Fluorescent compound and fluorescently labeled biological substance using the same

Abstract

To provide: a fluorescent compound that is excellent in suppressing the decrease in fluorescence intensity (fluorescence quantum yield) accompanying an increase in the fluorescent labeling rate upon labeling a biological substance or the like; and fluorescent-labeled biological substance based thereon.SOLUTION: The invention relates to a fluorescent compound represented by the general formula (1), and a fluorescent-labeled biological material using the compound. [In the general formula (1), FL is an n-valent fluorophore moiety; n is an integer equal to or greater than 1; and Q is a group represented by the general formula (B), where R1 to R3 are each H, an alkyl group, or the like, and L is a single bond or a linking group such as a group represented by the formulas at the bottom of the figure, and the symbol * indicates a bond.]SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a fluorescent compound and a fluorescently labeled biomolecule using the same.

Background Art

[0002] In order to observe changes in the living body in response to various stimuli (such as diseases and environmental changes), fluorescently labeled biomolecules in which a biomolecule (such as an antibody) having binding properties to a target substance to be detected is labeled with a fluorescent compound (fluorescent dye) are frequently used. For example, in Western blotting (hereinafter also abbreviated as WB) for detecting a specific protein from a protein mixture, a fluorescence method is used to detect the presence or absence or abundance of the specific protein using a fluorescently labeled antibody having binding properties to the protein. In addition, in bioimaging technology for analyzing the dynamics and functions of biomolecules, cells, tissues, etc. in a living body, bioluminescence imaging for observing a specific site of the living body visualized by fluorescent labeling is used as one of the techniques for observing the living body.

[0003] In the above fluorescent labeling, generally, organic fluorescent dye molecules are used, and usually, the fluorescence intensity is increased by using a fluorescently labeled biomolecule to which a plurality of fluorescent dye molecules are bound. As fluorescent dyes having a high fluorescence quantum yield and high light resistance (photobleaching resistance), fluorescein dyes, rhodamine dyes, etc. are known. In addition, research on fluorescent dyes in which the oxygen atom, which is a ring-constituting atom of the xanthene skeleton possessed by rhodamine or rhodol, is replaced with a silicon atom or the like to shift the absorption wavelength and fluorescence wavelength to longer wavelengths is also in progress. In addition to the above fluorescently labeled biomolecules, research on fluorescent probes for detecting specific bioactive molecules has also been conducted. Fluorescent probes are often compounds that do not exhibit fluorescence by themselves, and change into compounds that emit fluorescence as the molecular structure changes due to the reaction with the target bioactive molecule. For example, as a fluorescent probe that exhibits fluorescence upon cleavage of β-galactose by β-galactosidase, Patent Document 1 and Non-Patent Document 1 describe a compound in which β-galactose is bonded to the hydroxy group of Si-rhodol, and Non-Patent Document 2 describes a compound in which β-galactose is bonded to the hydroxy group of fluorescein. Further, Non-Patent Document 3 describes a compound in which β-galactose is introduced as a substituent on the amino group of Si-rhodamine.

[0004] However, most of the organic dyes that exhibit fluorescence, such as rhodamine dyes and rhodol dyes, have an aromatic chromophore with high planarity, so interactions between the dyes are likely to occur. As a result, a decrease in fluorescence intensity due to interactions such as self-association between the dyes after labeling is likely to occur, and as the number of fluorescent dye molecules per biomolecule (fluorescent labeling ratio: DOL) increases, the fluorescence intensity tends to decrease more due to self-association and the like.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] It becomes important to search for a chemical structure capable of suppressing a decrease in fluorescence intensity (fluorescence quantum yield) accompanying an increase in fluorescence labeling rate across all organic fluorescent dyes. An object of the present invention is to provide a fluorescent compound excellent in suppressing a decrease in fluorescence intensity (fluorescence quantum yield) accompanying an increase in fluorescence labeling rate when labeling a biological substance or the like. Another object of the present invention is to provide a fluorescently labeled biological substance formed by binding this fluorescent compound and a biological substance.

Means for Solving the Problems

[0008] That is, the above problems of the present invention have been solved by the following means. [1] A fluorescent compound represented by the following general formula (1). [Chemical formula] In the above formula, FL represents an n-valent phosphor moiety, and n is an integer of 1 or more. Q represents a group represented by the following general formula (B). [Chemical formula] In the above formula, R 1 ~R 3 represents a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group. L represents a single bond, or a linking group formed by combining one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and each group represented by the following formulas (1-1) to (1-8). * represents a bonding portion. However, L and R 1 ~R 3 do not combine with any one of them to form a glycosyl structure. [Chemical formula] In the above formula, R 31 and R32 represents a hydrogen atom or a substituent. * represents a bonding site. 〔2〕 The fluorescent compound according to 〔1〕, wherein the FL is a structural part composed of a dipyrromethene compound, a rhodamine compound, a xanthene compound, a rhodol compound, a pyrrolopyrrole compound, a pyrene compound, a coumarin compound or a cyanine compound. 〔3〕 The fluorescent compound according to 〔2〕, wherein the FL is a structural part composed of a rhodamine compound, a xanthene compound or a rhodol compound. 〔4〕 The fluorescent compound according to 〔3〕, which is represented by the following general formula (2).

Chemical formula

Advantages of the Invention

[0009] When the fluorescent compound of the present invention labels a biological substance or the like, it can exhibit an excellent inhibitory effect on the decrease in fluorescence intensity (fluorescence quantum yield) accompanying the increase in the fluorescence labeling rate. Further, the fluorescently labeled biological substance of the present invention is excellent in the inhibitory effect on the decrease in fluorescence intensity (fluorescence quantum yield) accompanying the increase in the fluorescence labeling rate.

Embodiments for Carrying Out the Invention

[0010] In the present invention, when there are a plurality of substituents or linking groups (hereinafter referred to as substituents, etc.) represented by specific symbols or formulas, or when a plurality of substituents, etc. are defined simultaneously, unless otherwise specified, each of the substituents, etc. may be the same as or different from each other. This also applies to the definition of the number of substituents, etc. Further, when a plurality of substituents, etc. are close to each other (especially when adjacent), unless otherwise specified, they may be linked to each other to form a ring. Further, when forming a ring, an unsaturated ring or an aromatic ring may be formed by removing a hydrogen atom. Further, unless otherwise specified, rings such as alicyclic rings, aromatic rings and heterocyclic rings may be further condensed to form a fused ring. In the present invention, unless otherwise specified, for a double bond, when both E-type and Z-type exist in the molecule, either one or a mixture thereof may be used. Further, unless otherwise specified, when an asymmetric carbon atom or an asymmetric center exists in the compound and diastereomers and enantiomers exist as the compound, either one or a mixture thereof may be used.

[0011] In the present invention, regarding the representation of compounds and substituents, in addition to the compound itself and the substituent itself, it is used in the meaning including its salt and its ion. For example, a carboxy group, a sulfo group, a phosphono group (-P(=O)(OH)2), etc. may have a hydrogen atom dissociated to take an ionic structure or may take a salt structure. That is, in the present invention, the "carboxy group" is used in the meaning including a carboxylate ion or its salt, the "sulfo group" is used in the meaning including a sulfonate ion or its salt, and the "phosphono group" is used in the meaning including a phosphonate ion or its salt. The monovalent or polyvalent cation when constituting the above salt structure is not particularly limited, and examples include an inorganic cation, an organic cation, etc. Specifically, Na + 、Li + and K + and other cations of alkali metals such as 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. In the case of a salt structure, the type of the salt may be one kind, two or more kinds may be mixed, a salt type and a group having a free acid structure may be mixed in the compound, and a compound having a salt structure and a compound having a free acid structure may be mixed. The compounds of the present invention are all electrically neutral compounds. For example, in the compound represented by the general formula (2), Y 2 is =N + R 17 R 18When this is the case, for the sake of convenience, it is specified and shown as a structure in which this nitrogen atom has a positive charge. However, since the FL (phosphor part) in the compound of the present invention has a conjugated system, actually, other atoms than the above nitrogen atom may take a positive charge, and as long as it is a compound that can take a structure represented by the general formula (2) as one of the chemical structures, it is included in the compound represented by the general formula (2). The same applies to the negative charge. The same also applies to the compounds represented by each of the other general formulas. Also, it means including those in which a part of the structure is changed within a range that does not impair the effects of the present invention. Further, for compounds in which substitution or non-substitution is not specified, it means that they may have any substituent within a range that does not impair the effects of the present invention. This also applies to substituents (groups expressed as, for example, "alkyl group", "methyl group", "methyl", etc.) and linking groups (groups expressed as, for example, "alkylene group", "methylene group", "methylene", etc.). Among such arbitrary substituents, the preferred substituents in the present invention are substituents selected from the substituent group T described later. In the present invention, the rhodamine compound means a rhodamine compound in which the 3-position and 6-position of the xanthene ring are substituted with an amino group and an imino group, and the rhodol compound means a rhodol compound in which the 3-position or 6-position of the xanthene ring is substituted with an amino group or an imino group. Also, in the present invention, the xanthene compound means a compound having a xanthene ring other than the above rhodamine compound and rhodol compound. For example, a fluorescein compound in which the 3-position and 6-position of the xanthene ring are substituted with a hydroxy group (the hydrogen atom in the hydroxy group may be replaced by an alkyl group or the like) and an oxo group can be mentioned. In the present invention, the rhodamine compound, rhodol compound, and xanthene compound are used in the meaning of including the rhodamine compound, rhodol compound, and xanthene compound in which the oxygen atom, which is a ring-constituting atom of the xanthene skeleton, is replaced with a silicon atom, a phosphorus atom, or a sulfur atom, respectively. 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 means the number of carbon atoms of the entire group. That is, when this group is in a form having further substituents, it means the total number of carbon atoms including this substituent.

[0012] Also, in the present invention, the numerical range represented by "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value.

[0013] The fluorescent compound of the present invention is represented by the following general formula (1). Although the reason why the fluorescent compound of the present invention is excellent in suppressing the decrease in fluorescence intensity (fluorescence quantum yield) accompanying the increase in the fluorescence labeling rate when labeling a biological substance or the like is not clear, it is presumed as follows. It is considered that the interaction between the phosphor moieties FL increases due to the increase in the concentration of the phosphor moiety FL accompanying the increase in the fluorescence labeling rate. As a result, it is presumed that self-association or aggregation of the fluorescent compound occurs, and the fluorescence quantum yield decreases. The group represented by the general formula (B) bonded to the phosphor moiety FL disclosed by the present invention has one hydroxy group on each of two adjacent carbon atoms. This is considered to be one of the reasons for suppressing at least the interaction between the phosphor moieties FL intermolecularly and suppressing the self-association or aggregation of the fluorescent compound of the present invention.

[0014] Hereinafter, the fluorescent compound represented by the general formula (1) of the present invention will be described in detail.

[0015] <Fluorescent compound represented by the general formula (1)> The fluorescent compound represented by the general formula (1) of the present invention (hereinafter, also referred to as "the compound of the present invention") is as follows.

[0016] [Chemical formula]

[0017] In the above formula, FL represents an n-valent phosphor moiety, and n is an integer of 1 or more. Q represents a group represented by the following general formula (B).

[0018]

Chemical formula

[0019] In the above formula, R 1 ~R 3 represents a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group. L represents a single bond, or a linking group formed by combining one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and each group represented by the following formulas (1-1) to (1-8). That is, L may be a single bond; also, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, the group represented by the following formula (1-1), the group represented by formula (1-2), the group represented by formula (1-3), the group represented by formula (1-4), the group represented by formula (1-5), the group represented by formula (1-6), the group represented by formula (1-7), or the group represented by formula (1-8) may be used; also, a group formed by combining two or more groups selected from an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, the group represented by the following formula (1-1), the group represented by formula (1-2), the group represented by formula (1-3), the group represented by formula (1-4), the group represented by formula (1-5), the group represented by formula (1-6), the group represented by formula (1-7), and the group represented by formula (1-8) may be used. * represents a bonding site.

[0020] However, L and R 1 ~R 3None of them combines with any one of the following to form a glycosyl structure. Here, the glycosyl structure means a structure obtained by removing the anomeric hydroxy group from a cyclic sugar, and generally includes glycosyl structures formed by O-glycoside bonds, N-glycoside bonds, or C-glycoside bonds. Taking specific examples, the structures shown below correspond to glycosyl structures in which the hydrogen atom at the anomeric hydroxy group of glucose is removed to form an O-glycoside bond. * indicates the bonding site.

[0021] [Chemical formula]

[0022] In addition, for the fluorescent compound, both of the above-mentioned FL and L do not have a structure represented by >C(OH)C(OH)<, and among the above-mentioned L, the site bonded to the carbon atom to which R 1 is bonded should not have a structure represented by >C(OH)-, and it is applied to the above general formula (1).

[0023] Hereinafter, the substituents and the like in the general formula (1) will be described in detail.

[0024] [Q: Group represented by general formula (B)] In the above general formula (B), R 1 ~R 3 represent a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group. R 1 ~R 3 The alkyl groups, aryl groups, and heteroaryl groups that can be taken as R R 1 ~R 3The substituents that the above-mentioned alkyl group, aryl group, and heteroaryl group may have are not particularly limited, and examples thereof include the substituents in the substituent group T described later. Among these, a hydroxy group, an alkoxy group, a cycloalkyloxy group, an aryloxy group, or a heterocyclic oxy group is preferable, and a hydroxy group, an alkoxy group, a cycloalkyloxy group, or an aliphatic heterocyclic oxy group is more preferable. The substituents that the above-mentioned alkoxy group, aryloxy group, cycloalkyloxy group, and heterocyclic oxy group may have include the substituents in the substituent group T described later, and a hydroxy group and an alkyl group are preferably mentioned. For example, as a group in which a heterocyclic oxy group (preferably an aliphatic heterocyclic oxy group) has a hydroxy group or a hydroxyalkyl group as a substituent, a glycosyloxy group (a heterocyclic oxy group obtained by removing a hydrogen atom from the anomeric hydroxy group in an aldose or a ketose) can be mentioned.

[0025] R 1 and R 2 is preferably a hydrogen atom. R 3 is preferably a hydrogen atom or an alkyl group, and more preferably a hydrogen atom or an alkyl group containing a hydroxy group as a substituent.

[0026] In the above general formula (B), L represents a single bond, or a linking group formed by combining one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and each group represented by any of the following formulas (1-1) to (1-8). The alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that can be adopted as L may be unsubstituted groups or groups having substituents, and groups obtained by removing one hydrogen atom from the alkyl group, alkenyl group, alkynyl group, aryl group, and heteroaryl group in the substituent group T described later can be applied respectively. The substituents that the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that can be taken as L may have are not particularly limited, and are preferably selected from the substituent group T described below.

[0027] L is a group that connects to the phosphor part FL and is not particularly limited as long as the effects of the present invention are achieved. Specifically, a linking group formed by combining two or more of an alkylene group, or an alkylene group, an arylene group, a heteroarylene group, and each group represented by any of the following formulas (1-3) to (1-4) is preferred, and a linking group formed by combining two or more of an alkylene group, or an alkylene group and each group represented by any of the following formulas (1-3) to (1-4) is more preferred, and an alkylene group is even more preferred.

[0028] The group represented by the above general formula (B) is preferably a group represented by the following general formula (B1) or (B2).

[0029]

Chemical formula

[0030] In the above formula, R 3a ~R 3f represents a hydrogen atom, an alkyl group, an aliphatic heterocyclic group, an aryl group, or a heteroaryl group. L, R 1 and R 2 have the same meanings as the above L, R 1 and R 2 respectively. * indicates the bonding part. However, L and any one of R 1 , R 2 and R 3a ~R 3f do not combine to form a glycosyl structure.

[0031] R 3a ~R 3frepresents a hydrogen atom, an alkyl group, an aliphatic heterocyclic group, an aryl group or a heteroaryl group. R 3a ~R 3f The alkyl group, aliphatic heterocyclic group, aryl group and heteroaryl group that can be taken as R 3a to R 3f may be unsubstituted groups or groups having substituents, and the alkyl group, aliphatic heterocyclic group, aryl group and heteroaryl group in the substituent group T described later can be respectively applied. R 3a ~R 3f The substituents that the above alkyl group, aliphatic heterocyclic group, aryl group and heteroaryl group that can be taken as R 3a to R 3f may have are not particularly limited, and examples include the substituents in the substituent group T described later. Among these, a hydroxy group or an alkyl group is preferably mentioned. For example, as a group in which an aliphatic heterocyclic group has a hydroxy group or a hydroxyalkyl group as a substituent, a glycosyl group (an aliphatic heterocyclic group obtained by removing an anomeric hydroxy group in an aldose or ketose) can be mentioned. R 3a ~R 3c , R 3e and R 3f are preferably hydrogen atoms. R 3d is preferably a hydrogen atom or an aliphatic heterocyclic group, more preferably a hydrogen atom or an aliphatic heterocyclic group having a group containing a hydroxy group as a substituent, and even more preferably a hydrogen atom or a glycosyl group.

[0032] [[FL, n]] FL represents an n-valent phosphor part, and n is an integer of 1 or more. The phosphor part that can be taken as FL can be used without particular limitation as long as it is a structural part composed of an organic compound that exhibits fluorescence. As FL, for example, a structural part composed of a dipyrromethene compound, a rhodamine compound, a xanthene compound, a rhodol compound, a pyrrolopyrrole compound, a pyrene compound, a coumarin compound or a cyanine compound can be mentioned, and is preferable. As the above dipyrromethene compound, rhodamine compound, xanthene compound, rhodol compound, pyrrolopyrrole compound, pyrene compound, coumarin compound or cyanine compound, compounds (fluorescent dyes) commonly known as these dyes can be used without particular limitation. n is an integer of 1 or more, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, and even more preferably an integer of 2 to 4.

[0033] From the viewpoint of versatility in bioimaging, the compound of the present invention preferably has at least one carboxy group or a substituent capable of binding to a biological substance described later, and more preferably has at least one carboxy group or a substituent capable of binding to a biological substance described later in FL. The compound of the present invention can bind to a biological substance through the above carboxy group or a substituent capable of binding to a biological substance to obtain a target labeled biological substance. The carboxy group can be easily induced into a substituent capable of binding to a biological substance by a conventional method. In the present invention, the "substituent capable of binding to a biological substance" includes a substituent capable of binding to a biological substance derived from a carboxy group. The total number of the carboxy group or the substituent capable of binding to a biological substance possessed by the compound of the present invention is preferably at least 1 or more, more preferably 1 to 3, even more preferably 1 or 2, and particularly preferably 1 from the viewpoint of quantification of the substance to be detected.

[0034] The compound of the present invention is preferably a compound in which the above FL is a structural part composed of a rhodamine compound, a xanthene compound or a rhodol compound, and more preferably a compound represented by the following general formula (2).

[0035]

Chemical formula

[0036] In the above formula, X is -O-, -S-, >SO, >SO2, >SiR 10 R 11 or >P(=O)R12 is shown. Y 1 is -OR 13 or -NR 14 R 15 is shown, and Y 2 is =O, =NR 16 or =N + R 17 R 18 is shown. Z represents a group represented by the following formula (A). R 4 ~R 9 represents a halogen atom, a cyano group, or a group represented by the following formula (A). R 10 and R 11 represent a group represented by the following formula (A), and R 10 and R 11 may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring. However, when R 10 and R 11 are not bonded to each other to form a ring, at least one of R 10 and R 11 is an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group. R 12 ~R 18 represent a group represented by the following formula (A).

[0037] However, at least one of R 13 ~R 18 in the above compound is a group represented by the above general formula (B). That is, it means that at least one of the groups Y 1 and Y 2 in the compound has at least one group represented by the above general formula (B) as a substituent.

[0038] 1) R 4 ~R 9 The above R 4 ~R 9 represent a halogen atom, a cyano group, or a group represented by the following formula (A), a halogen atom or a group represented by the following formula (A) is preferred, a hydrogen atom, a halogen atom, or a sulfo group is more preferred, and a hydrogen atom is even more preferred. R 4 ~R 9 Examples of halogen atoms that can be taken include fluorine atom, chlorine atom, bromine atom, and iodine atom, and a fluorine atom is preferred. R 4 ~R 9 Examples of alkyl groups that can be taken include those described for R 111 that can be taken below. R 4 ~R 9 The alkyl group that can be taken may be unsubstituted or may have a substituent, and preferred examples of the substituent that may be present include a sulfo group. R 4 ~R 9 Specific examples of include a hydrogen atom, a methyl group, an ethyl group, a fluorine atom, or a sulfo group.

[0039] 2) Z The above Z represents a group represented by the following formula (A), and a group in the preferred form of (1) or (2) below is preferred, and an alkyl group, an alkoxy group, an amino group, an aryl group, or a heteroaryl group is preferred, an alkyl group, an aryl group, or a heteroaryl group is more preferred, and an aryl group is even more preferred. Examples of the alkyl group, alkoxy group, amino group, aryl group, and heteroaryl group that Z can take include those described for the alkyl group, alkoxy group, amino group, aryl group, and heteroaryl group as the group represented by the following formula (A).

[0040] Z preferably has a carboxy group or a substituent capable of binding to a biological substance described below. In the description regarding each group that can be taken as Z below, a form having a substituent capable of binding to a biological substance described below instead of the carboxy group is also preferred. The alkyl group, alkoxy group, and amino group that Z can take preferably have a carboxy group, -(CH2) u COOH, -(CH2) u -phenylene-COOH, -cycloalkylene-COOH, -O(CH2) uCOOH or -NH(CH2) u COOH is more preferred. Here, u is an integer from 1 to 15, preferably an integer from 1 to 10, and more preferably an integer from 1 to 5. The heteroaryl group that Z can take preferably has a carboxy group. As the heteroaryl group, a monocyclic group is preferred, a thiophene ring group, an imidazole ring group, a pyrazole ring group or a pyridine ring group is more preferred, and an imidazole ring group, a pyrazole ring group or a pyridine ring group is even more preferred.

[0041] The aryl group that Z can take may be a monocyclic group or a condensed ring group, but a monocyclic group is preferred, and a group represented by the following formula (C) is more preferred.

[0042]

Chemical formula

[0043] In the formula, R 41 ~R 45 represents a hydrogen atom or a substituent. * represents a bonding site. However, at least one of the following (C-1) to (C-3) is satisfied. (C-1) At least one of R 42 ~R 44 has a group having either a carboxy group or a substituent capable of binding to a biological substance. (C-2) R 41 and R 45 are an alkyl group or a halogen atom. (C-3) At least one of R 41 and R 45 is a fluorine atom or an alkoxy group.

[0044] When the above (C-1) is satisfied, non-radiative deactivation is suppressed. When the above (C-2) is satisfied, thermal deactivation due to the free rotation of the aryl group is suppressed by steric hindrance. When the above (C-3) is satisfied, thermal deactivation due to the free rotation of the aryl group is suppressed by intramolecular hydrogen bonding. By satisfying at least any one of these (C-1) to (C-3), the fluorescence quantum yield is further improved.

[0045] R 41 ~R 45 Examples of the substituent that R and ~R can take include a halogen atom, a cyano group, an alkyl group, an alkoxy group, a carboxy group, an alkoxycarbonyl group, a carbamoyl group, an amide group, a sulfo group, a sulfonamide group, an amino group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aliphatic heterocyclic group, or a substituent capable of binding to a biological substance described later. A halogen atom, an alkyl group, an alkoxy group, a carboxy group, an alkoxycarbonyl group, a carbamoyl group, an alkynyl group, or a substituent capable of binding to a biological substance described later is preferable. R 41 ~R 45 As the halogen atom, alkyl group, alkoxy group, alkoxycarbonyl group, carbamoyl group, amide group, sulfonamide group, amino group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and aliphatic heterocyclic group that R and ~R can take, the corresponding groups in the substituent group T described later can be applied respectively. R 41 ~R 45 The alkyl group, alkoxy group, alkoxycarbonyl group, carbamoyl group, amide group, sulfonamide group, amino group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and aliphatic heterocyclic group that R and ~R can take may have a substituent. For example, the groups in the substituent group T described later can be mentioned, and a poly(alkyleneoxy) group, a sulfo group, a carboxy group, or a substituent capable of binding to a biological substance is preferably mentioned. R 41 and R 45 From the viewpoint of suppressing the rotation of the benzene ring in the formula (C), the substituent in R and R is preferably a halogen atom, an alkyl group, or an alkoxy group. R42 ~R 44 The substituents that can be adopted as ~R are not particularly limited, and are preferably selected from the substituent group T described below, and preferably have a carboxy group or a substituent capable of binding to a biological substance. The carboxy group or the substituent capable of binding to a biological substance may be directly bonded to the benzene ring in the general formula (C), or may be bonded via a linking group, but is preferably directly bonded. Examples of the linking group preferably include an alkylene group, a poly(alkyleneoxy) group, an alkoxycarbonyl group, an amide group, or a group formed by combining these. Examples of the alkylene group, the poly(alkyleneoxy) group, the alkoxycarbonyl group, the amide group, or the group formed by combining these include -C(=O)O(CH2CH2O) m CH2CH2-, -CONH(CH2CH2O) m CH2CH2-, -CO-piperidinediyl-. m means the average number of repetitions, is 1 to 30, preferably 1 to 15, and more preferably 1 to 10. Among them, it is preferable that R 43 has a carboxy group or a substituent capable of binding to a biological substance from the viewpoint of the binding property to the biological substance. R 42 ~R 44 is preferably a hydrogen atom or a group having a carboxy group or a substituent capable of binding to a biological substance, and more preferably a hydrogen atom, a carboxy group or a substituent capable of binding to a biological substance.

[0046] 3) Y 1 and Y 2 Y 1 represents -OR 13 or -NR 14 R 15 and Y 2 represents =O, =NR 16 or =N + R 17 R 18 and R 13 ~R 18 represents a group represented by the following formula (A). R 13 ~R 18As the basis, the preferred forms described in (1) or (2) below can be preferably applied. However, R in the above compound 13 ~R 18 At least one of them is a group represented by the above general formula (B).

[0047] R 14 and R 15 may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring, and R 17 and R 18 may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring. Further, R 13 ~R 18 may each be bonded to adjacent R 5 ~R 8 to form a 5- to 7-membered aliphatic heterocyclic ring or a 5- to 7-membered aromatic heterocyclic ring. Regarding the above ring that R 14 and R 15 may form by bonding to each other, the ring that R 17 and R 18 may form by bonding to each other, or the ring that R 13 ~R 18 may form by bonding to adjacent R 5 ~R 8 each has, as ring-constituting atoms, in addition to the nitrogen atom to which R 13 ~R 18 is bonded, 1 to 3 hetero atoms selected from an oxygen atom, a nitrogen atom, and a sulfur atom.

[0048] R 17 and R 18 are preferably a hydrogen atom, an alkyl group, or an aryl group, and more preferably an alkyl group. As the alkyl group or aryl group that R 17 and R 18 can take, the description of the alkyl group or aryl group as the group represented by the following formula (A) can be applied. R 17 and R 18Examples of the substituent that an alkyl group or aryl group may have include electron-withdrawing groups such as a halogen atom and a carbonyl group, which will be described later. Among them, R 17 and R 18 are preferably an alkyl group, and more preferably an alkyl group substituted with a fluorine atom or a carbonyl group. R 17 and R 18 When not bonded to any group, R 17 and R 18 may be the same or different, but are preferably the same.

[0049] R 17 and R 18 The 4- to 7-membered aliphatic heterocyclic ring that can be formed by bonding with each other is not particularly limited, but is preferably a saturated aliphatic heterocyclic ring, and the number of ring members is preferably 4 to 6. This aliphatic heterocyclic ring may be a monocyclic ring or may have a condensed ring structure such as a structure in which at least one atom in the norbornane structure is replaced by a nitrogen atom. Further, it is also preferable to have >SO2 as a ring-constituting atom, and it is also preferable to have an electron-withdrawing group such as a halogen atom (preferably a fluorine atom) or a carbonyl group, which will be described later, as a substituent.

[0050] R 17 or R 18 When R 7 or R 8 bonds to R 7 or R 8 to form a ring, any of them may be aliphatic or aromatic as long as it is a heterocyclic ring having 5 to 7 ring members. Among these rings, the number of ring members is preferably 5 or 6, and it is preferable that there is no heteroatom other than the nitrogen atom substituted by R 7 or R 8 as a ring-constituting atom. Further, it may be a monocyclic ring or a condensed ring, but the ring formed by the bond is preferably a monocyclic ring.

[0051] R 14 and R 15 As for the above R 17 and R 18The description in [reference] can be applied. However, for R 17 and R 18 the nitrogen atom to which they are attached has a positive charge, while for R 14 and R 15 the nitrogen atom to which they are attached has no positive charge, which is different. Also, the ring that R 17 or R 18 can form by bonding with R 7 or R 8 is read as the ring that R 14 or R 15 can form by bonding with R 5 or R 6 .

[0052] As R 16 an alkyl group is preferred. As the alkyl group that R 16 can take, the description of the alkyl group that the above R 17 and R 18 can take can be applied. The ring that R 17 or R 18 can form by bonding with R 7 or R 8 is read as the ring that R 16 adjacent to R 7 or R 8 can form by bonding.

[0053] As R 13 a hydroxy group, an acyl group or a sulfonyl group is preferred. As the acyl group or sulfonyl group that R 13 can take, the description of the acyl group or sulfonyl group as the group represented by the following formula (A) can be applied. The ring that R 17 or R 18 can form by bonding with R 7 or R 8 is read as the ring that R 13 adjacent to R 5 or R 6 can form by bonding.

[0054] 4) X X is -O-, -S-, >SO, >SO2, >SiR10 R 11 or >P(=O)R 12 is shown. The above R 10 and R 11 represent a group represented by the following formula (A), and the groups in the following preferred forms (1) or (2) can be preferably applied. R 10 and R 11 may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring. However, when R 10 and R 11 are not bonded to each other to form the above 4- to 7-membered aliphatic heterocyclic ring, at least one of R 10 and R 11 is an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group. The descriptions regarding these R 10 and R 11 are the same as the descriptions regarding the subsequent R 10 and R 11 in the following. R 10 and R 11 When at least one of them has an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group, these substituents are sterically bulky and can improve the stability of the silicon atom by suppressing the approach of photodegradation factor substances such as active oxygen to the silicon atom, and it is considered that excellent light resistance can be exhibited. R 10 and R 11 are preferably an alkyl group, an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group. R 10 and R 11 As the alkyl group, alkenyl group, alkynyl group, aryl group or heteroaryl group that can be adopted, the descriptions of the alkyl group, alkenyl group, alkynyl group, aryl group or heteroaryl group as the group represented by the following formula (A) can be applied. R 10 and R 11Examples of the substituent that an alkyl group, alkenyl group, alkynyl group, aryl group or heteroaryl group may have include a carboxy group, an alkoxy group or an aryl group. Among them, an alkenyl group substituted with a carboxy group or an alkoxy group, and an alkenyl group or an alkynyl group substituted with an aryl group are preferable. R 10 and R 11 Examples of the combination of 10 and 11 include a combination of an alkyl group and an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group, a combination of an alkenyl group and an alkenyl group, a combination of an alkynyl group and an alkynyl group, and a combination of a heteroaryl group and a heteroaryl group. A combination of an alkyl group and an alkenyl group or an aryl group is more preferable, and a combination of an alkyl group and an alkenyl group is even more preferable.

[0055] The above R 12 represents a group represented by the following formula (A), and is preferably a hydroxy group, an alkoxy group, an aryl group or an alkyl group. R 12 When R is a hydroxy group, the hydrogen atom in this hydroxy group may be dissociated to have an ionic structure or a salt structure. This is the same for the hydroxy group that can be adopted as R hereinafter. 12 The same applies to the hydroxy group that can be adopted as R hereinafter.

[0056] The above X is preferably -O-, >SO2 or >SiR 10 R 11 is preferable, -O- or >SiR 10 R 11 is more preferable, and >SiR 10 R 11 is even more preferable.

[0057] The compound represented by the above general formula (2) is preferably represented by the following general formula (3) or (4).

[0058]

Chemical formula

[0059] In the above formula, X is -O- or >SiR 10 R 11 represents. R 1 ~R 11 , L, and Z are the same as the above R 1 ~R 11 , L, and Z in meaning.

[0060] The compound represented by the general formula (3) is such that X in the compound represented by the above general formula (2) is -O- or >SiR 10 R 11 , Y 1 is -NHR 15 , Y 12 is =NR 16 , and R 15 and R 16 are both groups represented by the general formula (B). Similarly, the compound represented by the general formula (4) is such that X in the compound represented by the above general formula (2) is -O- or >SiR 10 R 11 , Y 1 is -OR 13 , Y 12 is =O, and R 13 is a group represented by the general formula (B). Therefore, regarding the structure corresponding to the group represented by the general formula (B) in the compound represented by the general formula (3) or (4), it is the same in meaning as the description regarding the group represented by the above general formula (B).

[0061] Specific examples of the compounds of the present invention are shown below, but the present invention is not limited to these compounds.

[0062]

Chemical formula

[0063]

Chemical formula

[0064] When the compound of the present invention has a substituent capable of binding to a biological substance, at least one substituent capable of binding to a biological substance in the compound can bind to biological substances such as proteins (including peptides), amino acids, nucleic acids, sugar chains, and lipids, and can be used as a labeled biological substance. As the substituent capable of binding to a biological substance, any group that acts (including attachment) or binds to a biological substance can be used without particular limitation, and examples thereof include the substituents described in International Publication No. 2002 / 026891 and the like. Among them, an NHS ester structure (N-hydroxysuccinimide ester), a succinimide structure, a maleimide structure, an azide group, an acetylene group, a peptide structure (polyamino acid structure), a long-chain alkyl group (preferably having 12 to 30 carbon atoms), and a quaternary ammonium group are preferably mentioned.

[0065] Among the compounds of the present invention, specific examples of the compound having at least one substituent capable of binding to a biological substance include, for example, a form in which the carboxy group in the exemplified compound of the compound of the present invention is appropriately replaced with a substituent capable of binding to a biological substance described later. The present invention is not limited to these compounds. For example, in these specific examples, for groups having dissociable hydrogen atoms such as carboxy groups and sulfo groups, the hydrogen atom may dissociate to adopt a salt structure.

[0066] The compound of the present invention can be synthesized with reference to conventional and known methods, except that the compound structure is defined by the general formula (1). The organic compound (fluorescent dye) constituting the phosphor part FL can be synthesized by a conventional method. For example, for the synthesis of a rhodamine compound, a xanthene compound, or a rhodol compound (preferably, a structure other than the group represented by general formula (B) in the above general formula (2)), for example, WO 2020 / 033681, WO 2014 / 106957, WO 2014 / 144793, US Patent Application Publication No. 2018 / 0284105, US Patent Application Publication No. 2019 / 0100653, US Patent No. 8506655, or the method described in WO 2018 / 043579 can be mentioned. For the synthesis of introducing the group represented by general formula (B) into the phosphor part FL, for example, a method using a reductive amination reagent such as 2-picoline-borane, or a method using an alkylating agent such as an alkyl halide can be mentioned. Further, it can be synthesized according to the synthesis method of each compound described in the examples described later. In particular, the compound represented by the above general formula (2) can be easily synthesized with reference to the synthesis examples described in the examples of the present application and based on common general technical knowledge as appropriate. A compound having a substituent capable of binding to a biological substance can be synthesized by a known method except that the compound structure is set to the structure defined by general formula (1). For example, Bioconjugate Techniques (Third Edition, written by Greg T. Hermanson) can be referred to.

[0067] <<Labeled biological substance>> The fluorescently labeled biological substance of the present invention (also simply referred to as a labeled biological substance) is a substance in which the compound of the present invention and a biological substance are bound. Since the compound of the present invention has fluorescence, exhibits high hydrophilicity and excellent fluorescence intensity, and exhibits excellent light resistance, it can be preferably used for the labeled biological substance. The binding between the compound of the present invention and the biological substance may be in a form in which the compound of the present invention and the biological substance are directly bound, or may be in a form in which they are linked via a linking group.

[0068] As the biological substance, proteins, amino acids, nucleic acids, nucleotides, sugar chains, and lipids are preferably mentioned. In the present invention, the term "protein" is used to include peptides, and refers to a compound formed by binding of two or more amino acids through peptide bonds. As the protein, an antibody is preferably mentioned. As the lipid, phospholipids, fatty acids, and sterols are preferably mentioned, and phospholipids are more preferable. Among the above biological substances, substances clinically and pathologically useful are not particularly limited. For example, immunoglobulins such as IgG (Immunoglobulin) G, 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), prostatic acid phosphatase (PAP), tumor markers such as CA (carbohydrate antigen) 19-9, CA-125 and their antibodies, hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen, insulin and their antibodies, hepatitis B virus (HBV)-related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), virus infection-related substances such as adult T-cell leukemia (ATL) and their antibodies, etc. can be mentioned. Furthermore, bacteria such as Corynebacterium diphtheriae, 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, drugs such as antiepileptic drugs like phenytoin and phenobarbital, cardiovascular drugs such as quinidine and digoxin, anti-asthma drugs such as theophylline, antibiotics such as chloramphenicol and gentamicin and their antibodies, other enzymes, exotoxins (such as streptolysin O) and their antibodies, etc. can also be mentioned. Also, antibody fragments such as Fab’2, Fab, and Fv can be used.

[0069] As specific forms in which the compound of the present invention interacts with and binds to a biological substance, for example, the forms described below can be mentioned. i) A non-covalent bond (for example, an ionic bond including a hydrogen bond and chelation formation) or a covalent bond between a peptide in the compound of the present invention and a peptide in the biological substance, ii) Van der Waals forces between a long-chain alkyl group in the compound of the present invention and a lipid bilayer and lipids in the biological substance, iii) An amide bond formed by the reaction between an NHS ester (N-hydroxysuccinimide ester) in the compound of the present invention and an amino group in the biological substance, iv) A thioether bond formed by the reaction between a maleimide group in the compound of the present invention and a sulfanyl group (-SH) in the biological substance, v) Formation of a triazole ring by a Click reaction between an azide group in the compound of the present invention and an acetylene group in the biological substance or a Click reaction between an acetylene group in the compound of the present invention and an azide group in the biological substance, can be mentioned. In addition to the forms of i) to v) above, for example, it can be bound by the forms 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. Also, in the preparation of the labeled biological substance of the present invention, methods and the like described in the same document can be appropriately referred to.

[0070] Among the compounds of the present invention, for the labeled biological substance of the present invention obtained from a compound having a substituent capable of binding to a biological substance and a biological substance that binds to the former by interaction, in the description of the compound examples and products in paragraph 0038 of JP-A-2019-172826, a compound in which a part other than the substituent capable of binding to a biological substance is replaced with the dye part in the compound of the present invention and its products are mentioned. However, the present invention is not limited to these compounds and the like.

[0071] <Reagent containing a labeled biological substance> In the reagent containing the labeled biological substance of the present invention, the labeled biological substance of the present invention is not particularly limited, and can be appropriately selected according to the purpose of use, etc., such as in the form of a solution dissolved in an aqueous medium such as physiological saline and phosphate buffer, and in solid forms such as particulate powder and freeze-dried powder. For example, when using the labeled biological substance of the present invention as a fluorescent labeling reagent, it can also be used as a reagent containing the labeled biological substance in any of the above forms.

[0072] <Use of the labeled biological substance> The labeled biological substance of the present invention obtained from the compound of the present invention can exhibit excellent fluorescence intensity, and the fluorescence emitted from the labeled biological substance excited by light irradiation can be stably detected. Therefore, the labeled biological substance of the present invention can be applied to various techniques using fluorescent labeling, and can be preferably used, for example, as a fluorescent labeling reagent or a bioluminescent imaging reagent in multicolor WB or dot blotting.

[0073] Fluorescence detection performed using the labeled biological substance of the present invention usually includes the following steps (i) to (iii) or (iv) to (vii). Fluorescence detection including steps (i) to (iii) corresponds to the direct method using a primary antibody fluorescently labeled with the compound of the present invention, and fluorescence detection including steps (iv) to (vii) corresponds to the indirect method using a secondary antibody fluorescently labeled with the compound of the present invention. (i) Step of preparing the following (a) and (b) respectively (a) A sample containing a target biological substance (hereinafter also referred to as "target biological substance"). (b) A labeled biological substance of the present invention (hereinafter also referred to as "labeled biological substance A of the present invention") in which a biological substance capable of binding to the target biological substance in (a) above (hereinafter also referred to as "primary biological substance") and the compound of the present invention are bound. (ii) A step of preparing a conjugate (hereinafter also referred to as "fluorescently labeled conjugate A") in which the target biological substance in (a) above and the primary biological substance in the labeled biological substance A of the present invention in (b) above are bound. (iii) A step of irradiating the above fluorescently labeled conjugate A with light in the wavelength range absorbed by the labeled biological substance A of the present invention and detecting the fluorescence emitted by the labeled biological substance A of the present invention. (iv) A step of preparing each of the following (c) to (e). (c) A sample containing a target biological substance. (d) A biological substance capable of binding to the target biological substance in (c) above (hereinafter also referred to as "primary biological substance"). (e) A labeled biological substance of the present invention (hereinafter also referred to as "labeled biological substance B of the present invention") in which a biological substance capable of binding to the primary biological substance in (d) above (hereinafter also referred to as "secondary biological substance") and the compound of the present invention are bound. (v) A step of preparing a conjugate (hereinafter also referred to as "conjugate b") in which the target biological substance in (c) above and the primary biological substance in (d) above are bound. (vi) A step of preparing a conjugate (hereinafter also referred to as "fluorescently labeled conjugate B2") in which the primary biological substance in the conjugate b and the secondary biological substance in the labeled biological substance B of the present invention are bound. (vii) A step of irradiating the above fluorescently labeled conjugate B2 with light in the wavelength range absorbed by the labeled biological substance B of the present invention and detecting the fluorescence emitted by the labeled biological substance B of the present invention.

[0074] Examples of the biological substance (primary biological substance) capable of binding to the above-described target biological substance and the biological substance (secondary biological substance) capable of binding to the primary biological substance include the biological substances in the labeled biological substance of the present invention. They can be appropriately selected according to the target biological substance (biological substance in the test sample) or the primary biological substance, and a biological substance capable of specifically binding to the biological substance or primary biological substance in the test sample can be selected.

[0075] Among the above-described target biological substances, examples of the protein include so-called disease markers. The disease markers are not particularly limited, and examples include 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), serial Tn antigen (STN), cyfra (cytokeratin: CYFRA) pepsinogen (PG), C-reactive protein (CRP), serum amyloid A protein (SAA), myoglobin, creatine kinase (CK), troponin T, ventricular myosin light chain I, and the like.

[0076] The above-described target biological substance may be a bacterium. Examples of the bacterium include bacteria targeted for cellular microbiological examination, and are not particularly limited. Examples include Escherichia coli, Salmonella, Legionella, and bacteria that cause problems in public health.

[0077] The above-mentioned target biological substance may be a virus. Although there is no particular limitation on this virus, for example, hepatitis virus antigens such as hepatitis C virus and hepatitis B virus antigens, p24 protein antigen of HIV virus, pp65 protein antigen of CMV (cytomegalovirus), E6 and E7 proteins of HPV (human papillomavirus), etc. can be mentioned.

[0078] In the above (i) or (iv), the sample containing the target biological substance can be prepared according to a conventional method without particular limitation. Also, the labeled biological substance of the present invention can be prepared by binding a biological substance capable of binding to the target biological substance and the compound of the present invention according to a conventional method without particular limitation. The form of binding and the reaction forming the binding are as described for the labeled biological substance of the present invention above.

[0079] In the above (v), the target biological substance and the primary biological substance may be directly bound, or may be bound 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 conjugate b and the secondary biological substance in the labeled biological substance B of the present invention may be directly bound, or may be bound via another biological substance different from the primary biological substance and the secondary biological substance. The labeled biological substance of the present invention can be used as a fluorescently labeled antibody in both the direct method and the indirect method, but it is preferably used as a fluorescently labeled antibody in the indirect method. In the above (ii) or (v) and (vi), the binding between the labeled biological substance of the present invention and the target biological substance can be carried out according to a conventional method without particular limitation.

[0080] In the above (iii) or (vii), the wavelength for exciting the labeled biological substance of the present invention is not particularly limited as long as it is an emission wavelength (wavelength light) capable of exciting the labeled biological substance of the present invention. Usually, 300 to 1000 nm is preferable, and 400 to 800 nm is more preferable.

[0081] As the fluorescence excitation light source used in the present invention, there is no particular limitation as long as it emits an emission wavelength (wavelength light) capable of exciting the labeled biological substance of the present invention. For example, various laser light sources can be used. Further, by using various optical filters, a preferable excitation wavelength can be obtained, or only fluorescence can be detected.

[0082] Regarding the other matters in the above (i) to (vii), there are no particular restrictions, and conditions such as methods, reagents, and apparatuses usually used in fluorescence detection using a fluorescence label can be appropriately selected. Also, regarding the steps other than the above (i) to (vii), in accordance with various methods using a fluorescence label, conditions such as methods, reagents, and apparatuses usually used can be appropriately selected.

[0083] For example, in the case of multi-color WB using the labeled biological substance of the present invention, a blot membrane is prepared by a method usually used as a target biological substance (separation of proteins by electrophoresis, blotting onto a membrane, blocking of the membrane), and by using the labeled biological substance of the present invention as a labeled antibody (preferably, a secondary antibody), fluorescence emitted from the target biological substance can be stably detected with excellent fluorescence intensity. Also, regarding dot blotting using the labeled biological substance of the present invention, similar to multi-color WB, a blot nitrocellulose membrane or a blot PVDF (polyvinylidene fluoride) membrane, etc. is prepared by a method usually used as a target biological substance, and by using the labeled biological substance of the present invention as a labeled antibody (preferably, a secondary antibody), fluorescence emitted from the target biological substance can be stably detected with excellent fluorescence intensity.

[0084] - Substituent group T - In the present invention, preferable substituents include substituents selected from the following substituent group T. Further, in the present invention, when only described as a substituent, this refers to substituent group T, and when only each group, for example, an alkyl group, is described, the corresponding group of this substituent group T is preferably applied. Furthermore, in this specification, when an alkyl group is described separately from a cyclic (cyclo)alkyl group, the alkyl group is used in the sense of including linear alkyl groups and branched alkyl groups. On the other hand, when an alkyl group is not described separately from a cyclic alkyl group and there is no particular note, the alkyl group is used in the sense of including linear alkyl groups, branched alkyl groups, and cycloalkyl groups. This also applies to groups containing a group capable of adopting a cyclic structure (such as an alkyl group, alkenyl group, alkynyl group, etc.) (such as an alkoxy group, alkylthio group, alkenyloxy group, etc.) and compounds containing a group capable of adopting 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 3 or more, preferably 5 or more, regardless of the lower limit of the number of atoms specifically described below for the group capable of adopting this structure. In the description of the following substituent group T, for example, in order to clarify a linear or branched structure group and a cyclic structure group such as an alkyl group and a cycloalkyl group, they may be described separately.

[0085] Examples of the groups included in the substituent group T include the following groups. An alkyl group (preferably having 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms, even more preferably 1 to 8 carbon atoms, particularly preferably 1 to 6 carbon atoms, and most preferably 1 to 3 carbon atoms. For example, methyl, ethyl, isopropyl, t-butyl, pentyl, heptyl, 1-ethylpentyl, benzyl, 2-ethoxyethyl, 1-carboxymethyl, trifluoromethyl, etc.), an alkenyl group (preferably having 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms, even more preferably 2 to 10 carbon atoms, particularly preferably 2 to 8 carbon atoms. For example, vinyl, allyl, oleyl, etc.), an alkynyl group (preferably having 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms, even more preferably 2 to 10 carbon atoms, particularly preferably 2 to 8 carbon atoms. For example, ethynyl, butadiynyl, phenylethynyl, etc.), a cycloalkyl group (preferably having 3 to 20 carbon atoms, such as cyclopropyl, cyclopentyl, cyclohexyl, 4-methylcyclohexyl, etc.), a cycloalkenyl group (preferably having 5 to 20 carbon atoms, such as cyclopentenyl, cyclohexenyl, etc.), an aryl group (which may be a monocyclic group or a condensed ring group (preferably a condensed ring group of 2 to 6 rings). The number of carbon atoms is preferably 6 to 26, more preferably 6 to 18, and even more preferably 6 to 12. For example, phenyl, 1-naphthyl, 4-methoxyphenyl, 2-chlorophenyl, 3-methylphenyl, etc.), a heterocyclic group (preferably having 2 to 20 carbon atoms. It has at least one oxygen atom, sulfur atom or nitrogen atom as a ring-constituting atom. It may be a monocyclic group or a condensed ring group (preferably a condensed ring group of 2 to 6 rings). When it is a monocyclic group, the number of ring members is preferably 5 to 7 members, more preferably 5 or 6 members. The heterocyclic group includes an aromatic heterocyclic group (heteroaryl group) and an aliphatic heterocyclic group (aliphatic heterocyclic group). For example, 2-pyridyl, 4-pyridyl, 2-imidazolyl, 2-benzimidazolyl, 2-thiazolyl, 2-oxazolyl, 2-tetrahydropyranyl, etc.), an alkoxy group (preferably having 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms. For example, methoxy, ethoxy, isopropyloxy, benzyloxy, etc.), an alkenyloxy group (preferably having 2 to 20 carbon atoms, more preferably 1 to 12 carbon atoms. For example, vinyloxy, allyloxy, etc.), an alkynyloxy group (preferably having 2 to 20 carbon atoms, more preferably 1 to 12 carbon atoms.For example, 2-propynyloxy, 4-butynyloxy, etc.), cycloalkyloxy group (preferably having 3 to 20 carbon atoms, for example, cyclopropyloxy, cyclopentyloxy, cyclohexyloxy, 4-methylcyclohexyloxy, etc.), aryloxy group (the number of carbon atoms is preferably 6 to 26, more preferably 6 to 14. For example, phenoxy, 1-naphthyloxy, 3-methylphenoxy, 4-methoxyphenoxy, etc.), heterocyclic oxy group (for the heterocyclic group part in the heterocyclic oxy group, the description of the above-mentioned heterocyclic group can be preferably applied. For example, imidazolyl oxy, benzimidazolyl oxy, thiazolyl oxy, benzothiazolyl oxy, triazinyl oxy, purinyl oxy, tetrahydropyranyl oxy), poly(alkyleneoxy) group (the number of carbon atoms is preferably 2 to 40, more preferably 2 to 20. For example, poly(ethyleneoxy) group),

[0086] An alkoxycarbonyl group (preferably having 2 to 20 carbon atoms, such as ethoxycarbonyl, 2-ethylhexyloxycarbonyl, etc.), a cycloalkoxycarbonyl group (preferably having 4 to 20 carbon atoms, such as cyclopropyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, etc.), an aryloxycarbonyl group (preferably having 6 to 20 carbon atoms, such as phenyloxycarbonyl, naphthyloxycarbonyl, etc.), an amino group (preferably having 0 to 20 carbon atoms, including alkylamino group, alkenylamino group, alkynylamino group, cycloalkylamino group, cycloalkenylamino group, arylamino group, heterocyclic amino group. Each of the groups substituting the unsubstituted amino group is synonymous with the corresponding group in the substituent group T. For example, amino, N,N-dimethylamino, N,N-diethylamino, N-ethylamino, N-allylamino, N-(2-propynyl)amino, N-cyclohexylamino, N-cyclohexenylamino, anilino, pyridylamino, imidazolylamino, benzimidazolylamino, thiazolylamino, benzothiazolylamino, triazinylamino, etc.), a sulfamoyl group (preferably having 0 to 20 carbon atoms, preferably an alkyl, cycloalkyl or aryl sulfamoyl group, such as N,N-dimethylsulfamoyl, N-cyclohexylsulfamoyl, N-phenylsulfamoyl, etc.), an acyl group (preferably having 1 to 20 carbon atoms, such as acetyl, cyclohexylcarbonyl, benzoyl, etc.), an acyloxy group (preferably having 1 to 20 carbon atoms, such as acetyloxy, cyclohexylcarbonyloxy, benzoyloxy, etc.), a carbamoyl group (preferably having 1 to 20 carbon atoms, preferably an alkyl, cycloalkyl or aryl carbamoyl group, such as N,N-dimethylcarbamoyl, N-cyclohexylcarbamoyl, N-phenylcarbamoyl, etc.),

[0087] An acylamino group (preferably an acylamino group having 1 to 20 carbon atoms, such as acetylamino, cyclohexylcarbonylamino, benzoylamino, etc.), an amide group (aminocarbonyl group), a sulfonamide group (preferably having 0 to 20 carbon atoms, and an alkyl, cycloalkyl or aryl sulfonamide group is preferred, such as methanesulfonamide, benzenesulfonamide, N-methylmethanesulfonamide, N-cyclohexylsulfonamide, N-ethylbenzenesulfonamide, etc.), an alkylthio group (preferably having 1 to 20 carbon atoms, such as methylthio, ethylthio, isopropylthio, benzylthio, etc.), a cycloalkylthio group (preferably having 3 to 20 carbon atoms, such as cyclopropylthio, cyclopentylthio, cyclohexylthio, 4-methylcyclohexylthio, etc.), an arylthio group (preferably having 6 to 26 carbon atoms, such as phenylthio, 1-naphthylthio, 3-methylphenylthio, 4-methoxyphenylthio, etc.), an alkyl, cycloalkyl or arylsulfonyl group (preferably having 1 to 20 carbon atoms, such as methylsulfonyl, ethylsulfonyl, cyclohexylsulfonyl, benzenesulfonyl, etc.),

[0088] A silyl group (preferably having 1 to 20 carbon atoms, and a silyl group substituted with alkyl, aryl, alkoxy and aryloxy is preferred, such as triethylsilyl, triphenylsilyl, diethylbenzylsilyl, dimethylphenylsilyl, etc.), a silyloxy group (preferably having 1 to 20 carbon atoms, and a silyloxy group substituted with alkyl, aryl, alkoxy and aryloxy is preferred, such as triethylsilyloxy, triphenylsilyloxy, diethylbenzylsilyloxy, dimethylphenylsilyloxy, etc.), a hydroxy group, a cyano group, a nitro group, a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and a fluorine atom is preferred.), an oxygen atom (specifically, >CH2 constituting the ring is replaced by >C=O), a carboxy group (-CO2H), a phosphono group [-PO(OH)2], a phosphonoxy group [-O-PO(OH)2], a sulfo group (-SO3H), a boric acid group [-B(OH)2], an onio group (an ammonio group containing a cyclic ammonio, a sulfonio group (-SH2+ ) containing a phosphonio group (-PH3 + ), preferably having 0 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, and examples thereof include a sulfanyl group (-SH), an amino acid residue, or a polyamino acid residue. Further, the above alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, heterocyclic group, alkoxy group, alkenyloxy group, alkynyloxy group, cycloalkyloxy group, aryloxy group, heterocyclic oxy group, alkoxycarbonyl group, cycloalkoxycarbonyl group, aryloxycarbonyl group, amino group, sulfamoyl group, acyl group, acyloxy group, carbamoyl group, acylamino group, alkylthio group, cycloalkylthio group, arylthio group, heterocyclic thio group, alkyl, cycloalkyl or arylsulfonyl group having a carboxy group, phosphono group, sulfo group, onio group, amino acid residue, or polyamino acid residue as a substituent are included. The substituent selected from the substituent group T is more preferably an alkyl group, alkenyl group, cycloalkyl group, aryl group, heterocyclic group, alkoxy group, cycloalkoxy group, aryloxy group, alkoxycarbonyl group, cycloalkoxycarbonyl group, the above amino group, acylamino group, cyano group or halogen atom, and particularly preferably an alkyl group, alkenyl group, heterocyclic group, alkoxy group, alkoxycarbonyl group, amino group, acylamino group or cyano group.

[0089] Unless otherwise specified, the substituent selected from the substituent group T also includes a group formed by combining a plurality of the above groups. For example, when a compound or a substituent contains an alkyl group, an alkenyl group, etc., these may be linear or branched, and these may be substituted or unsubstituted. Further, when an aryl group, a heterocyclic group, etc. are included, they may be monocyclic or condensed rings, and may be substituted or unsubstituted.

[0090] - Group represented by formula (A) -

Chemical formula

[0091] In the formula, L 3 represents a single bond, or a linking group formed by combining one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and each group represented by any one of the following formulas (1-1) to (1-8). That is, L 3 may be a single bond; it may also be an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, the group represented by formula (1-1) described below, the group represented by formula (1-2), the group represented by formula (1-3), the group represented by formula (1-4), the group represented by formula (1-5), the group represented by formula (1-6), the group represented by formula (1-7), or the group represented by formula (1-8); it may also be a group formed by combining two or more groups selected from an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, the group represented by formula (1-1) described below, the group represented by formula (1-2), the group represented by formula (1-3), the group represented by formula (1-4), the group represented by formula (1-5), the group represented by formula (1-6), the group represented by formula (1-7), and the group represented by formula (1-8). R 111 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group. However, when the hydrogen atom in R 111 is a dissociable hydrogen atom, this dissociable hydrogen atom may be dissociated. In addition, each group in L 3 and R 111 may further have a substituent. * indicates a bonding site.

[0092] Regarding the group represented by formula (A), L 3 and R 111 shall be interpreted based on the following rules (i) and (ii). (i) When the group represented by formula (A) has a group represented by any one of general formulas (1-1) to (1-8) * (bonding site) to the group represented by any one of general formulas (1-1) to (1-8) is interpreted as L 3 ​ For example, when the group represented by formula (A) is a carboxyphenyl group, the group represented by formula (A) is L 3 : a linking group formed by combining a phenylene group, a group represented by formula (1-4), and a group represented by formula (1-1), and R 111 : a group represented by a hydrogen atom. When the group represented by formula (A) is a sulfoalkyl group, the group represented by formula (A) is L 3 : a linking group formed by combining an alkylene group, a group represented by formula (1-7), and a group represented by formula (1-1), and R 111 : a group represented by a hydrogen atom. However, the groups represented by any of general formulas (1-1) to (1-8) do not form a ring as ring-constituting atoms. In addition, when the group represented by formula (A) contains two or more groups represented by any of general formulas (1-1) to (1-8), in the group represented by formula (A), up to the group represented by any of general formulas (1-1) to (1-8) located at the position where the shortest number of bonding atoms from the *(bonding part) to the group represented by any of general formulas (1-1) to (1-8) is maximized is taken as L 3 and interpreted. (ii) When the group represented by formula (A) does not have a group represented by any of general formulas (1-1) to (1-8) An alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or monovalent aliphatic heterocyclic group located at the terminal structure in the group represented by formula (A) is taken as R 111 and interpreted. For example, when the group represented by formula (A) is a phenyl group, the group represented by formula (A) is L 3 : a single bond, and R 111 : a group represented by a phenyl group. When the group represented by formula (A) is -CH2=CHPh, the group represented by formula (A) is L 3 : -CH2=CH-, and R 111 : Ph, where Ph means a phenyl group. However, the "terminal structure in the group represented by formula (A)" means the structure located at the outermost end among the longest bonding chains in the group represented by formula (A), counted from the *(bonding part). In the above (i) and (ii), when the group represented by formula (A) is a group having a substituent capable of binding to a biological substance described later, it is read with respect to the group in which the substituent capable of binding to the biological substance described later in the group represented by formula (A) is replaced with a hydrogen atom. That is, regarding the substituent capable of binding to the biological substance described later, L 3 or R 111 is interpreted as a substituent that each group in may further have. Also, within the scope conforming to the provisions of the above (i) and (ii), R 111 or L 3 The groups defined by may further have a substituent.

[0093] (L 3 ) L 3 The alkylene group that can be taken as is synonymous with the group obtained by further removing one hydrogen atom from the alkyl group selected from the substituent group T described above, and the preferred ones are the same. L 3 The alkenylene group that can be taken as is synonymous with the group obtained by further removing one hydrogen atom from the alkenyl group selected from the substituent group T described above, and the preferred ones are the same. L 3 The alkynylene group that can be taken as is synonymous with the group obtained by further removing one hydrogen atom from the alkynyl group selected from the substituent group T described above, and the preferred ones are the same. L 3 The arylene group that can be taken as is synonymous with the group obtained by further removing one hydrogen atom from the aryl group selected from the substituent group T described above, and the preferred ones are the same. L 3 The heteroarylene group that can be taken as is synonymous with the group obtained by further removing one hydrogen atom from the heteroaryl group selected from the substituent group T described above, and the preferred ones are the same. L 3The alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that can be adopted may be unsubstituted groups or groups having substituents. L 3 The substituents that the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that can be adopted may have are not particularly limited, and are preferably selected from the above-mentioned substituent group T, and more preferably include a halogen atom, an alkyl group, or an alkoxy group. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and preferably a fluorine atom or a chlorine atom. Also, L 3 The number of substituents that the above-mentioned alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that can be adopted may have is not particularly limited as long as the structure can be adopted, and can be at least 1 or more, and the upper limit is not particularly limited. For example, all hydrogen atoms in the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group may be substituted with substituents.

[0094] L 3 Among the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and linking group formed by combining one or more of the groups represented by any of the following formulas (1-1) to (1-8) that can be adopted, the types of groups to be combined are not particularly limited as long as a reasonable chemical structure is obtained. For example, L 3 does not include a group in which two or more groups represented by any of the following formulas (1-1) to (1-3) are consecutive. L 3 Among the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and linking group formed by combining one or more of the groups represented by any of the following formulas (1-1) to (1-8) that can be adopted, the types of groups to be combined are not particularly limited, but for example, 1 to 6 types are preferable, and 1 to 4 types are more preferable. L 3As an adoptable linking group, one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and each group represented by any of the following formulas (1-1) to (1-8) are combined. The number of the combined groups is not particularly limited, but for example, 1 to 10 are preferable, 1 to 6 are more preferable, and 1 to 4 are even more preferable.

[0095] L and L 3 The group represented by any of the formulas (1-1) to (1-8) that can be adopted is as follows.

Chemical formula

[0096] In the formula, R 31 and R 32 represent a hydrogen atom or a substituent. * represents a bonding portion.

[0097] R 31 The substituent that can be adopted as R is not particularly limited and is preferably selected from the aforementioned substituent group T. R 31 is preferably a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an acyl group, or a sulfonyl group, more preferably a hydrogen atom or an alkyl group, and even more preferably a hydrogen atom. Note that the above alkyl group, aryl group, heteroaryl group, acyl group, and sulfonyl group that can be adopted as R may all be unsubstituted groups or may be groups having substituents. 31 R 32 The substituent that can be adopted as R is not particularly limited and is preferably selected from the aforementioned substituent group T. R 32 is preferably a hydrogen atom, a hydroxy group, an alkoxy group, an aryloxy group, an alkyl group, an aryl group, or a heteroaryl group, more preferably a hydroxy group, an alkoxy group, or an aryloxy group, and even more preferably a hydroxy group.

[0098] As the group formed by combining the groups represented by any of the above formulas (1-1) to (1-8), groups represented by any of the following formulas (1A-1) to (1A-9) are preferably mentioned.

[0099]

Chemical formula

[0100] R 31 and R 32 are synonymous with the above R 31 and R 32 is synonymous. * and ** indicate bonding sites. Note that ** indicates the bonding site with the R 3 side in the case of a group that can be taken as L 111 side. Formula (1A-2) can be bonded to R 3 on either * side in L 111 side.

[0101] As the linking group formed by combining each group represented by any of the above formulas (1-1) to (1-8) that can be taken as the above L, a group represented by any of the above formulas (1A-1), (1A-2) or (1A-4) is preferable, and a group represented by the above formula (1A-1) or (1A-2) is more preferable. The above L 3 As the linking group formed by combining each group represented by any of the above formulas (1-1) to (1-8) that can be taken as, a group represented by any of the above formulas (1A-1), (1A-2) or (1A-4) is preferable, a group represented by the above formula (1A-1) or (1A-2) is more preferable, and a group represented by the above formula (1A-1) is even more preferable. For example, in the group represented by formula (A), the group represented by the above formula (1A-1) and the hydrogen atom as R 111 is equivalent to a carboxy group, and the group represented by the above formula (1A-4) and R 111The group represented by a hydrogen atom as such corresponds to a sulfo group. Further, the group in which a hydrogen atom dissociates from this carboxy group as a dissociable hydrogen atom corresponds to a carboxy group having an ionic structure or a salt structure, and the group in which a hydrogen atom dissociates from this sulfo group as a dissociable hydrogen atom corresponds to a sulfo group having an ionic structure or a salt structure.

[0102] Further, L 3 may be a linking group formed by combining each group represented by any one of Formula (1-1) to Formula (1-8) or a linking group formed by combining these groups with at least one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group, or may be a linking group in which two or more of each group represented by any one of Formula (1-1) to Formula (1-8) or a linking group formed by combining these groups are linked via one or two or more kinds of groups formed by combining an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group. L 3 Specific examples of the linking group formed by combining two or more kinds that L can take include, for example, a group formed by combining at least two (preferably 2 to 4 kinds) of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group, and a linking group formed by combining at least one (preferably 1 to 4 kinds) of an alkylene group, an arylene group, and a heteroarylene group with at least one (preferably 1 to 4 kinds) represented by any one of Formula (1-1) to Formula (1-8).

[0103] (R 111 ) R 111 When the hydrogen atom that R can take is a dissociable hydrogen atom, the group represented by Formula (A) may have the dissociable hydrogen atom dissociated. That is, it may form an ionic structure or a salt structure. This is the same in the description regarding R hereinafter. 111 This also applies to the description regarding R hereinafter. The fact that a hydrogen atom is dissociable means, for example, that the acid dissociation constant (pKa) is 10 or less, preferably 7 or less, and more preferably 5 or less. The above acid dissociation constant means the value at 25 °C in water.

[0104] R 111 Examples of the alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and monovalent aliphatic heterocyclic group that can be adopted as R are the same as the corresponding groups in the aforementioned substituent group T, and the preferred ones are also the same. R 111 Any of the above alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and monovalent aliphatic heterocyclic group that can be adopted as R may be an unsubstituted group or a group having a substituent. R 111 The substituents that the above groups that can be adopted as R may have are not particularly limited, and examples include groups selected from the aforementioned substituent group T, and preferably a halogen atom. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and preferably a fluorine atom. Further, it may have a substituent capable of binding to a biological substance described later. R 111 The number of substituents that the above groups that can be adopted as R may have is not particularly limited as long as the structure can be adopted, and can be at least 1 or more. The upper limit is not particularly limited. For example, all hydrogen atoms in the alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and monovalent aliphatic heterocyclic group may be substituted with substituents. R 111 Among the alkyl groups that can be adopted as R and have substituents, a halogenoalkyl group is preferably mentioned. R 111 The halogenoalkyl group that can be adopted as R has the same meaning as the alkyl group in the aforementioned substituent group T, and the preferred ones are also the same, except that at least one hydrogen atom in the alkyl group in the aforementioned substituent group T is substituted with a halogen atom. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and preferably a fluorine atom. There is no particular limitation on the number of halogen atoms constituting the halogenoalkyl group, and for example, it may be a perhalogenoalkyl group.

[0105] As the group represented by the formula (A), specifically, the following groups (1) or (2) are mentioned as preferred forms. However, in these examples, the substituents in L 3 and R 111 are not excluded from having substituents, and they may be unsubstituted or have substituents. The substituents that L 3 and R 111 may have are, respectively, preferably applicable to the description of the substituents that may be had by L 3 and R 111 as described above. (1) When having a group represented by any of the general formulas (1-1) to (1-8) The group represented by the formula (A) is such that L 3 is a single bond, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, or a linking group formed by combining one or more of each of the groups represented by the above formulas (1-1) to (1-8), and R 111 is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group. However, it has at least one of the groups represented by any of the formulas (1-1) to (1-8). This also applies to the description related to (1) hereinafter. In this case, L 3is preferably a single bond, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, or a linking group formed by combining one or more of the groups represented by the above formulas (1-1), (1-3), (1-4), and (1-7). More preferably, it is a single bond, an alkylene group, an arylene group, a heteroarylene group, or a linking group formed by combining one or more of the groups represented by the above formulas (1-1), (1-3), (1-4), and (1-7). Even more preferably, it is a linking group formed by combining an alkylene group, an arylene group, a heteroarylene group, or one or more of the groups represented by the above formulas (1-1), (1-3), (1-4), and (1-7). Particularly preferably, it is a linking group formed by combining an alkylene group, an arylene group, a heteroarylene group, or one or more of the groups represented by the above formulas (1-1), (1-3), and (1-4). In these cases, R 111 is preferably a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group. More preferably, it is a hydrogen atom or an alkyl group. Even more preferably, it is a hydrogen atom. (2) When not having a group represented by any of the general formulas (1-1) to (1-8) The group represented by the formula (A) is L 3 which is a single bond, or a linking group formed by combining one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group, and 111 R is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group. In this case, L 3 is a single bond, and 111 R is a hydrogen atom, an alkyl group, an alkenyl group, an aryl group, or a heteroaryl group, or L 3 is an alkenylene group or an alkynylene group, and 111 R is preferably an alkyl group or an aryl group. More preferably, L 3 is a single bond, and 111 R is a hydrogen atom, an alkyl group, an alkenyl group, or an aryl group.

Examples

[0106] The present invention will be described in more detail based on the following examples, but the present invention is not limited thereto. Note that room temperature means 25°C.

[0107] [Examples] Compounds (2) to (4) and Comparative Compound (1) used in the examples are shown below.

[0108] [Chemical formula]

[0109] The synthesis methods of the compounds and labeled antibodies are described in detail below, but the starting materials, dye intermediates, and synthesis routes are not limited thereto.

[0110] Unless otherwise specified, the carrier in silica gel column chromatography was SNAP KP-Sil Cartridge (trade name, manufactured by Biotage), High Flash Column W001, W002, W003, W004 or W005 (trade name, manufactured by Yamazen). For NH silica, SNAP KP-NH Cartridge (manufactured by Biotage) was used. The mixing ratio in the eluent is by volume ratio. For example, "ethyl acetate:normal hexane = 0:100 → 100:0" means that the eluent of "ethyl acetate:normal hexane = 0:100" was changed to the eluent of "ethyl acetate:normal hexane = 100:0". The MS spectrum was measured using an ACQUITY SQD LC / MS System [trade name, manufactured by Waters, ionization method: ESI (ElectroSpray Ionization)] or LCMS-2010EV [trade name, manufactured by Shimadzu Corporation, ionization method: an ionization method that simultaneously performs ESI and APCI (Atomospheric Pressure Chemical Ionization)]. In addition, when the synthesized compound and labeled antibody were not used immediately after preparation without special description, those stored under light-shielded conditions were used. Also, for commercially available compounds and labeled antibodies, those stored under light-shielded conditions were used until they were used after purchase.

[0111] The abbreviations used are summarized below. DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Pic·BH3: borane-2-methylpyridine complex WSC·HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride DMSO: dimethyl sulfoxide THF: tetrahydrofuran Ac: acetyl group nBu: normal butyl group Et: ethyl group Me: methyl group Ph: phenyl group

[0112] [Synthesis Example 1: Synthesis of Compound (2) and Labeled Antibody (2)] Based on the following scheme, Compound (2) and Labeled Antibody (2) were synthesized. [Chemical Formula]

[0113] [Synthesis of Compound (2-A)] To a 300 mL three-necked flask, 3-bromo-aniline (13.1 g, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and acetonitrile (76 mL, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) were added. Allyl bromide (19.8 mL, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and potassium carbonate (21.0 g, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) were mixed and stirred at 80 °C. The completion of the reaction was confirmed by LC / MS, and unnecessary substances were removed by filtration. After distilling off the solvent under reduced pressure from the filtrate, the obtained residue was dissolved in methylene chloride and purified by silica gel column chromatography (ethyl acetate:normal hexane), and the target product was recovered and the solvent was distilled off under reduced pressure to obtain compound (2-A) (17.2 g, yield 90%). [M+H + + :253

[0114] <Synthesis of Compound (2-B)> To a 100 mL three-necked flask, compound (2-A) (2.55 g) and tetrahydrofuran (30 mL, super dehydrated, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) were added. After purging with nitrogen, it was cooled to -78 °C, and n-butyllithium (7.23 mL, 1.6 M, n-hexane solution, manufactured by Kanto Chemical Co., Inc.) was added dropwise and stirred for 30 minutes. Subsequently, dichloromethylvinylsilane (0.700 mL, manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred for 1 hour. The completion of the reaction was confirmed by LC / MS. Under ice-cooling, a saturated aqueous ammonium chloride solution was added, extracted with ethyl acetate, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in methylene chloride and purified by silica gel column chromatography (ethyl acetate:normal hexane = 0:100 → 10:90), and the target product was recovered and the solvent was distilled off under reduced pressure to obtain colorless oily compound (2-B) (1.98 g, yield 49%). [M+H + + :415

[0115] <Synthesis of Compound (2-C)> ​​In a 5 mL test tube type reaction vessel, compound (2-B) (250 mg), 3,5-difluoro-4-formylbenzoic acid (112 mg, manufactured by COMBI-BLOCKS), zinc chloride (II) (247 mg, manufactured by FUJIFILM Wako Pure Chemical Corporation), and ethanol (2.1 mL, ultra-dehydrated, manufactured by FUJIFILM Wako Pure Chemical Corporation) were added. Microwaves were irradiated and heated at 140 °C for 30 minutes. The completion of the reaction was confirmed by LC / MS. Water was added to the reaction solution, extracted with ethyl acetate, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in methylene chloride and purified by silica gel column chromatography (ethyl acetate:normal hexane = 0:100 → 30:70) to recover the target product. The solvent was distilled off under reduced pressure to obtain a pale green gum-like compound (2-C) (150 mg, yield 38%). [M+H + + :583

[0116] <Synthesis of Compound (2-D)> Compound (2-C) (232 mg) and dichloromethane (4.0 mL, manufactured by FUJIFILM Wako Pure Chemical Corporation) were added to a 50 mL three-necked flask. Tetrakis(triphenylphosphine)palladium(0) (46 mg, manufactured by FUJIFILM Wako Pure Chemical Corporation) and 1,3-dimethylbarbituric acid (249 mg, manufactured by Tokyo Chemical Industry Co., Ltd.) were mixed and stirred at room temperature for 2 hours. The completion of the reaction was confirmed by LC / MS. The reaction solution was directly purified by silica gel column chromatography (ethyl acetate:normal hexane, and ethyl acetate:methanol) to recover the target product. The solvent was distilled off under reduced pressure to obtain compound (2-D) (59 mg, yield 35%). [M+H + + :423

[0117] <Synthesis of Compound (2-E)> ​​In a 5 mL test tube type reaction vessel, compound (2-D) (20 mg), 4,6-O-ethylidene-α-D-glucopyranose (32 mg, manufactured by Tokyo Chemical Industry Co., Ltd.), borane-2-methylpyridine complex (38 mg, manufactured by Tokyo Chemical Industry Co., Ltd.), acetic acid (20 μL, manufactured by Fujifilm Wako Pure Chemical Corporation), and ethanol (2.0 mL, ultra-dehydrated, manufactured by Fujifilm Wako Pure Chemical Corporation) were added, irradiated with microwaves, and heated at 80 °C for 30 minutes. The completion of the reaction was confirmed by LC / MS. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the solvent was distilled off under reduced pressure. The obtained residue was purified by reverse-phase silica gel column chromatography (C18, manufactured by Biotage, acetonitrile: distilled water = 0:100 → 70:30), the target product was recovered, the solvent was distilled off by freeze-drying, and compound (2-E) (13 mg, yield 35%) as a dark blue powder was obtained. [M+H + + :803

[0118] <Synthesis of Compound (2)> To a 10 mL flask, compound (2-E) (13 mg), dichloromethane (0.6 mL, manufactured by Fujifilm Wako Pure Chemical Corporation), distilled water (0.6 mL), and tetrahydrofuran (0.2 mL, manufactured by Fujifilm Wako Pure Chemical Corporation) were added. Under ice-cooling, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (25 mg, manufactured by Fujifilm Wako Pure Chemical Corporation) was added, and after warming to room temperature, the mixture was stirred for 1.5 hours. The completion of the reaction was confirmed by LC / MS. An aqueous saturated sodium thiosulfate solution was added to the reaction solution. Subsequently, tetrahydrofuran (3.0 mL, manufactured by Fujifilm Wako Pure Chemical Corporation) and aqueous hydrochloric acid solution (1.2 mL, 5.0 mol / L, manufactured by Fujifilm Wako Pure Chemical Corporation) were added, and the mixture was heated at 60 °C for 1 hour. The completion of the reaction was confirmed by LC / MS, and the solvent was distilled off under reduced pressure. The residue was purified by reverse-phase silica gel column chromatography (C18, manufactured by Biotage, acetonitrile: distilled water = 0:100 → 80:20), the target product was recovered, the solvent was distilled off by freeze-drying, and compound (2) (7.9 mg, yield 64%) as a dark blue powder was obtained. [M+H + + :749

[0119] <Synthesis of Compound (2-NHS)>​​ In a 5 mL test tube type reaction vessel, compound (2) (2.3 mg), N-hydroxysuccinimide (6.4 mg, manufactured by Fujifilm Wako Pure Chemical Corporation), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (10.8 mg, manufactured by Fujifilm Wako Pure Chemical Corporation), and DMSO (330 μL, manufactured by Fujifilm Wako Pure Chemical Corporation) were added, and the mixture was stirred at room temperature for 1 hour. The completion of the reaction was confirmed by LC / MS, and the reaction solution was directly purified by reverse-phase silica gel column chromatography (C18, manufactured by Biotage, acetonitrile: aqueous solution containing 0.1% trifluoroacetic acid = 0:100 → 80:20) to recover the target product. The solvent was distilled off by lyophilization to obtain compound (2-NHS) as a dark blue powder (1.4 mg, yield 53%). [M+H + + :846

[0120] <Synthesis of Labeled Antibody (2)> To 208 μL of anti-rabbit IgG antibody [host: goat, 2.4 mg / mL, catalog number: 111-005-003, manufactured by Jackson ImmunoResearch], 20.8 μL of carbonate pH standard solution (pH = 10.01) (manufactured by Fujifilm Wako Pure Chemical Corporation) and 13.0 μL of a DMSO solution of compound (2-NHS) with a concentration of 20 mM were added and stirred, and then left standing at room temperature for 1 hour. Subsequently, the reaction solution was directly charged onto a Sephadex G-25 column [catalog number: 17085101, manufactured by GE Healthcare] and purified with PBS [pH = 7.4, manufactured by Fujifilm Wako Pure Chemical Corporation] to obtain labeled antibody (2).

[0121] 〔Synthesis Example 2: Synthesis of Compound (3) and Labeled Antibody (3)〕 <Synthesis of Compound (3)> Compound (3) as a dark blue powder was obtained in the same manner as in the synthesis method of compound (2), except that N-acetyl-D-galactosamine (manufactured by Fujifilm Wako Pure Chemical Corporation) was used instead of 4,6-O-ethylidene-α-D-glucopyranose. [M+H + + :831 <Synthesis of Compound (3-NHS)> ​​In the synthesis method of compound (2-NHS), a deep blue powder compound (3-NHS) was obtained in the same manner except that compound (3) was used instead of compound (2). [M+H + + :928 <Synthesis of labeled antibody (3)> In the synthesis method of labeled antibody (2), labeled antibody (3) was obtained in the same manner except that compound (3-NHS) was used instead of compound (2-NHS).

[0122] [Synthesis Example 3: Synthesis of compound (4) and labeled antibody (4)] <Synthesis of compound (4)> In the synthesis method of compound (2-E) using 3-bromo-aniline as the starting compound, a deep blue powder compound (4) was obtained in the same manner except that maltose (manufactured by Fujifilm Wako Pure Chemical Corporation) was used instead of 4,6-O-ethylidene-α-D-glucopyranose. [M+H + + :1073 <Synthesis of compound (4-NHS)> In the synthesis method of compound (2-NHS), a deep blue powder compound (4-NHS) was obtained in the same manner except that compound (4) was used instead of compound (2). [M+H + + :1170 <Synthesis of labeled antibody (4)> In the synthesis method of labeled antibody (2), labeled antibody (4) was obtained in the same manner except that compound (4-NHS) was used instead of compound (2-NHS).

[0123] [Synthesis Example 4: Synthesis of comparative compound (1) and comparative labeled antibody (1)] <Synthesis of comparative compound (1)> In the synthesis method of compound (2-D) using 3-bromo-aniline as the starting compound, a yellow oily comparative compound (1) was obtained in the same manner except that 3-bromo-N-methylaniline (manufactured by Fujifilm Wako Pure Chemical Corporation) was used instead of 3-bromo-aniline. [M+H + + :449​​​​ <Synthesis of Comparative Compound (1-NHS)> In the synthesis method of Compound (2-NHS), a deep blue powder of Comparative Compound (1-NHS) was obtained in the same manner except that Comparative Compound (1) was used instead of Compound (2). [M+H + + :546 <Synthesis of Comparative Labeled Antibody (1)> In the synthesis method of Labeled Antibody (2), Comparative Labeled Antibody (1) was obtained in the same manner except that Comparative Compound (1-NHS) was used instead of Compound (2-NHS).

[0124] 〔Evaluation〕 The water solubility, maximum molar absorption coefficient and fluorescence quantum yield of each compound shown above, and the decrease rate of the fluorescence quantum yield accompanying the increase in the fluorescence labeling rate of each obtained labeled antibody were evaluated. The results are summarized in Table 1.

[0125] [1] Evaluation of water solubility In a 1.5 mL Eppendorf tube, 5 μL of a DMSO solution containing the above compound (hereinafter referred to as the sample) at a concentration of 20 mM and 495 μL of PBS (Phosphate Buffered Saline, pH 7.4) were added and mixed, and stirred at 2000 rpm for 30 minutes using a high-speed shaker CM-1000 (trade name, manufactured by Tokyo Rika Kikai Co., Ltd.). The obtained mixture was allowed to stand for 60 minutes in the dark. For the filtrate filtered using a filter with a pore size of 0.20 μm, the sample concentration (when all the sample was dissolved, the sample concentration would be 200 μM) was measured using Prominence XR [trade name, manufactured by Shimadzu Corporation, column: Shim-pack XR-ODSII]. In this test, if the sample concentration of the filtrate is 1 μM or more, it is preferable from the viewpoint of practicality as a fluorescent compound, and for each of the compounds (2) to (4) shown above, the sample concentration of the filtrate was 150 μM or more.

[0126] [2] Evaluation of maximum molar absorption coefficient ​The PBS solution (pH 7.4) of the above compound was evaluated by the method described in the following reference materials. The unit of the maximum molar extinction coefficient in the following evaluation criteria is mol -1 Lcm -1 is. “TECH TIP #6 Extinction Coefficients” (available from the materials of Thermo Scientific, https: / / assets.thermofisher.com / TFS-Assets / LSG / Application-Notes / TR0006-Extinction-coefficients.pdf) Regarding the maximum molar extinction coefficient, a higher evaluation rank is preferable because it indicates better performance in the maximum molar extinction coefficient. In this test, it is preferable that the maximum molar extinction coefficient meets the evaluation rank of “C” or higher from the perspective of the practicality of the fluorescent compound. - Evaluation criteria for the maximum molar extinction coefficient - A: 100,000 or more B: 50,000 or more and less than 100,000 C: 10,000 or more and less than 50,000 D: Less than 10,000 E: Unable to evaluate due to low water solubility

[0127] [3] Evaluation of fluorescence quantum yield The PBS solution (pH 7.4) of the above compound was evaluated by the method described in the following reference materials. “A Guide to Recording Fluorescence Quantum Yields” (available from the materials of HORIBA Scientific, https: / / www.horiba.com / fileadmin / uploads / Scientific / Documents / Fluorescence / quantumyieldstrad.pdf) Regarding the fluorescence quantum yield, a higher evaluation rank is preferable because it indicates better performance in the fluorescence quantum yield. In this test, it is preferable that the fluorescence quantum yield meets the evaluation rank of “C” or higher from the perspective of the practicality of the fluorescent compound. - Evaluation Criteria for Fluorescence Quantum Yield - A: 0.5 or more B: 0.3 or more and less than 0.5 C: 0.1 or more and less than 0.3 D: Less than 0.1 E: Evaluation not possible due to low water solubility

[0128] [4] Evaluation of the Decrease Rate of Fluorescence Quantum Yield with the Increase in Fluorescence Labeling Rate of Labeled Antibody The slope a of the approximate straight line obtained by plotting the relationship between the fluorescence labeling rate and the fluorescence quantum yield of the labeled antibody calculated by the following method was applied to the following evaluation criteria to evaluate the decrease rate of the fluorescence quantum yield with the increase in the fluorescence labeling rate of the labeled antibody. A high evaluation rank is very preferable because the decrease rate of the fluorescence quantum yield with the increase in the fluorescence labeling rate is small. In this test, the decrease rate of the fluorescence quantum yield is at the passing level when the evaluation rank is "C" or higher. - Evaluation Criteria for the Decrease Rate of Fluorescence Quantum Yield of Labeled Antibody - A: a is less than 0.05. B: a is 0.05 or more and less than 0.10. C: a is 0.10 or more and less than 0.20. D: a is 0.20 or more. The fluorescence labeling rate of the labeled antibody was evaluated by the following method. 217 μL of anti-rabbit IgG antibody (2.3 mg / ml) and 21.7 μL of carbonate buffer were placed in a microtube, and after shaking and stirring, a dimethyl sulfoxide solution of the above compound was added to the antibody in a molar equivalent ratio to make it 10, 20, 40, or 80, and further shaking and stirring was performed. The reaction solution was allowed to stand at room temperature for 1 hour, and the labeled antibody was obtained by purifying it using gel filtration column chromatography PD10 (manufactured by GE Healthcare Life Sciences) and PBS solution (phosphate buffered saline). For each of the obtained labeled antibodies, the fluorescence labeling rate was calculated by the method shown below. (Calculation of Fluorescence Labeling Rate) The method for calculating the fluorescence labeling rate used a general method as shown below. The descriptions in [ ] indicate units, and [-] means no unit. In this test, the protein means anti-rabbit IgG antibody. Fluorescence labeling rate = Concentration of fluorescent dye / Concentration of protein The concentration of the fluorescent dye means the total molar concentration [M] of the labeled fluorescent dye, and the concentration of the protein means the molar concentration [M] of the fluorescently labeled protein, and each is calculated by the following formula. Concentration of fluorescent dye = Dye max / ε dye Concentration of protein = (IgG 280 - (Dye max × CF)) / ε protein Each symbol in the above formula is as follows. Dye max ; Absorbance [-] at the maximum absorption wavelength of the fluorescent dye ε dye ; Molar extinction coefficient [M -1 cm -1 IgG 280 ; Absorbance [-] at 280 nm of the fluorescently labeled protein Dye 280 ; Absorbance [-] at 280 nm of the fluorescent dye ε protein ; Molar extinction coefficient [M -1 cm -1 CF (Correction Factor); Dye 280 / Dye max [-] (Evaluation of fluorescence quantum yield) For the labeled antibody for which the fluorescence labeling rate was calculated as described above, in the evaluation of the fluorescence quantum yield in [3] above, the fluorescence quantum yield was evaluated in the same manner except that the above-labeled antibody was used instead of the above compound. ​​Regarding the fluorescence labeling rate and fluorescence quantum yield of four samples in which the molar equivalent ratio of the compound to the antibody obtained in this way was 10, 20, 40, or 80, the vertical axis Y was the fluorescence quantum yield, and the horizontal axis X was the fluorescence labeling rate, and the slope a of the approximate straight line (that is, a in the linear approximation represented by Y = -aX + b) was calculated.

[0129]

Table 1

[0130] (Note to the table) Compounds (2) to (4) and comparative compound (1) are as shown above. In the column of the decrease rate of fluorescence quantum yield (labeled antibody), the evaluation results of the decrease rate of fluorescence quantum yield accompanying the increase in fluorescence labeling rate for the corresponding labeled antibody are described.

[0131] From the results in Table 1 above, the following can be seen. In the compound represented by the general formula (2) having a structural part composed of a Si-rhodamine compound as FL, the comparative compound (1) having no group represented by the general formula (B) had a large decrease rate of fluorescence quantum yield accompanying the increase in fluorescence labeling rate of the corresponding labeled antibody and was inferior. In contrast, all of the compounds (2) to (4) having a group represented by the general formula (B) suppressed the decrease rate of fluorescence quantum yield accompanying the increase in fluorescence labeling rate of the corresponding labeled antibody to a level exceeding twice that of the comparative compound (1), and were excellent in suppressing the decrease in fluorescence quantum yield accompanying the increase in fluorescence labeling rate.

Claims

1. A fluorescent compound represented by the following general formula (2) (however, compounds Ia-100 to Ia-105 below are excluded). 【Chemical 1】 In the above formula, X represents -O-, -S-, >SO, >SO₂, >SiR₁₀R₁₁ or >P(=O)R₁₂. Y₁ represents -OR₁₃ or -NR₁₄R₁₅, and Y₂ represents =O, =NR₁₆ or =N⁺R₁₇R₁₈. Z represents a group represented by the following formula (A). R₄ to R₉ represent a halogen atom, a cyano group or a group represented by the following formula (A). R₁₀ and R₁₁ represent a group represented by the following formula (A), and R₁₀ and R₁₁ may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring. However, when R₁₀ and R₁₁ are not bonded to each other to form a ring, at least one of R₁₀ and R₁₁ is an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group. R₁₂ to R₁₈ represent a group represented by the following formula (A). However, the above compound has a group represented by the following general formula (B) as at least one of the above R₁₃ to R₁₈. ​ In the formula, L₃ represents a single bond, or a linking group formed by combining one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group and each group represented by the following formulas (1-1) to (1-8). R₁₁₁ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or a monovalent aliphatic heterocyclic group. However, when the hydrogen atom in R₁₁₁ is a dissociable hydrogen atom, this dissociable hydrogen atom may be dissociated. * represents a bonding site. [Chemical Formula 3] In the above formula, R １ to R ３ represent a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group. L represents a single bond, or a linking group formed by combining one or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroaryl group and each group represented by the following formulas (1-1) to (1-8). * represents a bonding site. However, L and R １ ~R ３ do not combine with any one of them to form a glycosyl structure. [Chemical Formula 4] In the above formula, R ３１ and R ３２ each represents a hydrogen atom or a substituent. * represents a bonding site. 【Chemical Formula 5】

2. The fluorescent compound according to Claim 1, represented by the following general formula (3) or (4). 【Chemical Formula 6】 In the above formula, X represents -O- or >SiR １０ R １１ and is shown as such. R １ to R １１ , L, and Z are the same as the aforementioned R １ to R １１ , L, and Z are synonymous with

3. A fluorescently labeled biological substance formed by binding the compound according to Claim 1 or 2 to a biological substance.

4. The fluorescently labeled biological substance according to Claim 3, wherein the biological substance is any one of a protein, an amino acid, a nucleic acid, a nucleotide, a sugar chain and a lipid.

5. The fluorescently labeled biological substance according to claim 4, wherein the binding between the compound and the biological substance is a binding according to any one of the following i) to v). i) A non-covalent bond or a covalent bond between peptides, ii) A van der Waals interaction between a long-chain alkyl group in the compound and a lipid bilayer or lipid in the biological substance, iii) An amide bond formed by reacting an N-hydroxysuccinimide ester in the compound with an amino group in the biological substance, iv) A thioether bond formed by reacting a maleimide group in the compound with a sulfanyl group in the biological substance, v) A bond involving the formation of a triazole ring, which is formed by subjecting an azide group in the compound and an acetylene group in the biological substance, or an acetylene group in the compound and an azide group in the biological substance, to a Click reaction

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