Fluorescent compound

Abstract

This fluorescent compound is represented by general formula (1).　Z represents a group represented by general formula (Z1) or (Z2).　R1 and R9 represent an alkyl group or an aryl group. X represents an oxygen atom, a sulfur atom, or > C(R10)(R11). R2, R3, R10, and R11 represent alkyl groups, and at least one of them contains a neutral water-soluble unit. R4 to R8, and R12 to R20 represent a hydrogen atom or a substituent. Q represents an alkoxy group or an amino group. * represents a bond.　R1 to R20 and Q are not positively or negatively charged in an aqueous solution of pH 6 to 8.

Description

fluorescent compounds

[0001] The present invention relates to fluorescent compounds.

[0002] In the fields of research and medicine, bioimaging technology is used to analyze the dynamics and functions of biomolecules, cells, tissues, etc. in living organisms. In vivo fluorescence imaging, which observes specific parts of a living organism that have been made visible by fluorescent labeling using fluorescent compounds (fluorescent dyes), is used as one of the techniques for observing living organisms.

[0003] Flow cytometry, which allows for the rapid analysis of individual cells in a diverse cell population, is a well-known technique for counting, identifying, and separating specific cells from a particle population, such as 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 upon irradiation with laser light is dispersed by an optical filter (spectral filter) and measured as the amount of fluorescence (photons) (voltage). This allows for specific and quantitative detection of fluorescently labeled cells.

[0004] Among the above-mentioned biofluorescence imaging methods, fluorescent dyes used in fluorescent imaging of living cells must have excellent water solubility, as well as the ability to penetrate the cell membrane of living cells and reach the cells (hereinafter referred to as "cell membrane permeability"), and must also suppress non-specific adsorption to targets other than the target (hereinafter also referred to as "non-specific adsorption"). Among the fluorescent dyes that have both the above-mentioned cell membrane permeability and the suppression of non-specific adsorption, dyes that exhibit fluorescence in the green to red region (fluorescence maximum wavelength (λ ｍａｘ Regarding dyes that exhibit fluorescence in the deep red to near infrared (NIR) region (fluorescence maximum wavelength (λ ) in the range of approximately 500 nm to less than 600 nm), there have been reports focusing on rhodamine dyes. ｍａｘ There are few fluorescent dyes having a molar absorption coefficient ε in the range of 600 nm or more and approximately 800 nm or less, and it has been pointed out that commonly used Si-rhodamine fluorescent dyes such as SiR650 (trade name, manufactured by Spirochrome) have a low molar absorption coefficient ε and are insufficient in brightness.

[0005] On the other hand, although not intended for application to the above-mentioned live cell imaging, fluorescent dyes used for various fluorescent labels, such as cyanine dyes, dipyrromethene dyes, and squarylium dyes, are known, and studies have been conducted to improve the properties of these fluorescent dyes, such as water solubility and inhibition of dye aggregation. For example, Patent Document 1 describes various fluorescent dyes, such as cyanine dyes and squarylium dyes, as anionic or cationic compounds into which a PEG (polyethylene glycol) group and a sulfo group have been introduced to improve water solubility and brightness. Furthermore, in the field of dye-sensitized solar cells, Patent Document 2 describes a squarylium dye, a carboxyl group-containing zwitterionic compound into which a long-chain alkyl group has been introduced to inhibit aggregation and improve brightness, as a dye that exhibits fluorescence in the NIR region.

[0006] US Patent Application Publication No. 2016 / 0033520 US Patent Application Publication No. 2019 / 0270889

[0007] The present inventors have investigated fluorescent dyes with properties applicable to live cell imaging, such as dyes having a squarylium skeleton that fluoresce in the near-infrared region, as a new fluorescent dye with a skeleton different from rhodamine that fluoresces in the deep red to near-infrared region. As a result of their investigations, the inventors have found that the anionic squarylium dyes having sulfo groups, carboxy groups, etc., described in Patent Document 1 cannot penetrate the negatively charged cell membranes of live cells, and that the cationic squarylium dyes described in Patent Document 1 can penetrate the cell membranes of live cells but nonspecifically adsorb to mitochondria and other organelles within the cells due to electrostatic interactions resulting from the positive charge. Furthermore, the zwitterionic squarylium dyes containing a long-chain alkyl group and a carboxy group described in Patent Document 2 are uncharged and therefore can penetrate the cell membranes of live cells, but are poorly water-soluble, resulting in aggregation in water and / or within the cells, and furthermore, nonspecific adsorption to organelles such as the endoplasmic reticulum due to hydrophobic interactions. An object of the present invention is to provide a fluorescent compound that exhibits fluorescence in the deep red to near infrared region, has excellent water solubility and cell membrane permeability, and exhibits reduced nonspecific adsorption.

[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): In the formula, Z represents a group represented by the following general formula (Z1) or (Z2). In the formula, R １ and R ９ represents an alkyl group or an aryl group; X represents an oxygen atom, a sulfur atom, or >C(R １０ ) (R １１ ) indicates. ２ , R ３ , R １０ and R １１ represents an alkyl group. ２ , R ３ , R １０ and R １１ At least one alkyl group in R contains a neutral water-soluble unit. ４ ~R ８and R １２ ~R ２０ represents a hydrogen atom, a halogen atom, a cyano group, a nitro group, a hydroxy group, an alkyl group, an alkoxy group, an aryl group, a heteroaryl group, an acyloxy group, an acylamino group, an oxycarbonyl group, a carbamoyl group, or a sulfonamido group. Q represents an alkoxy group or an amino group. * represents a bond. However, R １ ~R ２０ and Q are not positively or negatively charged in an aqueous solution having a pH of 6 to 8. [2] The fluorescent compound according to [1], which is represented by the following general formula (2): In the formula, R １ ~R １６ is R in the above general formula (1). １ ~R １６ However, R １ ~R １６ does not become positively or negatively charged in an aqueous solution of pH 6 to 8. [3] The above R ２ , R ３ , R １０ and R １１ [4] The fluorescent compound according to [3], which is represented by the following general formula (3): In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ and R ４ ~R １６ is R in the above general formula (2). １ , R ２ and R ４ ~R １６ However, R１ , R ２ , R ４ ~R １６ , R ２１ and R ２２ does not become positively or negatively charged in an aqueous solution of pH 6 to 8. [5] The fluorescent compound according to [1], which is represented by the following general formula (4): In the formula, X １ represents an oxygen atom or a sulfur atom. １ ~R ９ and R １２ ~R １６ is R in the above general formula (1). １ ~R ９ and R １２ ~R １６ However, R １ ~R ９ and R １２ ~R １６ does not become positively or negatively charged in an aqueous solution of pH 6 to 8. [6] The above R ２ and R ３ [7] The fluorescent compound according to [6], which is represented by the following general formula (5): In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ , R ４ ~R ９ , R １２ ~R １６ and X １ is R in the above general formula (4). １ , R ２ , R ４ ~R ９, R １２ ~R １６ and X １ However, R １ , R ２ , R ４ ~R ９ , R １２ ~R １６ , R ２１ and R ２２ does not become positively or negatively charged in an aqueous solution of pH 6 to 8. [8] The fluorescent compound according to [1], which is represented by the following general formula (6): In the formula, R １ ~R ８ , R １７ ~R ２０ and Q is R in the above general formula (1). １ ~R ８ , R １７ ~R ２０ and Q. However, R １ ~R ８ , R １７ ~R ２０ and Q are not positively or negatively charged in an aqueous solution of pH 6 to 8. [9] The above R ２ and R ３

[10] The fluorescent compound according to [9], which is represented by the following general formula (7): In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ , R ４ ~R ８ , R １７ ~R ２０ and Q is R in the above general formula (6).１ , R ２ , R ４ ~R ８ , R １７ ~R ２０ and Q. However, R １ , R ２ , R ４ ~R ８ , R １７ ~R ２２ and Q are not positively or negatively charged in an aqueous solution of pH 6 to 8.

[11] The above R １７

[11] The fluorescent compound according to any one of [8] to

[10] , wherein Q is a hydroxy group.

[12] The fluorescent compound according to any one of [8] to

[11] , wherein Q is an amino group.

[13] The fluorescent compound according to any one of [1] to

[12] , which has at least one biological substance-binding group.

[0009] The fluorescent compound of the present invention exhibits fluorescence in the deep red to near infrared region, has excellent water solubility, excellent cell membrane permeability, and further has reduced nonspecific adsorption.

[0010] 1 shows confocal microscope images (images obtained by detecting fluorescence in the wavelength range of 650 to 750 nm using an excitation laser with a wavelength of 638 nm) obtained in Evaluation 2 of the example. (a) shows the confocal microscope image of Example No. 101, (b) to (e) show the confocal microscope images of Comparative Examples No. c11 to c14, respectively, and (f) shows the confocal microscope image (Reference Example) when no fluorescent compound was used.

[0011] In the present invention, when there are a plurality of substituents, linking groups, structural units, etc. (hereinafter referred to as "substituents, etc.") represented by a specific symbol or formula, or when a plurality of substituents, etc. are simultaneously specified, the respective substituents, etc. may be the same or different from each other, unless otherwise specified. This also applies to the specification of the number of substituents, etc. Furthermore, 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. For example, R in general formula (1) ４ ~R ８ and R １２ ~R ２０In the present invention, adjacent substituents may be linked to each other to form a fused ring. Furthermore, unless otherwise specified, rings, such as alicyclic rings, aromatic rings, and heterocyclic rings, may be further fused to form a fused ring. In the notation of groups (atomic groups) in the present invention, notations that do not specify whether they are substituted or unsubstituted include groups (atomic groups) that have no substituents as well as groups (atomic groups) that have a substituent. For example, the term "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. Preferred examples of optional substituents possessed by such substituted groups (atomic groups) include substituents selected from the substituent group T described below. In the present invention, when the number of carbon atoms of a certain group is specified, this number of carbon atoms refers to the number of carbon atoms of the entire group, unless otherwise specified in the present invention or this specification. In other words, if the group further has a substituent, it refers to the total number of carbon atoms including the substituent. In addition, if the substituent is a group containing a neutral water-soluble unit, the total number of carbon atoms of the substituent including the neutral water-soluble unit is specified separately.

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

[0013] When the fluorescent compound of the present invention exists as a stereoisomer such as an optical isomer, a geometric isomer, or a rotational isomer, or a tautomer, 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 compounds whose structure has been partially modified as long as the effect of the present invention is not impaired.

[0015] <Fluorescent Compound of the Present Invention>

[0016] The fluorescent compound of the present invention is represented by the general formula (1) shown below. As represented by the general formula (1) shown below, the fluorescent compound of the present invention has a squarylium (also called squaraine) structure. The fluorescent compound of the present invention is a novel fluorescent compound that exhibits fluorescence in the deep red to near infrared region, and has excellent water solubility, excellent cell membrane permeability, and suppressed nonspecific adsorption. The reason for this is not clear, but is thought to be as follows. The fluorescent compound of the present invention represented by the general formula (1) is a squarylium dye having a group represented by the general formula (Z1) or (Z2) and an indolenine ring, and is a fluorescent compound that exhibits fluorescence in the deep red to near infrared region. Furthermore, the fluorescent compound of the present invention has a squarylium skeleton having a positively charged nitrogen atom (R １ In the fluorescent compound of the present invention, both a nitrogen atom to which R is bonded and a negatively charged oxygen atom (oxygen atom derived from squaric acid) are bonded. Therefore, the properties of both the positive and negative charges are more effectively cancelled out compared to zwitterionic compounds in which neither a positively charged atom nor a negatively charged atom is bonded to the π-conjugated structure. In addition, the fluorescent compound of the present invention is １ ~R ２０ and Q are not positively or negatively charged in an aqueous solution at pH 6 to 8, and thus are zwitterionic compounds that have no charge at pH 6 to 8 other than the positive and negative charges of the atoms bonded to the π-conjugated structure. As a result, the fluorescent compound of the present invention has excellent cell membrane permeability, and moreover, nonspecific adsorption to mitochondria and the like is suppressed by electrostatic interactions. Furthermore, the fluorescent compound of the present invention is a compound represented by the general formula (1) ２ , R ３ , R １０ and R １１ At least one of the groups is an alkyl group containing a neutral water-soluble unit, which provides water solubility while suppressing hydrophobicity, thereby suppressing nonspecific adsorption to endoplasmic reticulum, etc. It is believed that the fluorescent compound of the present invention has excellent water solubility, excellent cell membrane permeability, and suppressed nonspecific adsorption due to these combined effects.

[0017] The fluorescent compound of the present invention is a compound represented by the general formula (1) R１ ~R ２０ and Q are not positively or negatively charged in an aqueous solution of pH 6 to 8. Therefore, the positive and negative charges on the squarylium dye skeleton, i.e., R １ The fluorescent compound of the present invention is a compound that does not have any group having a charge (anionic group or cationic group) requiring a counter ion or any group having a zwitterionic structure in an aqueous solution at pH 6 to 8, other than the positive charge on the nitrogen atom to which R is bonded and the negative charge on the oxygen atom derived from the squaric acid skeleton. Therefore, it can be said that the fluorescent compound of the present invention is a zwitterionic compound at pH 6 to 8. "A zwitterionic compound at pH 6 to 8" means a compound that has both a positive charge and a negative charge in the same molecule at pH 6 to 8, but has no charge as a whole. Therefore, the fluorescent compound of the present invention does not include cationic or anionic compounds that have a positive or negative charge as a whole molecule at pH 6 to 8, but whose charge becomes zero due to the presence of a counter ion. In other words, the fluorescent compound of the present invention is not a compound having a counter ion. As with the fluorescent compound represented by general formula (1), the fluorescent compounds represented by general formulas (2) to (7) described below also have a substituent (R １ ~R ２０ and Q) are neither positively nor negatively charged in aqueous solution at pH 6-8 and can be said to be zwitterionic compounds at pH 6-8.

[0018] The chemical structures of the fluorescent compounds of the present invention are described in detail below based on the general formulas. As mentioned above, the fluorescent compounds of the present invention represented by any of the following general formulas (1) to (7) are zwitterionic compounds at pH 6 to 8.

[0019] (Fluorescent Compound Represented by General Formula (1)) The fluorescent compound of the present invention is represented by the following general formula (1).

[0020]

[0021] In the formula, Z represents a group represented by the following general formula (Z1) or (Z2).

[0022]

[0023] In the formula, R １ and R ９ represents an alkyl group or an aryl group; X represents an oxygen atom, a sulfur atom, or >C(R １０ ) (R １１ ) indicates. ２ , R ３ , R １０ and R １１ represents an alkyl group. ２ , R ３ , R １０ and R １１ At least one alkyl group in R contains a neutral water-soluble unit. ４ ~R ８ and R １２ ~R ２０ represents a hydrogen atom, a halogen atom, a cyano group, a nitro group, a hydroxy group, an alkyl group, an alkoxy group, an aryl group, a heteroaryl group, an acyloxy group, an acylamino group, an oxycarbonyl group, a carbamoyl group, or a sulfonamido group. Q represents an alkoxy group or an amino group. * represents a bond. However, R １ ~R ２０ and Q are not positively or negatively charged in aqueous solutions of pH 6-8.

[0024] R １ and R ９ represents an alkyl group or an aryl group. １ and R ９ The alkyl and aryl groups that can be used as R can be those described below in relation to the alkyl and aryl groups in the group T of substituents. １ and R ９ The number of carbon atoms in the alkyl group that can be taken as R is preferably 1 to 21, more preferably 1 to 17, still more preferably 1 to 13, and particularly preferably 1 to 9. １ and R ９ As the substituent that the alkyl group and aryl group may have, for example, an acylamino group or a carbamoyl group in the substituent group T described below is preferred, and an alkylcarbonylamino group or an alkylcarbamoyl group is more preferred.

[0025] X is an oxygen atom, a sulfur atom, or >C(R １０ ) (R １１ ) indicates that X is >C(R １０ ) (R １１ ), then R １０ and R １１ It is preferred that at least one alkyl group among these groups contains a neutral water-soluble unit as described below.

[0026] R ２ , R ３ , R １０ and R １１ represents an alkyl group. ２ , R ３ , R １０ and R １１ The number of carbon atoms in the alkyl group that can be taken as R is preferably 1 to 21, more preferably 1 to 17, even more preferably 1 to 13, particularly preferably 1 to 9, and most preferably 1 to 6. ２ , R ３ , R １０ and R １１ When the alkyl group which can be taken as the alkyl group containing a neutral water-soluble unit described later, the number of carbon atoms is preferably 2 to 200, more preferably 2 to 100, still more preferably 4 to 50, and particularly preferably 4 to 26.

[0027] R ２ , R ３ , R １０ and R １１ Examples of the substituent that the alkyl group may have include an acylamino group, a carbamoyl group, an acyloxy group, an oxycarbonyl group, a sulfonamide group, an alkoxy group, and a hydroxy group in the substituent group T described later, as well as the neutral water-soluble unit described later. ２ -O) ｎ -R ２１ is preferred, and an acylamino group, a carbamoyl group or —O—(L ２ -O) ｎ -R ２１ More preferably, -O-(L ２ -O) ｎ -R ２１The above "-O-(L ２ -O) ｎ -R ２１ " is "-O-(L ２ -O) ｎ -R ２１ This also applies to the following general formulae. ２ , R ３ , R １０ and R １１ The acylamino group, carbamoyl group, acyloxy group, oxycarbonyl group, sulfonamide group and alkoxy group as the substituents which the alkyl group may have may be further substituted with a substituent, and for example, it is also preferable that they are substituted with an aminosulfonyl group in the substituent group T described later and a neutral water-soluble unit described later.

[0028] R ２ , R ３ , R １０ and R １１ Preferred examples of the alkyl group that can be taken as the alkyl group include methyl, ethyl, isopropyl, propyl, tert-butyl, pentyl, and heptyl, as well as groups in which these alkyl groups are substituted with the above-mentioned acylamino group, carbamoyl group, acyloxy group, oxycarbonyl group, sulfonamide group, alkoxy group, hydroxy group, or neutral water-soluble unit.

[0029] However, R ２ , R ３ , R １０ and R １１ In the fluorescent compound of the present invention, at least one alkyl group of R ２ , R ３ , R １０ and R １１ is a substituent perpendicular to the plane of the squarylium dye skeleton. ２ , R ３ , R １０ and R １１By including a neutral water-soluble unit in at least one of the alkyl groups, it is possible to effectively suppress nonspecific adsorption of the compound while improving water solubility. In the present invention, the term "neutral water-soluble unit" refers to a water-soluble unit that is not positively or negatively charged in an aqueous solution of pH 6 to 8. Examples of the neutral water-soluble unit include a polyalkyleneoxy group (-O-(L)) in the substituent group T described below. ２ -O) ｎ -R ２１ a polyol residue and a sugar residue; a polyalkyleneoxy group (-O-(L ２ -O) ｎ -R ２１ Among them, R ２ , R ３ , R １０ and R １１ Among these, it is preferable that at least one alkyl group contains a polyalkyleneoxy group, and R ２ and R ３ At least one alkyl group among R contains a polyalkyleneoxy group, and １０ and R １１ More preferably, at least one alkyl group among these groups comprises a polyalkyleneoxy group.

[0030] R ４ ~R ８ and R １２ ~R ２０ represents a hydrogen atom, a halogen atom, a cyano group, a nitro group, a hydroxy group, an alkyl group, an alkoxy group, an aryl group, a heteroaryl group, an acyloxy group, an acylamino group, an oxycarbonyl group, a carbamoyl group, or a sulfonamide group. ４ ~R ８ and R １２ ~R ２０As the halogen atom, alkyl group, alkoxy group, aryl group, heteroaryl group, acyloxy group, acylamino group, carbamoyl group and sulfonamide group that can be taken as R, the descriptions of the halogen atom, alkyl group, alkoxy group, aryl group, heteroaryl group, acyloxy group, acylamino group, carbamoyl group and sulfonamide group in the substituent group T described below can be applied. ４ ~R ８ and R １２ ~R ２０ Examples of the oxycarbonyl group that can be taken as R include an alkoxycarbonyl group, a cycloalkoxycarbonyl group, and an aryloxycarbonyl group in the substituent group T described below, and these descriptions can be applied. ４ ~R ８ and R １２ ~R ２０ is preferably a hydrogen atom or a hydroxy group. ４ ~R ８ and R １２ ~R １８ , R １９ and R ２０ is more preferably a hydrogen atom, and R １７ is more preferably a hydroxy group.

[0031] Q represents an alkoxy group or an amino group, preferably an amino group, and more preferably an alkyl-substituted amino group. The alkyl-substituted amino group may be either a monoalkyl-substituted amino group or a dialkyl-substituted amino group. The number of carbon atoms in the alkyl group in the alkoxy group or alkyl-substituted amino group that can be taken as Q is preferably 1 to 21, more preferably 1 to 17, even more preferably 1 to 13, particularly preferably 1 to 9, and most preferably 1 to 6. When the alkoxy group that can be taken as Q is an alkoxy group containing the aforementioned neutral water-soluble unit, the number of carbon atoms in the alkyl group in the alkyl-substituted amino group is preferably 2 to 200, more preferably 2 to 100, even more preferably 4 to 50, and particularly preferably 4 to 26. When the alkyl-substituted amino group that can be taken as Q is an alkyl-substituted amino group containing the aforementioned neutral water-soluble unit, the number of carbon atoms in the alkyl group in the alkyl-substituted amino group is preferably 2 to 200, more preferably 2 to 100, even more preferably 4 to 50, and particularly preferably 4 to 26.

[0032] Examples of the substituent that the alkoxy group and amino group that can be taken as Q may have include an acylamino group, a carbamoyl group, an acyloxy group, an oxycarbonyl group, a sulfonamide group, an alkoxy group, and a hydroxy group in the substituent group T described below, as well as the neutral water-soluble unit described above. ２ -O) ｎ -R ２１ is preferred, and an acylamino group, a carbamoyl group or —O—(L ２ -O) ｎ -R ２１ More preferably, -O-(L ２ -O) ｎ -R ２１ is more preferred.

[0033] However, R １ ~R ２０and Q are not positively or negatively charged in an aqueous solution of pH 6 to 8. In the present invention, "not positively or negatively charged in an aqueous solution of pH 6 to 8" means that the compound is not positively or negatively charged in an aqueous solution of pH 6 to 8 and does not have a zwitterionic structure in an aqueous solution of pH 6 to 8. The "zwitterionic structure" means a structure in which a positive charge and a negative charge are present at non-adjacent positions within the same substituent, no dissociable hydrogen atom is bonded to the positively charged atom, and the substituent as a whole has no charge. Therefore, for example, an azide group (-N) having a positive charge and a negative charge on adjacent nitrogen atoms as formal charges is not a suitable structure. ３ ) does not correspond to a group having a zwitterionic structure.

[0034] Also, R １ ~R ２０ At least one of R and Q may be a substituent containing an amide group or a biological substance-binding group, as described below. In this case, １ , R ９ , R １０ , R １７ and R ２０ At least one of the above is preferably a substituent containing an amide group or a biological substance-binding group, as described below.

[0035] The number of neutral water-soluble units contained in the fluorescent compound of the present invention is, for example, preferably 1 to 8, more preferably 1 to 6, and even more preferably 1 to 4.

[0036] (Fluorescent compound represented by general formula (2)) The fluorescent compound represented by the general formula (1) is preferably represented by the following general formula (2). The compound represented by the following general formula (2) is a compound represented by the general formula (1) in which Z is a group represented by general formula (Z1) and X is >C(R １０ ) (R １１ ) corresponds to a compound

[0037]

[0038] In the formula, R １ ~R １６ is R in the above general formula (1). １ ~R １６ However, R １~R １６ does not become positively or negatively charged in aqueous solution at pH 6-8.

[0039] R ２ , R ３ , R １０ and R １１ Preferably, the neutral water-soluble unit contained in at least one alkyl group among R is a polyalkyleneoxy group. １ ~R １６ At least one of the groups may be a substituent containing an amide group or a group capable of binding to a biological substance, as described below.

[0040] (Fluorescent compound represented by general formula (3)) The fluorescent compound represented by the general formula (2) is preferably represented by the following general formula (3). The compound represented by the following general formula (3) is a compound represented by the general formula (2) in which R ３ Ga-L １ -O-(L ２ -O) ｎ -R ２１ This corresponds to a compound which is a group represented by the formula:

[0041]

[0042] In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ and R ４ ~R １６ is R in the above general formula (2). １ , R ２ and R ４ ~R １６ However, R １ , R ２ , R ４ ~R１６ , R ２１ and R ２２ does not become positively or negatively charged in aqueous solution at pH 6-8.

[0043] L １ is R ２ is bonded to a carbon atom of which the bonding site is an alkylene group, and which is composed of 1 to 100 atoms. The above "linking group composed of 1 to 100 atoms" means a linking group in which the total number of atoms other than hydrogen atoms among the atoms constituting the linking group is 1 to 100. In a linking group composed of 1 to 100 atoms, the number of atoms constituting the linking group (the total number of atoms other than hydrogen atoms among the atoms constituting the linking group) is preferably 1 to 50, more preferably 1 to 20, and even more preferably 2 to 10. L １ The atoms constituting L may include, in addition to carbon atoms and hydrogen atoms, at least one of nitrogen atoms and oxygen atoms. １ Examples of the groups constituting the formula include an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, >C═O, and >NR Ａ Examples include: Ａ represents a hydrogen atom or a substituent. Ａ Examples of the substituents that can be used as R include the substituent group T described below. Ａ is preferably a hydrogen atom, an alkyl group or an aryl group. １ As the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group that can constitute L, a divalent group obtained by removing one hydrogen atom from an alkyl group, alkenyl group, alkynyl group, aryl group and heteroaryl group in the substituent group T described below can be applied. １ The structure of R ２ an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, —O—, >C═O, and >NR Ａ Examples of linking groups include one or a combination of two or more of the following: １ The structure of R ２an alkylene group, —O—, >C═O, and >NR Ａ The linking group is preferably one or a combination of two or more of the following: an alkylene group, or -alkylene-NR Ａ C(=O)-alkylene- is more preferred.

[0044] In the general formula (3), "-(L ２ -O) ｎ -R ２１ " in the group of substituents T described later, ２ -O) ｎ -R ２１ " applies. However, R １ , R ２ , R ４ ~R １６ , R ２１ and R ２２ does not become positively or negatively charged in an aqueous solution of pH 6 to 8. １ , R ２ , R ４ ~R １６ , R ２１ and R ２２ At least one of the groups may be a substituent containing an amide group or a group capable of binding to a biological substance, as described below.

[0045] (Fluorescent compound represented by general formula (4)) The fluorescent compound represented by the above general formula (1) is also preferably represented by the following general formula (4). The compound represented by the following general formula (4) corresponds to the compound represented by the above general formula (1) in which Z is a group represented by general formula (Z1) and X is an oxygen atom or a sulfur atom.

[0046]

[0047] In the formula, X １ represents an oxygen atom or a sulfur atom. １ ~R ９ and R １２ ~R １６ is R in the above general formula (1). １ ~R ９ and R １２ ~R １６However, R １ ~R ９ and R １２ ~R １６ does not become positively or negatively charged in aqueous solution at pH 6-8.

[0048] R ２ and R ３ Preferably, the neutral water-soluble unit contained in at least one alkyl group among R is a polyalkyleneoxy group. １ ~R ９ and R １２ ~R １６ At least one of the groups may be a substituent containing an amide group or a group capable of binding to a biological substance, as described below.

[0049] (Fluorescent compound represented by general formula (5)) The fluorescent compound represented by the general formula (4) is preferably represented by the following general formula (5). The compound represented by the following general formula (5) is a compound represented by the general formula (4) in which R ３ Ga-L １ -O-(L ２ -O) ｎ -R ２１ This corresponds to a compound which is a group represented by the formula:

[0050]

[0051] In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ , R ４ ~R ９ , R １２ ~R １６ and X １ is R in the above general formula (4). １ , R２ , R ４ ~R ９ , R １２ ~R １６ and X １ However, R １ , R ２ , R ４ ~R ９ , R １２ ~R １６ , R ２１ and R ２２ does not become positively or negatively charged in aqueous solution at pH 6-8.

[0052] L １ is L in the above general formula (3). １ The meaning of "-(L ２ -O) ｎ -R ２１ " in the group of substituents T described later, ２ -O) ｎ -R ２１ " can be applied. １ , R ２ , R ４ ~R ９ , R １２ ~R １６ , R ２１ and R ２２ At least one of the groups may be a substituent containing an amide group or a group capable of binding to a biological substance, as described below.

[0053] (Fluorescent compound represented by general formula (6)) The fluorescent compound represented by the above general formula (1) is also preferably represented by the following general formula (6). The compound represented by the following general formula (6) corresponds to the compound represented by the above general formula (1) in which Z is a group represented by general formula (Z2).

[0054]

[0055] In the formula, R １ ~R ８ , R １７ ~R ２０ and Q is R in the above general formula (1). １ ~R ８ , R １７ ~R２０ and Q. However, R １ ~R ８ , R １７ ~R ２０ and Q are not positively or negatively charged in aqueous solutions of pH 6-8.

[0056] R ２ and R ３ Preferably, the neutral water-soluble unit contained in at least one alkyl group among R is a polyalkyleneoxy group. １ ~R ８ , R １７ ~R ２０ At least one of Q may be a substituent containing an amide group or a biological substance-binding group, as described below.

[0057] (Fluorescent compound represented by general formula (7)) The fluorescent compound represented by the general formula (6) is preferably represented by the following general formula (7). The compound represented by the following general formula (7) is a compound represented by the general formula (6) in which R ３ Ga-L １ -O-(L ２ -O) ｎ -R ２１ This corresponds to a compound which is a group represented by the formula:

[0058]

[0059] In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ , R ４ ~R ８ , R １７ ~R ２０ and Q is R in the above general formula (6). １ , R２ , R ４ ~R ８ , R １７ ~R ２０ and Q. However, R １ , R ２ , R ４ ~R ８ , R １７ ~R ２２ and Q are not positively or negatively charged in aqueous solutions of pH 6-8.

[0060] L １ is L in the above general formula (3). １ The meaning of "-(L ２ -O) ｎ -R ２１ " in the group of substituents T described later, ２ -O) ｎ -R ２１ " applies. １７ is preferably a hydroxy group. Q is preferably an amino group. １ , R ２ , R ４ ~R ８ , R １７ ~R ２２ At least one of Q may be a substituent containing an amide group or a biological substance-binding group, as described below.

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

[0062]

[0063] The fluorescent compound of the present invention may have a substituent or a biological substance-binding group containing an amide group. When the fluorescent compound of the present invention has a biological substance-binding group, the biological substance-binding group of the fluorescent compound of the present invention can bind to a target biological substance and fluorescently label the biological substance, as described below. When the fluorescent compound of the present invention has a biological substance-binding group, the number of biological substance-binding groups it has may be at least one. From the viewpoint of quantifying the target substance, the number of biological substance-binding groups is preferably one to three, more preferably one or two, and even more preferably one. Furthermore, when the fluorescent compound of the present invention has a substituent containing an amide group, it can be used as a model compound that can easily evaluate the excellent water solubility and cell membrane permeability, as well as the effect of suppressing nonspecific adsorption, possessed by the fluorescent compound of the present invention. In this case, it is preferable to evaluate the compound by introducing a substituent containing an amide group into the site where the biological substance-binding group is to be introduced. The substituent containing an amide group may be any substituent containing an acylamino group or a carbamoyl group at its terminal portion, and is preferably a substituent containing an acylamino group or a carbamoyl group at its terminal portion, which may be substituted with an alkyl group (preferably an alkyl group having 1 to 3 carbon atoms, more preferably an alkyl group having 1 or 2 carbon atoms). When the fluorescent compound of the present invention has a substituent containing an amide group, the number of the substituents containing an amide group can be the same as the number of the above-mentioned biological substance-binding groups.

[0064] (Biological Substance-Binding Group) By introducing a biological substance-binding group into the fluorescent compound of the present invention, the introduced biological substance-binding group can be bound to biological substances such as proteins (including peptides), amino acids, nucleic acids, nucleotides, sugar chains, and lipids, thereby fluorescently labeling the biological substances. The biological substance-binding group can be any group that acts on (including attaches to) or binds to a biological substance, and is not particularly limited. For example, the electrophilic and nucleophilic groups described in Table 2 of WO 2002 / 026891, as well as 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 substance-binding group" include the following structures:

[0065]

[0066] In the specific examples of the "biological substance-binding group" above, 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, which do not become positively or negatively charged in aqueous solutions of pH 6 to 8; the same applies below), long-chain alkyl groups, and the like can also be used as the "biological substance-binding group". Among these, NHS ester structures (N-hydroxysuccinimide ester structures), succinimide structures, maleimide structures, azide groups, acetylene groups, peptide structures (polyamino acid structures), and long-chain alkyl groups (preferably having 12 to 30 carbon atoms) are preferred.

[0067] In addition to the above, examples of the biological substance binding group include ligands that are commonly used in cell imaging applications. For example, HaloTag (registered trademark) ligand (chloroalkane derivative ligand) that forms a covalent bond with HaloTag (registered trademark) and SNAP-tag (registered trademark) ligand (O ６ Examples of suitable biological substance-binding groups include a cytosine-benzylguanine derivative ligand, and a CLIP-tag® ligand (benzylcytosine derivative ligand) that forms a covalent bond with CLIP-tag®. The fluorescent compound of the present invention having the above-described ligand as a biological substance-binding group can be used as a fluorescently labeled ligand for exogenous fluorescent protein tags such as HaloTag®, SNAP-tag®, and CLIP-tag®. Note that an exogenously fluorescent protein (extrinsically fluorescent protein) refers to a non-fluorescent protein that is imparted with fluorescent properties by the addition of an exogenous fluorescently labeled ligand. In addition to the above, ligands for other tag systems may also be used as biological substance-binding groups, provided that the effects of the present invention are not impaired.

[0068] The fluorescent compound of the present invention can be synthesized by conventional methods. 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, U.S. Patent Application Publication No. 2010 / 0266507, U.S. Patent Application Publication No. 2016 / 0033520, and U.S. Patent Application Publication No. 2019 / 0270889 can also be referenced and applied. Compounds having a biological substance binding group can also be synthesized by conventional methods. For example, Bioconjugate Techniques (Third Edition, by Greg T. Hermanson) can be referenced. In addition, compounds having a ligand for a tag system for cell imaging applications, etc. as a biological substance binding group can also be synthesized by conventional methods.

[0069] <<Fluorescently Labeled Biological Substances>> The fluorescent compound of the present invention may be bound to a biological substance to form a fluorescently labeled biological substance. The fluorescent compound of the present invention has fluorescence, excellent water solubility, excellent cell membrane permeability, and furthermore, suppressed nonspecific adsorption, and therefore can be preferably used as a fluorescently labeled biological substance. For example, the compound can be suitably used in cell-based assays and / or imaging assays such as immunofluorescence staining (IF), immunoprecipitation (IP), protein purification, and the like.

[0070] Preferred examples of the biological substances include proteins (including peptides), amino acids, nucleic acids, nucleotides, sugar chains, and lipids. Examples of proteins include antibodies, and examples of lipids include phospholipids, fatty acids, and sterols. Among the biological substances, clinically useful substances include, but are not limited to, immunoglobulins such as Ig (Immunoglobulin) G, IgM, IgE, IgA, and IgD, complement, C-reactive protein (CRP), ferritin, α １ Microglobulin, beta ２Examples of the antibodies include plasma proteins such as microglobulin and antibodies thereof; tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA (carbohydrate antigen) 19-9, CA-125 and antibodies thereof; hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen, insulin and antibodies thereof; and viral infection-related substances such as hepatitis B virus (HBV)-related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), adult T-cell leukemia (ATL) and antibodies thereof. Further examples include bacteria such as Corynebacterium diphtheriae, Clostridium botulinum, Mycoplasma, and Treponema pallidum, and antibodies thereto; protozoa such as Toxoplasma, Trichomonas, Leishmania, Trypanosoma, and Plasmodium, and antibodies thereto; ES cells (embryonic stem cells) such as ELM3, HM1, KH2, v6.5, v17.2, and v26.2 (derived from mice 129, 129 / SV, C57BL / 6, and BALB / c) and antibodies thereto; antiepileptic drugs such as phenytoin and phenobarbital, cardiovascular drugs such as quinidine and digoxin, antiasthmatic drugs such as theophylline, and antibiotics such as chloramphenicol and gentamicin, and antibodies thereto; other enzymes; and exotoxins (such as styrelidin O) and antibodies thereto. Antibody fragments such as Fab'2, Fab, and Fv can also be used. Other examples include protein tags such as HaloTag (registered trademark), SNAP-tag (registered trademark), and CLIP-tag (registered trademark).

[0071] Specific examples of the form in which the fluorescent compound of the present invention and a biological substance interact and bind to each other include the forms described below. i) non-covalent bonds (e.g., hydrogen bonds, ionic bonds including chelate formation) or covalent bonds between peptides in the fluorescent compounds of the present invention and peptides in biological materials; ii) van der Waals forces between long-chain alkyl groups in the fluorescent compounds of the present invention and lipid bilayers and lipids in biological materials; iii) amide bonds formed by reaction between NHS esters (N-hydroxysuccinimide esters) in the fluorescent compounds of the present invention and amino groups in biological materials; iv) thioether bonds formed by reaction between maleimide groups in the fluorescent compounds of the present invention and sulfanyl groups (—SH) in biological materials; v) formation of triazole rings by Click reaction between azide groups in the fluorescent compounds of the present invention and acetylene groups in biological materials, or by Click reaction between acetylene groups in the fluorescent compounds of the present invention and azide groups in biological materials; and vi) formation of covalent bonds between the biological material-binding groups in the fluorescent compounds of the present invention as ligands and protein tags such as HaloTag (registered trademark), SNAP-tag (registered trademark), CLIP-tag (registered trademark), etc. However, in the above form i), the peptide in the fluorescent compound of the present invention is not particularly limited as long as it is a peptide that can form a non-covalent or covalent bond with a peptide in a biological material. In addition to the above forms i) to vi), for example, Lucas C. D. de Rezende and Flavio da Silva Emery., A Review of the Synthetic Strategies for the Development of BODIPY Dyes for Conjugation with Proteins, Orbital: The Electronic Journal of Chemistry, 2013, Vol. 5, No. 1, p. 62-83. Furthermore, in preparing a fluorescently labeled biological substance using the fluorescent compound of the present invention, the method described in the same document can be appropriately referred to.

[0072] Among the fluorescent compounds of the present invention, fluorescently labeled biological substances obtained from compounds having a biological substance-binding group and a biological substance that binds to the compound through interaction include, for example, compounds in which the portions other than the biological substance-binding group are replaced with the fluorescent compound of the present invention and their products, as described in 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 substances, etc.

[0073] <Reagents Comprising Fluorescently Labeled Biological Substances> In reagents containing fluorescently labeled biological substances obtained from the fluorescent compounds of the present invention, the fluorescently labeled biological substances 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 intended use, etc. For example, when a fluorescently labeled biological substance obtained from the fluorescent compound of the present invention is used as a fluorescently labeled reagent, it can also be used as a reagent containing a fluorescently labeled biological substance in any of the above forms.

[0074] <Uses of Fluorescently Labeled Biological Substances> Fluorescently labeled biological substances obtained from the fluorescent compounds of the present invention have fluorescence due to the fluorescent compounds of the present invention, excellent water solubility, excellent cell membrane permeability, and reduced nonspecific adsorption. Therefore, they can be suitably used for, for example, cell-based assays such as immunofluorescence staining (IF) and / or imaging assays, immunoprecipitation (IP), protein purification, and other intracellular or biochemical analyses of various fusion proteins.

[0075] Fluorescence detection using a fluorescently labeled biological material obtained from the fluorescent compound of the present invention generally comprises the following steps (i) to (iii) or (iv) to (vii): Fluorescence detection comprising 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 comprising steps (iv) to (vii) corresponds to an indirect method using a secondary antibody fluorescently labeled with the fluorescent compound of the present invention. (i) a step of preparing the following (a) and (b): (a) a sample containing a target biological substance (hereinafter also referred to as "target biological substance"); (b) a fluorescently labeled biological substance (hereinafter also referred to as "fluorescently labeled biological substance A") in which a biological substance capable of binding to the target biological substance in (a) above (hereinafter also referred to as "primary biological substance") is bound to the fluorescent compound of the present invention; (ii) a step of preparing a conjugate (hereinafter also referred to as "fluorescently labeled conjugate A") in which the target biological substance in (a) above is bound to the primary biological substance in the fluorescently labeled biological substance A 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 substance A and detecting the fluorescence emitted by the fluorescently labeled biological substance A; (iv) a step of preparing the following (c) to (e): (c) a sample containing a target biological substance (d) a biological substance capable of binding to the target biological substance in (c) above (hereinafter also referred to as "primary biological substance"); (e) a fluorescently labeled biological substance in which a biological substance capable of binding to the primary biological substance in (d) above (hereinafter also referred to as "secondary biological substance") is bound to a fluorescent compound of the present invention (hereinafter also referred to as "fluorescently labeled biological substance B"); (v) a step of preparing a conjugate in which the target biological substance in (c) above is bound to the primary biological substance in (d) above (hereinafter also referred to as "conjugate b"); (vi) a step of preparing a conjugate in which the primary biological substance in conjugate b is bound to the secondary biological substance in fluorescently labeled biological substance B (hereinafter also referred to as "fluorescently labeled conjugate B2"); and (vii) a step of irradiating the fluorescently labeled conjugate B2 with light in a wavelength range absorbed by the fluorescently labeled biological substance B and detecting the fluorescence emitted by the fluorescently labeled biological substance B.

[0076] Examples of the biological substance (primary biological substance) capable of binding to the target biological substance and the biological substance (secondary biological substance) capable of binding to the primary biological substance include biological substances in the fluorescently labeled biological substances obtained from the fluorescent compound of the present invention. These can be appropriately selected according to the target biological substance (biological substance in a test specimen) or the primary biological substance, and biological substances capable of specifically binding to the biological substance in a test specimen or the primary biological substance can be selected.

[0077] Among the target biological substances, proteins include so-called disease markers. Disease markers are not particularly limited, but include, for example, α-fetoprotein (AFP), PIVKA-II (protein induced by vitamin K absorption 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, gamma-seminoprotein (gamma-Sm), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), neuron-specific enolase (NSE), Iba1, amyloid beta, Examples of antigens 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.

[0078] The target biological material may be a bacterium, including, but not limited to, bacteria that are the subject of cellular microbiological testing, such as Escherichia coli, Salmonella, Legionella, and bacteria that cause public health problems.

[0079] The target biological substance 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.

[0080] When a protein tag such as HaloTag (registered trademark), SNAP-tag (registered trademark), or CLIP-tag (registered trademark) is used as the primary biological material, steps (i) and (ii) above should be interpreted as steps of preparing a sample containing a tag fusion protein in which the protein tag has been genetically introduced and expressed, and then preparing a conjugate a in which the tag fusion protein is conjugated to the fluorescent compound of the present invention (hereinafter referred to as "fluorescently labeled conjugate a"). Furthermore, in step (iii) above, fluorescently labeled conjugate A should be interpreted as fluorescently labeled conjugate a, and fluorescently labeled biological material A should be interpreted as the fluorescent compound of the present invention. The tag fusion protein can be prepared by expressing it in cells or animals by transfecting (transfecting) a plasmid encoding the tag fusion protein using standard methods. Furthermore, conjugate a in which the tag fusion protein is conjugated to the fluorescent compound of the present invention can be prepared in cells or animals by adding the fluorescent compound of the present invention to a sample containing the tag fusion protein and culturing the sample.

[0081] In the above (i) or (iv), the sample containing the target biological substance can be prepared according to a conventional method without any particular limitation. Furthermore, the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention can also be prepared according to a conventional method by binding a biological substance capable of binding to the target biological substance to the fluorescent compound of the present invention without any particular limitation. The bond type and the bond-forming reaction are as described above for the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention.

[0082] In (v) above, the target biological substance and the primary biological substance may be bound directly or via another biological substance different from the target biological substance and the primary biological substance. Furthermore, in (vi) above, the primary biological substance in the conjugate b and the secondary biological substance in the fluorescently labeled biological substance B may be bound directly or via another biological substance different from the primary biological substance and the secondary biological substance. The fluorescently labeled biological substance obtained from the fluorescent compound 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 (ii) or (v) and (vi) above, the binding of the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention to the target biological substance can be carried out according to a conventional method without any particular limitations.

[0083] In the above (iii) or (vii), the wavelength for exciting the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention is not particularly limited as long as it is light of a wavelength that can excite the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention. Generally, a wavelength of 600 to 800 nm is preferred, and a wavelength of 650 to 750 nm is more preferred.

[0084] The fluorescence excitation light source used in the present invention is not particularly limited as long as it emits light of a wavelength that can excite the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention, and various laser light sources can be used, for example. In addition, various optical filters can be used to obtain a preferred excitation wavelength or to detect only fluorescence in a specific wavelength range.

[0085] Other items in (i) to (vii) above are not particularly limited, and conditions such as techniques, reagents, and devices that are commonly used in fluorescence detection using fluorescent labels can be appropriately selected. Furthermore, for steps other than (i) to (vii) above, conditions such as techniques, reagents, and devices that are commonly used can be appropriately selected in accordance with various techniques that use fluorescent labels.

[0086] For example, flow cytometry using a fluorescently labeled biological substance obtained from the fluorescent compound of the present invention involves preparing a cell suspension in which the target biological substance is dispersed into single cells by a commonly used method, and using the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention as a labeled antibody (preferably a secondary antibody), thereby effectively suppressing leakage of fluorescence to be detected in one detection channel into another detection channel, and can also be used for multicolor simultaneous detection. Multicolor WB (Western blotting) using a fluorescently labeled biological substance obtained from the fluorescent compound of the present invention involves preparing a blot membrane by a commonly used method for target biological substances (protein separation by electrophoresis, blotting to a membrane, membrane blocking), and using the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention as a labeled antibody (preferably a secondary antibody), thereby enabling the detection of the target biological substance with high discrimination against target biological substances using other fluorescent dyes. In dot blotting using a fluorescently labeled biological substance obtained from the fluorescent compound of the present invention, as in multicolor WB, a blot nitrocellulose membrane or a blot PVDF (polyvinylidene fluoride) membrane is prepared by a commonly used method for the target biological substance, and the fluorescently labeled biological substance obtained from the fluorescent compound of the present invention is used as a labeled antibody (preferably a secondary antibody), thereby making it possible to detect the target biological substance with high discrimination compared to target biological substances using other fluorescent dyes.

[0087] - Substituent Group T - In the present invention, when a substituent is simply described as a substituent, the description of the corresponding substituent in Substituent Group T can be referenced and applied. For example, when an "alkyl group" is simply described, the description of the "alkyl group" in Substituent Group T can be referenced and applied. The same applies to substituents other than "alkyl groups." Furthermore, in the present invention, examples of substituents that a certain substituent such as an "alkyl group" may have include substituents selected from Substituent Group T below. Furthermore, 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 substituents formed by combining two or more substituents selected from Substituent Group T below. Furthermore, in the present invention, when an alkyl group is described separately from a cyclic (cyclo)alkyl group, the term "alkyl group" is used to encompass both linear and branched alkyl groups. On the other hand, when an alkyl group is not described separately from a cyclic alkyl group, or unless otherwise specified, the term "alkyl group" is used to encompass both linear and branched alkyl groups, and cycloalkyl groups. This also applies to groups (alkoxy groups, alkylthio groups, alkoxycarbonyl groups, alkylthiocarbonyl groups, alkylsulfonyl groups, etc.) containing groups capable of forming a cyclic structure (alkyl groups, alkenyl groups, alkynyl groups, etc.) and compounds containing groups capable of forming a cyclic structure. When a group can form a cyclic skeleton, the lower limit of the number of atoms in the group forming 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 capable of forming this structure. In the description of the substituent group T below, for example, groups having a linear or branched structure and groups having a cyclic structure are sometimes described separately, such as alkyl groups and cycloalkyl groups, in order to clarify these.

[0088] The groups included in the substituent group T include the following groups: 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), an aryl group (which may be a monocyclic group or a fused ring group (preferably a fused ring group of 2 to 6 rings). In the case of a fused ring group, it is preferably a 6- or 8-membered ring. Preferably, the monocycle is a 6-membered ring. The aryl group preferably has 6 to 40 carbon atoms, more preferably 6 to 30 carbon atoms, even more preferably 6 to 26 carbon atoms, and particularly preferably 6 to 10 carbon atoms), and may be 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, more preferably 5 or 6. 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).), alkoxy groups (preferably having 1 to 20 carbon atoms, 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), alkenyloxy groups (preferably having 2 to 20 carbon atoms, more preferably having 2 to 12 carbon atoms), alkynyloxy groups (preferably having 2 to 20 carbon atoms, more preferably having 2 to 12 carbon atoms), cycloalkyloxy groups (preferably having 3 to 20 carbon atoms), aryloxy groups (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), heterocyclic oxy groups (preferably having 2 to 20 carbon atoms), polyalkyleneoxy groups,

[0089] Alkoxycarbonyl groups (preferably having 2 to 20 carbon atoms, more preferably having 2 to 12 carbon atoms, and even more preferably having 2 to 8 carbon atoms), cycloalkoxycarbonyl groups (preferably having 4 to 20 carbon atoms, more preferably having 4 to 14 carbon atoms, and even more preferably having 4 to 10 carbon atoms), alkoxythiocarbonyl groups (preferably having 2 to 20 carbon atoms), aryloxycarbonyl groups (preferably having 6 to 20 carbon atoms, more preferably having 6 to 16 carbon atoms, and even more preferably having 6 to 12 carbon atoms), arylthiocarbonyl groups (preferably having 6 to 20 carbon atoms), amino groups (preferably having 0 to 20 carbon atoms, more preferably having 0 to 12 carbon atoms, more preferably having 0 to 8 carbon atoms, even more preferably having 0 to 6 carbon atoms, and particularly preferably having 0 to 3 carbon atoms), and unsubstituted amino groups (-NH ２), 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. The above groups substituting the unsubstituted amino group have the same meanings as the corresponding groups in 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, more preferably having 1 to 12 carbon atoms, and even more preferably having 1 to 8 carbon atoms), a carbamoyl group (preferably having 1 to 20 carbon atoms, more preferably having 1 to 12 carbon atoms, and even more preferably having 1 to 8 carbon atoms, and preferably an alkyl, cycloalkyl, or aryl carbamoyl group),

[0090] an acylamino group (preferably having 1 to 20 carbon atoms, more preferably having 1 to 12 carbon atoms, and even more preferably having 1 to 8 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, more preferably having 0 to 12 carbon atoms, and even more preferably having 0 to 8 carbon atoms, and an alkyl, cycloalkyl, or aryl sulfonamido group is preferred); 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); an aminosulfonyl group (with regard to the amino group in the aminosulfonyl group, the descriptions of the amino group in the above-mentioned substituent group T can be applied).

[0091] 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 alkyl, aryl, alkoxy or aryloxy), silyloxy group (preferably a silyloxy group having 1 to 20 carbon atoms and substituted with alkyl, aryl, alkoxy or aryloxy), hydroxy group, cyano group, nitro group, halogen atom (for example, fluorine atom, chlorine atom, bromine atom or iodine atom), oxygen atom (specifically, a >CH ２ is replaced by >C=O), boric acid group [-B(OH) ２ ], sulfanyl group (—SH), guanidino group (—NHC(═NH)NH ２ ), a polyol residue, or a sugar residue.

[0092] (Polyalkyleneoxy group) In the present invention, the polyalkyleneoxy group is a group represented by —O—(L ２ -O) ｎ -R ２１ It is sufficient that the group is a group represented by the following formula: Among polyol residues, those that fall under the category of polyalkyleneoxy groups are classified as polyalkyleneoxy groups. ２ may be linear or branched, preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, still more preferably 2 to 4 carbon atoms, and particularly preferably 2 or 3 carbon atoms; L ２ The number of carbon atoms contained in the shortest chain connecting the two carbon atoms that are the bonds of L is preferably 0 to 2, more preferably 0 or 1, and even more preferably 0. ２Specific examples of R 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. An ethylene group, a 1,2-propanediyl group, or a 1,2-butanediyl group is preferred, and an ethylene group is more preferred. n means the average repeat number (also simply referred to as the repeat number), and is not particularly limited, but is preferably 1 to 50, more preferably 1 to 23, even more preferably 1 to 17, and particularly preferably 1 to 11. Of these, 2 to 9 is preferred, and 3 to 8 is most preferred. In the present invention, the "average" in the average repeat number 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 repeat number is small, such as n being 1, it is possible to exhibit appropriate hydrophilicity and the effect of suppressing nonspecific adsorption by reducing hydrophobic interactions. R ２１ is a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. ２１ alkyl groups, aryl groups and heteroaryl groups which may be taken as R ２２ The alkyl group and aryl group that can be used as R can be the same as those described above for the corresponding substituents (alkyl group, aryl group, and heteroaryl group) in the substituent group T. ２１ is preferably a hydrogen atom or an alkyl group.

[0093] (Polyol Residue) In the present invention, the term "polyol residue" refers to a group obtained by removing one hydrogen atom from a polyol compound having two or more hydroxy groups in the molecule, and does not fall under either the sugar residue or polyalkyleneoxy group described below. The polyol residue may be a chain group or a group having a cyclic structure, such as a cyclodextrin residue. (Sugar Residue) In the present invention, the term "sugar residue" refers to a group obtained by removing one hydrogen atom from a sugar compound. The sugar compound may be any of monosaccharides, polysaccharides in which two or more sugars are bonded, sugar alcohols, and chemically modified sugars in which epichlorohydrin or the like is copolymerized with a sugar. Among polyol residues, those that fall under the category of sugar residues are classified as sugar residues. Examples of sugar residues include glucose, sucrose, maltose, lactose, trehalose, ribitol, sorbitol, mannitol, maltitol, lactitol, xylitol, fructose, 1-kestose, nystose trihydrate, fucose, dulcitol, galactooligosaccharide, 4'-galactosyllactose, isomaltooligosaccharide, lactulose, palatinite, palatinose monohydrate, raffinose pentahydrate, arabinose, dihydroxyacetone dimer, galactose, glyceraldehyde dimer, mannose, ribose, xylose, and lactositol. Examples of such sugar compounds include 1-deoxynojirimycin, ...

[0094] 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, alkoxycarbonyl groups, cycloalkoxycarbonyl groups, aryloxycarbonyl groups, amino groups, acyloxy groups, carbamoyl groups, acylamino groups, and sulfonamide groups, all of which have a substituent containing a polyalkyleneoxy group as a substituent.

[0095] The substituents selected from the substituent group T include not only groups formed by combining a plurality of substituents selected from the above-mentioned substituent group T, but also groups formed by combining a plurality of the above groups, unless otherwise specified. For example, when a compound or a substituent contains an alkyl group, an alkenyl group, or the like, it may be substituted or unsubstituted. Furthermore, when it contains an aryl group, a heterocyclic group, or the like, it may be a monocyclic or fused ring, and may be substituted or unsubstituted.

[0096] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. Note that room temperature means 25°C.

[0097] The fluorescent compound of the present invention, Compound (1), and comparative compounds (1) to (4) are shown below.

[0098]

[0099] (Comparative Compounds) Comparative compound (2) is a fluorescent compound known as sulfo-Cy5 (CAS No. 1144107-82-3), comparative compound (3) is a fluorescent compound known as HIDC (CAS No. 36536-22), and comparative compound (4) is a fluorescent compound known as Cy5 acid (CAS No. 195867-59-5).

[0100] The synthesis methods for each compound are explained in detail below, but the starting materials, dye intermediates and synthesis routes are not limited to these.

[0101] 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 reverse-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 of "acetonitrile:water = 0:100" was changed to an eluent of "acetonitrile:water = 20:80". For preparative HPLC (High Performance Liquid Chromatography), 2767 (trade name, manufactured by Waters) was used.

[0102] 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: ESI and APCI (Atmospheric Pressure Chemical Ionization) simultaneously performed).

[0103] <Synthesis of Compounds> Synthesis Example 1: Synthesis of Compound (1)

[0104] (Synthesis of Compound (1-A)) 200 ml of tert-butanol (t-BuOH) and 12 g of potassium tert-butoxide (t-BuOK) were placed in a 500 ml three-neck flask purged with nitrogen, and while stirring, 14.4 g of ethyl 2-methylacetoacetate was added dropwise and stirred for a while. Next, triethylene glycol 2-bromoethyl methyl ether (Br-mPEG ４32.5 g of HCl (HClaq.) was added dropwise and the mixture was heated and stirred. After stirring at 80°C for 1 hour, the solvent was distilled off under reduced pressure, and a liquid separation operation was performed using ethyl acetate and distilled water, and a crude product was extracted with distilled water. 30 ml of 30% aqueous hydrochloric acid solution (HClaq.) was added to the obtained crude product, and the mixture was stirred at 100°C for 3 hours. Thereafter, the solvent was distilled off under reduced pressure, and the product was purified by normal phase column chromatography (eluent: hexane:ethyl acetate = 50:50 to 30:70) to obtain 11.0 g of compound (1-A).

[0105] (Synthesis of Compound (1-B)) 554 mg of compound (1-A), 203 ml of phenylhydrazine, and 2.77 ml of acetic acid (AcOH) were placed in a pressure test tube and heated with stirring at 120°C for 2 hours under microwave irradiation. The solvent was evaporated under reduced pressure, and ethyl acetate and a 1N aqueous NaOH solution were added. Extraction was carried out by a liquid separation operation, and the residue was dried over sodium sulfate, after which the solvent was evaporated under reduced pressure. The obtained brown oily compound (1-B) was used in the next step without purification.

[0106] (Synthesis of Compound (1-C)) 168 mg of compound (1-B), 201 mg of 6-bromohexanoic acid, and 0.5 ml of acetonitrile (MeCN) were placed in a pressure test tube, and the mixture was heated and stirred at 100° C. for 6.5 hours under microwave irradiation. Subsequently, ethyl acetate and water were added, and the aqueous phase was extracted and purified by reverse phase column chromatography (eluent: water:acetonitrile=100:0 to 70:30), yielding 28 mg of compound (1-C).

[0107] (Synthesis of Compound (1-D)) 200 mg of compound (1-B), 222 mg of methyl-p-toluenesulfonate, and 1.4 ml of acetonitrile (MeCN) were placed in a pressure test tube, and the mixture was heated and stirred at 120° C. for 0.5 hours under microwave irradiation. Subsequently, 1.4 ml of water was added, and the reaction solution was directly loaded onto a column. The mixture was purified by reverse phase column chromatography (eluent: water:acetonitrile=100:0 to 50:50) to obtain 200 mg of compound (1-D).

[0108] (Synthesis of Compound (1-E)) 18 mg of compound (1-C), 14 mg of compound (1-D), 2.9 mg of squaric acid, 0.24 ml of 1-butanol, and 0.24 ml of toluene were placed in a pressure test tube, and the mixture was heated and stirred at 100° C. for 1.5 hours under microwave irradiation. Subsequently, the solvent was distilled off, and the mixture was purified by preparative HPLC (eluent: 20 mM aqueous ammonium formate:acetonitrile=95:5 to 5:95) to obtain 2.5 mg of compound (1-E).

[0109] (Synthesis of Compound (1)) 2.1 mg of compound (1-E), 2.4 μl of methylamine (9.8 M methanol solution), 2.8 mg of HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), 2.1 μL of diisopropylethylamine, and 0.5 ml of dimethyl sulfoxide were added to a test tube and stirred at room temperature for one hour. Subsequently, 0.5 ml of saturated aqueous sodium bicarbonate was added, and the mixture was purified by preparative HPLC (eluent: 20 mM aqueous ammonium formate:acetonitrile = 95:5 to 5:95) to obtain 0.5 mg of compound (1). ESI-MS [M+H ＋ ] ＋ =891

[0110] <Synthesis of Comparative Compound (1)>

[0111] (Synthesis of Comparative Compound (1-A)) 539 mg of comparative compound (1-A) was obtained in the same manner as in the synthesis of compound (1-C) above, except that 2,3,3-trimethylindolenine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of compound (1-B).

[0112] (Synthesis of Comparative Compound (1-B)) 2.1 mg of comparative compound (1-B) was obtained in the same manner as in the synthesis of compound (1-E) above, except that comparative compound (1-A) was used instead of compound (1-C) and 1,2,3,3-tetramethyl-3H-indolium iodide (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of compound (1-D).

[0113] (Synthesis of Comparative Compound (1)) 1.1 mg of comparative compound (1) was obtained in the same manner as in the synthesis of compound (1) above, except that comparative compound (1-B) was used instead of compound (1-E).

[0114] The water solubility, cell membrane permeability, and inhibition of nonspecific adsorption of each of the above compounds were evaluated as follows. The results are shown in Table 1.

[0115] [Evaluation 1: Water Solubility] 0.1 mg of each compound (powder) was weighed into a vial, and 10 μl (adjusted sample concentration: 10 mg / ml), 20 μl (adjusted sample concentration: 5 mg / ml), or 100 μl (adjusted sample concentration: 1 mg / ml) of water was added to prepare a sample solution of each sample concentration. The sample solution at 20°C was stirred with a vortex mixer for 1 minute, and then visually confirmed to see if the compound (powder) had dissolved. A solubility in water at 20°C of 10 mg / ml or more can be said to have water solubility suitable for bioimaging in both in vitro and in vivo tests. Since comparative compounds (2) to (4) are commercially available fluorescent compounds, their water solubility was evaluated based on the solubility information in water or PBS (phosphate-buffered saline) published by each distributor.

[0116] [Evaluation 2: Cell Membrane Permeability and Inhibition of Nonspecific Adsorption] HeLa cells were seeded into a 24-well plate and cultured at 37°C for 1 day, after which they were washed twice with HBSS (Hanks' Balanced Salts Solution). Subsequently, a 0.5 μM HBSS solution of each compound was added to the wells and incubated at 37°C for 30 minutes. For the obtained samples, XY images (magnification 63x) of live cells were observed using a confocal microscope (Leica, product name: Stellaris 5) without washing. Fluorescence in the wavelength range of 650-750 nm was detected using an excitation laser with a wavelength of 638 nm. Figure 1 shows the XY images obtained using the confocal microscope. Cell membrane permeability and inhibition of nonspecific adsorption were evaluated by applying the observation results of fluorescent signals inside and outside the cells to the following evaluation criteria. - Evaluation results (cell membrane permeability) - A: Fluorescent signals were observed inside the cells. B: Fluorescent signals were not observed inside the cells, and were observed only outside the cells. - Evaluation results (inhibition of non-specific adsorption) - A: Fluorescent signals were observed both inside and outside the cells. B: Fluorescent signals were not observed outside the cells, and were observed only inside the cells.

[0117]

[0118] (Notes for the table) Compound (1) and comparative compounds (1) to (4): These correspond to the above-mentioned compound (1) and comparative compounds (1) to (4), respectively. The water solubility of comparative compounds (2) to (4) is a reference value based on the following description. １） K in comparative compound (2) ＋ H ＋ The water solubility value of the compound (Cy5, CAS No. 146368-11-8) replaced by (https: / / www.glpbio.com / jp / gc35769.html) ２） Cl in comparative compound (3) － Ga I －The solubility value in PBS of the compound (DiIC1(5), CAS No. 36536-22-8) replaced by (https: / / www.glpbio.com / jp / diic1-5.html). Note that since there is thought to be little difference between the solubility in PBS and in water, the solubility value in PBS is used as the reference. ３） Solubility value in water of comparative compound (4) (CAS No. 195867-59-5) (https: / / www.aladdinsci.com / c171352.html) "-": Comparative compound (2) did not exhibit cell membrane permeability, so its ability to inhibit nonspecific adsorption could not be evaluated.

[0119] The results in Table 1 and Figure 1 reveal the following: Comparative compound (2), a negatively charged fluorescent compound, had no cell membrane permeability at all (see No. c12 and Figure 1(c)). Comparative compound (3), a positively charged fluorescent compound, had low solubility in water to begin with, and although it permeated the cell membrane, it was significantly adsorbed to mitochondria-like granular sites. Because it was not washed, no extracellular fluorescent signal that would normally be observed was detected, and nonspecific adsorption was not suppressed (see No. c13 and Figure 1(d)). Furthermore, comparative compound (4), a zwitterionic fluorescent compound having a cyanine skeleton rather than a squarylium skeleton, also had low solubility in water to begin with, and although it permeated the cell membrane, it was significantly adsorbed to mitochondria-like granular sites, and nonspecific adsorption was not suppressed (see No. c14 and Figure 1(e)). It is believed that the comparative compound (4) was unable to suppress the positive charge property because no charged atom was bonded to the π-conjugated structure. Furthermore, although it is a fluorescent compound having a zwitterionic squarylium skeleton, the R ２ , R ３ , R １０ and R １１ However, the comparative compound (1) which is not an alkyl group containing a neutral water-soluble unit has low solubility in water to begin with, and moreover, nonspecific adsorption which is different in appearance from the mitochondria-like granules was observed. This is because the comparative compound (1) is ２, R ３ , R １０ and R １１ Since none of the substituents in R is an alkyl group having a neutral water-soluble unit, it is thought that they are adsorbed to the endoplasmic reticulum etc. by hydrophobic interactions (see No. c11 and FIG. 1(b)). In contrast, compound (1), which is a fluorescent compound of the present invention, is excellent in water solubility and cell membrane permeability, and furthermore, nonspecific adsorption is suppressed. This is because both a positively charged nitrogen atom and a negatively charged oxygen atom are bonded to the π-conjugated structure, so that the properties of both the positive and negative charges are more effectively canceled out, and ２ , R ３ , R １０ and R １１ At least one of the groups is an alkyl group containing a neutral water-soluble unit, which reduces hydrophobic interactions. As a result, the compound has excellent water solubility and cell membrane permeability, and nonspecific adsorption is suppressed (see No. 101 and Figure 1(a)).

[0120] This application claims priority based on Japanese Patent Application No. 2024-045371, filed on March 21, 2024, the contents of which are incorporated herein by reference as part of the present specification.

Claims

1. A fluorescent compound represented by the following general formula (1): In the formula, Z represents a group represented by the following general formula (Z1) or (Z2). In the formula, R １ and R ９ represents an alkyl group or an aryl group; X represents an oxygen atom, a sulfur atom, or >C(R １０ ) (R １１ ) indicates. ２ , R ３ , R １０ and R １１ represents an alkyl group. ２ , R ３ , R １０ and R １１ At least one alkyl group in R contains a neutral water-soluble unit. ４ ~R ８ and R １２ ~R ２０ represents a hydrogen atom, a halogen atom, a cyano group, a nitro group, a hydroxy group, an alkyl group, an alkoxy group, an aryl group, a heteroaryl group, an acyloxy group, an acylamino group, an oxycarbonyl group, a carbamoyl group, or a sulfonamido group. Q represents an alkoxy group or an amino group. * represents a bond. However, R １ ~R ２０ and Q are not positively or negatively charged in aqueous solutions of pH 6-8.

2. The fluorescent compound according to claim 1, which is represented by the following general formula (2): In the formula, R １ ~R １６ is R in the general formula (1). １ ~R １６ However, R １ ~R １６ does not become positively or negatively charged in aqueous solution at pH 6-8.

3. The above R ２ , R ３ , R １０ and R １１ 3. The fluorescent compound according to claim 2, wherein the neutral water-soluble unit contained in at least one of the alkyl groups is a polyalkyleneoxy group.

4. The fluorescent compound according to claim 3, which is represented by the following general formula (3): In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ and R ４ ~R １６ is R in the general formula (2). １ , R ２ and R ４ ~R １６ However, R １ , R ２ , R ４ ~R １６ , R ２１ and R ２２ does not become positively or negatively charged in aqueous solution at pH 6-8.

5. The fluorescent compound according to claim 1, which is represented by the following general formula (4): In the formula, X １ represents an oxygen atom or a sulfur atom. １ ~R ９ and R １２ ~R １６ is R in the general formula (1). １ ~R ９ and R １２ ~R １６ However, R １ ~R ９ and R １２ ~R １６ does not become positively or negatively charged in aqueous solution at pH 6-8.

6. The above R ２ and R ３ 6. The fluorescent compound according to claim 5, wherein the neutral water-soluble unit contained in at least one of the alkyl groups is a polyalkyleneoxy group.

7. The fluorescent compound according to claim 6, which is represented by the following general formula (5): In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ , R ４ ~R ９ , R １２ ~R １６ and X １ is R in the general formula (4). １ , R ２ , R ４ ~R ９ , R １２ ~R １６ and X １ However, R １ , R ２ , R ４ ~R ９ , R １２ ~R １６ , R ２１ and R ２２ does not become positively or negatively charged in aqueous solution at pH 6-8.

8. The fluorescent compound according to claim 1, which is represented by the following general formula (6): In the formula, R １ ~R ８ , R １７ ~R ２０ and Q is R in the general formula (1). １ ~R ８ , R １７ ~R ２０ and Q. However, R １ ~R ８ , R １７ ~R ２０ and Q are not positively or negatively charged in aqueous solutions of pH 6-8.

9. The above R ２ and R ３ 9. The fluorescent compound according to claim 8, wherein the neutral water-soluble unit contained in at least one of the alkyl groups is a polyalkyleneoxy group.

10. The fluorescent compound according to claim 9, which is represented by the following general formula (7): In the formula, L １ is R ２ represents a linking group consisting of 1 to 100 atoms, the bonding site to the carbon atom to which L is bonded being an alkylene group; ２ represents an alkylene group having 2 to 4 carbon atoms. n is an integer of 1 to 23. R ２１ represents a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, a propargyl group, or —C(═O)R ２２ indicates R ２２ represents an alkyl group or an aryl group. １ , R ２ , R ４ ~R ８ , R １７ ~R ２０ and Q is R in the general formula (6). １ , R ２ , R ４ ~R ８ , R １７ ~R ２０ and Q. However, R １ , R ２ , R ４ ~R ８ , R １７ ~R ２２ and Q are not positively or negatively charged in aqueous solutions of pH 6-8.

11. The above R １７ The fluorescent compound of claim 8 , wherein is a hydroxy group.

12. The fluorescent compound of claim 8, wherein Q is an amino group.

13. The fluorescent compound according to any one of claims 1 to 12, which has at least one group capable of binding to a biological substance.

Images