as it is a structural part composed of a compound that is metabolized in vivo to change into a physiologically active substance, it can be used without particular limitation. As the prodrug, for example, the description in paragraph

[0003] of JP-A-2020-105187 (prodrug mode of 2-pyrroline doxorubicin) can be applied.

[0339] As a radioisotope containing part that can be adopted as M, as long as it is a structural part containing a radioisotope that can be used in the medical field, it can be used without particular limitation. As a radioisotope, for example, iodine 131, indium 111, yttrium 90 and lutetium 177, copper 64 can be cited, but are not limited to these. The record of paragraph

[0225] of Japanese Patent Application Laid-Open No. 2021-11483 can be applied. As a structural part containing a radioisotope, a structural part obtained by bonding or coordination of the above-mentioned radioisotope with a nitrogen atom, a sulfanyl group, an aryl group or a heteroaryl group of an amino group or a tertiary amine can be cited. Examples of the structural part in which the above radioactive isotope is coordinated to the nitrogen atom of the tertiary amine include a structural part such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) coordinated to the above radioactive isotope to form a complex. Examples of the structural part in which the above radioactive isotope is coordinated to the sulfanyl group include a structural part composed of a complex such as diacetylbis(N(4)-methylthiosemicarbazone)copper(II).

[0340] <Compound represented by general formula (III)>

[0341] The compound represented by the above general formula (II) is preferably represented by the following general formula (III).

[0342] [Chemical formula 10]

[0343]

[0344] Where Y 1 ~Y 3 , Z 1 ~Z 3 and W 1 ~W 3 It represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxyl group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group or an anionic group.

[0345] LL 3 and LL 4 represents a divalent linking group.

[0346] s, t, and u are integers greater than or equal to 0.

[0347] R 4 ~R 7 , L 1 , L 2 , L 5 , L 6 , X 1 ~X 3 , M, n and m are the same as R in the above general formula (II) 4 ~R 7 , L1 , L 2 , L 5 , L 6 , X 1 ~X 3 , M, n, and m have the same meaning.

[0348] Among them, the structure enclosed by s, t, or u will not be the structure represented by the above general formula (I). That is, Y 1 will not bond with W 1 or Z 1 to form a structure represented by the general formula (I), or Y 2 will not bond with W 2 or Z 2 to form a structure represented by the general formula (I), or Y 3 will not bond with W 3 or Z 3 to form a structure represented by the general formula (I).

[0349] Y 1 ~Y 3 , Z 1 ~Z 3 , and W 1 ~W 3 represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxyl group, an alkoxy group, a thioalkyl group, an aryl group, a heteroaryl group, or an anionic group.

[0350] The alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group, heteroaryl group, and anionic group that can be used for Y 1 ~Y 3 , Z 1 ~Z 3 , or W 1 ~W 3 have the same meaning as the alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group, heteroaryl group, and anionic group in the substituent group T described later, and the preferred ranges are also the same.

[0351] The alkyl group, alkenyl group, alkynyl group, acyl group, amino group, alkoxy group, aryl group, and heteroaryl group that can be used for Y 1 ~Y 3 , Z 1 ~Z 3 , or W 1 ~W 3 can be unsubstituted or can have substituents. As the substituents that can be had, the substituents in the substituent group T described later can be enumerated. For example, an aryl group, a halogen atom, or an anionic group is preferred.

[0352] And, it can form Y 1 ~Y 3 , Z 1 ~Z 3 , or W1 to W 3 The substituents that the above-mentioned alkyl, alkenyl, alkynyl, acyl, amino, alkoxy, aryl and heteroaryl groups may have can be further substituted by substituents selected from the following substituent group T. For example, an anionic group is preferred.

[0353] Y 1 to Y 3 It is preferably a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group, more preferably a hydrogen atom or an alkyl group.

[0354] W 1 to W 3 It is preferably a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group, more preferably a hydrogen atom.

[0355] Z 1 to Z 3 It is preferably an alkyl group, an aryl group or a heteroaryl group, more preferably an alkyl group.

[0356] In the compound represented by the general formula (III), it is preferable to contain a group having a charge repulsive action to suppress the interaction between the structures represented by the general formula (I). Therefore, from this viewpoint, Z 1 to Z 3 At least one of them is preferably a group containing an anionic group, more preferably an alkyl group containing an anionic group.

[0357] Among them, "at least one of Z 1 to Z 3 is a group containing an anionic group" means that at least one of the following conditions (Z1) to (Z3) is satisfied.

[0358] Condition (Z1): The above s is an integer of 1 or more, and the above Z 1 is a group containing an anionic group.

[0359] Condition (Z2): The above t is an integer of 1 or more, and the above Z 2 is a group containing an anionic group.

[0360] Condition (Z3): The above u is an integer of 1 or more, and the above Z 3 is a group containing an anionic group.

[0361] And, "at least one of Z 1 to Z 3 is an alkyl group containing an anionic group" means that on the basis of changing "a group containing an anionic group" in the above conditions (Z1) to (Z3) to "an alkyl group containing an anionic group", at least one of the above conditions (Z1) to (Z3) is satisfied.

[0362] LL3 and LL 4 represent a divalent linking group, preferably a linking group formed by combining one or more of alkylene, alkenylene, alkynylene, arylene, heteroarylene, -O-, >C=O and >NR A . R A represents a hydrogen atom or a substituent.

[0363] As the alkylene, alkenylene, alkynylene, arylene, heteroarylene and >NR that can form LL 3 and LL 4 , the descriptions of the alkylene, alkenylene, alkynylene, arylene, heteroarylene and >NR that can form L A ~L 1 ~L 7 can preferably be applied. A The description.

[0364] LL 3 is preferably >C=O, >NR A , alkylene, arylene or heteroarylene, more preferably >C=O or NR A . In addition, R A is preferably a hydrogen atom.

[0365] LL 4 is preferably >C=O, >NR A , alkylene, arylene or heteroarylene, more preferably >C=O or >NR A . In addition, R A is preferably a hydrogen atom.

[0366] s, t and u are integers of 0 or more.

[0367] There is no particular limitation on the upper limit values of s, t and u. For example, they can be set to 20 or less, preferably 10 or less, more preferably 5 or less. Among them, s, t and u are preferably integers of 0 or 1.

[0368] In addition, when it can be classified into either the structure enclosed by t or the structure enclosed by u, it is preferably classified into the structure enclosed by u.

[0369] <Compound represented by general formula (IV)>

[0370] The compound represented by the above general formula (III) is preferably represented by the following general formula (IV).

[0371] [Chemical formula 11]

[0372]

[0373] In the formula, Y 4 and Y 5It represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxyl group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group or an anionic group.

[0374] R 4 ~R 7 , L 2 , L 5 , X 1 ~X 3 , Y 1 ~Y 3 , Z 1 ~Z 3 and W 1 ~W 3 , M, n, m, s, t and u are the same as R in the above general formula (III) 4 ~R 7 , L 2 , L 5 , X 1 ~X 3 , Y 1 ~Y 3 , Z 1 ~Z 3 and W 1 ~W 3 , M, n, m, s, t and u have the same meaning.

[0375] In addition, when the R 7 When the terminal of the side is a carboxyl group or a substituent capable of bonding to a biological substance of the type bonded via a carbonyl group or a substituent capable of bonding to a solid support of the type bonded via a carbonyl group, the "-C=OR 7 " is interpreted as a carboxyl group, a substituent capable of bonding to a biological substance of the type bonding through a carbonyl group, or a substituent capable of bonding to a solid support of the type bonding through a carbonyl group.

[0376] Y 4 and Y 5 It represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an acyl group, an amino group, a hydroxyl group, an alkoxy group, a sulfanyl group, an aryl group, a heteroaryl group or an anionic group.

[0377] Can be used as Y 4 or Y 5 The alkyl, alkenyl, alkynyl, acyl, amino, alkoxy, aryl, heteroaryl and anionic groups have the same meanings as the alkyl, alkenyl, alkynyl, acyl, amino, alkoxy, aryl, heteroaryl and anionic groups in the substituent group T described later, and the preferred ranges are also the same.

[0378] In addition, Y 4 or Y 5The above-mentioned alkyl, alkenyl, alkynyl, acyl, amino, alkoxy, aryl and heteroaryl groups can be unsubstituted groups or groups with substituents.

[0379] Y 4 and Y 5 are preferably a hydrogen atom or an alkyl group, more preferably a hydrogen atom.

[0380] <Compound represented by general formula (V)>

[0381] The compound represented by the above general formula (IV) is preferably represented by the following general formula (V).

[0382] [Chemical formula 12]

[0383]

[0384] In the formula, R 6A and R 7A represent a hydrogen atom, a hydroxyl group, a thioalkyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group or Q. Among them, R 6A and R 7A at least one of them represents Q.

[0385] L 8 and L 9 represent a linking group. Among them, when R 6A is Q, L 9 is a linking group with a shortest number of atoms for connecting >NY 4 to R 6A of 3 or more, and when R 7A is Q, L 8 is a linking group with a shortest number of atoms for connecting >C=O to R 7A of 3 or more.

[0386] R 4 , R 5 , L 2 , L 5 , X 1 ~X 3 , Y 1 ~Y 5 , Z 1 ~Z 3 , W 1 ~W 3 , M, Q, n, m, s, t and u are the same as R 4 , R 5 , L 2 , L 5 , X 1 ~X 3 , Y 1~Y 5 , Z 1 ~Z 3 , W 1 ~W 3 , M, Q, n, m, s, t, and u have the same meaning.

[0387] R 6A or R 7A and L 9 or L 8 are respectively determined as R 6A or R 7A is an unsubstituted group, and L 9 or L 8 becomes the longest group. Among them, when the group represented by -L 9 R 6A or -L 8 R 7A has an anionic group, a cationic group, or Q, it is determined that the anionic group, cationic group, or Q located on the most terminal side (the R side in -L 9 R 6A , the R in -L 6A R 8 side) becomes R 7A , the R in -L 7A side) becomes R 6A or R 7A .

[0388] R 6A and R 7A represent a hydrogen atom, a hydroxyl group, a thioalkyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group, or Q. Among them, at least one of R 6A and R 7A represents Q.

[0389] The alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, heteroaryl group, amino group, acyl group, anionic group, and cationic group that can be used as R 6A and R 7A have the same meaning as the alkyl group, alkenyl group, alkynyl group, aryl group, alkoxy group, heteroaryl group, amino group, acyl group, anionic group, and cationic group in the substituent group T described later, and the preferred ranges are also the same. Among them, they are all unsubstituted groups.

[0390] The Q that can be used as R 6A and R 7A has the same meaning as the aforementioned Q, and the preferred range is also the same.

[0391] R 6A is preferably an alkyl group, a thioalkyl group, an aryl group, a heteroaryl group, or Q, and more preferably an alkyl group or Q.

[0392] R7A Preferably an alkyl group, a thioalkyl group, an aryl group, a heteroaryl group or Q, more preferably an alkyl group or Q.

[0393] L 8 and L 9 represent a linking group.

[0394] As the L 8 and L 9 The linking groups that can be used, for example, are preferably an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, -O-, >C=O and >NR A One of these or a linking group formed by combining two or more of them. R A represents a hydrogen atom or a substituent.

[0395] The alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group and >NR 8 or L 9 that can form can preferably be applied to the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group and >NR A described above that can form L 1 ~L 7 The alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group and >NR A is described.

[0396] As the alkylene group, alkenylene group, alkynylene group, arylene group or heteroarylene group that can form L 8 or L 9 The substituents that can be possessed are not particularly limited, and are preferably selected from the substituent group T described later, and more preferably a halogen atom, an aryl group or an alkyl group can be listed. And, the alkylene group, alkenylene group, alkynylene group, arylene group or heteroarylene group that can form L 8 or L 9 The substituents that can be possessed by the above alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group can be further substituted by substituents selected from the substituent group T described later, for example, an anionic group is preferred.

[0397] And, regarding the number of substituents that the above alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group that can form L 8 or L 9 can have, as long as it is a structure that can be adopted, there is no particular limitation, and it can be set to at least 1 or more, and there is no particular limitation as the upper limit value. For example, all hydrogen atoms in the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group can be substituted by substituents.

[0398] Among them, when R 6A is Q, L 9 is a linking group in which the shortest number of atoms connecting >NY 4 and R 6A is 3 or more. When R 7A is Q, L 8is a linking group with the shortest number of atoms of 3 or more for connecting >C=O and R 7A The shortest number of atoms of the linking group for connecting >C=O and R is 3 or more.

[0399] Regarding the divalent linking group L described above 9 "the shortest number of atoms for connecting >NY 4 and R 6A " refers to the number of atoms constituting the shortest chain for connecting >NY 4 and R 6A Regarding the divalent linking group L described above 8 "the shortest number of atoms for connecting >C=O and R 7A " refers to the number of atoms constituting the shortest chain for connecting >C=O and R 7A In addition, >NY 4 refers to >NY directly bonded to L 9 , and >C=O refers to >C=O directly bonded to L 4 . For example, in the compound (4) used in the following examples, L 8 is -NH(C2H4O)4C2H4-, and R 8 is -COOH. Therefore, the number of atoms constituting the shortest chain for connecting >C=O and R 7A is 15. 7A In the compound represented by the above general formula (V), at least one of R

[0400] and R 6A is Q. Through this Q, it is connected to >C=O or >NY 7A via a linking group with the shortest number of atoms of 3 or more. The distance from the main chain connecting R 4 and R 6A to the bonding point of M (that is, the carbon atom bonded to L 7A and R 5 or the carbon atom bonded to L 5 and R 2 and R 4 ) becomes farther, and the steric hindrance around Q decreases. As a result, the reactivity with an antibody of a pigment multimer having 2 or more phosphor parts M in the compound can be improved, and the fluorescence labeling rate (DOL) can be increased.

[0401] At least one of the above R 6A and R 7A is Q. The shortest number of atoms of the linking group for connecting this Q to >C=O or >NY 4 (the shortest number of atoms for connecting >NY 6A and R 4 when R 6A is Q and the shortest number of atoms for connecting >C=O and R 7A when R 7AThe shortest number of atoms for connection) is preferably from 3 to 60, more preferably from 12 to 40, still more preferably from 15 to 40.

[0402] In the present invention, from the viewpoint of ease of synthesis, L 8 and L 9 is preferably any one of the groups containing -(L-O) g -, more preferably L 8 is a group containing -(L-O) g -.

[0403] L 8 is more preferably an alkylene group, -O-, >C=O and >NR A or a linking group formed by combining two or more of them, and is still more preferably an alkylene group, -O-, >C=O, >NR A , a group in which -NR A -alkylene-C(=O)]-represents a repeating unit (the repeating number is preferably from 1 to 20) and -NR A -alkylene is bonded to the right side, -NR A -(L-O) g -alkylene or -C(=O)-(L-O) g -alkylene.

[0404] L 9 is more preferably an alkylene group, -O-, >C=O and >NR A or a linking group formed by combining two or more of them, and is still more preferably an alkylene group, -O-, >C=O, >NR A , a group in which -NR A -alkylene-C(=O)]-represents a repeating unit (the repeating number is preferably from 1 to 20) and -NR A -alkylene is bonded to the right side, -NR A -(L-O) g -alkylene or -C(=O)-(L-O) g -alkylene.

[0405] <Compound represented by general formula (VI)>

[0406] The compound represented by the above general formula (II) is also preferably represented by the following general formula (VI). The compound represented by the following general formula (VI) corresponds to the compound represented by the above general formula (II) in which L 1 and L 4 is >NH, L 3 and L 6 is >C=O, L 7 is a single bond, R 4 and R 5 is a hydrogen atom, L1 With L 2 And L 4 With L 5 Compounds that are respectively bonded to each other to form a specific 5 - membered ring.

[0407] [Chemical formula 13]

[0408]

[0409] In the formula, X 4 ~X 9 Represents -O-, -S-, >NR 101 Or >CR 102 R 103 .

[0410] Among them, one of X 4 ~X 6 Is >NR 101 Or >CR 102 R 103 And when one of X 4 ~X 6 Is >NR 101 , R 101 Is -L 10 -M, when one of X 4 ~X 6 Is >CR 102 R 103 , R 102 Or R 103 Is -L 10 -M. One of X 7 ~X 9 Is >NR 101 Or >CR 102 R 103 And when one of X 7 ~X 9 Is >NR 101 , R 101 Is -L 11 -M, when one of X 7 ~X 9 Is >CR 102 R 103 , R 102 Or R 103 Is -L 11 -M.

[0411] Not -L 10 -M and -L 11 -M any of the R 101 ~R 103 Represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an acyl group, -NR 8 R9 、 -OR 10 or an anionic group.

[0412] L 10 and L 11 represent a single bond or a divalent linking group.

[0413] n1 is an integer of 2 or more.

[0414] R 6 ~R 10 、X 1 ~X 3 、M and m have the same meanings as R 6 ~R 10 、X 1 ~X 3 、M and m in the general formula (II).

[0415] In addition, when the terminal on the R 7 side of the compound is a carboxyl group or a substituent capable of bonding to a biological substance of a type that bonds through a carbonyl group or a substituent capable of bonding to a solid support of a type that bonds through a carbonyl group, the entire label of “-C=O-R 7 ” in the formula can be interpreted as a carboxyl group, a substituent capable of bonding to a biological substance of a type that bonds through a carbonyl group, or a substituent capable of bonding to a solid support of a type that bonds through a carbonyl group.

[0416] As X 4 ~X 6 ,one of X 4 ~X 6 is >NR 101 or >CR 102 R 103 ,and when one of X 4 ~X 6 is >NR 101 ,R 101 is -L 10 -M, and when one of X 4 ~X 6 is >CR 102 R 103 ,R 102 or R 103 is -L 10 -M. Unless otherwise specified, the description of X 1 ~X 3 in the general formula (I) above can be applied.

[0417] And, as X 7 ~X 9 ,one of X 7 ~X 9One of them is >NR 101 or >CR 102 R 103 , and when X 7 ~X 9 One of them is >NR 101 R 101 is -L 11 -M, when X 7 ~X 9 One of them is >CR 102 R 103 R 102 or R 103 is -L 11 -M, unless otherwise specified, the description of X 1 ~X 3 in the aforementioned general formula (I) can be applied.

[0418] That is, as X 4 and X 7 the description of the aforementioned X 1 can be applied, as X 5 and X 8 the description of the aforementioned X 2 can be applied, as X 6 and X 9 the description of the aforementioned X 3 can be applied. As R 10 -M and -L 11 -M, neither of which is R 101 , R 102 and R 103 , the description of R 1 , R 2 and R 3 in the aforementioned general formula (I) can be applied respectively.

[0419] X 4 ~X 9 does not have -L 10 -M and -L 11 -M, neither of which is >NR 101 and >CR 102 R 103 in, R 101 is preferably an alkyl group, R 102 and R 103 are preferably hydrogen atoms.

[0420] As the two groups that do not have the aforementioned -L 4 ~X 6 -M in X 10 , preferably at least one is >CR 102 R 103, more preferably, at least one is >CR 102 R 103 and the remaining one is -O-, -S- or >CR 102 R 103 , further preferably, both are >CR 102 R 103 .

[0421] As two groups that do not have the above -L 7 ~X 9 -M in X, preferably at least one is >CR 11 102 R 103 , more preferably, at least one is >CR 102 R 103 and the remaining one is -O-, -S- or >CR 102 R 103 , further preferably, both are >CR 102 R 103 .

[0422] As >NR 4 ~X 6 that has -L 10 -M as any one of R 101 ~R 103 or >CR 101 102 R 103 , preferably R 103 is -L 10 -M and the group represented by >CR 102 R 103 , more preferably, R 102 is a hydrogen atom and R 103 is -L 10 -M and the group represented by >CR 102 R 103 .

[0423] As >NR 7 ~X 9 that has -L 11 -M as any one of R 101 ~R 103 or >CR 101 102 R 103 , preferably R 103 is -L 11 -M and the group represented by >CR 102 R 103 , more preferably, R 102 is a hydrogen atom and R 103 is -L 11 -M and the group represented by >CR​​​102 R 103 The group represented by

[0424] X 4 ~X 6 The group having -L 10 -M is not particularly limited, and is preferably X 5 .

[0425] X 7 ~X 9 The group having -L 11 -M is not particularly limited, and is preferably X 8 .

[0426] L 10 and L 11 are preferably a single bond, or an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, -O-, -S-, >C=O and >NR A in one kind or a group formed by combining two or more kinds thereof, more preferably an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, -O-, -S-, >C=O and >NR A in one kind or a group formed by combining two or more kinds thereof, and further preferably a group represented by *-L x1 -L y1 -**

[0427] Regarding the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group and >NR 10 that can form L 11 and L A , unless otherwise specified, the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group and >NR 2 that can form L 5 and L A can be applied to the description thereof.

[0428] L x1 is a single bond, or a group formed by combining one kind or two or more kinds of an alkylene group, an alkenylene group, an alkynylene group, an arylene group and a heteroarylene group, and L y1 is a single bond, -O-, -S-, >C=O or >NR A . In addition, in *-L x1 -L y1 -**, * represents a bonding bond to X 4 ~X 9 and ** represents a bonding bond to M.

[0429] Regarding L 10 and L 11 , the above-mentioned *-L x1 -L y1-Among the groups represented, L is also preferably y1 a single bond, -S-, >C=O or >NR A group, more preferably L y1 is >C=O or >NR A group, further preferably L x1 is a single bond, L y1 is >C=O or >NR A group.

[0430] In addition, in the compound represented by the above general formula (VI), for the connecting chain connecting M to any one of X 4 ~X 9 (including the connecting chain containing L 10 and the connecting chain containing L 11 ), the shortest number of atoms can be, for example, 1 to 60, preferably 1 to 40. When M is a phosphor part, the above shortest number of atoms refers to the number of atoms in the shortest chain in the connecting chain that forms the conjugated structure part for fluorescence display in the phosphor part M and connects to any one of X 4 ~X 9 .

[0431] In addition, in the compound represented by the above general formula (VI), it is also preferred that in the structure represented by the conjugated structure part of M - the aforementioned connecting group ZZZ-L 10 -, any part of the connecting chain represented by "-connecting group ZZZ-L 10 -", and in the structure represented by the conjugated structure part of M - the aforementioned connecting group ZZZ-L 11 -, any part of the connecting chain represented by "-connecting group ZZZ-L 11 -" has the aforementioned structure represented by -(CH2-CH2-O) b - (b is also as described above).

[0432] n1 is an integer of 2 or more.

[0433] Compared with the compound represented by any one of the aforementioned general formulas (III) to (V), since the main chain of the linker connecting between two phosphor parts is straight and pigment association can be further suppressed, as the lower limit value of n1, an integer of 3 or more is preferred, and when it is an integer of 5 or more, a sufficient effect of increasing fluorescence intensity can be obtained, and it is more preferred in this regard. This is the same when the number of the above phosphor parts is 2 or more.

[0434] The upper limit value of n1 is preferably an integer of 36 or less, more preferably an integer of 24 or less, and still more preferably an integer of 18 or less. That is, n1 is preferably an integer of 3 to 36, more preferably an integer of 5 to 24.

[0435] <Compound represented by the general formula (VII)>

[0436] The compound represented by the above general formula (VI) is preferably represented by the following general formula (VII).

[0437] [Chemical formula 14]

[0438]

[0439] In the formula, R 6A and R 7A represent a hydrogen atom, a hydroxyl group, a thioalkyl group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, a heteroaryl group, an amino group, an acyl group, an anionic group, a cationic group, or Q. Among them, at least one of R 6A and R 7A represents Q.

[0440] L 12 and L 13 represent a linking group.

[0441] na and nb are integers of 0 or more.

[0442] L 10 , L 11 , X 1 ~X 9 , M, Q, n1 and m have the same meanings as L 10 , L 11 , X 1 ~X 9 , M, Q, n1 and m in the above general formula (VI).

[0443] In addition, when the terminal on the R 7A side of the compound is a carboxyl group, or a substituent capable of bonding to a biological substance of a type that bonds through a carbonyl group or a substituent capable of bonding to a solid support of a type that bonds through a carbonyl group, the entire label of "-C=O-L 12 -R 7A " in the formula can be interpreted as a carboxyl group, a substituent capable of bonding to a biological substance of a type that bonds through a carbonyl group, or a substituent capable of bonding to a solid support of a type that bonds through a carbonyl group.

[0444] Unless otherwise specified, R 6A and R 7A have the same meanings as R 6A and R 7A in the above general formula (V). That is, as R6A and R 7A is applicable to R in the aforementioned general formula (V) 6A and R 7A as described.

[0445] R 6A or R 7A and L 12 or L 13 is respectively determined as R 6A or R 7A is an unsubstituted group, and L 12 or L 13 becomes the longest group. Among them, when the group represented by -L 13 R 6A or -L 12 R 7A has an anionic group, a cationic group or Q, it is determined that the anionic group, cationic group or Q located on the most terminal side (-L 13 R 6A side, -L 6A R 12 side) becomes R 7A side, -L 7A side) becomes R 6A or R 7A .

[0446] L 12 and L 13 represent a linking group.

[0447] As the linking group that can be adopted as L 12 and L 13 for example, it is preferably an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, -O-, >C=O and >NR A or a linking group formed by combining two or more of them. R A represents a hydrogen atom or a substituent.

[0448] As the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group and >NR 12 and L 13 that can form L A , it is applicable to the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group and >NR 8 or L 9 that can form L A as described in the aforementioned general formula (V).

[0449] Among the compounds represented by the above general formula (VII), preferably (A) na is an integer of 1 or more and R 6A is Q, and / or (B) nb is an integer of 1 or more and R 7A is Q.

[0450] Among them, in the case of the above (A), the minimum number of connecting atoms of L 13 is 7 or less, and in the case of the above (B), the minimum number of connecting atoms of L 12 is 7 or less.

[0451] Regarding the above divalent linking group L 13 ,"the minimum number of connecting atoms of L 13 " means, when na is an integer of 1 or more, the number of atoms in the shortest chain that forms the connection between the N directly bonded to L na and R 13 shown in the structure enclosed by (). When na is 0, it means the number of atoms in the shortest chain that forms the connection between the N directly bonded to L 6A and R m shown in the structure enclosed by (). 13 and R 6A shown in the structure enclosed by ().

[0452] And regarding the above divalent linking group L 12 ,"the minimum number of connecting atoms of L 12 " means, when nb is an integer of 1 or more, the number of atoms in the shortest chain that forms the connection between the >C=O directly bonded to L nb and R 12 shown in the structure enclosed by (). When nb is 0, it means the number of atoms in the shortest chain that forms the connection between the >C=O shown on the left side of the structure enclosed by () 7A and R nb shown in the general formula (VII). 7A shown in the general formula (VII).

[0453] For example, in the compounds (1) to (3) used in the following examples, L 12 is -NHC2H4-, and R 7A is -COOH. Therefore, the minimum number of connecting atoms of L 12 is 3.

[0454] In the compound represented by the above general formula (VII), at least one of R 6A and R 7A is Q. In particular, it is considered that by satisfying the above (A) and / or (B), the mobility of the linker main chain is reduced, thereby further suppressing pigment association.

[0455] The minimum number of connecting atoms of the above L 12 and the minimum number of connecting atoms of L 13 are preferably 1 to 5, more preferably 1 to 4.

[0456] In the present invention, from the viewpoint of ease of synthesis, L is also preferably 12 and L 13 any one of which is a group containing -(L-O) g -, more preferably L 12 is a group containing -(L-O) g -.

[0457] L 12 is more preferably an alkylene group, -O-, >C=O, and >NR A a linking group formed by combining one or two or more of them, more preferably -NR A -alkylene or -NR A -(L-O) g -alkylene, and further preferably -NR A -alkylene.

[0458] L 13 is more preferably an alkylene group, -O-, >C=O, and >NR A a linking group formed by combining one or two or more of them, more preferably >NR A or >C=O.

[0459] na and nb are integers of 0 or more.

[0460] When R 6A is Q, na is preferably an integer of 0 to 20, more preferably an integer of 2 to 20, and further preferably an integer of 4 to 18.

[0461] When R 7A is Q, nb is preferably an integer of 0 to 20, more preferably an integer of 2 to 20, and further preferably an integer of 4 to 18. In addition, the lower limit value of nb can be 0, in which case nb is preferably an integer of 0 to 20, more preferably an integer of 0 to 18.

[0462] When R 6A is not Q, na is preferably an integer of 0 to 20, more preferably an integer of 0 to 10, and further preferably an integer of 0 to 5.

[0463] When R 7A is not Q, nb is preferably an integer of 0 to 20, more preferably an integer of 0 to 10, and further preferably an integer of 0 to 5.

[0464] When obtaining the compound represented by any one of the general formulas (II) to (VII) in the fluorescent dye (F) by the peptide synthesis method, usually the right side on the paper is the C-terminal structure, and the left side on the paper is the N-terminal structure.

[0465] The fluorescent dye (F) preferably contains at least one substituent represented by Q as described above, namely, a carboxyl group, a substituent capable of bonding to a biological substance, or a substituent capable of bonding to a solid support.

[0466] Fluorescent dyes such as the fluorescent dye (F) can be bonded to a biological substance through a carboxyl group or a substituent capable of bonding to a biological substance described later to obtain a target fluorescent dye-labeled biomolecule. In addition, a carboxyl group can be easily derivatized into a substituent capable of bonding to a biological substance by a conventional method.

[0467] Furthermore, a fluorescent dye can be bonded to a solid support such as a microparticle through a carboxyl group or a substituent capable of bonding to a solid support described later to obtain a target labeled microparticle or the like. The microparticle is not particularly limited, and examples thereof include small particles useful for bonding to a fluorescent dye, including non-polymer beads such as glass beads and magnetic beads, and polymer beads. In a certain embodiment, the microparticle includes polystyrene beads. Regarding the small particles, as long as they are of a size commonly used in fluorescence labeling, there is no particular limitation, and generally, the average particle diameter is 10 nm to 10 μm. In addition, a carboxyl group can be easily derivatized into a substituent capable of bonding to a solid support by a conventional method.

[0468] In the present invention, for convenience, the substituent capable of bonding to a biological substance and the substituent capable of bonding to a solid support do not include a carboxyl group. The "substituent capable of bonding to a biological substance" includes a substituent derived from a carboxyl group and capable of bonding to a biological substance, and the "substituent capable of bonding to a solid support" includes a substituent derived from a carboxyl group and capable of bonding to a solid support. Among them, as described above, bonding to a biological substance or a solid support can also be achieved through a carboxyl group.

[0469] In the fluorescent dye (F), the position of the substituent represented by Q as described above is not particularly limited, and it is preferably present in the structure represented by the general formula (I) and the structure other than the phosphor part. In the compound represented by the general formula (II), it is preferably present in at least one of R 6 and R 7 .

[0470] The total number of the substituents represented by Q in the fluorescent dye (F) may be at least 1 or more. From the viewpoint of quantifying the substance to be detected, 1 to 3 are preferred, 1 or 2 are more preferred, and 1 is further preferred.

[0471] Furthermore, from the viewpoint of imparting sufficient hydrophilicity to the compound, the fluorescent dye (F) also preferably has an anionic group described later at a position other than the phosphor part, for example, preferably has 1 or more, more preferably has 1 to 8, and further preferably has 1 to 6.

[0472] Unless otherwise specified, the position of the anionic group is not particularly limited. As the group having the above anionic group, for example, in the compound represented by the general formula (III), Z 1 ~Z 3 or X 1 ~X 3 .

[0473] Specific examples of the fluorescent dye (F) are shown below, but the present invention is not limited to these compounds. In the following specific examples, the sulfo group and the phosphonyloxy group may take a salt structure by dissociation of a hydrogen ion. In the following specific examples, Dye represents a fluorescent moiety.

[0474] [Chemical formula 15]

[0475]

[0476] [Chemical formula 16]

[0477]

[0478] Regarding fluorescent dyes such as the fluorescent dye (F), it can be bonded to a biological substance such as a protein (including a peptide), an amino acid, a nucleic acid, a nucleotide, a sugar chain, and a lipid through at least one substituent capable of bonding to a biological substance possessed by the compound, and can be used as a fluorescent dye-labeled biomolecule.

[0479] As the substituent capable of bonding to a biological substance, any group can be used without particular limitation as long as it is a group for acting (including attaching) or bonding to a biological substance. For example, substituents described in International Publication No. 2002 / 026891 etc. can be cited. Specifically, the electrophilic groups and nucleophilic groups described in Table 2 of International Publication No. 2002 / 026891, and the description of the reactive group Rx described on pages 18, line 16 to page 19, line 13 of International Publication No. 2002 / 026891 can be applied to the present invention.

[0480] Specifically, the following structures can be cited as the "substituent capable of bonding to a biological substance".

[0481] [Chemical formula 17]

[0482]

[0483] X refers to a halogen atom such as an iodine atom or a bromine atom. * represents a connecting bond.

[0484] In addition to the above, as the "substituent capable of bonding to a biological substance", a peptide structure (polyamino acid structure), a long-chain alkyl group, etc. can be used.

[0485] Among them, preferred examples include NHS ester structure (N-hydroxysuccinimide ester structure), succinimide structure, maleimide structure, azide group, ethynyl group, peptide structure (polyamino acid structure), long-chain alkyl group (preferably having 12 to 30 carbon atoms), and quaternary ammonium group.

[0486] As a specific example of a compound having at least one substituent capable of bonding to a biological substance in the fluorescent dye (F), for example, a mode in which a carboxyl group in the exemplified compound of the above fluorescent dye (F) is appropriately substituted with the above substituent capable of bonding to a biological substance can also be cited as a specific example. In addition, the present invention is not limited to these compounds. For example, in these specific examples, for groups having dissociable hydrogen atoms such as carboxyl group and sulfo group, a salt structure can be taken by dissociation of a hydrogen atom.

[0487] Regarding fluorescent dyes such as the fluorescent dye (F), it can be bonded to a solid support such as the above microparticles through at least one substituent capable of bonding to a solid support possessed by the compound, and can be used as a solid support reagent.

[0488] As a substituent capable of bonding to a solid support, as long as it is a group used for acting (including adhering) or bonding to a solid support, it can be used without particular limitation, and preferred examples include the above substituents cited as substituents capable of bonding to a biological substance. Among them, NHS ester structure (N-hydroxysuccinimide ester), succinimide structure or maleimide structure can be preferably cited.

[0489] As a specific example of a compound having at least one substituent capable of bonding to a solid support in the fluorescent dye (F), for example, a mode in which a carboxyl group in the exemplified compound of the above fluorescent dye (F) is appropriately substituted with the above substituent capable of bonding to a solid support can also be cited as a specific example. In addition, the present invention is not limited to these compounds. For example, in these specific examples, for groups having dissociable hydrogen atoms such as carboxyl group and sulfo group, a salt structure can be taken by dissociation of a hydrogen atom.

[0490] The fluorescent dye (F) can be synthesized by a conventional method. For example, it can be synthesized based on peptide synthesis such as peptide solid-phase synthesis, and a method using a peptide automatic synthesizer described in International Publication No. 2018 / 174078 can also be preferably applied. Regarding the phosphor part, bioactive substance part, prodrug part, and radioactive isotope-containing part, they can also be synthesized according to a conventional method and introduced into the fluorescent dye (F).

[0491] Compounds having substituents capable of bonding to biomaterials can also be synthesized by conventional methods. For example, Bioconjugate Techniques (Third Edition, written by Greg T. Hermanson) can be referred to.

[0492] <Fluorescent Dye-Labeled Biomolecules>

[0493] A fluorescent dye-labeled biomolecule refers to a substance formed by the bonding of a fluorescent dye to a biomaterial. In particular, since the fluorescent dye (F) has fluorescence due to the fluorescent moiety and exhibits excellent fluorescence intensity, it can be preferably used for fluorescent dye-labeled biomolecules. The bonding between the fluorescent dye and the biomaterial can be in a manner of direct bonding between the fluorescent dye and the biomaterial or in a manner of being connected via a linking group.

[0494] As the above-mentioned biomaterials, proteins (including peptides), amino acids, nucleic acids, nucleotides, sugar chains, and lipids can be preferably listed. As proteins, antibodies can be preferably listed, and as lipids, phospholipids, fatty acids, and sterols can be listed, with phospholipids being more preferred.

[0495] Among the above-mentioned biomaterials, as substances useful in clinical pathology, there is no particular limitation. For example, immunoglobulins such as IgG (Immunoglobulin), IgM, IgE, IgA, IgD, complements, C-reactive protein (CRP), ferritin, plasma proteins such as α1-microglobulin and β2-microglobulin and their antibodies, α-fetoprotein, carcinoembryonic antigen (CEA), prostate acid phosphatase (PAP), tumor markers such as CA (carbohydrate antigen) 19-9 and CA-125 and their antibodies, hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen, insulin and their antibodies, virus infection-related substances such as hepatitis B virus (HBV)-related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), adult T-cell leukemia (ATL) and their antibodies, etc. can be listed.

[0496] In addition, bacteria such as diphtheria, Clostridium botulinum, Mycoplasma, Treponema pallidum and their antibodies, protozoa such as Toxoplasma, Trichomonas, Leishmania, Trypanosoma, Plasmodium and their antibodies, ES cells (Embryonic Stem Cell) such as ELM3, HM1, KH2, v6.5, v17.2, v26.2 (derived from mouse 129, 129 / SV, C57BL / 6, BALB / c) and their antibodies, antiepileptic drugs such as Phenytoin and Phenobarbital, cardiovascular drugs such as Quinidine and Digoxin, anti-asthma drugs such as Theophylline, antibiotics such as chloramphenicol and Gentamicin, and other drugs and their antibodies, other enzymes, bacterial exotoxins (such as streptolysin O), and their antibodies, etc. Moreover, antibody fragments such as Fab’2, Fab, and Fv can also be used.

[0497] As specific ways in which a fluorescent pigment binds through interaction with a biological substance, for example, the ways described below can be cited.

[0498] i) Non-covalent bonds (e.g., hydrogen bonds, ionic bonds including chelation formation) or covalent bonds between the peptide in the fluorescent pigment and the peptide in the biological substance,

[0499] ii) Van der Waals forces between the long-chain alkyl group in the fluorescent pigment and the lipid bilayer membrane and lipids in the biological substance,

[0500] iii) Amide bonds formed by the reaction of NHS ester (N-hydroxysuccinimide ester) in the fluorescent pigment with the amino group in the biological substance,

[0501] iv) Thioether bonds formed by the reaction of the maleimide group in the fluorescent pigment with the sulfanyl group (-SH) in the biological substance,

[0502] v) Triazole rings formed by the click reaction of the azide group in the fluorescent pigment with the ethynyl group in the biological substance or the click reaction of the ethynyl group in the fluorescent pigment with the azide group in the biological substance.

[0503] Among them, in the method of the above i), the peptide in the fluorescent pigment is not particularly limited as long as it can form a non-covalent bond or a covalent bond with the peptide in the biological substance. As the position having such a peptide, for example, R in the aforementioned general formula (II) can be preferably cited. 6 or R 7 .

[0504] In addition to the above methods i) to v), for example, it is also possible to bond by the method described in Lucas C.D. de Rezende and Flavio da Silva Emery, A Review of the Synthetic Strategies for the Development of BODIPY Dyes for Conjugation with Proteins, Orbital: The Electronic Journal of Chemistry, 2013, Vol 5, No.1, p.62 - 83. And in the production of fluorescent pigment-labeled biomolecules, the methods described in this document can also be appropriately referred to.

[0505] Regarding the fluorescent pigment-labeled biomolecules obtained from a fluorescent pigment such as a fluorescent pigment (F) having a substituent capable of bonding to a biological substance and a biological substance bonded by interacting with it, examples of compounds and products are described in the

[0038] section of Japanese Patent Application Laid-Open No. 2019-172826, where the part other than the substituent capable of bonding to the biological substance is replaced with the fluorescent pigment (F), and their products. However, it is not limited to these fluorescent pigment-labeled biomolecules, etc.

[0506] The fluorescent pigment-labeled biomolecules obtained from the fluorescent pigment (F) can exhibit excellent fluorescence intensity, and thus can stably detect the fluorescence released from the fluorescent pigment-labeled biomolecules excited by light irradiation. Therefore, the fluorescent pigment-labeled biomolecules obtained from the fluorescent pigment (F) can preferably be used in combination with the fluorescence intensity enhancer of the present invention.

[0507] In addition, commercially available primary antibodies and secondary antibodies can also be used as fluorescent dye-labeled biomolecules without particular limitation. For example, Alexa Fluor Plus 405, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 594, Alexa Fluor Plus 647, Alexa Fluor Plus 680, and Alexa Fluor Plus 800 (all are product names manufactured by Thermo Fisher Scientific) can be cited.

[0508] In addition, fluorescent dye-labeled biomolecules obtained from commercially available fluorescent dyes can also be used without particular limitation.

[0509] <Fluorescence Detection Using Fluorescent Dye-Labeled Biomolecules>

[0510] Fluorescence detection using fluorescent dye-labeled biomolecules generally includes the following steps (i) to (iii) or (iv) to (vii). The fluorescence detection including steps (i) to (iii) corresponds to the direct method using a primary antibody fluorescently labeled with a fluorescent dye, and the fluorescence detection including steps (iv) to (vii) corresponds to the indirect method using a secondary antibody fluorescently labeled with a fluorescent dye.

[0511] (i) Steps of separately preparing the following (a) and (b)

[0512] (a) A sample containing a biological substance as a target (hereinafter, also referred to as "target biological substance").

[0513] (b) A fluorescent dye-labeled biomolecule (hereinafter, also referred to as "fluorescent dye-labeled biomolecule A") formed by bonding a biological substance (hereinafter, also referred to as "primary biological substance") capable of bonding to the target biological substance in the above (a) with a fluorescent dye.

[0514] (ii) Step of preparing a conjugate (hereinafter, also referred to as "fluorescently labeled conjugate A") formed by bonding the target biological substance in the above (a) with the primary biological substance in the fluorescent dye-labeled biomolecule A in the above (b).

[0515] (iii) Step of irradiating the fluorescently labeled conjugate A with light in the wavelength region that can be absorbed by the fluorescent dye-labeled biomolecule A to detect the fluorescence emitted by the fluorescent dye-labeled biomolecule A.

[0516] (iv) Steps of separately preparing the following (c) to (e)

[0517] (c) A sample containing a target biological substance

[0518] (d) A biological substance capable of binding to the target biological substance in (c) above (hereinafter, also referred to as "primary biological substance").

[0519] (e) A fluorescent dye-labeled biomolecule formed by binding a biological substance capable of binding to the primary biological substance in (d) above (hereinafter, also referred to as "secondary biological substance") to a fluorescent dye (hereinafter, also referred to as "fluorescent dye-labeled biomolecule B").

[0520] (v) A step of preparing a conjugate formed by binding the target biological substance in (c) above to the primary biological substance in (d) above (hereinafter, also referred to as "conjugate b").

[0521] (vi) A step of preparing a conjugate formed by binding the primary biological substance in the conjugate b above to the secondary biological substance in the fluorescent dye-labeled biomolecule B (hereinafter, also referred to as "fluorescently labeled conjugate B2").

[0522] (vii) A step of irradiating the fluorescently labeled conjugate B2 above with light in the wavelength region that can be absorbed by the fluorescent dye-labeled biomolecule B to detect the fluorescence emitted by the fluorescent dye-labeled biomolecule B.

[0523] As the biological substance (primary biological substance) capable of binding to the target biological substance and the biological substance (secondary biological substance) capable of binding to the primary biological substance, the biological substance in the above fluorescent dye-labeled biomolecule can be cited. It can be appropriately selected according to the target biological substance (biological substance in the subject) or the primary biological substance, and a biological substance capable of specifically binding to the biological substance in the subject or the primary biological substance can be selected.

[0524] Among the above-mentioned target biological substances, as proteins, so-called disease markers can be cited. As disease markers, there are no particular restrictions. For example, α-fetoprotein (AFP), PIVKA-II (protein induced by vitamin K absence or antagonist II), BCA (breast carcinoma-associated antigen) 225, basic fetoprotein (BFP), CA (carbohydrate antigen) 15-3, CA19-9, CA72-4, CA125, CA130, CA602, CA54 / 61 (CA546), carcinoembryonic antigen (CEA), DUPAN-2, elastase 1, immunosuppressive acidic protein (IAP), NCC-ST-439, γ-seminoprotein (γ-Sm), prostate-specific antigen (PSA), prostate acid phosphatase (PAP), neuron-specific enolase (NSE), Iba1, amyloid-β, Tau, flotillin, squamous cell carcinoma-related antigen (SCC antigen), sialyl LeX-i antigen (SLX), SPan-1, tissue polypeptide antigen (TPA), sialyl Tn antigen (STN), cytokeratin (CYFRA), pepsinogen (PG), C-reactive protein (CRP), serum amyloid A (SAA), myoglobin, creatine kinase (CK), troponin T, ventricular myosin light chain I, etc. can be cited.

[0525] The above-mentioned target biological substances can be bacteria. As such bacteria, bacteria that are the objects of cellular microbiological examination can be cited. There are no particular restrictions. For example, Escherichia coli, Salmonella, Legionella, bacteria that cause public health problems, etc. can be cited.

[0526] The above-mentioned target biological substances can be viruses. As antigens of such viruses, there are no particular restrictions. For example, hepatitis virus antigens such as antigens of hepatitis C and B viruses, p24 protein antigen of HIV virus, pp65 protein antigen of CMV (cytomegalovirus), E6 and E7 proteins of HPV (human papillomavirus), etc. can be cited.

[0527] In the above (i) or (iv), the sample containing the target biological substance is not particularly restricted and can be prepared by a conventional method.

[0528] Moreover, the fluorescent dye-labeled biomolecule is not particularly restricted and can be prepared by bonding a biomolecule capable of bonding to the target biological substance to a fluorescent dye by a conventional method. The bonding method and the reaction for forming the bond are as described in the above fluorescent dye-labeled biomolecule.

[0529] In the above (v), the target biological substance and the primary biological substance can be directly bonded, or can be bonded via another biological substance different from the target biological substance and the primary biological substance. Also, in the above (vi), the primary biological substance in the bond b and the secondary biological substance in the fluorescent pigment-labeled biomolecule B can be directly bonded, or can be bonded via another biological substance different from the primary biological substance and the secondary biological substance.

[0530] The fluorescent pigment-labeled biomolecule obtained from the fluorescent pigment (F) can be used as a fluorescent pigment-labeled antibody in either the direct method or the indirect method, and is preferably used as a fluorescent pigment-labeled antibody in the indirect method.

[0531] In the above (ii) or (v) and (vi), the bonding of the fluorescent pigment-labeled biomolecule and the like to the target biological substance is not particularly limited and can be carried out according to a conventional method.

[0532] In the above (iii) or (vii), the wavelength of the light irradiated to excite the fluorescent pigment-labeled biomolecule is not particularly limited as long as it is a wavelength capable of exciting the fluorescent pigment-labeled biomolecule. Generally, 300 to 1000 nm is preferred, and 400 to 800 nm is more preferred.

[0533] As the light source used in the above (iii) or (vii), as long as it is a light source that emits light having a wavelength capable of exciting the fluorescent pigment-labeled biomolecule, it is not particularly limited. For example, various laser light sources can be used. Also, various filters can be used to adjust the emission wavelength from the light source to a wavelength in the preferred wavelength region, or to a wavelength that can only detect the fluorescence emitted from the fluorescent pigment-labeled biomolecule.

[0534] Regarding other matters in the above (i) to (vii), there is no particular limitation, and conditions such as methods, reagents, and devices commonly used in fluorescence detection using a fluorescent label can be appropriately selected.

[0535] Also, regarding processes other than the above (i) to (vii), conditions such as methods, reagents, and devices commonly used can be appropriately selected according to various methods using a fluorescent label.

[0536] For example, in multi-color WB for labeling biomolecules with fluorescent dyes, a blot membrane is prepared as a target biomaterial by commonly used methods (separation of proteins based on electrophoresis, blotting onto a membrane, blocking of the membrane), and the fluorescent dye-labeled biomolecule is used as a fluorescent dye-labeled antibody (among the fluorescent dye-labeled antibodies obtained from the fluorescent dye (F), the second antibody is preferably used). Thus, the target biomaterial can be detected with excellent fluorescence intensity. Regarding the dot blot method using a fluorescent dye-labeled biomolecule, similar to multi-color WB, a nitrocellulose blot membrane or a PVDF (polyvinylidene fluoride) blot membrane, etc. is prepared as a target biomaterial by commonly used methods, and the fluorescent dye-labeled biomolecule is used as a fluorescent dye-labeled antibody (among the fluorescent dye-labeled antibodies obtained from the fluorescent dye (F), the second antibody is preferably used). Thus, the target biomaterial can be detected with excellent fluorescence intensity.

[0537] - Substituent group T -

[0538] In the present invention, as preferred substituents, substituents selected from the following substituent group T can be cited.

[0539] Moreover, in the present invention, when only a substituent is described, the description of the corresponding substituent in this substituent group T can be referred to and applied. For example, when only "alkyl" is described, the description of "alkyl" in this substituent group T can be referred to and applied. The same applies to other substituents other than "alkyl".

[0540] Furthermore, in the present invention, regarding the substituents that a certain substituent such as "alkyl" can have, substituents selected from the following substituent group T can be cited. And when a certain substituent such as "alkyl" has a substituent and further has a substituent, as the substituent that a certain substituent has, a substituent formed by combining two or more substituents selected from the following substituent group T can be cited.

[0541] In addition, in the present invention, when alkyl and cyclic (cyclo)alkyl are distinguished and described, alkyl is used in the sense of including linear alkyl and branched alkyl. On the other hand, in the case where alkyl and cycloalkyl are not distinguished and described and in the case where no other explanation is given, alkyl is used in the sense of including linear alkyl, branched alkyl, and cycloalkyl. The same applies to groups (alkoxy, alkylthio, alkenyloxy, etc.) containing a group (alkyl, alkenyl, alkynyl, etc.) that can adopt a cyclic structure and to compounds containing a group that can adopt a cyclic structure. When a group can form a cyclic skeleton, the lower limit of the number of atoms of the group forming the cyclic skeleton is not related to the lower limit of the number of atoms specifically described below for the group that can adopt this structure, and is 3 or more, preferably 5 or more.

[0542] In the description of the following substituent group T, for example, in the case of an alkyl group and a cycloalkyl group, in order to clarify the group having a linear or branched structure and the group having a cyclic structure, they are sometimes described separately.

[0543] As the groups included in the substituent group T, the following groups are included.

[0544] Examples thereof include an alkyl group (preferably having 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, further preferably 1 to 12 carbon atoms, further preferably 1 to 8 carbon atoms, further preferably 1 to 6 carbon atoms, and particularly preferably 1 to 3 carbon atoms), an alkenyl group (preferably having 2 to 30 carbon atoms, more preferably 2 to 20 carbon atoms, further preferably 2 to 12 carbon atoms, further preferably 2 to 6 carbon atoms, and further preferably 2 to 4 carbon atoms), an alkynyl group (preferably having 2 to 30 carbon atoms, more preferably 2 to 20 carbon atoms, further preferably 2 to 12 carbon atoms, further preferably 2 to 6 carbon atoms, and further preferably 2 to 4 carbon atoms), a cycloalkyl group (preferably having 3 to 20 carbon atoms), a cycloalkenyl group (preferably having 5 to 20 carbon atoms), an aryl group (which may be a monocyclic group or a fused ring group (preferably a 2- to 6-ring fused ring group). In the case of a fused ring group, it is composed of a 5- to 7-membered ring or the like. The aryl group preferably has 6 to 40 carbon atoms, more preferably 6 to 30 carbon atoms, further preferably 6 to 26 carbon atoms, and particularly preferably 6 to 10 carbon atoms), a heterocyclic group (having at least one nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom or selenium atom as a ring-constituting atom, which may be a monocyclic group or a fused ring group (preferably a 2- to 6-ring fused ring group). In the case of a monocyclic group, the number of ring members is preferably 5 to 7, more preferably 5 or 6. The carbon atoms of the heterocyclic group preferably have 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms. The heterocyclic group includes an aromatic heterocyclic group (heteroaryl) and an aliphatic heterocyclic group (aliphatic heterocyclic group).), an alkoxy group (preferably having 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms), an alkenyloxy group (preferably having 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms), an alkynyloxy group (preferably having 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms), a cycloalkoxy group (preferably having 3 to 20 carbon atoms), an aryloxy group (preferably having 6 to 40 carbon atoms, more preferably 6 to 26 carbon atoms, further preferably 6 to 14 carbon atoms), a heteroaryloxy group (preferably having 2 to 20 carbon atoms), a polyalkyleneoxy,

[0545] Alkoxycarbonyl (preferably having 2 to 20 carbon atoms), cycloalkoxycarbonyl (preferably having 4 to 20 carbon atoms), aryloxycarbonyl (preferably having 6 to 20 carbon atoms), amino (preferably having 0 to 20 carbon atoms, including unsubstituted amino (-NH2), (mono- or di-) alkylamino, (mono- or di-) alkenylamino, (mono- or di-) alkynylamino, (mono- or di-) cycloalkylamino, (mono- or di-) cycloalkenylamino, (mono- or di-) arylamino, (mono- or di-) heterocyclic amino. The corresponding group meanings of the above groups substituting the unsubstituted amino are the same as those of the groups in substituent group T.), sulfamoyl (preferably having 0 to 20 carbon atoms, preferably sulfamoyl of alkyl, cycloalkyl or aryl), acyl (preferably having 1 to 20 carbon atoms, more preferably having 2 to 15 carbon atoms, including -C(=O)H, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclic carbonyl), acyloxy (preferably having 1 to 20 carbon atoms), carbamoyl (preferably having 1 to 20 carbon atoms, preferably carbamoyl of alkyl, cycloalkyl or aryl),

[0546] Acylamino (preferably having 1 to 20 carbon atoms), sulfonamido (preferably having 0 to 20 carbon atoms, preferably sulfonamido of alkyl, cycloalkyl or aryl), alkylthio (preferably having 1 to 20 carbon atoms, more preferably having 1 to 12 carbon atoms), cycloalkylthio (preferably having 3 to 20 carbon atoms), arylthio (preferably having 6 to 40 carbon atoms, more preferably having 6 to 26 carbon atoms, further preferably having 6 to 14 carbon atoms), heterocyclic thio (preferably having 2 to 20 carbon atoms), alkyl, cycloalkyl or arylsulfonyl (preferably having 1 to 20 carbon atoms),

[0547] Silyl (preferably having 1 to 30 carbon atoms, more preferably having 1 to 20 carbon atoms, preferably silyl substituted by alkyl, aryl, alkoxy or aryloxy), silyloxy (preferably having 1 to 20 carbon atoms, preferably silyloxy substituted by alkyl, aryl, alkoxy or aryloxy), hydroxyl, cyano, nitro, halogen atom (such as fluorine atom, chlorine atom, bromine atom or iodine atom), oxygen atom (specifically, >CH2 constituting the ring is substituted by >C=O), carboxyl (-CO2H), phosphonyl [-PO(OH)2], phosphonyloxy [-O-PO(OH)2], sulfo (-SO3H), boronic acid group [-B(OH)2], onium group (also called cationic group. Including ammonium group containing cyclic ammonium, sulfonium group, phosphonium group, preferably having 0 to 30 carbon atoms, more preferably 1 to 20), thioalkyl (-SH), guanidino (-NHC(=NH)NH2), amino acid residue or polyamino acid residue.

[0548] (Anionic group)

[0549] In the present invention, the anionic group only needs to be a group having an anion. Examples of such anionic groups include a carboxyl group, a phosphonyl group (phosphonic acid group, -PO(OH)2), a phosphonyloxy group (phosphoric acid group, -OPO(OH)2), and a sulfo group, etc. A phosphonyl group, a phosphonyloxy group, or a sulfo group is preferred, and a phosphonyloxy group or a sulfo group is more preferred.

[0550] The anionic group can dissociate a hydrogen ion to adopt an ionic structure or can adopt a salt structure. As the monovalent or polyvalent cation when the anionic group has a salt structure, the description of the monovalent or polyvalent cation in the description of the aforementioned salt structure can be preferably applied.

[0551] (Cationic group)

[0552] In the present invention, the cationic group only needs to be a group having a cation. Examples of such cationic groups include a group having a quaternary ammonium ion (quaternary ammonium group) and a group having a quaternary phosphonium ion (quaternary phosphonium group), etc. A group having a quaternary ammonium ion is preferred. In addition, as the substituent of N in the group having a quaternary ammonium ion + and the substituent of P in the group having a quaternary phosphonium ion + the substituents can preferably include an alkyl group and an aryl group, and more preferably the substituents of N + and P + are all alkyl groups.

[0553] In addition to the ionic structure, the cationic group can also adopt a salt structure. As the monovalent or polyvalent anion when the cationic group has a salt structure, for example, fluoride ions such as F - , Cl - and other halide ions, BF4 - , PF6 - and monovalent or polyvalent organic anions such as bis(trifluoromethylsulfonyl)imide ion.

[0554] (Polyalkyleneoxy)

[0555] In the present invention, the above polyalkyleneoxy only needs to be a group represented by -(L-O) g R E .

[0556] The above L represents an alkylene group obtained by removing one hydrogen atom from the alkyl group in the above substituent group T. The number of carbon atoms is preferably 2 to 4, more preferably 2 or 3, and further preferably 2. The number of carbon atoms included in the shortest chain connecting the two carbon atoms of the connecting bond of the alkylene group is preferably 0 to 2, more preferably 0 or 1, and further preferably 0. That is, L is most preferably an ethylene group.

[0557] The above-mentioned g refers to the average number of repetitions (abbreviated as the number of repetitions), preferably 1 to 24, more preferably 1 to 12, and further preferably 4 to 12. When the number of repetitions such as g is 1, appropriate hydrophilicity and appropriate excluded volume effect can be exhibited.

[0558] The above-mentioned R E represents a hydrogen atom or an alkyl group. The alkyl group that can be adopted as R E can preferably apply the description of the alkyl group in the above-mentioned substituent group T, and among them, an alkyl group having 1 to 3 carbon atoms is preferred. The alkyl group that can be adopted as R E may have a substituent.

[0559] And, as a group formed by combining a plurality of substituents selected from the substituent group T, for example, an anionic group (carboxyl group, phosphonyl group, sulfo group), a cationic group (onium group), an amino acid residue, a polyamino acid residue, or -(CH2-CH2-O) b -alkyl (b has the same meaning as b in the above general formula (α) for R 1 ~R 4 in.) as a substituent of the above-mentioned alkyl group (Alkyl Group), alkenyl group, alkynyl group, cycloalkyl group (Cycloalkyl Group), cycloalkenyl group, aryl group, heterocyclic group, alkoxy group, alkenyloxy group, alkynyloxy group, cycloalkoxy group, aryloxy group, heterocyclic oxy group, alkoxycarbonyl group, cycloalkoxycarbonyl group, aryloxycarbonyl group, amino group, sulfamoyl group, acyl group, acyloxy group, carbamoyl group, acylamino group, sulfonamide group, alkylthio group, cycloalkylthio group, arylthio group, heterocyclic thio group, alkyl (Alkyl), cycloalkyl (Cycloalkyl) or arylsulfonyl group.

[0560] The substituents selected from the substituent group T are more preferably an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an acyl group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a carbamoyl group, a cyano group, a halogen atom, an anionic group or a cationic group, and particularly preferably an alkyl group, an alkenyl group, an aryl group, a heterocyclic group, an alkoxy group, an acyl group, an alkoxycarbonyl group, an amino group, an acylamino group, a carbamoyl group, a cyano group, an anionic group or a cationic group.

[0561] The substituents selected from the substituent group T, in addition to including groups formed by combining a plurality of substituents selected from the above-mentioned substituent group T, unless otherwise specified, also include groups formed by combining the above-mentioned groups. For example, when a compound or a substituent, etc. contains an alkyl group, an alkenyl group, etc., they may be substituted or unsubstituted. And when containing an aryl group, a heterocyclic group, etc., they may be monocyclic or fused rings, and may be substituted or unsubstituted.

[0562] Examples

[0563] Hereinafter, the present invention will be described in further detail based on examples, but the present invention is not limited thereto. In addition, room temperature means 25°C.

[0564] Compounds (1) to (4) corresponding to the fluorescent dye (F) are shown below.

[0565] Moreover, in each compound, unless otherwise specified, the sulfo group and phosphonyloxy group may also include a salt structure (for example, potassium salt, sodium salt, TEA (triethylammonium) salt, or DIPEA (N,N - diisopropylethylamine) salt).

[0566] [Chemical formula 18]

[0567]

[0568] [Chemical formula 19]

[0569]

[0570] Hereinafter, the synthesis methods of each compound will be described in detail, but the starting materials, pigment intermediates, and synthesis routes are not limited to these.

[0571] In addition, the abbreviations used in the synthesis of each compound shown below are as follows.

[0572] DBU: 1,8 - diazabicyclo[5.4.0]-7 - undecene

[0573] PyAOP: (7 - azabenzotriazol - 1 - yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0574] DIC: Diisopropylcarbodiimide

[0575] EDCI: 1 - ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide hydrochloride

[0576] HOBt: 1 - Hydroxybenzotriazole

[0577] Pd(dppf)Cl2: [1,1’ - bis(diphenylphosphino)ferrocene]palladium(II) dichloride

[0578] Pd(Amphos)Cl2: Bis(di - tert - butyl(4 - dimethylaminophenyl)phosphine)palladium(II) dichloride

[0579] DMT - MM: 4 - (4,6 - dimethoxy - 1,3,5 - triazin - 2 - yl)-4 - methylmorpholine hydrochloride

[0580] NMP: N - methyl - 2 - pyrrolidone

[0581] DMF: Dimethylformamide

[0582] DMAP: 4-Dimethylaminopyridine

[0583] DIPEA: N-Diisopropylethylamine

[0584] TFA: Trifluoroacetic acid

[0585] THF: Tetrahydrofuran

[0586] TFE: 2,2,2-Trifluoroethanol

[0587] Me: Methyl

[0588] Ms: Methanesulfonyl

[0589] Et: Ethyl

[0590] Ac: Acetyl

[0591] Ts: p-Toluenesulfonyl

[0592] Trt: Triphenylmethyl

[0593] Fmoc: 9-Fluorenylmethyloxycarbonyl

[0594] Boc: tert-Butyloxycarbonyl

[0595] Ala: Alanine

[0596] Lys: Lysine

[0597] Pro: Proline

[0598] Resin: Resin

[0599] mPEG p : represents the following structure, PEG p where p in PEG is the average number of repeats. * represents the linking bond.

[0600] EO p : represents the following structure, EO p where p in EO is the average number of repeats. Among them, EO p is bonded to a nitrogen atom or an oxygen atom on the carbon atom side. * represents the linking bond.

[0601] [Chemical formula 20]

[0602]

[0603] And, % v / v refers to volume percentage.

[0604] The MS spectra were measured using an ACQUITY SQD LC / MS System [product name, manufactured by Waters Corporation, ionization method: ESI (ElectroSpray Ionization)] or an LCMS-2010EV [product name, manufactured by Shimadzu Corporation, ionization method: an ionization method that simultaneously performs ESI and APCI (Atmospheric Pressure Chemical Ionization)].

[0605] In addition, when synthesizing each compound, the peptide chain was synthesized according to the general method of the peptide solid-phase method described in International Publication No. 2018 / 174078.

[0606] [General method for peptide solid-phase synthesis using a peptide automatic synthesizer]

[0607] Peptide solid-phase synthesis was carried out using a peptide automatic synthesizer (manufactured by Biotage Japan Ltd., product name: Syro I). Rink Amide-ChemMatrix (registered trademark, manufactured by Biotage Japan Ltd.), an N-methyl-2-pyrrolidone (NMP) solution of Fmoc amino acid (0.5 mol / L), an NMP solution of cyano-hydroxyimino-ethyl acetate (1.0 mol / L) and diisopropylamine (0.1 mol / L), an NMP solution of diisopropylcarbodiimide (1.0 mol / L), an NMP solution of piperidine (20% v / v), and an NMP solution of acetic anhydride (20% v / v) were set in the synthesizer, and the synthesis was carried out according to the instructions. Deprotection of Fmoc (20 minutes), washing with NMP, condensation of Fmoc amino acid (1 hour), and washing with NMP were repeated as one cycle, and the peptide chain was extended by repeating this cycle.

[0608] [Synthesis of compound (M2-1)]

[0609] Compound (M2-1) was synthesized according to the following scheme. The MS measurement results of compound (M2-13) are as follows.

[0610] MS (ESI m / z): (M + H + ) + = 1384, (M - H + ) - = 1382

[0611] [Chemical formula 21]

[0612]

[0613] <Synthesis of Compounds (1) and (1-NHS)>

[0614] 1) Synthesis of Compound (1-8)

[0615] Compound (1-8) was synthesized according to the following scheme.

[0616] [Chemical Formula 22]

[0617]

[0618] (i) Synthesis of Compound (1-1)

[0619] Using H-Pro-Trt(2-Cl)-Resin (manufactured by WATANABE CHEMICAL INDUSTRIES, LTD., 0.93 mmol / g, 53.8 mg) as the starting material, solid-phase peptide synthesis was carried out. N-(9-Fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) and (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)) were elongated, and the elongation using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 3 cycles. (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)) was elongated, and the elongation using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated for 3 cycles. (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxy)carbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)) and N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) were elongated. After the elongation was completed, the resin was washed with dichloromethane, and then the solvent was removed by distillation under reduced pressure. 2.0 mL of a mixed solution of TFA:triisopropylsilane:water = 95:2.5:2.5 was added, and peptide cleavage and deprotection were carried out. After 2 hours, the resin was filtered out, and methyl tert-butyl ether (12 mL) was added to the filtrate to form a solid. Centrifugation was carried out to precipitate the solid, and then the supernatant was removed. The solid was washed with methyl tert-butyl ether, and then the solvent was removed by distillation under reduced pressure to obtain 51.2 mg of white solid Compound (1-1).

[0620] (ii) Synthesis of Compound (1-2)

[0621] 352 mg of compound (1-1), 3.5 mL of chloroform (CHCl₃), 423 μL of N-diisopropylethylamine (DIPEA), and 115 μL of acetic anhydride (Ac₂O) were added to a 50 mL eggplant-shaped flask and stirred at room temperature for 1 hour. Then, the reaction solution was concentrated and purified by preparative HPLC, and freeze-dried to obtain 314 mg of compound (1-2).

[0622] (iii) Synthesis of compound (1-3)

[0623] 500 mg of the compound (4-1) described in International Publication No. 2020 / 175473 as compound (2-1), 5 mL of tetrahydrofuran (THF), 339.3 mg of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-β-alanine (Fmoc-βAla-OH), 171 μL of diisopropylcarbodiimide, and 13.3 mg of 4-dimethylaminopyridine were added to a 50 mL eggplant-shaped flask and stirred at room temperature for 2 hours. The precipitated solid was filtered by adding acetonitrile (50 mL) and dried under reduced pressure to obtain 593 mg of compound (1-3).

[0624] (iv) Synthesis of compound (1-4)

[0625] 108 mg of compound (1-3), 2.2 mL of chloroform (CHCl₃), and 26.8 μL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were added to a 50 mL eggplant-shaped flask and stirred at 35 °C for 1 hour. 11.7 μL of methanesulfonic acid (MsOH), 93.3 μL of N,N-diisopropylethylamine (DIPEA), 169 mg of compound (1-2), and 141 mg of (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were added and stirred at 35 °C for 3 hours. The precipitated solid was filtered by adding acetonitrile (10 mL) and dried under reduced pressure to obtain 219 mg of compound (1-4).

[0626] (v) Synthesis of compound (1-5)

[0627] In the synthesis of the above compounds (1-4), (2S,4S)-(tert-butoxycarbonyl)-4-amino-1-(9H-fluoren-9-ylmethoxycarbonyl)-pyrrolidine-2-carboxylic acid (Fmoc-L-Pro(4-NHBoc)-OH(2S,4S)) was used instead of compound (1-2), and compound (1-4) was used instead of compound (1-3). Otherwise, in the same manner, 233 mg of the above compound (1-5) was synthesized from 219 mg of compound (1-4).

[0628] (vi) Synthesis of compound (1-6)

[0629] The same operations as those for the synthesis of the above compounds (1-4) and (1-5) were repeated twice, and 190 mg of compound (1-6) was synthesized from 100 mg of compound (1-5).

[0630] (vii) Synthesis of compound (1-7)

[0631] 40.2 mg of compound (1-6), 5 mL of chloroform (CHCl3), and 3.1 μL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) were added to a 100 mL eggplant-shaped flask, and the mixture was stirred at 35 °C for 1 hour. 1.35 μL of methanesulfonic acid (MsOH), 7.2 μL of N,N-diisopropylethylamine (DIPEA), and 1.9 μL of acetic anhydride (Ac2O) were added, and the mixture was stirred at 35 °C for 1 hour. The precipitated solid was filtered by adding acetonitrile (50 mL) and dried under reduced pressure, whereby 33.7 mg of compound (1-7) was obtained.

[0632] (viii) Synthesis of compound (1-8)

[0633] 30.1 mg of compound (1-7) and 1.0 mL of a mixture of TFA:triisopropylsilane:water = 95:2.5:2.5 were added to a 10 mL eggplant-shaped flask, and the mixture was stirred at room temperature for 1 hour. After adding 10 mL of methanol (MeOH) to the reaction solution to form a solid, the solid was removed by filtration. After concentrating the filtrate, it was purified by preparative HPLC and freeze-dried to obtain 10.6 mg of compound (1-8).

[0634] 2) Synthesis of compounds (1) and (1-NHS)

[0635] Compounds (1) and (1-NHS) were synthesized according to the following scheme.

[0636] In addition, although the labeled forms of the repeating structures of the polypeptide chains of compound (1-8), compound (1), and (1-NHS) are different, they have the same structure as the repeating structure of the polypeptide chain.

[0637] [Chemical formula 23]

[0638]

[0639] (i) Synthesis of compound (1)

[0640] To a 10 mL eggplant-shaped flask, 2.33 mg of compound (1-8), 110 μL of N,N-dimethylformamide (DMF), 7.3 μL of triethylamine (Et3N), and 3.1 mg of compound (M2-1) were added, and the mixture was stirred at room temperature for 1 hour. Subsequently, the reaction solution was concentrated and purified by preparative HPLC, and freeze-dried to obtain 0.86 mg of compound (1). The MS measurement results of compound (1) are as follows.

[0641] MS(ESI m / z): (M+H + ) + = 8571, (M-H + ) - = 8569

[0642] (ii) Synthesis of compound (1-NHS)

[0643] To 1.26 mg of compound (1), 126 μL of N,N-dimethylformamide (DMF), 2.64 mg of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate, and 2.1 μL of triethylamine (Et3N) were added, and the mixture was stirred for 1 hour. Subsequently, the solvent was distilled off under reduced pressure, ethyl acetate was added to remove the supernatant, and vacuum drying was performed to obtain compound (1-NHS).

[0644] <Synthesis of compound (2)>

[0645] 1) Synthesis of compound (M3-1) Compound (M3-1) was synthesized according to the following scheme.

[0646] [Chemical formula 24]

[0647]

[0648] (i) Synthesis of compound (2-2)

[0649] To a 100-ml eggplant-shaped flask, 2.9 g of compound (2-1) and 15 ml of ethanol were added. After dropping 15 ml of ethanol in which 2.1 mL of aniline was dissolved at 0 °C, the mixture was stirred under a nitrogen atmosphere at 60 °C for 4 hours. The solvent was distilled under reduced pressure to make the solvent volume 15 ml, and it was air-cooled to 0 °C. The precipitated yellow crystals were filtered to obtain 2.6 g of compound (2-2).

[0650] (ii) Synthesis of compound (2-4)

[0651] To a 100-ml three-necked eggplant-shaped flask, 100 mg of compound (2-3), 0.3 ml of acetic acid (AcOH), 124 mg of compound (1-C), and 31 mg of potassium acetate (AcOK) were added. The mixture was stirred under a nitrogen atmosphere at 130 °C for 1 hour. The solvent was distilled under reduced pressure and purified by reverse-phase column chromatography (eluent: acetonitrile / water = 0 / 100 to 20 / 80) to obtain 58 mg of compound (2-4).

[0652] (iii) Synthesis of compound (2-5)

[0653] To a 50-ml eggplant-shaped flask, 470 mg of compound (2-4), 0.15 mL of 1,3-propane sultone, 2 ml of sulfolane, and 254 mg of potassium acetate (AcOK) were added. The mixture was heated and stirred at 110 °C for 1 hour. 20 mL of ethyl acetate was added to the reaction solution to cause precipitation. The precipitate was purified by reverse-phase column chromatography (eluent: acetonitrile / water = 0 / 100 to 30 / 70) to obtain 475 mg of compound (2-5).

[0654] (iv) Synthesis of compound (2-6)

[0655] To a test tube, 171 mg of compound (2-5), 48 mg of compound (2-2), 71 μL of triethylamine (Et3N), 5 μL of methanol (MeOH), and 0.48 mL of acetic anhydride (Ac2O) were added. The mixture was stirred under a nitrogen atmosphere at 50 °C for 2 hours. After the reaction converged, distilled water was added and purified by reverse-phase column chromatography (eluent: acetonitrile / water = 0 / 100 to 25 / 75) to obtain 150 mg of compound (2-6).

[0656] (v) Synthesis of compound (2-8)

[0657] 10 mg of compound (2-6), 10 mg of potassium carbonate (K2CO3), 300 μL of ethanol (EtOH), and 100 μL of distilled water were added to a test tube and stirred at 50 °C. A solution prepared by mixing 6 mg of compound (2-7) and palladium(II) dichloride [1,1'-bis(diphenylphosphino)ferrocene] (Pd(dppf)Cl2) in 200 μL of distilled water was added dropwise to this solution, and the mixture was stirred at 50 °C for 30 minutes. The reaction solution was cooled to room temperature, purified by preparative HPLC, and freeze-dried to obtain 3.6 mg of compound (2-8). The MS measurement results of compound (2-8) are as follows.

[0658] MS(ESI m / z): (M+H + ) + = 1491, (M-H + ) - = 1489

[0659] (vi) Synthesis of compound (2-9)

[0660] In the same manner as the synthesis of the above compound (M2-1), 3.5 mg of compound (2-9) was synthesized from 3.6 mg of compound (2-8). The MS measurement results of compound (2-9) are as follows.

[0661] MS(ESI m / z): (M+H + ) + = 1588, (M-H + ) - = 1586

[0662] (vii) Synthesis of compound (2-10)

[0663] 7.53 mg of compound (2-9), 261 μL of N,N-dimethylformamide (DMF), 3.6 μL of triethylamine (Et3N), and 11.3 mg of β-alanine were added to a test tube and stirred at room temperature for 3 hours. Then, the reaction solution was concentrated and purified by preparative HPLC, and freeze-dried to obtain 4.49 mg of compound (2-10). The MS measurement results of compound (2-10) are as follows.

[0664] MS(ESI m / z): (M+H + ) + = 1562, (M-H + ) - = 1560

[0665] (viii) Synthesis of compound (M3-1)

[0666] In the same manner as the synthesis of the above compound (M2-1), 4.21 mg of compound (M3-1) was synthesized from 4.49 mg of compound (2-10). The MS measurement results of compound (M3-1) are as follows.

[0667] MS(ESI m / z):(M+H + ) + =1659、(M-H + ) - =1657

[0668] 2) Synthesis of compound (2)

[0669] According to the following scheme, in the same manner as compound (1), 1.64 mg of compound (2) was synthesized from 3.0 mg of compound (1-8). The MS measurement results of compound (2) are as follows.

[0670] MS(ESI m / z):(M+H + ) + =9112、(M-H + ) - =9110

[0671] [Chemical formula 25]

[0672]

[0673] <Synthesis of compound (3)>

[0674] 1) Synthesis of compound (M4-1)

[0675] According to the following scheme, compound (M4-1) was synthesized in the same manner as the synthesis of the above compound (M3-1). The MS measurement results of compound (M4-1) are as follows.

[0676] MS(ESI m / z):(M+H + ) + =1688、(M-H + ) - =1686

[0677] In addition, compound (3-2) was synthesized as follows. 300 mg of compound (3-1), 185 mg of tetrahydroxy diborane, 1.9 mg of bis[di-tert-butyl-(4-dimethylaminophenyl)phosphine] dichloropalladium(II), 457 μL of N,N-diisopropylethylamine (DIPEA), 3 mL of tetrahydrofuran (THF), and 1.5 mL of methanol (MeOH) were added to a 50 mL three-necked eggplant-shaped flask, and the mixture was stirred under a nitrogen atmosphere at 55 °C for 3 hours. The solvent was distilled off under reduced pressure, and purification was carried out by reverse-phase column chromatography to obtain 254 mg of compound (3-2).

[0678] [Chemical formula 26]

[0679]

[0680] 2) Synthesis of compound (3)

[0681] According to the following scheme, 1.57 mg of compound (3) was synthesized from 3.0 mg of compound (1-8) in the same manner as the synthesis of the above compound (1). The MS measurement results of compound (3) are as follows.

[0682] MS(ESI m / z):(M+H + ) + =9194、(M-H + ) - =9196

[0683] [Chemical formula 27]

[0684]

[0685] <Synthesis of compound (4)>

[0686] 1) Synthesis of compound (4-7)

[0687] Compound (4-7) was synthesized according to the following scheme.

[0688] [Chemical formula 28]

[0689]

[0690] (i) Synthesis of compound (4-2)

[0691] To a 50 mL eggplant-shaped flask, 300 mg of the compound (4-1) described in International Publication No. 2020 / 175473 as the compound (4-1), 3 mL of tetrahydrofuran (THF), 229.8 mg of Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine (Fmoc-Lys(Boc)-OH), 76.8 μL of diisopropylcarbodiimide, and 8.0 mg of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 2 hours. By adding acetonitrile (30 mL), the precipitated solid was filtered and dried under reduced pressure to obtain 350 mg of the compound (4-2).

[0692] (ii) Synthesis of compound (4-3)

[0693] Using H-Pro-Trt(2-Cl)-Resin (manufactured by WATANABE CHEMICAL INDUSTRIES, LTD., 0.94 mmol / g, 53.2 mg) as the starting material, peptide solid-phase synthesis was carried out. The elongation using N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) was repeated 11 cycles, and the elongation of Nε-(tert-butoxycarbonyl)-Nα-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine (Fmoc-Lys(Boc)-OH) was carried out. Then, a solution of piperidine (20% v / v) in NMP was added and allowed to react for 20 minutes to deprotect the Fmoc group, and a solution of acetic anhydride (20% v / v) in NMP was added and allowed to react for 10 minutes to acetylate the amino group at the N-terminus. After the elongation was completed, the resin was washed with dichloromethane, and then the solvent was removed by distillation under reduced pressure. 2.0 mL of a mixture of hexafluoro-2-propanol (HFIP):dichloromethane (CH2Cl2) = 1:4 was added for peptide cleavage. After 30 minutes, the resin was filtered out and the filtrate was concentrated, and purified by reverse-phase column chromatography to obtain 59.3 mg of the compound (4-3) as a white solid.

[0694] (iii) Synthesis of compound (4-4)

[0695] To a 10 mL eggplant-shaped flask, 20 mg of compound (4-2), 400 μL of chloroform (CHCl₃), and 4.4 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were added, and the mixture was stirred at 35 °C for 1 hour. 1.9 μL of methanesulfonic acid (MsOH), 15.3 μL of N,N-diisopropylethylamine (DIPEA), 25.5 mg of compound (4-3), and 23.1 mg of (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were added, and the mixture was stirred at 35 °C for 1 hour. By adding acetonitrile (4 mL), the precipitated solid was filtered and dried under reduced pressure, thereby obtaining 34.0 mg of compound (4-4).

[0696] (iv) Synthesis of compound (4-5)

[0697] To a 10 mL eggplant-shaped flask, 30.0 mg of compound (4-4), 600 μL of chloroform (CHCl₃), 60 μL of 2,2,2-trifluoroethanol (TFE), and 6 μL of trifluoroacetic acid (TFA) were added, and the mixture was stirred at room temperature for 4 hours. Then, the reaction solution was filtered, and the precipitate formed by adding 6 mL of methanol (MeOH) to the filtrate was centrifuged. The recovered compound was purified by preparative HPLC and freeze-dried to obtain 16.5 mg of compound (4-5).

[0698] (v) Synthesis of compound (4-6)

[0699] To a 10 mL eggplant-shaped flask, 10 mg of compound (4-5), 200 μL of water, 200 μL of N,N-dimethylformamide (DMF), 3.9 mg of tert-butyl 1-amino-3,6,9,12-tetraoxapentadecane-15-carboxylate, and 6.7 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) were added, and the mixture was stirred at room temperature for 2 hours. Then, the reaction solution was concentrated and purified by preparative HPLC and freeze-dried to obtain 8.9 mg of compound (4-6).

[0700] (vi) Synthesis of compound (4-7)

[0701] To a 10 mL eggplant-shaped flask, 7.0 mg of compound (4-6) and 200 μL of trifluoroacetic acid (TFA) were added, and the mixture was stirred at room temperature for 1 hour. Then, the reaction solution was concentrated and purified by preparative HPLC and freeze-dried to obtain 5.9 mg of compound (4-7).

[0702] 2) Synthesis of compound (M1-1)

[0703] Compound (M1-1) was synthesized according to the following scheme. In addition, compound (1-C) is the same as compound (1-C) in the synthesis of compound (M2-1) described below. The MS measurement results of compound (M1-8) are as follows.

[0704] MS(ESI m / z):(M+H + ) + =1723、(M-H + ) - =1721

[0705] [Chemical formula 29]

[0706]

[0707] [Chemical formula 30]

[0708]

[0709] 3) Synthesis of compound (4)

[0710] According to the following scheme, 3.2 mg of compound (4) was synthesized from 2.0 mg of compound (4-7) in the same manner as the synthesis of the above compound (1). The MS measurement results of compound (4) are as follows.

[0711] MS(ESI m / z):(M+H + ) + =5136、(M-H + ) - =5134

[0712] [Chemical formula 31]

[0713]

[0714] <Synthesis of compounds (2-NHS), (3-NHS) and (4-NHS)>

[0715] In the synthesis of the above compound (1-NHS), compounds (2), (3) or (4) were used instead of compound (1), and in other respects, compounds (2-NHS), (3-NHS) and (4-NHS) were synthesized in the same manner, which were obtained by converting the terminal carboxyl group in the peptide chain of compounds (2), (3) or (4) into an NHS ester structure.

[0716] <Example 1>

[0717] For each of the above compounds (1-NHS) to (4-NHS), the fluorescence labeling rate and the fluorescence intensity of immunoblotting were evaluated.

[0718] [1] Preparation of fluorescently labeled antibody

[0719] 104 μL of anti-rabbit IgG antibody (2.3 mg / ml) and 10.4 μL of carbonate buffer were added to a microtube. After shaking and stirring, a dimethyl sulfoxide solution of compound (1-NHS) was added in a molar equivalent ratio (30 equivalent ratio) shown in Table A relative to 1 equivalent of the antibody, and further shaking and stirring were performed. The mixture was allowed to stand at 4 °C for 24 hours, and the reaction solution was purified using a centrifugal ultrafiltration filter (product name: Amicon Ultra UFC510096, manufactured by Merck & Co., Inc.) and PBS solution (phosphate buffered saline) to obtain an IgG-labeled antibody of compound (1) (2.0 mg / mL). For compounds (2) to (4), compounds (2-NHS) to (4-NHS) were added in a molar equivalent ratio (20, 30, or 40 equivalent ratio) shown in Table A instead of compound (1-NHS), and the labeled antibody was obtained in the same manner. For the obtained fluorescent dye-labeled antibody, the fluorescence labeling rate (DOL) was calculated by the method shown below. The results are summarized in Table A.

[0720] The method for calculating the fluorescence labeling rate used the following general method. The notations in [] indicate units, and [-] means no unit. In this experiment, the protein refers to anti-rabbit IgG antibody in compounds (1) to (4).

[0721] Fluorescence labeling rate = Concentration of fluorescent dye / Concentration of protein

[0722] The concentration of the fluorescent dye refers to the total molar concentration [M] of the labeled fluorescent dye, and the concentration of the protein refers to the molar concentration [M] of the fluorescently labeled protein, which were calculated by the following equations respectively.

[0723] Concentration of fluorescent dye = Dye max / ε dye

[0724] Concentration of protein = (IgG 280 - (Dye max × CF)) / ε protein

[0725] Each symbol in the above equations is described as follows.

[0726] Dye max ; Absorbance of the fluorescent dye at the maximum absorption wavelength [-]

[0727] ε dye ; Molar extinction coefficient of the fluorescent dye [M -1 cm -1

[0728] IgG 280 ; Absorbance of fluorescently labeled protein at 280 nm [-]

[0729] Dye 280 ; Absorbance of fluorescent dye at 280 nm [-]

[0730] ε protein ; Molar extinction coefficient of protein [M -1 cm -1

[0731] CF (Correction Factor); Dye 280 / Dye max [-]

[0732] [Table A]

[0733] No. Fluorescent dye-labeled antibody 20 equivalents 30 equivalents 40 equivalents 001 Compound (1)-IgG - 4.3 - 002 Compound (2)-IgG - 5.5 - 003 Compound (3)-IgG - - 5.7 004 Compound (4)-IgG 5.8 - -

[0734] (Table Note)

[0735] In the column of fluorescent dye-labeled antibody, the labeling of compound (Z)-IgG refers to the IgG-labeled antibody of compound (Z-NHS). Z refers to the number of each compound. The same applies to the following tables.

[0736] Based on the results of Table A above, the following can be known.

[0737] Regarding compounds (1) and (2), when added at a molar equivalent ratio of 30 equivalents relative to 1 equivalent of antibody, the fluorescence labeling rates are 4.3 and 5.5 respectively. Regarding compound (3), when added at a molar equivalent ratio of 40 equivalents relative to 1 equivalent of antibody, the fluorescence labeling rate is 5.7. Regarding compound (4), when added at a molar equivalent ratio of 20 equivalents relative to 1 equivalent of antibody, the fluorescence labeling rate is 5.8, showing a sufficient level with no practical problems in terms of binding to the antibody.

[0738] [2] Immunoblot Fluorescence Intensity Evaluation

[0739] (1) Preparation of Dry Membrane

[0740] ​Transferrin (manufactured by Merck, quality about 76 kDa) was diluted with Fluorescent Compatible Sample Buffer (4X, non-reducing) (manufactured by Thermo Fisher Scientific) to make 1 ng / μL, 0.3 ng / μL, and 0.1 ng / μL, and heat treatment was carried out at 95 °C for 5 minutes. After loading the aforementioned transferrin sample and PageRuler Prestained NIR Protein Ladder (manufactured by Thermo Fisher Scientific) onto Novex 4-20% Tris-Glycine Mini Gels (manufactured by Thermo Fisher Scientific), electrophoresis was carried out at a constant voltage of 225 V for 45 minutes. After overlapping the electrophoresed gel and nitrocellulose membrane (manufactured by Cytiva), protein blotting was carried out under the conditions of a constant voltage of 12 V for 1 hour, and then the membrane was immersed in Western Blot Blocking Buffer (Fish Gelatin) (manufactured by Takara Bio Inc.) and left standing at 4 °C for 12 hours. After washing the membrane with TBS-T Buffer (manufactured by FUJIFILM Wako Pure Chemical Corporation), it was immersed in a liquid added with the first antibody for transferrin (manufactured by Abcam) (diluted 5000-fold), shaken for 1 hour, and after discarding the liquid added with the first antibody, the membrane was washed with TBS-T Buffer (manufactured by FUJIFILM Wako Pure Chemical Corporation). A 25000-fold dilution of the fluorescently labeled antibody prepared above or a 25000-fold dilution of the commercially available fluorescent secondary antibody described below was adjusted, and the membrane was immersed in the adjusted liquid, shaken for 1 hour in the dark, and the liquid of the fluorescently labeled antibody was discarded. Then, it was shaken in 20 mL of TBS-T (manufactured by FUJIFILM Wako Pure Chemical Corporation, Tween 20 content 0.1 (W / V)%) for 10 minutes, repeated 3 times, washed, and then shaken in 20 mL of TBS obtained by dissolving the water-soluble compounds described in Tables 1 to 5 at the addition amounts described in Tables 1 to 5 for 10 minutes once and washed. After washing, the membrane was dried in the dark in a 40 °C thermostat for 1 hour.

[0741] In addition, (W / V)% refers to the mass-to-volume percentage.

[0742] (2) Measurement of fluorescence intensity

[0743] (i) When using compound (1)-IgG (diluted 25,000-fold) as the fluorescent dye-labeled antibody

[0744] For the dry film prepared above, it was imaged using an Amersham Typhoon scanner (product name, manufactured by Cytiva). The measurement conditions were unified to an excitation light of 785 nm, and the region where the protein band was detected was measured at 3.24 mm 2 The integrated value of the fluorescence intensity in the range of 810 - 840 nm of the fluorescence wavelength of the fraction was taken as the "signal fluorescence intensity". Also, in the region where no protein band was detected below the measurement region of the above signal fluorescence intensity (the low-quality side with respect to the measurement region of the signal fluorescence intensity), it was measured at 3.24 mm 2 The integrated value of the fluorescence intensity in the range of 810 - 840 nm of the fluorescence wavelength of the fraction was taken as the "interference fluorescence intensity".

[0745] For the case of using compound (4)-IgG or Alexa Fluor Plus 800 (product name, manufactured by Thermo Fisher Scientific Inc., fluorescent secondary antibody) (both diluted 25,000-fold) as the fluorescent dye-labeled antibody, the measurement was also carried out in the same manner as the case of using compound (1)-IgG (diluted 25,000-fold).

[0746] (ii) When using compound (2)-IgG, compound (3)-IgG or Alexa Fluor Plus 680 (product name, manufactured by Thermo Fisher Scientific Inc., fluorescent secondary antibody) (all diluted 25,000-fold) as the fluorescent dye-labeled antibody

[0747] For the dry film prepared above, it was imaged using an Amersham Typhoon scanner (product name, manufactured by Cytiva). The measurement conditions were unified to an excitation light of 685 nm, and the region where the protein band was detected was measured at 3.24 mm 2 The integrated value of the fluorescence intensity in the range of 710 - 730 nm of the fluorescence wavelength of the fraction was taken as the "signal fluorescence intensity". Also, in the region where no protein band was detected below the measurement region of the above signal fluorescence intensity (the low-quality side with respect to the measurement region of the signal fluorescence intensity), it was measured at 3.24 mm 2 The integrated value of the fluorescence intensity in the range of 710 - 730 nm of the fluorescence wavelength of the fraction was taken as the "interference fluorescence intensity".

[0748] (3) Evaluation of fluorescence intensity

[0749] Among the fluorescent dye-labeled antibodies, the signal fluorescence intensities of No. c11, c21, c31, c41, c51 or c61, which were washed using TBS without water-soluble compounds instead of TBS dissolving water-soluble compounds, were set as reference values respectively, the ratios relative to the reference values (signal fluorescence intensity of fluorescent dye-labeled antibody / reference value) were calculated, and the evaluation was carried out according to the following evaluation criteria.

[0750] Moreover, among the fluorescent dye-labeled antibodies, the interference fluorescence intensities of No. c11, c21, c31, c41, c51 or c61, which were washed using TBS without water-soluble compounds instead of TBS dissolving water-soluble compounds, were set as reference values respectively, the ratios to the reference values (interference fluorescence intensity of fluorescent dye-labeled antibody / reference value) were calculated, and the evaluation was carried out according to the following evaluation criteria.

[0751] In addition, any evaluation was carried out by comparing fluorescent dye-labeled antibodies in a state with a dilution concentration of transferrin (manufactured by Merck) (1 ng / 10 μL, 0.3 ng / 10 μL or 0.1 ng / 10 μL). No difference was found in the above ratios relative to the reference value according to the dilution concentration of transferrin (manufactured by Merck).

[0752] The results were summarized and shown in Tables 1 to 6.

[0753] - Evaluation criteria for signal fluorescence intensity (integral value) -

[0754] A: The ratio of signal fluorescence intensity to the reference value is 2.0 times or more

[0755] B: The ratio of signal fluorescence intensity to the reference value is more than 1.9 times and less than 2.0 times

[0756] C: The ratio of signal fluorescence intensity to the reference value is more than 1.8 times and less than 1.9 times

[0757] D: The ratio of signal fluorescence intensity to the reference value is more than 1.7 times and less than 1.8 times

[0758] E: The ratio of signal fluorescence intensity to the reference value is more than 1.6 times and less than 1.7 times

[0759] F: The ratio of signal fluorescence intensity to the reference value is more than 1.5 times and less than 1.6 times

[0760] G: The ratio of signal fluorescence intensity to the reference value is more than 1.4 times and less than 1.5 times

[0761] H: The ratio of signal fluorescence intensity to the reference value is more than 1.3 times and less than 1.4 times

[0762] I: The ratio of the signal fluorescence intensity to the reference value is 1.2 times or more and less than 1.3 times.

[0763] J: The ratio of the signal fluorescence intensity to the reference value is 1.1 times or more and less than 1.2 times.

[0764] - Evaluation criteria for interfering fluorescence intensity (integral value) -

[0765] A: The ratio of the interfering fluorescence intensity to the reference value is less than 1.2 times.

[0766] B: The ratio of the interfering fluorescence intensity to the reference value is 1.2 times or more and less than 1.3 times.

[0767] C: The ratio of the interfering fluorescence intensity to the reference value is 1.3 times or more and less than 1.4 times.

[0768] D: The ratio of the interfering fluorescence intensity to the reference value is 1.4 times or more and less than 1.5 times.

[0769] E: The ratio of the interfering fluorescence intensity to the reference value is 1.5 times or more and less than 1.6 times.

[0770] F: The ratio of the interfering fluorescence intensity to the reference value is 1.6 times or more and 1.7 times or less.

[0771] [Table 1]

[0772]

[0773] [Table 2]

[0774]

[0775] [Table 3]

[0776]

[0777] [Table 4]

[0778]

[0779] [Table 5]

[0780]

[0781] [Table 6]

[0782]

[0783]

[0784] (Fluorescent dye-labeled antibody)

[0785] Compound (Z)-IgG: Compound (Z)-IgG as described above.

[0786] Alexa Fluor Plus 680: Product name, manufactured by Thermo Fisher Scientific, fluorescent secondary antibody

[0787] Alexa Fluor Plus 800: Product name, manufactured by Thermo Fisher Scientific, fluorescent secondary antibody

[0788] (Water-soluble compound)

[0789] Polyoxyethylene polyoxypropylene glycol (160 E.O.) (30 P.O.): Product name, manufactured by FUJIFILM Wako Pure Chemical Corporation

[0790] Resorcinol: Product name, manufactured by FUJIFILM Wako Pure Chemical Corporation, 1,3-dihydroxybenzene

[0791] Polyvinylpyrrolidone K30: Product name, manufactured by FUJIFILM Wako Pure Chemical Corporation

[0792] Betaine: Product name, manufactured by FUJIFILM Wako Pure Chemical Corporation, trimethylglycine

[0793] Dextran 40000: Product name, manufactured by FUJIFILM Wako Pure Chemical Corporation

[0794] Ficoll 400: Product name, manufactured by FUJIFILM Wako Pure Chemical Corporation, water-soluble polymer copolymerized from sucrose and epichlorohydrin

[0795] In addition, all the compounds listed in the water-soluble compound column are compounds that dissolve 1 g or more in 100 mL of water at 25°C.

[0796] Addition amount: Represents the proportion of the addition amount of the water-soluble compound based on the mass of the TBS solution before the addition of the water-soluble compound (expressed as 100%), unit is mass% (wt%).

[0797] Content: Refers to the proportion of the content of the water-soluble compound in the TBS solution containing the water-soluble compound based on the mass (expressed as 100%), unit is mass% (wt%).

[0798] Melting point: Determined as follows by simultaneous thermogravimetry-differential thermal analysis (TG-DTA).

[0799] In addition, as the simultaneous thermogravimetry-differential thermal analysis apparatus, a simultaneous differential thermal and thermogravimetric analyzer (Model: STA7200) manufactured by Hitachi High-Tech Science Corporation was used.

[0800] First, the measurement sample was held at a predicted 30 °C until the apparatus was stabilized, and then heated to 200 °C at a heating rate of 20 °C / minute to prepare a TG curve and a DTA curve.

[0801] The temperature at the intersection of the straight line obtained by extending the baseline on the low-temperature side of the DTA curve to the high-temperature side and the tangent line drawn at the point where the curve gradient at the stepwise change part at the melting point is maximum was defined as the melting point.

[0802] In addition, for water-soluble compounds other than polysucrose 400 among water-soluble compounds with a melting point exceeding 200 °C, the setting of the heating upper limit temperature was changed from 200 °C to a temperature at which the melting point could be measured, and the measured values were recorded.

[0803] From the results of Tables 1 to 6 above, the following can be known.

[0804] In Comparative Example No. c12 where TBS containing the water-soluble compound 1,6-hexanediol with a melting point of 41 °C was used for membrane cleaning, the signal fluorescence intensity and the interference fluorescence intensity both showed the same values as those in Comparative Example No. c11 (reference) where TBS not containing a water-soluble compound was used for membrane cleaning, and no enhancement effect of fluorescence intensity was obtained.

[0805] In contrast, in Examples No. 101 to 116 using a water-soluble compound having a melting point of 50°C or higher, the signal fluorescence intensity was at least increased to 1.2 times or more compared to No. c11 (reference) using TBS without a water-soluble compound for membrane washing. The increase in the interfering fluorescence intensity was also suppressed to 1.7 times or less. The increase in the signal fluorescence intensity was greater than the increase in the interfering fluorescence intensity, showing an excellent enhancement effect on the fluorescence intensity. Similarly, in the case of using different fluorescent dye-labeled antibodies or commercially available fluorescent secondary antibodies, in the examples using a water-soluble compound having a melting point of 50°C or higher, an excellent enhancement effect on the fluorescence intensity was also shown compared to the reference using TBS without a water-soluble compound for membrane washing (No. 201 to 207 relative to No. c21 (reference), No. 301 relative to No. c31 (reference), No. 401 relative to No. c41 (reference), No. 501 relative to No. c51 (reference), No. 601 relative to No. c61 (reference)).

[0806] Among them, it is known that when the water-soluble compound does not contain a salt structure, the signal fluorescence intensity can be further increased (refer to No. 101 to 113 and 116 compared to No. 114 and 115). When the melting point of the water-soluble compound is 80°C or higher, the signal fluorescence intensity can be further increased (refer to No. 103 to 113 and 116 compared to No. 101 and 102). When the water-soluble compound is a polyol compound or a betaine compound, the interfering fluorescence intensity can be suppressed (refer to No. 103 to 106, 108, 109, 111 to 113 and 116 compared to No. 107 and 110).

[0807] Furthermore, it is known that when the content of the water-soluble compound is 4.5% by mass or more, the signal fluorescence intensity can be further increased (refer to No. 202 to 207 compared to No. 201). When the content of the water-soluble compound is 20% by mass or more, the signal fluorescence intensity can be further increased (refer to No. 205 to 207 compared to No. 201 to 204).

[0808] The present invention has been described together with its embodiments. Unless otherwise specified, it is not intended to limit the present invention in any details of the description. It is considered that a broad interpretation should be made without departing from the spirit and scope of the invention shown in the appended claims.

[0809] This application claims the priority of Japanese Patent Application No. 2022-209083 filed in Japan on December 26, 2022. It is hereby incorporated by reference and its content is incorporated as a part of what is described in this specification.

Claims

1. A fluorescence intensity enhancer, which comprises a water-soluble compound having a melting point of 50 °C or higher.

2. The fluorescence intensity enhancer according to claim 1, wherein the content of the water-soluble compound is 4.5% by mass or more.

3. The fluorescence intensity enhancer according to claim 1, wherein the water-soluble compound does not contain a salt structure.

4. The fluorescence intensity enhancer according to claim 1, wherein the water-soluble compound is a polyol compound or a betaine compound.

5. The fluorescence intensity enhancer according to claim 2, wherein the content of the water-soluble compound is 20% by mass or more.

6. The fluorescence intensity enhancer according to claim 1, wherein the melting point is 80 °C or higher.

7. The fluorescence intensity enhancer according to claim 1, which is in a solution state.

8. The fluorescence intensity enhancer according to claim 1, which is used for fluorescence imaging.

9. A method for enhancing the fluorescence intensity of a fluorescently labeled target biological substance, comprising: A step of allowing the fluorescence intensity enhancer according to any one of claims 1 to 8 to act on a fluorescently labeled target biomolecule.

10. A method for enhancing the fluorescence intensity of a fluorescently labeled target biological substance, comprising: A step of allowing the fluorescence intensity enhancer according to any one of claims 1 to 8 to act on a fluorescently labeled target biomolecule to enhance the fluorescence intensity emitted from the fluorescent label.

11. A kit for fluorescence detection, which comprises the following (a) and (b), (a) A fluorescence intensity enhancer containing a water-soluble compound having a melting point of 50 °C or higher, (b) A reagent for producing a fluorescent dye-labeled biomolecule or a fluorescent dye-labeled biomolecule containing a fluorescent dye.

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