Fluorescent compound and fluorescently labeled biological material using the same

Abstract

To provide a fluorescent compound and a fluorescently labeled biological material that exhibit superior water solubility and effectively suppress fluorescence bleed-through.SOLUTION: The present invention provides a fluorescent compound and a fluorescently labeled biological material represented by general formula (1) or (2), wherein R1 to R2, R11 to R14, R19, and R24 to R25 represent alkyl groups, with at least one of R1 to R2 and R11 to R12, and at least one of R13 to R14, R19, and R24 to R25 including an alkylene glycol structure, and at least one remaining alkyl group including a carboxyl group or a biomolecule-binding group, where the fluorescent compound includes at least one sulfo group.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a fluorescent compound and a fluorescently labeled biological material using the same. [Background technology]

[0002] In the fields of research and medicine, fluorescently labeled biological materials in which biological molecules (such as antibodies) that specifically bind to a target substance to be detected are labeled with a fluorescent compound (fluorescent dye) are widely used. For example, in Western blotting (hereinafter also abbreviated as WB), which detects a specific protein from a protein mixture to observe changes in the body in response to various stimuli (diseases, environmental changes, etc.), a fluorescent method is used to detect the presence or absence or amount of the specific protein using a fluorescently labeled antibody that binds to the protein. In addition, in bioimaging technology that analyzes the dynamics and functions of biomolecules, cells, tissues, etc. in a living body, biofluorescence imaging, which observes specific parts of a living body that have been visualized by fluorescent labeling, is used as one of the techniques for observing living bodies.

[0003] Furthermore, flow cytometry is known as a technique that can count, identify, and separate specific cells from a particle population of cells, etc., and can rapidly analyze cells one by one from a population containing a wide variety of cells. In this technique, cells passing through a flow cell are irradiated with laser light, and the size and complexity of the internal structure of the cells are analyzed based on the scattered light from the cells. Furthermore, in fluorescently labeled cells, the fluorescence emitted from the fluorescent dye by irradiation with laser light is dispersed by an optical filter (spectral filter) and measured as the amount of fluorescence (photons) (voltage). This allows the fluorescently labeled cells to be detected specifically and quantitatively.

[0004] Known fluorescent dyes used for the above-mentioned various fluorescent labels include, for example, cyanine dyes, rhodamine dyes, dipyrromethene dyes, and squarylium dyes, and efforts are being made to improve the properties of these fluorescent dyes. For example, Patent Document 1 describes a luminescent compound having a rotaxane structure in which a squarylium dye molecule, which is an axis molecule, is included in a cyclic molecule such as cyclodextrin. According to Patent Document 1, it is said that the photostability of the squarylium dye molecule can be improved by the rotaxane structure. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent Application Publication No. 2010 / 0266507 Summary of the Invention [Problem to be solved by the invention]

[0006] In the above-mentioned flow cytometry, by using a combination of multiple types of laser light sources for exciting the fluorescent dyes, multiple types of fluorescent dyes, and multiple types of spectral filters, it is possible to perform simultaneous multicolor detection using, for example, more than ten types of detection channels at the same time. Recently, there has been a strong demand for the development of a technology that enables simultaneous multicolor detection using even more types of detection channels. The more detection channels there are, the more likely it is that the fluorescence to be detected in one detection channel will leak into another detection channel. As a result of the studies by the present inventors, it has been found that the above-mentioned leakage of fluorescence is significant in the case of conventional fluorescent dyes, such as commercially available fluorescent dyes and the compounds described in the above Patent Document 1, in simultaneous multi-color detection using multiple types of detection channels.

[0007] An object of the present invention is to provide a fluorescent compound that is highly water-soluble and can effectively suppress leakage of fluorescence, and to provide a fluorescently labeled biological material obtained by binding this fluorescent compound to a biological material. [Means for solving the problem]

[0008] That is, the above-mentioned object of the present invention has been achieved by the following means. [1] A fluorescent compound represented by the following general formula (1) or (2): [ka] In the formula, R 1 , R 2 , R 11 and R 12 represents an alkyl group. 1 , R 2 , R 11 and R 12 At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. R 3 , R 4 , R 9 and R 10 represents a hydrogen atom, a halogen atom, an alkyl group, an aryl group or a heteroaryl group. R 5 ~R 8 represents a hydrogen atom, a hydroxy group, an alkoxy group, an amino group, an acylamino group, or an alkyl group. However, the fluorescent compound represented by the above general formula (1) contains at least one sulfo group. [ka] In the formula, R 13 , R 14 , R 19 , R 24 and R 25 represents an alkyl group. 13 , R 14 , R 19 , R 24 and R 25 At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. R 15 and R 16 are the above R 3 and R 4 is synonymous with R 17 and R 18 are the above R5 and R 6 is synonymous with. R 20 ~R 23 represents a hydrogen atom, a halogen atom, a hydroxy group, a carboxy group, a sulfo group, a phosphonooxy group, a phosphono group, an alkyl group, an aryl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, a carbonyl group, a sulfonyl group, an amino group, or a silyl group. However, the fluorescent compound represented by the above general formula (2) contains at least one sulfo group. [2] The above R in the above general formula (1) 5 ~R 8 The fluorescent compound according to [1], wherein at least two of the above are hydroxy groups. [3] The above R in the above general formula (2) 17 and R 18 At least one of the above is a hydroxy group. [4] The fluorescent compound represented by the above general formula (1) is -(CH2-CH2-O) m -R 30 The fluorescent compound according to any one of [1] to [3], comprising two or more of the following: m is 1 to 10, R 30 is a hydrogen atom or an alkyl group. [5] The above R in the above general formula (1) 1 , R 2 , R 11 and R 12 [4] The fluorescent compound according to any one of [1] to [4], wherein at least one of the alkyl groups includes a sulfoalkyl group. [6] The above R in the above general formula (2) 24 and R 25 At least one alkyl group is -(CH2-CH2-O) m -R 30 and the above R in the above general formula (2) is a substituted alkyl group represented by 13 , R14 and R 19 The fluorescent compound according to any one of [1] to [5], wherein at least one of the alkyl groups contains a sulfo group. m is 1 to 10, R 30 is a hydrogen atom or an alkyl group. [7] A fluorescently labeled biological substance comprising the compound according to any one of [1] to [6] and a biological substance bound thereto. [8] The fluorescently labeled biological material according to [7], wherein the biological material is any one of a protein, a peptide, an amino acid, a nucleic acid, a sugar chain, and a lipid. [9] The fluorescently labeled biological substance according to [7] or [8], wherein the bond between the compound and the biological substance is any one of the bonds i) to v) below: i) non-covalent or covalent bonds between peptides; ii) van der Waals interactions between long-chain alkyl groups in the compound and lipid bilayers or lipids in biological materials; iii) an amide bond formed by reacting an N-hydroxysuccinimide ester in a compound with an amino group in a biological material; iv) a thioether bond formed by reacting a maleimide group in a compound with a sulfanyl group in a biological material; v) A bond involving the formation of a triazole ring, which is formed by a Click reaction between an azide group in a compound and an acetylene group in a biological substance, or between an acetylene group in a compound and an azide group in a biological substance. Effect of the Invention

[0009] The fluorescent compound of the present invention has excellent water solubility and can effectively suppress the leakage of fluorescence. Also, the fluorescently labeled biological material of the present invention can effectively suppress the leakage of fluorescence. [Brief description of the drawings]

[0010] [Figure 1]1 shows the absorption spectrum and fluorescence spectrum of a PBS solution of compound (1-1) in an example, in which the solid line indicates the absorption spectrum and the dashed line indicates the fluorescence spectrum. [Diagram 2] 2 shows the absorption spectrum and fluorescence spectrum of a PBS solution of compound (1-2) in an example, in which the solid line indicates the absorption spectrum and the dashed line indicates the fluorescence spectrum. [Diagram 3] 3 shows the absorption spectrum and fluorescence spectrum of a PBS solution of compound (1-3) in an example, in which the solid line indicates the absorption spectrum and the dashed line indicates the fluorescence spectrum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In the present invention, when there are a plurality of substituents, linking groups, or structural units, etc., represented by a specific symbol or formula (hereinafter referred to as "substituents, etc."), or when a plurality of substituents, etc., are specified at the same time, unless otherwise specified, the respective substituents, etc. may be the same or different from each other. This also applies to the definition of the number of substituents, etc. In addition, when a plurality of substituents, etc., are adjacent to each other (particularly, when adjacent), they may be linked to each other to form a ring, unless otherwise specified. In addition, unless otherwise specified, rings, such as alicyclic rings, aromatic rings, and heterocyclic rings, may be further condensed to form a condensed ring. In the description of groups (atomic groups) in the present invention, the description without indicating whether substituted or unsubstituted includes groups (atomic groups) that have no substituents as well as groups (atomic groups) that have a substituent. For example, "alkyl group" includes not only alkyl groups that have no substituents (unsubstituted alkyl groups) but also alkyl groups that have a substituent (substituted alkyl groups). This also applies to divalent linking groups such as alkylene groups. As the optional substituents that such groups (atomic groups) have, substituents selected from the substituent group T described below are preferably mentioned. In the present invention, when the number of carbon atoms of a certain group is specified, this carbon number means the total number of carbon atoms of the group unless otherwise specified in the present invention or this specification. In other words, when this group further has a substituent, it means the total number of carbon atoms including the substituent.

[0012] In the present invention, a numerical range expressed using "to" means a range including the numerical values ​​before and after "to" as the lower and upper limits. In addition, the chemical structural formula in the present invention may be described as a simplified structural formula in which hydrogen atoms are omitted.

[0013] In the present invention, the indication of compounds and substituents is used to include not only the compounds themselves and the substituents themselves, but also their salts and ions. For example, acidic groups such as carboxy, sulfo, phosphono, and phosphonooxy groups may have an ionic structure with dissociated hydrogen ions, or may have a salt structure. That is, in the present invention, the term "carboxy" refers to a carboxylate ion or a salt thereof, the term "sulfo" refers to a sulfonate ion or a salt thereof, the term "phosphono" refers to a phosphonate ion or a salt thereof, and the term "phosphonooxy" refers to a phosphate ion or a salt thereof. Examples of salts of acidic groups include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, ammonium salts, and salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, organic phosphonium salts. That is, in the present invention, the term "acidic group" is used to include salt-type groups. Salts of basic groups such as amino groups include, for example, salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid, and salts with phosphoric acid. When the fluorescent compound of the present invention has a salt structure, the type of salt may be one type or two or more types may be mixed, the compound may contain a mixture of salt-type and free acid-type groups, or the compound may contain a mixture of salt-type compounds and free acid-type compounds. When the fluorescent compound or a salt thereof of the present invention has stereoisomers such as optical isomers, geometric isomers or rotational isomers, or tautomers, the present invention encompasses these isomers, as well as anhydrates, solvates, hydrates, and various forms of crystals.

[0014] The fluorescent compound of the present invention includes those whose structure is partially modified within the scope that does not impair the effects of the present invention.

[0015] <Fluorescent Compound of the Present Invention>

[0016] The fluorescent compound of the present invention is a fluorescent compound represented by the general formula (1) or (2) described below. As represented by the general formula (1) or (2) described below, the fluorescent compound of the present invention has a squarylium (also called squaraine) structure. The reason why the fluorescent compound of the present invention has excellent water solubility and can sufficiently suppress fluorescence leakage is not clear, but it is thought to be as follows. The fluorescent compound represented by general formula (1) or (2) of the present invention is a squarylium dye having two aniline rings, or an aniline ring and an indolenine ring, and is a fluorescent compound that exhibits fluorescence in the red region. On the other hand, squarylium dyes are highly planar molecules, and are known to easily associate with each other in a solution, leading to aggregation. The fluorescent compound of the present invention has an alkyl group containing an alkylene glycol structure and an alkyl group containing a carboxy group or a biological substance-binding group (a substituent capable of binding to a biological substance) at a specific position that can contribute to suppression of intermolecular association (in general formula (1), the substituent R on the nitrogen atom of the aniline structure). 1 , R 2 , R 11 and R 12 In the general formula (2), at least two of the substituents R 13 and R 14 and the substituent R on the nitrogen atom of the indolenine ring 19 and sp 3 Substituent R on a carbon atom 24 and R 25 (at least two of the above) and further, the compound contains at least one sulfo group. Therefore, it is possible to achieve good water solubility required for fluorescent labeling and suppression of intermolecular association and aggregation, and as a result, the fluorescence (emission) at the shoulder of the fluorescent peak is suppressed. Therefore, it is considered that it has the desired maximum absorption wavelength and maximum fluorescence wavelength suitable for flow cytometry, and can realize a high level of suppression of fluorescence spillover. In contrast, in the technology described in Patent Document 1 in which a rotaxane structure is used to suppress association and aggregation between squarylium dyes, the maximum fluorescence wavelength is shifted to the long wavelength side by the rotaxane structure, and as shown in the experimental results using comparative compound (1) and its labeled antibody described below, the rate of fluorescence leakage increases in flow cytometry applications.

[0017] The fluorescent compound of the present invention will be described in detail below.

[0018] (Fluorescent compound represented by general formula (1)) [ka]

[0019] In the formula, R 1 , R 2 , R 11 and R 12 represents an alkyl group. 1 , R 2 , R 11 and R 12 At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. R 3 , R 4 , R 9 and R 10 represents a hydrogen atom, a halogen atom, an alkyl group, an aryl group or a heteroaryl group. R 5 ~R 8 represents a hydrogen atom, a hydroxy group, an alkoxy group, an amino group, an acylamino group, or an alkyl group. However, the fluorescent compound represented by the above general formula (1) contains at least one sulfo group.

[0020] R 1 , R 2 , R 11 and R 12 represents an alkyl group. R 1 , R 2 , R 11 and R 12 The alkyl group which can be taken as the alkyl group preferably has 1 to 21 carbon atoms, more preferably 1 to 17 carbon atoms, and further preferably 1 to 13 carbon atoms. In addition, R 1 , R 2 , R 11 and R 12 The alkyl group that can be taken as the alkylene glycol structure (-O-(alkylene-O) m In the case where R is a group containing -), the number of carbon atoms is preferably 2 to 201, more preferably 2 to 13, and most preferably 4 to 9. 1 , R 2 , R11 and R 12 The alkyl group that can be taken as the alkylene glycol structure (-O-(alkylene-O) m When the group does not contain --), the number of carbon atoms is particularly preferably 1 to 9, and most preferably 1 to 6.

[0021] R 1 , R 2 , R 11 and R 12 The substituents that the alkyl group may have include an aryl group, -O-(alkylene-O) m -R 30 , sulfo group, phosphonooxy group, phosphono group, carboxy group or a group capable of binding to a biological substance is preferred; aryl group, -O-(alkylene-O) m -R 30 , a sulfo group, a carboxy group or a group capable of binding to a biological substance is more preferred. Here, the above "-O-(alkylene-O) m -R 30 "-(alkylene-O)" m -R 30 " refers to "-(alkylene-O)" in the substituent group T described later. m -R 30 " may be applied. The above "biological substance-binding group" can be applied to the description of the "biological substance-binding group" described below. These also apply to the following description of substituents.

[0022] R 1 , R 2 , R 11 and R 12 The aryl group that may be included in the alkyl group that can be taken as is preferably a group represented by the following general formula (AR): By having a bulky group represented by the following general formula (AR) on the nitrogen atom of the aniline structure, it becomes possible to position a group represented by general formula (AR) having an association-inhibiting effect close to the center of the squarylium skeleton, and association and aggregation can be effectively inhibited.

[0023] [ka]

[0024] In the formula, R A1 and R A2 is an alkoxy group or -O-(alkylene-O) m -R 30 Shows. R A3 and R A4 represents a hydrogen atom, a sulfo group, a phosphonooxy group, or a phosphono group. R A5 represents a hydrogen atom, an alkyl group, an alkoxy group, a carbamoyl group, an acylamino group, a carboxy group or a group capable of binding to a biological substance, preferably a hydrogen atom, an alkyl group or an alkoxy group, more preferably a hydrogen atom.

[0025] R A1 and R A2 The alkoxy group which can be taken as the aryl group preferably has 1 to 13 carbon atoms, more preferably 1 to 9 carbon atoms, further preferably 1 to 6 carbon atoms, and particularly preferably 1 to 3 carbon atoms. R A1 and R A2 Examples of the alkoxy group which can be taken include methoxy, ethoxy, and isopropyloxy. R A1 and R A2 The alkoxy group which may be taken as the alkoxy group is also preferably substituted with a sulfo group. R A3 and R A4 is preferably a hydrogen atom or a sulfo group. R A5 The carbamoyl group and acylamino group which can be taken as the alkyl group preferably have 1 to 13 carbon atoms, more preferably 1 to 9 carbon atoms, and further preferably 1 to 6 carbon atoms. R A5 Examples of the carbamoyl group that can be taken as R include N,N-dimethylcarbamoyl, N-cyclohexylcarbamoyl, and N-phenylcarbamoyl. A5Examples of the acylamino group that can be taken include acetylamino, cyclohexylcarbonylamino, and benzoylamino. R A5 The alkoxy group, carbamoyl group and acylamino group which may be taken as the radical are also preferably substituted with a sulfo group.

[0026] R 1 , R 2 , R 11 and R 12 Examples of the alkyl group that can be taken as the alkyl group include methyl, ethyl, isopropyl, propyl, tert-butyl, pentyl and heptyl, and the alkyl groups that can be taken as the alkyl group in the above-mentioned general formula (AR), the above-mentioned -O-(alkylene-O) m -R 30 Preferred examples of the aryl group include those substituted with a sulfo group, a phosphonooxy group, a phosphono group, a carboxy group, or a group capable of binding to a biological substance. R 1 , R 2 , R 11 and R 12 The group represented by the above general formula (AR), -(CH2-CH2-O) m -R 30 More preferred groups include carboxyalkyl groups, alkyl groups substituted with a group capable of binding to a biological substance, and sulfoalkyl groups.

[0027] However, R 1 , R 2 , R 11 and R 12 At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. In the present invention, the term "alkylene glycol structure" refers to -(alkylene-O) m The above "-(alkylene-O)" means a structure represented by the formula: m The "-" is "-(alkylene-O)" listed in the substituent group T described later. m -R 30 "-(alkylene-O)" m"-" can be used. m means the average number of repetitions, and is not particularly limited, but is preferably 1 to 100, more preferably 1 to 10, further preferably 2 to 9, and particularly preferably 3 to 8. R 1 , R 2 , R 11 and R 12 It is also preferred that at least one of the alkyl groups comprises a sulfoalkyl group. Among them, R 1 , R 2 , R 11 and R 12 Among these, it is preferable that at least one alkyl group contains a carboxy group or a group capable of binding to a biological substance, one to three alkyl groups contain an alkylene glycol structure, and one to three alkyl groups contain a sulfoalkyl group. 1 , R 2 , R 11 and R 12 may contain only one, or two or three, of a carboxy group or a biosubstance-binding group, an alkylene glycol structure, and a sulfoalkyl group in one substituent.

[0028] R 3 , R 4 , R 9 and R 10 represents a hydrogen atom, a halogen atom, an alkyl group, an aryl group or a heteroaryl group. R 3 , R 4 , R 9 and R 10 Examples of the halogen atom which can be taken as a halogen atom include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, with a chlorine atom being preferred. R 3 , R 4 , R 9 and R 10 The aryl group which can be taken as the radical has preferably 6 to 13 carbon atoms, more preferably 6 to 10 carbon atoms, and further preferably 6 to 8 carbon atoms. R 3 , R 4 , R 9 and R10 Examples of the aryl group which can be taken as the radical include phenyl and 1-naphthyl. R 3 , R 4 , R 9 and R 10 The heteroaryl group which can be taken as is preferably a group having at least one oxygen atom, sulfur atom or nitrogen atom, is preferably a monocyclic group, preferably has 5 or 6 ring members, and has preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms. R 3 , R 4 , R 9 and R 10 Examples of the heteroaryl group which can be taken as include 2-pyridyl, 4-pyridyl, 2-imidazolyl, 2-thiazolyl, and 2-oxazolyl. R 3 , R 4 , R 9 and R 10 The alkyl group which can be taken as the alkyl group preferably has 1 to 13 carbon atoms, more preferably 1 to 9 carbon atoms, further preferably 1 to 6 carbon atoms, and particularly preferably 1 to 3 carbon atoms. R 3 , R 4 , R 9 and R 10 Examples of the alkyl group that can be taken include methyl, ethyl, isopropyl, t-butyl, pentyl, heptyl, and 1-ethylpentyl. R 3 , R 4 , R 9 and R 10 is preferably a hydrogen atom.

[0029] R 5 ~R 8 represents a hydrogen atom, a hydroxy group, an alkoxy group, an amino group, an acylamino group, or an alkyl group. R 5 ~R 8 The alkoxy group which can be taken as the aryl group preferably has 1 to 13 carbon atoms, more preferably 1 to 9 carbon atoms, further preferably 1 to 6 carbon atoms, and particularly preferably 1 to 3 carbon atoms. R 5 ~R8 Examples of the alkoxy group which can be taken include methoxy, ethoxy, and isopropyloxy. R 5 ~R 8 Examples of the amino group that can be taken as the amino group include an unsubstituted amino group (-NH2), as well as a mono-substituted amino group and a di-substituted amino group substituted with a group selected from an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, and a heterocyclic group. Of these, an unsubstituted amino group (-NH2) or a mono-substituted amino group substituted with an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, or a heterocyclic group is preferable. R 5 ~R 8 The amino group which can be taken as the aryl group preferably has 0 to 12 carbon atoms, more preferably 0 to 8 carbon atoms, further preferably 0 to 6 carbon atoms, and particularly preferably 0 to 3 carbon atoms. R 5 ~R 8 Examples of the amino group that can be taken as the aryl group include unsubstituted amino (-NH2), N,N-dimethylamino, N,N-diethylamino, N-ethylamino, N-allylamino, N-(2-propynyl)amino, N-cyclohexylamino, N-cyclohexenylamino, anilino, pyridylamino, imidazolylamino, benzimidazolylamino, thiazolylamino, benzothiazolylamino, and triazinylamino. R 5 ~R 8 The acylamino group which can be taken as the alkyl group preferably has 1 to 13 carbon atoms, more preferably 1 to 9 carbon atoms, further preferably 1 to 6 carbon atoms, and particularly preferably 1 to 3 carbon atoms. R 5 ~R 8 Examples of the acylamino group that can be taken include acetylamino, cyclohexylcarbonylamino, and benzoylamino. R 5 ~R 8 The alkyl group that can be used as R 3 , R 4 , R 9 and R 10The descriptions of the alkyl groups that can be taken as above can be applied.

[0030] The planarity of the squarylium skeleton is improved by forming hydrogen bonds with the oxygen atoms in the squaric acid moiety, and the waveforms of the absorption and fluorescence spectra become sharper, which can further reduce the rate of fluorescence leakage. 5 ~R 8 is preferably the following combination: R 5 ~R 8 Among these, it is preferable that at least one is a hydroxy group, -NH2, a mono-substituted amino group, or an acylamino group, it is more preferable that at least two are a hydroxy group, -NH2, a mono-substituted amino group, or an acylamino group, and it is even more preferable that at least two are hydroxy groups. R 5 ~R 8 As a combination of at least two of 5 and R 6 At least one of and R 7 and R 8 It is preferable that the composition further comprises at least one of the following: In addition, R 5 ~R 8 Of these, the remaining group which is not a hydroxy group, an amino group or an acylamino group is preferably a hydrogen atom.

[0031] The fluorescent compound represented by the above general formula (1) contains at least one sulfo group in order to achieve good water solubility. Specifically, R 1 ~R 12 At least one of R is a substituent containing a sulfo group. 1 , R 2 , R 11 and R 12 It is preferred that at least one of the alkyl groups comprises a sulfoalkyl group. There is no particular upper limit to the number of sulfo groups in the compound. For example, the number of sulfo groups is preferably 1 to 8, more preferably 1 to 6, and even more preferably 1 to 4.

[0032] In addition, the fluorescent compound represented by the above general formula (1) is a -(CH2-CH2-O) compound in order to further suppress association between fluorescent compounds and between fluorescent compounds in a fluorescently labeled biological material and to make the waveforms of the absorption spectrum and fluorescence spectrum sharper. m -R 30 It is preferable that the alkyl group contains two or more substituted alkyl groups represented by the following formula: -(CH2-CH2-O) m -R 30 The substituted alkyl group represented by R 1 , R 2 , R 11 and R 12 It is preferable that at least one of the alkyl groups R 1 , R 2 , R 11 and R 12 At least one alkyl group among the groups has two or more -(CH2-CH2-O) m -R 30 and R may contain a substituted alkyl group represented by 1 , R 2 , R 11 and R 12 At least two of the alkyl groups are -(CH2-CH2-O) m -R 30 and each of the substituted alkyl groups represented by the following formula (1) may contain at least one substituted alkyl group represented by the following formula (1): In the compound -(CH2-CH2-O) m -R 30 There is no particular upper limit to the number of, for example, -(CH2-CH2-O) m -R 30 The number is preferably 1 to 8, more preferably 1 to 6, and even more preferably 1 to 4.

[0033] (Fluorescent compound represented by general formula (2)) [ka]

[0034] In the formula, R 13 , R14 , R 19 , R 24 and R 25 represents an alkyl group. 13 , R 14 , R 19 , R 24 and R 25 At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. R 15 and R 16 are the above-mentioned R 3 and R 4 is synonymous with R 17 and R 18 are the above-mentioned R 5 and R 6 is synonymous with. R 20 ~R 23 represents a hydrogen atom, a halogen atom, a hydroxy group, a carboxy group, a sulfo group, a phosphonooxy group, a phosphono group, an alkyl group, an aryl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, a carbonyl group, a sulfonyl group, an amino group, or a silyl group. However, the fluorescent compound represented by the above general formula (2) contains at least one sulfo group.

[0035] R 13 , R 14 , R 19 , R 24 and R 25 represents an alkyl group. R 13 , R 14 , R 19 , R 24 and R 25 The alkyl group that can be used as R 1 , R 2 , R 11 and R 12 The descriptions of the alkyl groups that can be taken as above can be applied. R 13 and R 14 The group represented by the above general formula (AR), -(CH2-CH2-O)m -R 30 More preferred groups include carboxyalkyl groups, alkyl groups substituted with a group capable of binding to a biological substance, and sulfoalkyl groups. R 19 , R 24 and R 25 As an example, -(CH2-CH2-O) m -R 30 More preferred groups include carboxyalkyl groups, alkyl groups substituted with a group capable of binding to a biological substance, and sulfoalkyl groups.

[0036] However, R 13 , R 14 , R 19 , R 24 and R 25 At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. Regarding the "alkylene glycol structure", see above R 1 , R 2 , R 11 and R 12 The description of "alkylene glycol structure" in can be applied. Among them, R 24 and R 25 At least one alkyl group is -(CH2-CH2-O) m -R 30 Preferably, R is a substituted alkyl group represented by the formula 13 , R 14 and R 19 It is preferred that at least one alkyl group among R contains a sulfo group. 13 , R 14 and R 19 It is more preferable that at least one of the alkyl groups comprises a sulfoalkyl group. More preferably, R 13 , R 14 , R 19 , R 24 and R 25 In R 13 , R 14 , R19 , R 24 and R 25 At least one alkyl group among R contains a carboxy group or a group capable of binding to a biological substance. 24 and R 25 At least one alkyl group is -(CH2-CH2-O) m -R 30 R is a substituted alkyl group represented by the formula 13 , R 14 and R 19 At least one of the alkyl groups in R contains a sulfo group (preferably a sulfoalkyl group). 13 , R 14 , R 19 , R 24 and R 25 may contain only one, or two or three, of a carboxy group or a biosubstance-binding group, an alkylene glycol structure, and a sulfoalkyl group in one substituent.

[0037] R 15 and R 16 is the above R 3 and R 4 and represents a hydrogen atom, a halogen atom, an alkyl group, an aryl group, or a heteroaryl group. Therefore, R 15 and R 16 The halogen atom, alkyl group, aryl group and heteroaryl group that can be taken as R 3 and R 4 The descriptions of the halogen atoms, alkyl groups, aryl groups and heteroaryl groups which can be taken as the substituents can also be applied. R 15 and R 16 is preferably a hydrogen atom.

[0038] R 17 and R 18 is the above R 5 and R 6 and represents a hydrogen atom, a hydroxy group, an alkoxy group, an amino group, an acylamino group, or an alkyl group. Therefore, the above R 17and R 18 The alkoxy group, amino group, acylamino group and alkyl group that can be taken as R 5 and R 6 The descriptions of the alkoxy group, amino group, acylamino group and alkyl group that can be taken as the aryl group can be applied.

[0039] The planarity of the squarylium skeleton is improved, the absorption and fluorescence spectra are sharper, and the fluorescence leakage rate can be further reduced. 17 and R 18 is preferably the following combination: R 17 and R 18 At least one of R is preferably a hydroxy group, -NH2, a mono-substituted amino group, or an acylamino group, and more preferably a hydroxy group. 17 and R 18 Of these, the remaining group which is not a hydroxy group, -NH2, a mono-substituted amino group or an acylamino group is preferably a hydrogen atom.

[0040] R 20 ~R 23 represents a hydrogen atom, a halogen atom, a hydroxy group, a carboxy group, a sulfo group, a phosphonooxy group, a phosphono group, an alkyl group, an aryl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, a carbonyl group, a sulfonyl group, an amino group, or a silyl group. R 20 ~R 23 The halogen atom, alkyl group and aryl group that can be taken as R 3 , R 4 , R 9 and R 10 The descriptions of halogen atoms, alkyl groups and aryl groups which may be taken as the substituents can be applied. R 20 ~R 23 The alkoxy group that can be taken as R 5 ~R 8 The description of the alkoxy group which can be taken as above can be applied. R20 ~R 23 The aryloxy group which can be taken as the aryloxy group preferably has 6 to 13 carbon atoms, more preferably 6 to 10 carbon atoms, and further preferably 6 to 8 carbon atoms. R 20 ~R 23 Examples of the aryloxy group which can be taken include phenoxy and 1-naphthyloxy. R 20 ~R 23 The alkylthio group which can be taken as the alkyl group preferably has 1 to 13 carbon atoms, more preferably 1 to 9 carbon atoms, further preferably 1 to 6 carbon atoms, and particularly preferably 1 to 3 carbon atoms. R 20 ~R 23 Examples of the alkylthio group which can be taken as the alkylthio group include methylthio, ethylthio and isopropylthio. R 20 ~R 23 The arylthio group which can be taken as the aryl group preferably has 6 to 13 carbon atoms, more preferably 6 to 10 carbon atoms, and further preferably 6 to 8 carbon atoms. R 20 ~R 23 Examples of the arylthio group which can be taken as the radical include phenylthio and 1-naphthylthio. R 20 ~R 23 Examples of the carbonyl group that can be taken as include an alkoxycarbonyl group, an alkylthiocarbonyl group, an aryloxycarbonyl group, and an arylthiocarbonyl group in the below-described substituent group T. The above descriptions of the alkoxy group, alkylthio group, aryloxy group, and arylthio group constituting the alkoxycarbonyl group, alkylthiocarbonyl group, aryloxycarbonyl group, and arylthiocarbonyl group can be applied to the alkoxy group, alkylthio group, aryloxy group, and arylthio group. R 20 ~R 23 Examples of the carbonyl group that can be taken as the radical include ethoxycarbonyl, 2-ethylhexyloxycarbonyl, ethylthiocarbonyl, 2-ethylhexylthiocarbonyl, phenyloxycarbonyl, naphthyloxycarbonyl, phenylthiocarbonyl, and naphthylthiocarbonyl. R 20 ~R 23 The sulfonyl group that can be taken as the sulfonyl group includes a sulfonyl group substituted with an alkyl group, a cycloalkyl group, or an aryl group. The above description of the alkyl group and aryl group can be applied to the alkyl group and aryl group that constitute the sulfonyl group substituted with an alkyl group or an aryl group. The carbon number of the cycloalkyl group that constitutes the sulfonyl group substituted with a cycloalkyl group is preferably 3 to 12, more preferably 3 to 8. R 20 ~R 23 Examples of the sulfonyl group that can be taken include methylsulfonyl, ethylsulfonyl, cyclohexylsulfonyl, and benzenesulfonyl. R 20 ~R 23 Examples of the amino group that can be taken as the aryl group include an unsubstituted amino group (-NH2), as well as mono- and di-substituted amino groups substituted with a group selected from an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, and a heterocyclic group. R 20 ~R 23 The number of carbon atoms and specific examples of the amino group that can be used as R 5 ~R 8 The carbon number and specific examples of the amino group that can be taken as above can be applied. R 20 ~R 23 Examples of the silyl group that can be taken as R include a silyl group substituted with an alkyl group, an aryl group, an alkoxy group, or an aryloxy group. The alkyl group, aryl group, alkoxy group, and aryloxy group constituting the silyl group substituted with an alkyl group, an aryl group, an alkoxy group, or an aryloxy group are 20 ~R 23 The alkyl and aryl groups that can be taken as R 20 ~R 23 The descriptions of the alkoxy group and aryloxy group constituting the alkoxycarbonyl group and aryloxycarbonyl group which can be taken as the alkyl group and the aryl group can be applied. R 20 ~R 23 Examples of the silyl group that can be taken as the silyl group include triethylsilyl, triphenylsilyl, diethylbenzylsilyl, and dimethylphenylsilyl.

[0041] R 20 ~R 23 is preferably a hydrogen atom or a sulfo group, more preferably at least one is a sulfo group and the rest are hydrogen atoms; R 22 It is more preferable that is a sulfo group and the remainder is a hydrogen atom.

[0042] The fluorescent compound represented by the above general formula (2) contains at least one sulfo group in order to achieve good water solubility. Specifically, R 13 ~R 25 At least one of R is a group containing a sulfo group as a substituent, or R 20 ~R 23 means that at least one of R is a sulfo group; 13 , R 14 , R 19 , R 24 and R 25 Preferably, at least one alkyl group among R comprises a sulfoalkyl group; 13 , R 14 and R 19 It is more preferable that at least one of the alkyl groups comprises a sulfoalkyl group. There is no particular upper limit to the number of sulfo groups in the compound. For example, the number of sulfo groups is preferably 1 to 8, more preferably 1 to 6, and even more preferably 1 to 4.

[0043] In addition, the fluorescent compound represented by the above general formula (2) is a -(CH2-CH2-O) compound, which is capable of suppressing association between fluorescent compounds and between fluorescent compounds in a fluorescently labeled biological material and sharpening the waveforms of the absorption spectrum and fluorescence spectrum. m -R 30 It is preferable that the alkyl group contains two or more substituted alkyl groups represented by the following formula: -(CH2-CH2-O) m -R 30 The substituted alkyl group represented by R 13 , R 14 , R 19 , R 24 and R 25 It is preferable that at least one alkyl group of R 13 , R 14 , R 19 , R 24 and R 25 One of the alkyl groups has two or more -(CH2-CH2-O) m -R 30 and R may contain a substituted alkyl group represented by 13 , R 14 , R 19 , R 24 and R 25 At least two of the alkyl groups are -(CH2-CH2-O) m -R 30 and each of the substituted alkyl groups represented by the following formula (1) may contain at least one substituted alkyl group represented by the following formula (1): In the compound -(CH2-CH2-O) m -R 30 There is no particular upper limit to the number of, for example, -(CH2-CH2-O) m -R 30 The number is preferably 1 to 8, more preferably 1 to 6, and even more preferably 1 to 4.

[0044] As described above, the fluorescent compound of the present invention is a fluorescent compound represented by the above general formula (1), 1 , R 2 , R 11 and R 12 In the fluorescent compound represented by the general formula (2), R 13 , R 14 , R 19 , R 24 and R 25 At least one of the above contains at least one carboxy group or a group capable of binding to a biological material. The fluorescent compound of the present invention can bind to a biological substance via a carboxy group or a biological substance-binding group described below to obtain a desired fluorescently labeled biological substance. The biological substance-binding group can be easily derived from the carboxy group by a conventional method. In the present invention, for convenience, the term "biological substance-binding group" does not include a carboxy group, and the term "biological substance-binding group" includes a biological substance-binding group derived from a carboxy group, although it is also possible to bind to a biological substance via a carboxy group, as described below. The number of the above-mentioned carboxy groups or biological substance-binding groups in the fluorescent compound of the present invention may be at least one in total, and from the viewpoint of quantifying the substance to be detected, 1 to 3 is preferable, 1 or 2 is more preferable, and 1 is even more preferable.

[0045] It is preferable that the fluorescent compound of the present invention is not used in combination with a cyclic molecule, that is, it is not used as a rotaxane compound in which the fluorescent compound of the present invention is included by a cyclic molecule as an axis molecule.

[0046] Specific examples of the fluorescent compound of the present invention are shown below, but the present invention is not limited to these compounds. In the following specific examples, -SO3 - X + X in + is Et3NH + where Et is an ethyl group.

[0047] [ka]

[0048] [ka]

[0049] The fluorescent compounds of the present invention can be bound to biological substances such as proteins (including peptides), amino acids, nucleic acids, nucleotides, sugar chains, and lipids via at least one biological substance-binding group possessed by the compounds, and can be used as fluorescently labeled biological substances. The biological material-binding group can be any group that acts on (including attaches to) or binds to a biological material, without any particular limitation. For example, the electrophilic groups and nucleophilic groups described in Table 2 of WO 2002 / 026891, and the reactive groups Rx described on page 18, line 16 to page 19, line 13 of WO 2002 / 026891 can be applied to the present invention. Specific examples of the "biological material-binding group" include the following structures.

[0050] [ka]

[0051] In the above specific examples of the "biological substance-binding group", X represents a halogen atom such as an iodine atom or a bromine atom. * represents a bond. In addition to the above, peptide structures (polyamino acid structures), long-chain alkyl groups, etc. can also be used as the "biological substance-binding group". Among these, preferred examples include an NHS ester structure (N-hydroxysuccinimide ester structure), a succinimide structure, a maleimide structure, an azide group, an acetylene group, a peptide structure (a polyamino acid structure), a long-chain alkyl group (preferably having 12 to 30 carbon atoms), and a quaternary ammonium group.

[0052] Among the fluorescent compounds of the present invention, specific examples of compounds having at least one biological substance binding group include, for example, the above-mentioned exemplary compounds of the present invention in which the carboxy group is appropriately replaced with the above-mentioned biological substance binding group. Note that 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 form a salt structure.

[0053] The fluorescent compound of the present invention can be synthesized by a conventional method. In addition to the synthesis examples described in the examples below, for example, the methods described in Dyes and Pigments, 2020, Vol. 180, Article 108457 and U.S. Patent Application Publication No. 2010 / 0266507 can also be referenced and applied. Compounds having a group capable of binding to a biological substance can also be synthesized by a conventional method, for example, Bioconjugate Techniques (Third Edition, by Greg T. Hermanson).

[0054] <<Fluorescently labeled biological materials>> The fluorescently labeled biological material of the present invention is a material in which the fluorescent compound of the present invention is bound to a biological material. The fluorescent compound of the present invention has fluorescence due to the fluorescent body portion, is excellent in water solubility, and shows an excellent aggregation suppression effect in a solution state, so it can be preferably used for the fluorescently labeled biological material. In particular, it can be preferably used as a fluorescent dye that can effectively suppress the leakage of fluorescence in flow cytometry, for example, as a fluorescent dye excited by a red laser and detected by a 670 / 14 filter. The binding between the fluorescent compound of the present invention and the biological material may be in the form of a direct binding between the fluorescent compound of the present invention and the biological material, or may be in the form of a linkage via a linking group.

[0055] Preferred examples of the biological material include proteins (including peptides), amino acids, nucleic acids, nucleotides, sugar chains, and lipids. Preferred examples of the protein include antibodies, and preferred examples of the lipid include phospholipids, fatty acids, and sterols, with phospholipids being more preferred. Among the above biological materials, examples of clinically pathologically useful materials include, but are not limited to, immunoglobulins such as Ig (Immunoglobulin) G, IgM, IgE, IgA, IgD, etc.; complement, C-reactive protein (CRP), ferritin, α1 microglobulin, β2 microglobulin, etc., plasma proteins and their antibodies; tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA (carbohydrate antigen) 19-9, CA-125, etc., and their antibodies; hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen, insulin, etc., and their antibodies; viral infection-related substances such as hepatitis B virus (HBV)-related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), adult T-cell leukemia (ATL), etc., and their antibodies; Further examples include bacteria such as Corynebacterium diphtheriae, Clostridium botulinum, Mycoplasma, Treponema pallidum, and antibodies thereto, protozoa such as Toxoplasma, Trichomonas, Leishmania, Trypanosoma, Plasmodium, and antibodies thereto, ES cells (embryonic stem cells) such as ELM3, HM1, KH2, v6.5, v17.2, v26.2 (derived from mouse 129, 129 / SV, C57BL / 6, BALB / c) and antibodies thereto, antiepileptic drugs such as phenytoin, phenobarbital, cardiovascular drugs such as quinidine, digoxin, antiasthmatic drugs such as theophylline, antibiotics such as chloramphenicol, gentamicin, and antibodies thereto, other enzymes, exotoxins (such as styrelidin O), and antibodies thereto, etc. Antibody fragments such as Fab'2, Fab, Fv, etc. can also be used.

[0056] Specific examples of the form in which the fluorescent compound of the present invention and a biological substance are bound through interaction include the forms described below. i) a non-covalent bond (e.g., hydrogen bond, ionic bond including chelate formation) or covalent bond between a peptide in the fluorescent compound of the present invention and a peptide in a biological material; ii) van der Waals forces between the long-chain alkyl group in the fluorescent compound of the present invention and the lipid bilayer and lipids in biological materials, iii) An amide bond formed by reaction of an NHS ester (N-hydroxysuccinimide ester) in the fluorescent compound of the present invention with an amino group in a biological material; iv) A thioether bond formed by reaction between a maleimide group in the fluorescent compound of the present invention and a sulfanyl group (-SH) in a biological material; v) formation of a triazole ring by a Click reaction between an azide group in the fluorescent compound of the present invention and an acetylene group in a biological material, or a Click reaction between an acetylene group in the fluorescent compound of the present invention and an azide group in a biological material; Examples include: However, in the above embodiment i), the peptide in the fluorescent compound of the present invention is not particularly limited as long as it is a peptide capable of forming a non-covalent bond or a covalent bond with a peptide in a biological material. Examples of the position having such a peptide include R 6 or R 7 are preferred. In addition to the above forms i) to v), binding can be performed in the form described in, for example, Lucas CD 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, pp. 62-83. In addition, the method described in the same document can be appropriately referred to in the preparation of the fluorescently labeled biological material of the present invention.

[0057] Among the fluorescent compounds of the present invention, the fluorescently labeled biological material of the present invention obtained from a compound having a biological material binding group and a biological material that binds to the compound through interaction includes compounds in which the portions other than the biological material binding group are replaced with the fluorescent compound of the present invention and the products thereof in the description of the compound examples and products in paragraph 0038 of JP 2019-172826 A. However, the present invention is not limited to these fluorescently labeled biological materials.

[0058] <Reagents containing fluorescently labeled biological substances> In a reagent containing the fluorescently labeled biological material of the present invention, the fluorescently labeled biological material of the present invention may be in the form of a solution dissolved in an aqueous medium such as physiological saline or phosphate buffer, or in the form of a solid such as a fine particle powder or a freeze-dried powder, without any particular limitation, and the form can be appropriately selected depending on the purpose of use, etc. For example, when the fluorescently labeled biological material of the present invention is used as a fluorescently labeled reagent, it can be used as a reagent containing the fluorescently labeled biological material in any of the above forms.

[0059] <Applications of fluorescently labeled biological materials> The fluorescently labeled biological material of the present invention obtained from the fluorescent compound of the present invention can suppress the fluorescence (emission) of the shoulder of the fluorescent peak due to the excellent aggregation suppression effect of the fluorescent compound of the present invention in a solution state, and it is considered that the distinguishability against the target biological material using other fluorescent dyes can be improved in multicolor fluorescent labeling applications. Therefore, the fluorescently labeled biological material of the present invention can be applied to various techniques using fluorescent labels, and for example, since it can effectively suppress the leakage of fluorescence in flow cytometry, it can also be used for multicolor simultaneous detection, and can be suitably used as a fluorescent dye excited by a red laser and detected by a 670 / 14 filter. It can also be suitably used as a fluorescent labeling reagent in multicolor WB or dot blotting or a biological fluorescent imaging reagent.

[0060] Fluorescence detection using the fluorescently labeled biological material of the present invention generally includes the following steps (i) to (iii) or (iv) to (vii): Fluorescence detection including steps (i) to (iii) corresponds to a direct method using a primary antibody fluorescently labeled with the fluorescent compound of the present invention, and fluorescence detection including steps (iv) to (vii) corresponds to an indirect method using a secondary antibody fluorescently labeled with the fluorescent compound of the present invention. (i) preparing the following (a) and (b): (a) A sample containing a target biological material (hereinafter also referred to as "target biological material"). (b) A fluorescently labeled biological material of the present invention (hereinafter also referred to as "fluorescently labeled biological material A of the present invention") in which a biological material capable of binding to the target biological material in (a) above (hereinafter also referred to as "primary biological material") is bound to a fluorescent compound of the present invention. (ii) preparing a conjugate (hereinafter also referred to as "fluorescently labeled conjugate A") in which the target biological material in (a) above is bound to the primary biological material in the fluorescently labeled biological material A of the present invention in (b) above. (iii) A step of irradiating the fluorescently labeled conjugate A with light in a wavelength range absorbed by the fluorescently labeled biological material A of the present invention and detecting the fluorescence emitted by the fluorescently labeled biological material A of the present invention. (iv) preparing the following (c) to (e) (c) a sample containing the target biological material; (d) A biological material capable of binding to the target biological material in (c) above (hereinafter also referred to as the "primary biological material"). (e) A fluorescently labeled biological material of the present invention in which a biological material capable of binding to the primary biological material (d) above (hereinafter also referred to as a "secondary biological material") is bound to a fluorescent compound of the present invention (hereinafter also referred to as "fluorescently labeled biological material B of the present invention"). (v) preparing a conjugate (hereinafter also referred to as "conjugate b") in which the target biological material in (c) and the primary biological material in (d) are bound to each other. (vi) preparing a conjugate (hereinafter also referred to as "fluorescently labeled conjugate B2") in which the primary biological material in the conjugate b is conjugated to the secondary biological material in the fluorescently labeled biological material B of the present invention; (vii) a step of irradiating the fluorescently labeled conjugate B2 with light in a wavelength range absorbed by the fluorescently labeled biological material B of the present invention and detecting the fluorescence emitted by the fluorescently labeled biological material B of the present invention.

[0061] Examples of the biological material (primary biological material) capable of binding to the target biological material and the biological material (secondary biological material) capable of binding to the primary biological material include the biological material in the fluorescently labeled biological material of the present invention. They can be appropriately selected according to the target biological material (biological material in a specimen) or the primary biological material, and a biological material capable of specifically binding to the biological material in a specimen or the primary biological material can be selected.

[0062] Among the above target biological substances, the protein may be a so-called disease marker. The disease marker is not particularly limited, but may be, for example, α-fetoprotein (AFP), PIVKA-II (protein induced by vitamin K absence or antagonist II), BCA (breast carcinoma-associated antigen) 225, basic fetoprotein (BFP), CA (carbohydrate antigen) 15-3, CA19-9, CA72-4, CA125, CA130, CA602, CA54 / 61 (CA546), carcinoembryonic antigen (CEA), DUPAN-2, elastase 1, immunosuppressive acidic protein (IAP), NCC-ST-439, γ-seminoprotein (γ-Sm), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), neurospecific enolase (NSE), Iba1, amyloid β, ta These include flotillin, squamous cell carcinoma-associated antigen (SCC antigen), sialyl LeX-i antigen (SLX), SPan-1, tissue polypeptide antigen (TPA), sialyl Tn antigen (STN), cytokeratin (CYFRA), pepsinogen (PG), C-reactive protein (CRP), serum amyloid A protein (SAA), myoglobin, creatine kinase (CK), troponin T, and ventricular myosin light chain I.

[0063] The target biological material may be a bacterium, including, but not limited to, bacteria that are subject to cellular microbiological testing, such as E. coli, Salmonella, Legionella, and bacteria that cause public health problems.

[0064] The target biological material may be a viral antigen, and examples of such viral antigens include, but are not limited to, hepatitis virus antigens such as antigens of hepatitis C and B viruses, p24 protein antigen of HIV virus, pp65 protein antigen of CMV (cytomegalovirus), E6 and E7 protein antigens of HPV (human papillomavirus), etc.

[0065] In the above (i) or (iv), the sample containing the target biological material is not particularly limited and can be prepared according to a conventional method. The fluorescently labeled biological material of the present invention is also not particularly limited and can be prepared by binding a biological material capable of binding to a target biological material with the fluorescent compound of the present invention in a conventional manner. The form of binding and the reaction for forming the bond are as explained for the fluorescently labeled biological material of the present invention.

[0066] In the above (v), the target biological material and the primary biological material may be directly bonded to each other, or may be bonded to each other via another biological material different from the target biological material and the primary biological material. Also, in the above (vi), the primary biological material in the conjugate b and the secondary biological material in the fluorescently labeled biological material B of the present invention may be bonded to each other, or may be bonded to each other via another biological material different from the primary biological material and the secondary biological material. The fluorescently labeled biological material of the present invention can be used as a fluorescently labeled antibody in either the direct method or the indirect method, but is preferably used as a fluorescently labeled antibody in the indirect method. In the above (ii) or (v) and (vi), the binding between the fluorescently labeled biological substance of the present invention and the target biological substance is not particularly limited and can be carried out according to a conventional method.

[0067] In the above (iii) or (vii), the wavelength for exciting the fluorescently labeled biological material of the present invention is not particularly limited as long as the light has a wavelength capable of exciting the fluorescently labeled biological material of the present invention. In general, the wavelength is preferably 350 to 800 nm, and more preferably 405 to 650 nm.

[0068] The fluorescence excitation light source used in the present invention is not particularly limited as long as it emits light of a wavelength capable of exciting the fluorescently labeled biological material of the present invention, and for example, various laser light sources can be used. In addition, various optical filters can be used to obtain a preferred excitation wavelength or to detect only fluorescence in a specific wavelength range.

[0069] Other matters in the above (i) to (vii) are not particularly limited, and conditions such as techniques, reagents, and devices that are usually used in fluorescence detection using fluorescent labels can be appropriately selected. For steps other than the above (i) to (vii), conditions of commonly used techniques, reagents, devices, etc. can be appropriately selected in accordance with various techniques using fluorescent labels.

[0070] For example, flow cytometry using the fluorescently labeled biological material of the present invention can be used for simultaneous multi-color detection by preparing a cell suspension in which the target biological material is dispersed into single cells using a method commonly used, and using the fluorescently labeled biological material of the present invention as a labeled antibody (preferably, a secondary antibody), which can effectively suppress leakage of fluorescence to be detected in one detection channel into another detection channel. Multicolor WB using the fluorescently labeled biological material of the present invention involves preparing a blot membrane using techniques commonly used for the target biological material (protein separation by electrophoresis, blotting to a membrane, and blocking of the membrane), and using the fluorescently labeled biological material of the present invention as a labeled antibody (preferably, a secondary antibody), thereby enabling the detection of the target biological material with high discrimination against target biological materials using other fluorescent dyes. As with multicolor WB, in dot blotting using the fluorescently labeled biological material of the present invention, a blot nitrocellulose membrane or a blot PVDF (polyvinylidene fluoride) membrane is prepared by a commonly used method for the target biological material, and the fluorescently labeled biological material of the present invention is used as a labeled antibody (preferably a secondary antibody), thereby making it possible to detect the target biological material with high discrimination against target biological materials using other fluorescent dyes.

[0071] - Substituent group T - In the present invention, when a substituent is merely described, the description of the corresponding substituent in the substituent group T can be referred to and applied. For example, when an "alkyl group" is merely described, the description of the "alkyl group" in the substituent group T can be referred to and applied. The same applies to other substituents other than the "alkyl group". In the present invention, examples of the substituent that a certain substituent such as an "alkyl group" may have include substituents selected from the following substituent group T. In addition, when a certain substituent such as an "alkyl group" has a substituent and further has a substituent, examples of the substituent that the certain substituent has include a substituent consisting of a combination of two or more substituents selected from the following substituent group T. Furthermore, in the present invention, when the alkyl group is described in distinction from the cyclic (cyclo) alkyl group, the alkyl group is used to include linear alkyl groups and branched alkyl groups. On the other hand, when the alkyl group is not described in distinction from the cyclic alkyl group, and unless otherwise specified, the alkyl group is used to include linear alkyl groups, branched alkyl groups, and cycloalkyl groups. This also applies to groups (alkoxy groups, alkylthio groups, alkoxycarbonyl groups, alkylthiocarbonyl groups, alkylsulfonyl groups, etc.) containing groups that can have a cyclic structure (alkyl groups, alkenyl groups, alkynyl groups, etc.) and compounds containing groups that can have a cyclic structure. When a group can form a cyclic skeleton, the lower limit of the number of atoms of the group that forms the cyclic skeleton is 3 or more, preferably 4 or more, and more preferably 5 or more, regardless of the lower limit of the number of atoms specifically described below for groups that can have this structure. In the description of the substituent group T below, a group having a linear or branched structure and a group having a cyclic structure are sometimes described separately, for example, an alkyl group and a cycloalkyl group, in order to clarify the difference.

[0072] The groups contained in the substituent group T include the following groups.

[0043] The alkyl group may be an alkyl group (preferably having 1 to 30 carbon atoms, more preferably having 1 to 20 carbon atoms, even more preferably having 1 to 12 carbon atoms, even more preferably having 1 to 8 carbon atoms, even more preferably having 1 to 6 carbon atoms, and particularly preferably having 1 to 3 carbon atoms), an alkenyl group (preferably having 2 to 30 carbon atoms, more preferably having 2 to 20 carbon atoms, even more preferably having 2 to 12 carbon atoms, even more preferably having 2 to 6 carbon atoms, and even more preferably having 2 to 4 carbon atoms), an alkynyl group (preferably having 2 to 30 carbon atoms, more preferably having 2 to 20 carbon atoms, even more preferably having 2 to 12 carbon atoms, even more preferably having 2 to 6 carbon atoms, and even more preferably having 2 to 4 carbon atoms), a cycloalkyl group (preferably having 3 to 20 carbon atoms), a cycloalkenyl group (preferably having 4 to 20 carbon atoms), or an aryl group (which may be a monocyclic group or a condensed ring group (preferably a condensed ring group of 2 to 6 rings). In the case of a condensed ring group, it is preferably a 6- or 8-membered ring. The monocyclic ring is preferably a 6-membered ring. The aryl group is preferably a group having 6 to 40 carbon atoms, more preferably 6 to 30 carbon atoms, further preferably 6 to 26 carbon atoms, particularly preferably 6 to 10 carbon atoms), a heterocyclic group (a group having at least one nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom or selenium atom (preferably at least one oxygen atom, sulfur atom or nitrogen atom) as a ring-constituting atom, and may be a monocyclic group or a condensed ring group (preferably a condensed ring group of 2 to 6 rings). The number of ring members in the ring of the monocyclic group and the ring constituting the condensed ring group is preferably 5 to 7 members, more preferably 5 or 6 members. The number of carbon atoms in the heterocyclic group is preferably 2 to 40, more preferably 2 to 20. The heterocyclic group includes an aromatic heterocyclic group (heteroaryl group) and an aliphatic heterocyclic group (aliphatic heterocyclic group).), an alkoxy group (preferably having 1 to 20 carbon atoms, more preferably having 1 to 12 carbon atoms, even more preferably having 1 to 8 carbon atoms, still more preferably having 1 to 6 carbon atoms, and particularly preferably having 1 to 3 carbon atoms), an alkenyloxy group (preferably having 2 to 20 carbon atoms, more preferably having 2 to 12 carbon atoms), an alkynyloxy group (preferably having 2 to 20 carbon atoms, more preferably having 2 to 12 carbon atoms), a cycloalkyloxy group (preferably having 3 to 20 carbon atoms), an aryloxy group (preferably having 6 to 40 carbon atoms, more preferably having 6 to 26 carbon atoms, and still more preferably having 6 to 14 carbon atoms), a heterocyclic oxy group (preferably having 2 to 20 carbon atoms), and a polyalkyleneoxy group.

[0073] An alkoxycarbonyl group (preferably having 2 to 20 carbon atoms), a cycloalkoxycarbonyl group (preferably having 4 to 20 carbon atoms), an alkoxythiocarbonyl group (preferably having 2 to 20 carbon atoms), an aryloxycarbonyl group (preferably having 6 to 20 carbon atoms), an arylthiocarbonyl group (preferably having 6 to 20 carbon atoms), an amino group (preferably having 0 to 20 carbon atoms, more preferably having 0 to 12 carbon atoms, still more preferably having 0 to 8 carbon atoms, still more preferably having 0 to 6 carbon atoms, particularly preferably having 0 to 3 carbon atoms), an unsubstituted amino group (-NH2), and an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, and a heterocyclic group. The above-mentioned groups substituting the unsubstituted amino group include mono- and di-substituted amino groups substituted with a group. The above-mentioned groups substituting the unsubstituted amino group have the same meaning as the corresponding groups in the substituent group T.), a sulfamoyl group (preferably having 0 to 20 carbon atoms, and preferably an alkyl, cycloalkyl or aryl sulfamoyl group), an acyl group (preferably having 1 to 20 carbon atoms, more preferably having 2 to 15 carbon atoms, and including -C(=O)H, an alkylcarbonyl group, a cycloalkylcarbonyl group, an arylcarbonyl group or a heterocyclic carbonyl group), an acyloxy group (preferably having 1 to 20 carbon atoms), a carbamoyl group (preferably having 1 to 20 carbon atoms, and preferably an alkyl, cycloalkyl or aryl carbamoyl group),

[0074] acylamino group (preferably having 1 to 20 carbon atoms, including an alkylcarbonylamino group, a cycloalkylcarbonylamino group, an arylcarbonylamino group, or a heterocyclic carbonylamino group), a sulfonamido group (preferably having 0 to 20 carbon atoms, and preferably an alkyl, cycloalkyl, or aryl sulfonamido group), an alkylthio group (preferably having 1 to 20 carbon atoms, more preferably having 1 to 12 carbon atoms), a cycloalkylthio group (preferably having 3 to 20 carbon atoms), an arylthio group (preferably having 6 to 40 carbon atoms, more preferably having 6 to 26 carbon atoms, and even more preferably having 6 to 14 carbon atoms), a heterocyclic thio group (preferably having 2 to 20 carbon atoms), an alkyl, cycloalkyl, or arylsulfonyl group (preferably having 1 to 20 carbon atoms),

[0075] Silyl group (preferably a silyl group having 1 to 30 carbon atoms, more preferably a silyl group having 1 to 20 carbon atoms and substituted with an alkyl, aryl, alkoxy or aryloxy), silyloxy group (preferably a silyloxy group having 1 to 20 carbon atoms and substituted with an alkyl, aryl, alkoxy or aryloxy), hydroxy group, cyano group, nitro group, halogen atom (e.g., fluorine atom, chlorine atom, bromine atom or iodine atom), oxygen atom (specifically, a silyl group having 1 to 30 carbon atoms, more preferably a silyl group having 1 to 20 carbon atoms and substituted with an alkyl, aryl, alkoxy or aryloxy), Examples of the alkyl group include a carboxy group (-CO2H), a phosphono group [-PO(OH)2], a phosphonooxy group [-O-PO(OH)2], a sulfo group (-SO3H), a boric acid group [-B(OH)2], an onio group (also called a cationic group, which includes an ammonio group including a cyclic ammonio group, a sulfonio group, and a phosphonio group, and which preferably has 0 to 30 carbon atoms, and more preferably has 1 to 20 carbon atoms), a sulfanyl group (-SH), a guanidino group (-NHC(=NH)NH2), a betaine residue, a polyol residue, a sugar residue, or a polyamino acid residue.

[0076] (Polyalkyleneoxy group) In the present invention, the polyalkyleneoxy group is -(alkylene-O) m -R 30 The group may be any group represented by the following formula: Among the polyol residues, those which fall under the category of polyalkyleneoxy groups are classified as polyalkyleneoxy groups. "-(alkylene-O) m The alkylene group in "-" may be linear or branched, and preferably has 2 to 10 carbon atoms, more preferably has 2 to 6 carbon atoms, further preferably has 2 to 4 carbon atoms, and particularly preferably has 2 or 3 carbon atoms. "-(alkylene-O) m Examples of the alkylene group in "-" include an ethylene group, a 1,2-propanediyl group, a 1,3-propanediyl group, a 1,2-butanediyl group, a 1,3-butanediyl group, and a 1,4-butanediyl group. Of these, an ethylene group, a 1,2-propanediyl group, or a 1,2-butanediyl group is preferable, and an ethylene group is more preferable. m means the average number of repetitions, and is not particularly limited, but is preferably 1 to 100, more preferably 1 to 10, further preferably 2 to 9, and particularly preferably 3 to 8. In the present invention, the average in the average number of repetitions means a number average, and is a value measured and calculated, for example, by GPC (gel permeation chromatography) or NMR (nuclear magnetic resonance analysis). Even when the number of repetitions is small, such as m being 1, it is possible to exhibit appropriate hydrophilicity and appropriate excluded volume effect. R 30 R represents a hydrogen atom or an alkyl group. 30 The alkyl group that can be taken as R preferably has 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms. 30 A preferred example of the substituent that the alkyl group may have is a sulfo group.

[0077] Furthermore, examples of groups formed by combining a plurality of substituents selected from the substituent group T include the above-mentioned alkyl groups, cycloalkyl groups, aryl groups, heterocyclic groups, alkoxy groups, aryloxy groups, heterocyclic oxy groups, alkoxycarbonyl groups, aryloxycarbonyl groups, amino groups, carbamoyl groups, acylamino groups, alkylthio groups, arylthio groups, alkyl, cycloalkyl or arylsulfonyl groups, each of which has as a substituent at least one of groups selected from a carboxy group, a phosphono group, a sulfo group and a polyalkyleneoxy group.

[0078] The substituents selected from the substituent group T include groups formed by combining a plurality of the above-mentioned groups, unless otherwise specified. For example, when the compound or the substituent contains an alkyl group, an alkenyl group, etc., these may be substituted or unsubstituted. When the compound or the substituent contains an aryl group, a heterocyclic group, etc., these may be monocyclic or condensed, and may be substituted or unsubstituted. EXAMPLES

[0079] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited thereto. Room temperature means 25°C.

[0080] The compounds (1-1) to (1-3) and (2-1), which are the fluorescent compounds represented by general formula (1) or (2) of the present invention, are shown below. In the chemical structure and synthesis examples below, -SO3 - X + X in + is Et3NH + where Et is an ethyl group.

[0081] [ka]

[0082] (Comparative Compound) Comparative compound (1) is Squaraine-Rotaxane 10, a compound described in paragraph 0245 of U.S. Patent Application Publication No. 2010 / 0266507, whose structure is shown below.

[0083] [ka]

[0084] Comparative compound (2) is Alexa Fluor TM 647 (trade name, Invitrogen, cyanine fluorescent dye).

[0085] The synthesis method of each compound will be described in detail below, but the starting materials, dye intermediates and synthesis routes are not limited to these.

[0086] Additionally, %v / v means percent by volume, and %w / v means percent by weight by volume.

[0087] Unless otherwise specified, the carrier used in reverse phase column chromatography was Sfar C18 (trade name, manufactured by Biotage), and the carrier used in normal phase column chromatography was Hi-Flash Column (trade name, manufactured by Yamazen). The mixing ratio of the eluent used in reversed-phase column chromatography or normal-phase column chromatography is a volume ratio. For example, "acetonitrile:water = 0:100 to 20:80" means that the eluent "acetonitrile:water = 0:100" was changed to the eluent "acetonitrile:water = 20:80". For preparative HPLC (High Performance Liquid Chromatography), 2767 (trade name, manufactured by Waters) was used.

[0088] MS spectra were measured using an ACQUITY SQD LC / MS System (trade name, manufactured by Waters Corporation; ionization method: ESI (ElectroSpray Ionization)) or an LCMS-2010EV (trade name, manufactured by Shimadzu Corporation; ionization method: simultaneous ESI and APCI (Atmospheric Pressure Chemical Ionization)).

[0089] <Synthesis of Compounds> Synthesis Example 1-1: Synthesis of Compound (1-1) [ka]

[0090] 300 mg of 3-aminophenol, 0.57 g of potassium carbonate, and 2-propanol were placed in a 100 ml three-neck flask, immersed in an oil bath at 80°C, and heated to reflux. A solution of 1.86 g of triethylene glycol-2-bromoethyl methyl ether dissolved in 8 ml of 2-propanol was slowly added dropwise thereto, and reacted for 5 hours. The mixture was returned to room temperature, the solvent was removed under reduced pressure, and the mixture was dissolved in 2 ml of ethyl acetate. The mixture was purified by silica gel column chromatography using ethyl acetate and hexane as eluents, yielding 300 mg of compound (1-1A) and 50 mg of compound (1-2A). 100 mg of compound (1-1A) and 3 ml of 2-propanol were placed in a 100 ml three-neck flask, 0.03 ml of 1,3-propane sultone was added while stirring, and the mixture was heated to reflux in an oil bath at 85°C. After the reaction was completed, 0.093 ml of triethylamine was added and stirred, and the solvent was distilled off under reduced pressure. This was purified by reverse phase column chromatography using acetonitrile and water as eluents, to obtain 60 mg of compound (1-1B). The reaction was carried out in the same manner as in the synthesis of compound (1-1A), except that 1.66 g of 1,3-propane sultone was used instead of triethylene glycol-2-bromoethyl methyl ether and 1 g of 3-aminophenol was reacted therewith. The reaction was purified by reverse phase column chromatography using acetonitrile and water as an eluent to synthesize 667 mg of compound (1-1C). The reaction was carried out in the same manner as in the synthesis of compound (1-1B), except that 73 mg of 6-bromohexanoic acid was used instead of 1,3-propane sultone and 80 mg of compound (1-1C) was reacted instead of compound (1-1A). The reaction was purified by reverse phase column chromatography using acetonitrile and water as an eluent to synthesize 90 mg of compound (1-1D). 12 mg of compound (1-1B), 10 mg of compound (1-1D), 3,4-dihydroxy-3-cyclobutene-1,2-dione, and 3 ml of 1-butanol were placed in a 100 ml three-neck flask and heated to reflux for 3 hours using an oil bath heated to 130° C. The solvent was distilled off under reduced pressure, and the residue was purified by reverse phase column chromatography using acetonitrile / water as an eluent to obtain 3 mg of compound (1-1). [M-2Et3N+H + ] + =845

[0091] Synthesis Example 1-2: Synthesis of Compound (1-2) Compound (1-2) (3 mg) was synthesized in the same manner as in the synthesis of compound (1-1) above, except that compound (1-2A) was used instead of compound (1-1B). [M―Et3N+H + ] + =913

[0092] Synthesis Example 1-3: Synthesis of compound (1-3) [ka]

[0093] 2.0g of 2,6-dihydroxybenzaldehyde, 40ml of dichloromethane, and 20ml of acetonitrile were placed in a 300ml three-neck flask and cooled to 0°C while stirring. 1.15ml of chlorosulfonic acid was added dropwise, and after reacting for 50 minutes, 4.8ml of triethylamine was slowly added dropwise. The mixture was returned to room temperature, the solvent was distilled off under reduced pressure, and the mixture was purified by reverse phase column chromatography using acetonitrile and water as eluents to obtain 4.2g of compound (1-3A). 2 g of compound (1-3A), 15 ml of N,N-dimethylformamide, 2.45 g of potassium carbonate, and 2.88 ml of triethylene glycol-2-bromoethyl methyl ether were placed in a 100 ml three-neck flask and reacted for 5 hours using an oil bath at 120° C. The mixture was returned to room temperature and purified by reverse phase column chromatography using acetonitrile and water as eluents to obtain 1.39 g of compound (1-3B). 550 mg of compound (1-3B), 11 ml of tetrahydrofuran, 0.153 g of 3-aminophenol, and 53 μl of acetic acid were added to a 50 ml eggplant flask and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the mixture was purified by reverse phase column chromatography using acetonitrile and water as eluents to obtain 376 mg of compound (1-3C). 50 mg of compound (1-3C), 1 ml of 2-propanol, and 4 μl of 1,3-propane sultone were placed in a 50 ml eggplant flask and stirred, and 6 μl of triethylamine was added. The mixture was heated and stirred for 4 hours using a 60°C oil bath. The mixture was returned to room temperature and purified by reverse phase column chromatography using acetonitrile and water as eluents to obtain 20 mg of compound (1-3D). Compound (1-3) (2 mg) was synthesized in the same manner as in the synthesis of compound (1-1) above, except that compound (1-3D) was used instead of compound (1-1B). [M-3Et3N+H + ] + =1237

[0094] Synthesis Example 1-4: Synthesis of compound (2-1) [ka]

[0095] 100 mg of the compound (2-E) described in International Publication No. 2022 / 265043, 3 ml of ethanol, and 0.482 ml of 3,4-diethoxy-3-cyclobutene-1,2-dione were placed in a 100 ml three-necked flask, and 1 ml of triethylamine was added while stirring. Heating under reflux was carried out using an oil bath at 100 °C. The solvent was distilled off under reduced pressure, and purification was performed by reverse-phase column chromatography using acetonitrile and water as eluents to obtain 66 mg of the compound (2-1A). 66 mg of the compound (2-1A) and 1 ml of distilled water were placed in a 50 ml eggplant flask, 5.7 μl of a 50% aqueous sodium hydroxide solution was added while stirring, and the mixture was stirred at room temperature for 1.5 hours. Purification was performed by reverse-phase column chromatography using acetonitrile and water as eluents to obtain 48 mg of the compound (2-1B). 4.5 mg of the compound (2-1B), 3 mg of the compound (1-1D), 3 ml of 1-butanol, and 1.3 ml of pyridine were placed in a 100 ml three-necked flask, and the mixture was heated and stirred at 130 °C for 4 hours in an oil bath. The solvent was distilled off under reduced pressure, and purification was performed by reverse-phase column chromatography using acetonitrile and water as eluents to obtain 3 mg of the compound (2-1). [M - 3Et3N + H + + = 961

[0096] <Synthesis of NHS ester compounds> Synthesis Example 2-1: Synthesis of the compound (1-3-NHS) To a 50 mL eggplant flask, 2 mg of the compound (1-3), 40 μL of N,N-dimethylformamide (DMF), 3 μg of N-hydroxysuccinimide, and 7.8 μL of N,N'-diisopropylcarbodiimide (DIC) were added, and the mixture was stirred at room temperature for 3.5 hours. After completion of the reaction, ethyl acetate was added, and the compound (1-3-NHS) was obtained by filtration under reduced pressure.

[0097]

Chemical formula

[0098] Synthesis Example 2-2: Synthesis of the compound (2-1-NHS) ​Compound (2-1-NHS) was obtained in the same manner as in the preparation of compound (1-3-NHS) above, except that compound (2-1) was used instead of compound (1-3). Compound (2-1-NHS) is a compound in which the carboxy group in compound (2-1) is replaced with an NHS ester group, similar to the carboxy group in compound (1-3).

[0099] <Preparation of labeled antibodies> In a microtube, 217 μL of anti-rabbit IgG antibody (2.3 mg / ml) and 21.7 μL of carbonate buffer were placed and stirred by shaking, and then the dimethyl sulfoxide solution of the above compound (1-3-NHS) was added in an amount of 10 molar equivalents per equivalent of antibody, and further stirred by shaking. The mixture was left to stand at room temperature for 1 hour, and the reaction solution was purified using gel filtration column chromatography PD10 (GE Healthcare Life Sciences) and PBS solution (phosphate buffered saline), to obtain a labeled antibody of compound (1-3) in which the anti-rabbit IgG antibody was labeled with the above compound (1-3). A labeled antibody of compound (2-1) was obtained in the same manner as in the preparation of the labeled antibody of compound (1-3) above, except that compound (2-1-NHS) was used instead of compound (1-3-NHS). A labeled antibody of the comparative compound (1) was obtained in the same manner as in the preparation of the labeled antibody of the above compound (1-3), except that the compound 10-NHS described in paragraph 0247 of U.S. Patent Application Publication No. 2010 / 0266507 was used instead of the compound (1-3-NHS). In the preparation of the labeled antibody of the above compound (1-3), Alexa Fluor was used instead of the compound (1-3-NHS). TM A labeled antibody of comparative compound (2) was obtained in the same manner, except that 647 NHS ester (trade name, manufactured by Invitrogen, cyanine fluorescent dye) was used. For each of the labeled antibodies, the fluorescent labeling rate was calculated by the method for calculating the fluorescent labeling rate described in paragraph 0187 of WO 2022 / 265043. For all of the labeled antibodies, the values ​​were in the range of 2 to 5, and the binding to the antibody was at a sufficient level without practical problems.

[0100] The fluorescence leakage rate and water solubility of each of the above compounds and labeled antibodies were evaluated as follows. The results are shown in Table 1.

[0101] [Evaluation 1] Evaluation of fluorescence leakage rate The absorption spectrum of each compound or each labeled antibody in PBS solution was measured using a spectrophotometer UV-3600 (trade name) manufactured by Shimadzu Corporation, and the concentration of the solution was adjusted so that the absorbance (Abs) of the absorption maximum peak in the wavelength region of 600 nm or more was within the range of 0.1 to 0.2 to prepare a measurement solution. The absorption spectra of compounds (1-1) to (1-3) are shown by solid lines in Figures 1 to 3, respectively. Next, the fluorescence spectrum of the measurement solution was measured by irradiating it with 595 nm excitation light set to a bandwidth of 3 nm using a spectrofluorometer RF-6000 (trade name, excitation light source: xenon light source) manufactured by Shimadzu Corporation. The fluorescence spectra of compounds (1-1) to (1-3) are shown by dashed lines in Figures 1 to 3, respectively. Each compound and each labeled antibody has a maximum absorption peak in the wavelength range of 640 to 660 nm, and the Stokes shift of fluorescence is small. Therefore, in order to avoid scattering of the excitation light by the measurement sample, the measurement was performed using an excitation light of 595 nm, which is located on the short wavelength side of the maximum absorption peak. Using the obtained fluorescence spectrum, the integral value (area) of the fluorescence intensity in the wavelength range corresponding to the transmission wavelength range of each bandpass filter used in flow cytometry, S A ~S C was calculated. S A ~S C are as follows, respectively: S A :Integrated fluorescence intensity in the 663-677 nm wavelength range, which is the transmission wavelength range of the 670 / 14 filter S B :Integrated fluorescence intensity in the 705-735 nm wavelength range, which is the transmission wavelength range of the 720 / 30 filter S C :Integrated fluorescence intensity in the 750-810 nm wavelength range, which is the transmission wavelength range of the 780 / 60 filter Each compound and each labeled antibody exhibits a maximum fluorescence peak in the wavelength range of 660 to 675 nm, and can therefore be used for detection using a 670 / 14 filter. The obtained S A ~S C The leakage rate into the 720 / 30 filter and the leakage rate into the 780 / 60 filter were calculated using the following formula, and the results were evaluated based on the following evaluation ranks. [Leakage rate into 720 / 30 filter] = (S B / S A )×100% [Leakage rate into 780 / 60 filter] = (S C / S A )×100% - Rating Rank - SS: Less than 5% S: 5% to less than 10% AA: 10% to less than 15% A: 15% to less than 25% B: 25% or more but less than 30% C: 30% or more but less than 35% D: 35% or more but less than 40% E: 40% or more

[0102] [Evaluation 2] Evaluation of compound water solubility A PBS solution of each compound with a concentration of 1 mg / ml was prepared at room temperature, and the formation of precipitate was visually confirmed to evaluate water solubility. When no precipitate was observed, it means that the compound has good water solubility. In Table 1 below, when no precipitate was observed, it is indicated by "◯", and when precipitate was observed, it is indicated by "×". Labeled antibodies were not evaluated, so they are indicated by "-".

[0103] [Table 1]

[0104] (Table Notes) Binding site with biological substance: When a compound contains a carboxyl group or a group capable of binding to a biological substance, it is indicated by "〇". Note that for labeled antibodies, they are indicated by "-" since they are already bound to the antibody, which is a biological substance.

[0105] The results in Table 1 above reveal the following: Comparative compound (1) is R 1 , R 2 , R 11 and R 12 is a group not containing an alkylene glycol structure, and is therefore not a fluorescent compound represented by general formula (1) of the present invention. Comparative compound (2) is a commercially available cyanine fluorescent dye, and is not a squarylium fluorescent compound represented by general formula (1) or (2) of the present invention. These comparative compounds (1) and (2) and their labeled antibodies all had a large [leakage rate into a 720 / 30 filter], and when used as a fluorescent dye for detection with a 670 / 14 filter in flow cytometry, fluorescence leakage into other filters occurred. In contrast, the fluorescent compounds of the present invention, compounds (1-1) to (1-3) and (2-1), and the labeled antibodies of compounds (1-3) and (2-1), all exhibited good water solubility, had small [leakage rate into 720 / 30 filter] and [leakage rate into 780 / 60 filter], and could suppress the leakage of fluorescence into other filters as fluorescent dyes for detection with a 670 / 14 filter in flow cytometry. In particular, the labeled antibodies of fluorescent compounds (1-1) to (1-3) and compound (1-3) represented by general formula (1) had a smaller [leakage rate into 720 / 30 filter] than the labeled antibodies of fluorescent compounds (2-1) and compound (2-1) represented by general formula (2), and could suppress the leakage of fluorescence into the 720 / 30 filter, which has a closer wavelength range, as fluorescent dyes for detection with a 670 / 14 filter in flow cytometry.

Claims

1. A fluorescent compound represented by the following general formula (1) or (2): 【Chemistry 1】 In the formula, R １ , R ２ , R １１ and R １２ represents an alkyl group. １ , R ２ , R １１ and R １２ At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. R ３ , R ４ , R ９ and R １０ represents a hydrogen atom, a halogen atom, an alkyl group, an aryl group or a heteroaryl group. R ５ ~R ８ represents a hydrogen atom, a hydroxy group, an alkoxy group, an amino group, an acylamino group, or an alkyl group. However, the fluorescent compound represented by the above general formula (1) contains at least one sulfo group. 【Chemistry 2】 In the formula, R １３ , R １４ , R １９ , R ２４ and R ２５ represents an alkyl group. １３ , R １４ , R １９ , R ２４ and R ２５ At least one of the alkyl groups contains an alkylene glycol structure, and at least one of the remaining alkyl groups contains a carboxy group or a group capable of binding to a biological material. R １５ and R １６ are the above R ３ and R ４ is synonymous with R １７ and R １８ are the above R ５ and R ６ is synonymous with. R ２０ ~R ２３ represents a hydrogen atom, a halogen atom, a hydroxy group, a carboxy group, a sulfo group, a phosphonooxy group, a phosphono group, an alkyl group, an aryl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, a carbonyl group, a sulfonyl group, an amino group, or a silyl group. However, the fluorescent compound represented by the above general formula (2) contains at least one sulfo group.

2. The R in the general formula (1) ５ ~R ８ The fluorescent compound of claim 1 , wherein at least two of the are hydroxy groups.

3. The R in the general formula (2) １７ and R １８ The fluorescent compound of claim 1 , wherein at least one of is a hydroxy group.

4. The fluorescent compound represented by the general formula (1) is -(CH ２ -CH ２ -O) ｍ -R ３０ The fluorescent compound according to claim 1 , comprising two or more of the following: m is 1 to 10, R ３０ is a hydrogen atom or an alkyl group.

5. The R in the general formula (1) １ , R ２ , R １１ and R １２ 2. The fluorescent compound of claim 1, wherein at least one alkyl group comprises a sulfoalkyl group.

6. The R in the general formula (2) ２４ and R ２５ At least one alkyl group among the groups is -(CH ２ -CH ２ -O) ｍ -R ３０ and the R in the general formula (2) is a substituted alkyl group represented by the formula １３ , R １４ and R １９ 2. The fluorescent compound of claim 1, wherein at least one alkyl group of m is 1 to 10, R ３０ is a hydrogen atom or an alkyl group.

7. A fluorescently labeled biological substance comprising the compound according to any one of claims 1 to 6 bound to a biological substance.

8. 8. The fluorescently labeled biological material according to claim 7, wherein the biological material is any one of a protein, a peptide, an amino acid, a nucleic acid, a sugar chain, and a lipid.

9. 8. The fluorescently labeled biological material according to claim 7, wherein the bond between the compound and the biological material is any of the bonds in the following i) to v): i) a non-covalent or covalent bond between peptides; ii) van der Waals interactions between the long-chain alkyl group in the compound and the lipid bilayer or lipid in the biological material; iii) an amide bond formed by reacting an N-hydroxysuccinimide ester in a compound with an amino group in a biological material; iv) a thioether bond formed by reacting a maleimide group in a compound with a sulfanyl group in a biological material; v) A bond involving the formation of a triazole ring, which is formed by a Click reaction between an azide group in a compound and an acetylene group in a biological material, or between an acetylene group in a compound and an azide group in a biological material.

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