JP2023059377A - Fluorescent compound and fluorescent-labeled biological substance based thereon - Google Patents

Abstract

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

Description

TECHNICAL FIELD The present invention relates to a fluorescent compound and a fluorescently labeled biological material using the same.

Fluorescence in which biomolecules (antibodies, etc.) that bind to the substance to be detected are labeled with fluorescent compounds (fluorochromes) in order to observe changes in the body in response to various stimuli (disease, environmental changes, etc.) Labeled biomaterials are often used.
For example, in Western blotting (hereinafter also abbreviated as WB) for detecting a specific protein from a protein mixture, the presence or absence or abundance of the specific protein is detected using a fluorescence-labeled antibody that binds to this protein. A fluorescence method is used.
In bioimaging technology that analyzes the dynamics and functions of biomolecules, cells, tissues, etc. in living organisms, biofluorescence imaging that observes specific parts of the organism visualized by fluorescent labeling is one of the techniques for in vivo observation. used as one.

In the above fluorescent labeling, organic fluorescent dye molecules are generally used, and the brightness (fluorescence intensity) is generally increased by using a fluorescently labeled biological substance in which a plurality of fluorescent dye molecules are bound. Fluorescein dyes, rhodamine dyes, and the like are known as fluorescent dyes having a high fluorescence quantum yield and high light resistance (light fade resistance). Research is also underway on fluorescent dyes in which the absorption wavelength and fluorescence wavelength are lengthened by replacing the oxygen atom, which is a ring-constituting atom of the xanthene skeleton of rhodamine or rhodol, with a silicon atom or the like.
In addition to the fluorescently labeled biosubstances described above, research has also been conducted on fluorescent probes for detecting specific physiologically active molecules. Fluorescent probes are often compounds that do not exhibit fluorescence per se, and change into compounds that emit fluorescence as the molecular structure changes due to reaction with target physiologically active molecules.
For example, Patent Document 1 and Non-Patent Document 1 describe a compound in which β-galactose is bound to the hydroxyl group of Si-rodol as a fluorescent probe that exhibits fluorescence when β-galactose is cleaved by β-galactosidase. describe compounds in which β-galactose is attached to the hydroxy group of fluorescein. Also, in Non-Patent Document 3,
A compound is described in which β-galactose is introduced as a substituent on the amino group of Si-rhodamine.

However, most fluorescent organic dyes, such as rhodamine dyes and rhodol dyes, have highly planar aromatic chromophores, and are therefore likely to interact with each other. As a result, fluorescence intensity tends to decrease due to interactions such as self-association between dyes after labeling. Fluorescence intensity tends to be lower due to

WO2014/106957

Mako Kamiya et. al. , J. AM. CHEM. SOC. , 2007, 129, 3918-3929. Takahiro Egawa et. al. , Chem. Commun. , 2011, 47, 4162-4164. Hongxing Zhang et. al. , Biomaterials, 2018, 158, 10-22.

For all organic fluorescent dyes, it is important to search for a chemical structure that can suppress the decrease in fluorescence intensity (fluorescence quantum yield) that accompanies an increase in fluorescence labeling rate.
An object of the present invention is to provide a fluorescent compound that is excellent in suppressing a decrease in fluorescence intensity (fluorescence quantum yield) accompanying an increase in fluorescence labeling rate when a biological substance or the like is labeled. Another object of the present invention is to provide a fluorescence-labeled biological substance obtained by binding this fluorescent compound to a biological substance.

上記式中、ＦＬはｎ価の蛍光体部を示し、ｎは１以上の整数である。
Ｑは下記一般式（Ｂ）で表される基を示す。

上記式中、Ｒ^１～Ｒ^３は水素原子、アルキル基、アリール基又はヘテロアリール基を示す。
Ｌは、単結合、又は、アルキレン基、アルケニレン基、アルキニレン基、アリーレン基、ヘテロアリーレン基及び下記式（１－１）～式（１－８）のいずれかで表される各基のうちの１種又は２種以上を組み合わせた連結基を示す。
＊は結合部を示す。
ただし、ＬとＲ^１～Ｒ^３のいずれか１つとが結合して、グリコシル構造を形成することはない。

上記式中、Ｒ^３１及びＲ^３２は水素原子又は置換基を示す。
＊は結合部を示す。
〔２〕
上記ＦＬが、ジピロメテン化合物、ローダミン化合物、キサンテン化合物、ロードール化合物、ピロロピロール化合物、ピレン化合物、クマリン化合物又はシアニン化合物からなる構造部である、〔１〕に記載の蛍光性化合物。
〔３〕
上記ＦＬが、ローダミン化合物、キサンテン化合物又はロードール化合物からなる構造部である、〔２〕に記載の蛍光性化合物。
〔４〕
下記一般式（２）で表される、〔３〕に記載の蛍光性化合物。

上記式中、Ｘは、－Ｏ－、－Ｓ－、＞ＳＯ、＞ＳＯ_２、＞ＳｉＲ^１０Ｒ^１１又は＞Ｐ（＝Ｏ）Ｒ^１２を示す。
Ｙ^１は－ＯＲ^１３又は－ＮＲ^１４Ｒ^１５を示し、Ｙ^２は＝Ｏ、＝ＮＲ^１６又は＝Ｎ^＋Ｒ^１７Ｒ^１８を示す。Ｚは下記式（Ａ）で表される基を示す。
Ｒ^４～Ｒ^９は、ハロゲン原子、シアノ基又は下記式（Ａ）で表される基を示す。
Ｒ^１０及びＲ^１１は下記式（Ａ）で表される基を示し、Ｒ^１０とＲ^１１が互いに結合して４～７員環の脂肪族複素環を形成していてもよい。ただし、Ｒ^１０とＲ^１１が互いに結合して環を形成していない場合、Ｒ^１０及びＲ^１１の少なくとも一方はアルケニル基、アルキニル基、アリール基又はヘテロアリール基である。
Ｒ^１２～Ｒ^１８は下記式（Ａ）で表される基を示す。
ただし、上記化合物中のＲ^１３～Ｒ^１８のうちの少なくとも１つは、上記一般式（Ｂ）で表される基である。

式中、Ｌ^３は、単結合、又は、アルキレン基、アルケニレン基、アルキニレン基、アリーレン基、ヘテロアリーレン基及び上記式（１－１）～式（１－８）のいずれかで表される各基のうちの１種又は２種以上を組み合わせた連結基を示す。
Ｒ^１１１は、水素原子、アルキル基、アルケニル基、アルキニル基、アリール基、ヘテロアリール基又は１価の脂肪族複素環基を示す。
ただし、Ｒ^１１１における水素原子が解離性の水素原子である場合は、この解離性の水素原子は解離していてもよい。
＊は結合部を示す。
〔５〕
下記一般式（３）又は（４）で表される、〔４〕に記載の蛍光性化合物。

上記式中、Ｘは、－Ｏ－又は＞ＳｉＲ^１０Ｒ^１１を示す。
Ｒ^１～Ｒ^１１、Ｌ及びＺは上記のＲ^１～Ｒ^１１、Ｌ及びＺと同義である。
〔６〕
〔１〕～〔５〕のいずれか１つに記載の化合物と生体物質とが結合してなる蛍光標識生体物質。
〔７〕
上記生体物質がタンパク質、アミノ酸、核酸、ヌクレオチド、糖鎖及び脂質のいずれかである〔６〕に記載の蛍光標識生体物質。
〔８〕
上記化合物と上記生体物質との結合が下記ｉ）～ｖ）のいずれかによる結合である〔６〕に記載の蛍光標識生体物質。
ｉ）ペプチド間での非共有結合又は共有結合、
ｉｉ）化合物中の長鎖アルキル基と生体物質中の脂質二重膜又は脂質とのファンデルワールス相互作用、
ｉｉｉ）化合物中のＮ－ヒドロキシスクシンイミドエステルと生体物質中のアミノ基とを反応させてなるアミド結合、
ｉｖ）化合物中のマレイミド基と生体物質中のスルファニル基とを反応させてなるチオエーテル結合、
ｖ）化合物中のアジド基と生体物質中のアセチレン基、又は、化合物中のアセチレン基と生体物質中のアジド基とをＣｌｉｃｋ反応させてなる、トリアゾール環の形成を伴う結合 That is, the above problems of the present invention have been solved by the following means.
[1]
A fluorescent compound represented by the following general formula (1).

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

In the above formula, R ¹ to R ³ represent a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group.
L is a single bond, or an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and any of the groups represented by the following formulas (1-1) to (1-8) It represents a linking group of one type or a combination of two or more types.
* indicates a joint.
However, L and any one of R ¹ to R ³ do not combine to form a glycosyl structure.

In the above formula, R ³¹ and R ³² represent hydrogen atoms or substituents.
* indicates a joint.
[2]
The fluorescent compound according to [1], wherein FL is a structural moiety comprising a dipyrromethene compound, a rhodamine compound, a xanthene compound, a rhodol compound, a pyrrolopyrrole compound, a pyrene compound, a coumarin compound, or a cyanine compound.
[3]
The fluorescent compound according to [2], wherein the above FL is a structural moiety composed of a rhodamine compound, a xanthene compound or a rhodol compound.
[4]
The fluorescent compound according to [3], represented by the following general formula (2).

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

In the formula, L ³ is a single bond, or an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and any of the above formulas (1-1) to (1-8). A linking group consisting of one or a combination of two or more of the groups is shown.
R ¹¹¹ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or a monovalent aliphatic heterocyclic group.
However, when the hydrogen atom in R ¹¹¹ is a dissociative hydrogen atom, this dissociative hydrogen atom may be dissociated.
* indicates a joint.
[5]
The fluorescent compound according to [4], represented by the following general formula (3) or (4).

In the above formula, X represents —O— or >SiR ¹⁰ R ¹¹ .
R ¹ to R ¹¹ , L and Z have the same definitions as R ¹ to R ¹¹ , L and Z above.
[6]
A fluorescence-labeled biological material obtained by binding the compound according to any one of [1] to [5] to a biological material.
[7]
The fluorescence-labeled biomaterial of [6], wherein the biomaterial is any one of proteins, amino acids, nucleic acids, nucleotides, sugar chains and lipids.
[8]
The fluorescence-labeled biological material according to [6], wherein the binding between the compound and the biological material is by any one of the following i) to v).
i) non-covalent or covalent bonding between peptides,
ii) van der Waals interactions between long-chain alkyl groups in compounds and lipid bilayer membranes or lipids in biomaterials;
iii) an amide bond formed by reacting an N-hydroxysuccinimide ester in the compound with an amino group in the biological substance;
iv) a thioether bond formed by reacting a maleimide group in the compound with a sulfanyl group in the biological substance;
v) Bonding accompanied by formation of a triazole ring by Click reaction between an azide group in a compound and an acetylene group in a biological substance, or an acetylene group in a compound and an azide group in a biological substance

INDUSTRIAL APPLICABILITY The fluorescent compound of the present invention can exhibit an excellent effect of suppressing a decrease in fluorescence intensity (fluorescence quantum yield) accompanying an increase in fluorescence labeling rate when a biological substance or the like is labeled. In addition, the fluorescence-labeled biological material of the present invention is excellent in suppressing a decrease in fluorescence intensity (fluorescence quantum yield) accompanying an increase in fluorescence labeling rate.

In the present invention, when there are a plurality of substituents or connecting groups (hereinafter referred to as substituents, etc.) indicated by a specific symbol or formula, or when a plurality of substituents, etc. are defined at the same time, there is no particular notice. As long as the respective substituents and the like may be the same or different. This also applies to the number of substituents and the like. Moreover, when a plurality of substituents and the like are close to each other (especially when they are adjacent), they may be linked to each other to form a ring unless otherwise specified. Moreover, when forming a ring, a hydrogen atom may be removed to form an unsaturated ring or an aromatic ring. In addition, unless otherwise specified, rings such as alicyclic rings, aromatic rings and heterocyclic rings may be further condensed to form condensed rings.
In the present invention, the double bond may be either E-type or Z-type, or a mixture thereof, unless otherwise specified. In addition, unless otherwise specified, when a compound has an asymmetric carbon atom or an asymmetric center and the compound has diastereomers and enantiomers, any of them and mixtures thereof There may be.

In the present invention, the term "compound" and "substituent" includes not only the compound itself and the substituent itself, but also its salts and ions. For example, a carboxy group, a sulfo group, a phosphono group (--P(=O)(OH) ₂ ) and the like may have an ionic structure or a salt structure due to dissociation of a hydrogen atom. That is, in the present invention, the term "carboxy group" means a carboxylate ion or a salt thereof, the term "sulfo group" means a sulfonate ion or a salt thereof, and the term "phosphono group" means a phosphonate ion or a salt thereof. do. The monovalent or polyvalent cation for forming the above salt structure is not particularly limited, and includes inorganic cations, organic cations, etc. Specifically, alkali metals such as Na ⁺ , Li ⁺ and K ⁺ cations, alkaline earth metal cations such as Mg ²⁺ , Ca ²⁺ and Ba ²⁺ , and organic ammonium cations such as trialkylammonium cations and tetraalkylammonium cations.
In the case of a salt structure, the type of the salt may be one type, or two or more types may be mixed, and the salt type and free acid structure groups may be mixed in the compound. and a free acid structure compound may be mixed.
All of the compounds of the present invention are electrically neutral compounds. For example, in the compound represented by the general formula (2), when Y ² is =N ⁺ R ¹⁷ R ¹⁸ , this nitrogen atom is specifically shown as a positively charged structure for convenience. However, since the FL (phosphor moiety) in the compound of the present invention has a conjugated system, in fact, other atoms other than the nitrogen atom may take a positive charge, and one of the chemical structures is the general formula Any compound that can take the structure represented by (2) is included in the compound represented by general formula (2). This also applies to negative charges. The same applies to compounds represented by other general formulas.
Moreover, it is a meaning including the thing which changed a part of structure in the range which does not impair the effect of this invention. Furthermore, compounds that are not specified as substituted or unsubstituted are meant to have optional substituents within a range that does not impair the effects of the present invention. This includes substituents (e.g., groups expressed as "alkyl group", "methyl group", "methyl", etc.) and linking groups (e.g., "alkylene group", "methylene group", "methylene" The same applies to groups expressed as such. Among such optional substituents, preferred substituents in the present invention are substituents selected from the group T of substituents described below.
In the present invention, the rhodamine compound means a rhodamine compound in which the 3- and 6-positions of the xanthene ring are substituted with an amino group and an imino group. It means a rhodol compound substituted with an imino group. Further, the xanthene compound in the present invention means a compound having a xanthene ring other than the rhodamine compounds and rhodol compounds described above. Atoms may be substituted with alkyl groups, etc.) and fluorescein compounds substituted with oxo groups.
In the present invention, a rhodamine compound, a rhodol compound and a xanthene compound are respectively a rhodamine compound, a rhodol compound and a xanthene compound in which an oxygen atom that is a ring-constituting atom of the xanthene skeleton is replaced with a silicon atom, a phosphorus atom or a sulfur atom. Used in the sense of including.
In the present invention, when the number of carbon atoms in a certain group is defined, this number of carbon atoms means the number of carbon atoms in the entire group unless otherwise specified in the present invention or this specification. That is, when this group is in the form of further having a substituent, it means the total number of carbon atoms including this substituent.

Further, in the present invention, a numerical range represented by "-" means a range including the numerical values described before and after "-" as lower and upper limits.

The fluorescent compound of the present invention is represented by general formula (1) below. The reason why the fluorescent compound of the present invention is excellent in suppressing a decrease in fluorescence intensity (fluorescence quantum yield) accompanying an increase in the fluorescent labeling rate when a biological substance or the like is labeled is not clear, but is presumed as follows. ing.
It is considered that the interaction between the phosphor portions FL increases due to the increase in the concentration of the phosphor portion FL accompanying the increase in the fluorescent labeling rate. It is presumed that this causes self-association or aggregation of the fluorescent compound and reduces the fluorescence quantum yield. The group represented by the general formula (B) that binds to the phosphor portion FL disclosed by the present invention has one hydroxy group on each of two adjacent carbon atoms. As a result, at least intermolecular interaction between the phosphor portions FL is suppressed, and self-association or aggregation of the fluorescent compound of the present invention can be suppressed.

The fluorescent compound represented by formula (1) of the present invention is described in detail below.

<Fluorescent Compound Represented by Formula (1)>
The fluorescent compound represented by the general formula (1) of the present invention (hereinafter also referred to as "the compound of the present invention") is as follows.

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

In the above formula, R ¹ to R ³ represent a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group.
L is a single bond, or an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and any of the groups represented by formulas (1-1) to (1-8) described below. is a linking group obtained by combining one or more of That is, L may be a single bond; also, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, a group represented by formula (1-1) described later, formula (1-2) A group represented by the formula (1-3), a group represented by the formula (1-4), a group represented by the formula (1-5), represented by the formula (1-6) a group represented by formula (1-7), or a group represented by formula (1-8); also an alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, described later The group represented by the formula (1-1), the group represented by the formula (1-2), the group represented by the formula (1-3), the group represented by the formula (1-4), the formula selected from a group represented by (1-5), a group represented by formula (1-6), a group represented by formula (1-7), and a group represented by formula (1-8) A group formed by combining two or more groups may also be used.
* indicates a joint.

However, L and any one of R ¹ to R ³ do not combine to form a glycosyl structure. Here, the glycosyl structure means a structure obtained by removing an anomeric hydroxy group from a cyclic sugar, and is generally a glycosyl structure forming an O-glycosidic bond, an N-glycosidic bond or a C-glycosidic bond. are mentioned. As a specific example, the structure shown below corresponds to a glycosyl structure in which the hydrogen atom in the anomeric hydroxy group of glucose has been removed to form an O-glycosidic bond. * indicates the binding site.

In the fluorescent compound, neither of the above FL and L has a structure represented by >C (OH) C (OH) <, and among the above L, the carbon atom to which R ¹ is bonded The above general formula (1) shall be applied so that the site that binds to does not have a structure represented by >C(OH)-.

The substituents and the like in formula (1) are described in detail below.

[Q: group represented by general formula (B)]
In general formula (B) above, R ¹ to R ³ represent a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group.
Alkyl groups, aryl groups, and heteroaryl groups that can be used as R ¹ to R ³ may be unsubstituted groups or groups having substituents. Aryl and heteroaryl groups can be applied respectively.
The substituents that the above alkyl groups, aryl groups and heteroaryl groups that can be taken as R ¹ to R ³ may have are not particularly limited, and include substituents in the substituent group T described later. Among these, a hydroxy group, an alkoxy group, a cycloalkyloxy group, an aryloxy group or a heterocyclicoxy group is preferred, and a hydroxy group, an alkoxy group, a cycloalkyloxy group or an aliphatic heterocyclicoxy group is more preferred.
Examples of the substituents that the above alkoxy group, aryloxy group, cycloalkyloxy group and heterocyclic oxy group may have include substituents in the substituent group T described later, and hydroxy group and alkyl group are preferable. mentioned. For example, a heterocyclic oxy group (preferably an aliphatic heterocyclic oxy group) having a hydroxy group or a hydroxyalkyl group as a substituent includes a glycosyloxy group (an anomeric hydroxy group in an aldose or ketose excluding a hydrogen atom). obtained heterocyclic oxy group).

Hydrogen atoms are preferred as R ¹ and R ² .
R ³ is preferably a hydrogen atom or an alkyl group, more preferably a hydrogen atom or an alkyl group containing a hydroxy group as a substituent.

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

L is not particularly limited as long as it is a group that connects with the phosphor part FL and is within the range in which the effects of the present invention are exhibited.
Specifically, an alkylene group, or an alkylene group, an arylene group, a heteroarylene group, and two or more of each group represented by any of formulas (1-3) to (1-4) described below. A combined linking group is preferable, and an alkylene group, or a linking group obtained by combining two or more of each group represented by any of the following formulas (1-3) to (1-4) with an alkylene group. More preferred is an alkylene group.

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

In the above formula, R ^3a to R ^3f represent a hydrogen atom, an alkyl group, an aliphatic heterocyclic group, an aryl group or a heteroaryl group.
L, R ¹ and R ² have the same definitions as L, R ¹ and R ² above.
* indicates a joint.
However, L and any one of R ¹ , R ² and R ^3a to R ^3f do not combine to form a glycosyl structure.

R ^3a to R ^3f represent a hydrogen atom, an alkyl group, an aliphatic heterocyclic group, an aryl group or a heteroaryl group.
Alkyl groups, aliphatic heterocyclic groups, aryl groups and heteroaryl groups that can be taken as R ^3a to R ^3f may be unsubstituted groups or groups having substituents, and substituents described later. Alkyl groups, aliphatic heterocyclic groups, aryl groups and heteroaryl groups in group T can be applied respectively.
The substituents that the above alkyl groups, aliphatic heterocyclic groups, aryl groups and heteroaryl groups that can be taken as R ^3a to R ^3f are not particularly limited, and are substituted in the substituent group T described later. groups, and among these, a hydroxy group or an alkyl group is preferred. For example, the aliphatic heterocyclic group having a hydroxy group or a hydroxyalkyl group as a substituent includes a glycosyl group (an aliphatic heterocyclic group obtained by removing an anomeric hydroxy group in an aldose or ketose).
R ^3a to R ^3c , R ^3e and R ^3f are preferably hydrogen atoms.
R ^3d is preferably a hydrogen atom or an aliphatic heterocyclic group, more preferably a hydrogen atom or an aliphatic heterocyclic group having a hydroxyl group-containing group as a substituent, and still more preferably a hydrogen atom or a glycosyl group.

[FL, n]
FL indicates an n-valent phosphor portion, where n is an integer of 1 or more.
As the phosphor portion that can be used as the FL, any structure portion made of an organic compound exhibiting fluorescence can be used without particular limitation.
Examples of FL include structural units composed of dipyrromethene compounds, rhodamine compounds, xanthene compounds, rhodol compounds, pyrrolopyrrole compounds, pyrene compounds, coumarin compounds or cyanine compounds, and are preferred.
As the dipyrromethene compound, rhodamine compound, xanthene compound, rhodol compound, pyrrolopyrrole compound, pyrene compound, coumarin compound or cyanine compound, compounds (fluorescent dyes) commonly known as these dyes are used without particular limitation. be able to.
n is an integer of 1 or more, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, and even more preferably an integer of 2 to 4.

From the viewpoint of versatility in bioimaging, the compound of the present invention preferably has at least one carboxy group or a substituent capable of binding to a biological substance described later, and a carboxy group in FL or a biological substance described later. It is more preferable to have at least one substituent.
The compound of the present invention can bind to a biological substance via the carboxy group or the substituent capable of binding to the biological substance to obtain the desired labeled biological substance. A carboxyl group can be easily derived into a substituent capable of binding to a biological substance by a conventional method.
In the present invention, the "substituent capable of binding to a biological substance" includes a substituent capable of binding to a biological substance derived from a carboxy group.
The total number of carboxyl groups or substituents capable of binding to biological substances in the compound of the present invention is preferably at least 1 or more, and more preferably 1 to 3 from the viewpoint of quantification of the substance to be detected. One or two are more preferred, and one is particularly preferred.

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

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

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

1) ^R4 to ^R9
R ⁴ to R ⁹ above represent a halogen atom, a cyano group or a group represented by formula (A) described later, preferably a halogen atom or a group represented by formula (A) described later, a hydrogen atom, a halogen atom or a sulfo group is more preferred, and a hydrogen atom is even more preferred.
Halogen atoms which R ⁴ to R ⁹ can take include fluorine, chlorine, bromine and iodine atoms, preferably fluorine.
As the alkyl group that can be taken by R ⁴ to R ⁹ , the description of the alkyl group that can be taken as R ¹¹¹ described later can be applied.
The alkyl group which can be taken by R ⁴ to R ⁹ may be unsubstituted or may have a substituent, and the substituent which may have is preferably a sulfo group.
Specific examples of R ⁴ to R ⁹ include hydrogen atom, methyl group, ethyl group, fluorine atom and sulfo group.

2) Z.
The above Z represents a group represented by the formula (A) described later, preferably a group in the preferred form of (1) or (2) described later, an alkyl group, an alkoxy group, an amino group, an aryl group or a heteroaryl group An alkyl group, an aryl group or a heteroaryl group is preferred, and an aryl group is even more preferred.
The alkyl group, alkoxy group, amino group, aryl group and heteroaryl group that Z can take include the alkyl group, alkoxy group, amino group, aryl group and heteroaryl group represented by the formula (A) below. can be applied.

Z preferably has a carboxy group or a substituent capable of binding to a biosubstance, which will be described later.
In the description of each group that can be used as Z below, a form having a substituent capable of binding to a biosubstance described below instead of the carboxy group is also preferable.
The alkyl group, alkoxy group and amino group that Z can take preferably have a carboxy group, and are -(CH ₂ ) _u COOH, -(CH ₂ ) _u -phenylene-COOH, -cycloalkylene-COOH, —O(CH ₂ ) _u COOH or —NH(CH ₂ ) _u COOH are more preferred. Note that u is an integer of 1 to 15, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
The heteroaryl group that Z can take preferably has a carboxy group.
The heteroaryl group is preferably a monocyclic group, more preferably a thiophene ring group, an imidazole ring group, a pyrazole ring group or a pyridine ring group, and still more preferably an imidazole ring group, a pyrazole ring group or a pyridine ring group.

The aryl group that Z can take may be a monocyclic group or a condensed ring group, preferably a monocyclic group, and more preferably a group represented by the following formula (C).

In the formula, R ⁴¹ to R ⁴⁵ represent hydrogen atoms or substituents. * indicates a joint.
However, at least one of the following (C-1) to (C-3) is satisfied.
(C-1) At least one of R ⁴² to R ⁴⁴ is a group having either a carboxy group or a substituent capable of binding to a biological substance.
(C-2) R ⁴¹ and R ⁴⁵ are alkyl groups or halogen atoms.
(C-3) At least one of R ⁴¹ and R ⁴⁵ is a fluorine atom or an alkoxy group.

Non-radiative deactivation is suppressed when the above (C-1) is satisfied, and thermal deactivation due to free rotation of the aryl group is suppressed by steric hindrance when the above (C-2) is satisfied, and the above (C- When 3) is satisfied, heat deactivation due to free rotation of the aryl group is suppressed by intramolecular hydrogen bonding, and by satisfying at least one of these (C-1) to (C-3), Fluorescence quantum yield is further improved.

Substituents that R ⁴¹ to R ⁴⁵ can take include halogen atoms, cyano groups, alkyl groups, alkoxy groups, carboxy groups, alkoxycarbonyl groups, carbamoyl groups, amido groups, sulfo groups, sulfoamido groups, amino groups, alkenyl groups, An alkynyl group, an aryl group, a heteroaryl group, an aliphatic heterocyclic group, or a substituent capable of binding to a biosubstance described later includes a halogen atom, an alkyl group, an alkoxy group, a carboxy group, an alkoxycarbonyl group, a carbamoyl group, An alkynyl group or a substituent capable of binding to a biosubstance, which will be described later, is preferred.
Halogen atoms, alkyl groups, alkoxy groups, alkoxycarbonyl groups, carbamoyl groups, amide groups, sulfonamide groups, amino groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups and aliphatic heteroaryl groups that can be taken as R ⁴¹ to R ⁴⁵ As the cyclic group, corresponding groups in the group of substituents T to be described later can be applied.
Alkyl groups, alkoxy groups, alkoxycarbonyl groups, carbamoyl groups, amido groups, sulfonamido groups, amino groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups and aliphatic heterocyclic groups that can be used as R ⁴¹ to R ⁴⁵ are It may have a substituent, and examples thereof include groups in the substituent group T described later, and preferred examples include a poly(alkyleneoxy) group, a sulfo group, a carboxy group, or a substituent capable of binding to a biological substance.
The substituents for R ⁴¹ and R ⁴⁵ are preferably halogen atoms, alkyl groups or alkoxy groups from the viewpoint of suppressing rotation of the benzene ring in formula (C).
Substituents that can be used as R ⁴² to R ⁴⁴ are not particularly limited, and are preferably selected from the group of substituents T described later and preferably have a carboxy group or a substituent capable of binding to a biological substance. A carboxy group or a substituent capable of binding to a biological substance may be directly bonded to the benzene ring in general formula (C), or may be bonded via a linking group. preferable. Preferred examples of the linking group include an alkylene group, a poly(alkyleneoxy) group, an alkoxycarbonyl group, an amide group, and a group formed by combining these groups. Examples of the alkylene group, poly(alkyleneoxy) group, alkoxycarbonyl group, amido group, or group formed by combining these groups include -C(=O)O(CH ₂ CH ₂ O) _m CH ₂ CH ₂ -, -CONH(CH ₂ CH ₂ O) _m CH ₂ CH ₂ -, -CO-piperidinediyl-. m means the average number of repetitions, and is 1 to 30, preferably 1 to 15, more preferably 1 to 10.
Among them, it is preferable that R ⁴³ has a carboxy group or a substituent capable of binding to a biosubstance from the viewpoint of binding to a biosubstance.
R ⁴² to R ⁴⁴ are preferably a hydrogen atom, a carboxy group, or a group having a substituent capable of binding to a biological substance, and a hydrogen atom, a carboxy group, or a substituent capable of binding to a biological substance. is more preferred.

3) Y ¹ and Y ²
Y ¹ represents -OR ¹³ or -NR ¹⁴ R ¹⁵ , Y ² represents =O, =NR ¹⁶ or =N ⁺ R ¹⁷ R ¹⁸ , and R ¹³ to R ¹⁸ are represented by formula (A) described later. indicates a group.
As R ¹³ to R ¹⁸ , the preferred forms of groups (1) or (2) described below can be preferably applied.
However, at least one of R ¹³ to R ¹⁸ in the above compound is a group represented by the above general formula (B).

R ¹⁴ and R ¹⁵ may combine with each other to form a 4- to 7-membered aliphatic heterocyclic ring, and R ¹⁷ and R ¹⁸ may combine with each other to form a 4- to 7-membered aliphatic heterocyclic ring. You may have In addition, R ¹³ to R ¹⁸ may combine with adjacent R ⁵ to R ⁸ to form a 5- to 7-membered aliphatic heterocyclic ring or a 5- to 7-membered aromatic heterocyclic ring.
The ring which may be formed by combining R ¹⁴ and R ¹⁵ , the ring which may be formed by combining R ¹⁷ and R ¹⁸ , or R ¹³ to R ¹⁸ are adjacent to each other. Any ring that may be formed by bonding with R ⁵ to R ⁸ is selected from nitrogen atoms to which R ¹³ to R ¹⁸ are bonded, oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting atoms. may have 1 to 3 heteroatoms.

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

The 4- to 7-membered aliphatic heterocyclic ring that can be formed by combining R ¹⁷ and R ¹⁸ is not particularly limited, but is preferably a saturated aliphatic heterocyclic ring having 4 to 6 ring members. is preferred. This aliphatic heterocyclic ring may be a monocyclic ring, or may have a condensed ring structure such as a structure in which at least one atom in the norbornane structure is replaced with a nitrogen atom. It is also preferable to have >SO ₂ as a ring-constituting atom, and it is also preferable to have an electron-withdrawing group such as a halogen atom (preferably a fluorine atom) or a carbonyl group, which will be described later, as a substituent.

Any ring that can be formed by combining R ¹⁷ or R ¹⁸ with R ⁷ or R ⁸ may be aliphatic or aromatic as long as it is a heterocyclic ring having 5 to 7 ring members. . Among these rings, the number of ring members is preferably 5 or 6, and it is preferable that they do not have heteroatoms other than the nitrogen atoms substituted by R ⁷ or R ⁸ as ring-constituting atoms. Moreover, although it may be a single ring or a condensed ring, the ring formed by the bond is preferably a single ring.

As for R ¹⁴ and R ¹⁵ , the descriptions for R ¹⁷ and R ¹⁸ above can be applied. However, the difference is that the nitrogen atom to which R ¹⁷ and R ¹⁸ are bonded has a positive charge, whereas the nitrogen atom to which R ¹⁴ and R ¹⁵ are bonded does not have a positive charge. Also, the ring that can be formed by combining R ¹⁷ or R ¹⁸ with R ⁷ or R ⁸ is read as the ring that can be formed by combining R ¹⁴ or R ¹⁵ with R ⁵ or R ⁶ .

R ¹⁶ is preferably an alkyl group.
As the alkyl group that R ¹⁶ can take, the description of the alkyl group that R ¹⁷ and R ¹⁸ can take can be applied. A ring that can be formed by combining R ¹⁷ or R ¹⁸ with R ⁷ or R ⁸ should be read as a ring that can be formed by combining R ¹⁶ with adjacent R ⁷ or R ⁸ .

R ¹³ is preferably a hydroxy group, an acyl group or a sulfonyl group.
As the acyl group or sulfonyl group that R ¹³ can take, the description of the acyl group or sulfonyl group as the group represented by formula (A) below can be applied. A ring that can be formed by combining R ¹⁷ or R ¹⁸ with R ⁷ or R ⁸ should be read as a ring that can be formed by combining R ¹³ with adjacent R ⁵ or R ⁶ .

4) X
X represents -O-, -S-, >SO, >SO ₂ , >SiR ¹⁰ R ¹¹ or >P(=O)R ¹² ;
The above R ¹⁰ and R ¹¹ represent groups represented by the formula (A) described later, and preferred forms of groups (1) or (2) described later can be preferably applied.
R ¹⁰ and R ¹¹ may combine with each other to form a 4- to 7-membered aliphatic heterocyclic ring.
However, when R ¹⁰ and R ¹¹ are not bonded together to form the above 4- to 7-membered aliphatic heterocyclic ring, at least one of R ¹⁰ and R ¹¹ is an alkenyl group, alkynyl group, aryl group or heteroaryl is the base.
These descriptions of R ¹⁰ and R ¹¹ are the same as those of R ¹⁰ and R ¹¹ below.
When at least one of R ¹⁰ and R ¹¹ has an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group, these substituents are sterically bulky, and photodegradation factors such as active oxygen are attached to the silicon atom. It is thought that by suppressing the approach, the stability of silicon atoms can be improved, and excellent light resistance can be exhibited.
R ¹⁰ and R ¹¹ are preferably alkyl groups, alkenyl groups, alkynyl groups, aryl groups or heteroaryl groups.
Examples of the alkyl group, alkenyl group, alkynyl group, aryl group, or heteroaryl group that R ¹⁰ and R ¹¹ can take include the alkyl group, alkenyl group, alkynyl group, and aryl group represented by formula (A) described below. Or the description of the heteroaryl group can be applied.
Preferred substituents that the alkyl, alkenyl, alkynyl, aryl or heteroaryl groups that R ¹⁰ and R ¹¹ may have include a carboxy group, an alkoxy group and an aryl group. Among them, an alkenyl group substituted with a carboxy group or an alkoxy group, and an alkenyl group or alkynyl group substituted with an aryl group are preferable.
Combinations of R ¹⁰ and R ¹¹ include a combination of an alkyl group and an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group, a combination of an alkenyl group and an alkenyl group, a combination of an alkynyl group and an alkynyl group, heteroaryl A combination of a group and a heteroaryl group is preferred, a combination of an alkyl group and an alkenyl group or an aryl group is more preferred, and a combination of an alkyl group and an alkenyl group is even more preferred.

R ¹² above represents a group represented by formula (A) described later, and is preferably a hydroxy group, an alkoxy group, an aryl group or an alkyl group.
When R ¹² is a hydroxy group, the hydrogen atom in this hydroxy group may be dissociated to have an ionic structure or a salt structure. This is the same for the hydroxy group that can be used as R ¹² below.

X above is preferably —O—, >SO ₂ or >SiR ¹⁰ R ¹¹ , more preferably —O— or >SiR ¹⁰ R ¹¹ , and even more preferably >SiR ¹⁰ R ¹¹ .

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

In the above formula, X represents —O— or >SiR ¹⁰ R ¹¹ .
R ¹ to R ¹¹ , L and Z have the same definitions as R ¹ to R ¹¹ , L and Z above.

The compound represented by the general formula (3) is the compound represented by the general formula (2) above, wherein X is —O— or >SiR ¹⁰ R ¹¹ , Y ¹ is —NHR ¹⁵ , and Y ¹² is =NR ¹⁶ and both R ¹⁵ and R ¹⁶ are groups represented by general formula (B). Similarly, the compound represented by general formula (4) is the compound represented by general formula (2) above wherein X is —O— or >SiR ¹⁰ R ¹¹ and Y ¹ is —OR ¹³ . , Y ¹² is ═O and R ¹³ is a group represented by general formula (B).
Therefore, the structure corresponding to the group represented by the general formula (B) in the compound represented by the general formula (3) or (4) is the same as the description of the group represented by the general formula (B) above. Synonymous.

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

When the compound of the present invention has a substituent capable of binding to a biological substance, proteins (including peptides), amino acids, nucleic acids, sugar chains, lipids, etc. It can be bound to a biological substance and used as a labeled biological substance.
Substituents that can bind to biological substances are not particularly limited as long as they act (including attachment) or bind to biological substances, and are described in International Publication No. 2002/026891. Substituents of can be mentioned. Among them, NHS ester structure (N-hydroxysuccinimide ester), succinimide structure, maleimide structure, azide group, acetylene group, peptide structure (polyamino acid structure), long-chain alkyl group (preferably having 12 to 30 carbon atoms), 4 Preferred are tertiary ammonium groups.

Among the compounds of the present invention, specific examples of the compounds having at least one substituent capable of binding to a biological substance include, for example, a substituent capable of binding to a biological substance described below for the carboxy group in the exemplary compound of the compound of the present invention. As a specific example, a form in which is appropriately replaced with . However, the present invention is not limited to these compounds. For example, in these specific examples, a group having a dissociable hydrogen atom such as a carboxy group and a sulfo group may take a salt structure by dissociating the hydrogen atom.

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

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

Proteins, amino acids, nucleic acids, nucleotides, sugar chains, and lipids are preferred examples of the biological substances. In the present invention, the term "protein" is used to include peptides, and refers to compounds in which two or more amino acids are bound by peptide bonds. Antibodies are preferred as proteins, and phospholipids, fatty acids and sterols are preferred as lipids, and phospholipids are more preferred.
Of the above biosubstances, clinically pathologically useful substances are not particularly limited. plasma _proteins and their antibodies, α _- fetoprotein, carcinoembryonic antigen (CEA), prostate acid phosphatase (PAP), CA (carbohydrate antigen ) 19-9, CA-125 and other tumor markers and their antibodies, luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotrobin (hCG), estrogen, insulin and other hormones and their antibodies , hepatitis B virus (HBV)-related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), viral infection-related substances such as adult T-cell leukemia (ATL), and antibodies thereof.
Furthermore, bacteria such as Diphtheria, Clostridium botulinum, Mycoplasma, and Treponema pallidum and their antibodies, Toxoplasma, Trichomonas, Leishmania, Trivanosoma, Plasmodium and other protozoa and their antibodies, ELM3, HM1, KH2, v6.5, ES cells (Embryonic Stem Cell) such as v17.2, v26.2 (derived mouse 129, 129/SV, C57BL/6, BALB/c) and their antibodies, phenytoin, antiepileptic drugs such as phenobarbital, quinidine, Cardiovascular drugs such as digoquinisin, anti-asthma drugs such as theophylline, drugs such as antibiotics such as chloramphenicol and gentamicin, their antibodies, other enzymes, exotoxins (such as styrelidine O) and their antibodies etc. are also mentioned. Antibody fragments such as Fab'2, Fab and Fv can also be used.

Specific forms in which the compound of the present invention interacts with and binds to a biological substance include, for example, the forms described below.
i) non-covalent (e.g. hydrogen bonding, ionic bonding including chelation) or covalent bonding between a peptide in a compound of the invention and a peptide in a biological material;
ii) Van der Waals forces between long-chain alkyl groups in the compounds of the present invention and lipid bilayer membranes and lipids in biological materials, etc.;
iii) an amide bond by reaction of NHS ester (N-hydroxysuccinimide ester) in the compound of the invention with an amino group in the biomaterial;
iv) a thioether bond by reaction between a maleimide group in the compound of the present invention and a sulfanyl group (-SH) in the biomaterial;
v) Click reaction between an azide group in a compound of the present invention and an acetylene group in a biological substance or formation of a triazole ring by a Click reaction between an acetylene group in the compound of the present invention and an azide group in a biological substance;
are mentioned.
In addition to the forms i) to v) above, 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, 201 3, Vol 5, No. 1, p. 62-83. Also in the production of the labeled biological material of the present invention, the method described in the same document can be appropriately referred to.

Among the compounds of the present invention, the compound having a substituent capable of binding to a biological substance and the labeled biological substance of the present invention obtained from a biological substance that binds to it by interaction are described in JP-A-2019-172826. Examples of compounds and descriptions of products in paragraph 0038 include compounds and products thereof in which moieties other than the substituents that can bind to biological substances are replaced with dye moieties in the compounds of the present invention. However, the present invention is not limited to these compounds and the like.

<Reagent containing labeled biological substance>
In the reagent containing the labeled biological material of the present invention, the labeled biological material of the present invention can be, for example, in the form of a solution dissolved in an aqueous medium such as physiological saline or phosphate buffer, or in the form of particulate powder, freeze-dried powder, or the like. The form, such as a solid form, can be appropriately selected according to the purpose of use, etc., without being particularly limited.
For example, when the labeled biological substance of the present invention is used as a fluorescent labeling reagent, it can also be used as a reagent containing any of the above forms of labeled biological substance.

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

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

Examples of the biological material (primary biological material) capable of binding to the target biological material and the biological material (secondary biological material) capable of binding to the primary biological material include the biological material in the labeled biological material of the present invention. . Selecting a biological material that can be appropriately selected according to the target biological material (biological material in the subject) or the primary biological material and that can specifically bind to the biological material in the subject or the primary biological material can be done.

Among the above target biological substances, proteins include so-called disease markers. Examples of disease markers include, but are not limited to, α-fetoprotein (AFP), PIVKA-II (protein induced by vitamin K absorption or antagonist II), BCA (breast carcinoma-associated antigen) 225, base sex 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 acid protein (IAP), NCC-ST-439, γ-seminoprotein (γ-Sm), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), neuro-specific enolase (NSE), Iba1, amyloid β, tau, flotillin, squamous cell carcinoma-associated antigen (SCC antigen), sialyl LeX-i antigen (SLX), SPan-1, tissue polypeptide antigen (TPA), serial Tn antigen (STN), cytokeratin: CYFRA pepsinogen (PG), C-reactive protein (CRP), serum amyloid A protein (SAA), myoglobin, creatine kinase (CK), troponin T, ventricular muscle myosin light chain I and the like.

The target biological substance may be a bacterium, and examples of the bacterium include, but are not limited to, bacteria that are the subject of cell microbiological examination, but are not particularly limited. Problem-producing bacteria and the like can be mentioned.

The target biological substance may be a virus, and the virus is not particularly limited. pp65 protein antigen of megalovirus), E6 and E7 proteins of HPV (human papillomavirus), and the like.

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

In (v) above, the target biological material and the primary biological material may be directly bound, or may be bound via another biological material different from the target biological material and the primary biological material. In addition, in (vi) above, the primary biological material in the conjugate b and the secondary biological material in the labeled biological material B of the present invention are different from the primary biological material and the secondary biological material even if they are directly bound. may be bound via the biological material of
The labeled biological material of the present invention can be used as a fluorescence-labeled antibody in both direct and indirect methods, but is preferably used as a fluorescence-labeled antibody in the indirect method.
In (ii) or (v) and (vi) above, the binding of the labeled biological substance or the like of the present invention to the target biological substance is not particularly limited, and can be carried out according to a conventional method.

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

The fluorescence excitation light source used in the present invention is not particularly limited as long as it emits light having an emission wavelength (wavelength light) that can excite the labeled biological substance of the present invention. For example, various laser light sources can be used. Moreover, various optical filters can be used to obtain a preferable excitation wavelength or to detect only fluorescence.

The other items in (i) to (vii) above are not particularly limited, and conditions such as methods, reagents, and devices that are commonly used in fluorescence detection using fluorescent labels can be appropriately selected.
In addition, for steps other than the above (i) to (vii), conditions such as methods, reagents, equipment, etc. that are commonly used can be appropriately selected in accordance with various methods using fluorescent labeling.

For example, the multicolor WB using the labeled biological material of the present invention can be obtained by preparing a blot membrane by a technique commonly used as a target biological material (separation of proteins by electrophoresis, blotting onto a membrane, blocking the membrane). is used as a labeled antibody (preferably a secondary antibody), fluorescence emitted from the target biological substance can be stably detected with excellent fluorescence intensity. For dot blotting using the labeled biological material of the present invention, a blot nitrocellulose membrane or a blot PVDF (polyvinylidene fluoride) membrane or the like is prepared by a technique commonly used as a target biological material, similar to multicolor WB. By using a labeled biological substance as a labeled antibody (preferably, a secondary antibody), fluorescence emitted from the target biological substance can be stably detected with excellent fluorescence intensity.

- Substituent group T -
In the present invention, preferable substituents include substituents selected from the following substituent group T.
In addition, in the present invention, when only a substituent is described, this substituent group T is referred to, and when each group, for example, an alkyl group, is only described, this Corresponding groups of the substituent group T are preferably applied.
Furthermore, in this specification, when an alkyl group is described separately from a cyclic (cyclo)alkyl group, the alkyl group is used in the sense of including a straight-chain alkyl group and a branched alkyl group. On the other hand, when the alkyl group is not described separately from the cyclic alkyl group, and unless otherwise specified, the alkyl group is used in the sense of including a linear alkyl group, a branched alkyl group and a cycloalkyl group. This also applies to groups (alkoxy groups, alkylthio groups, alkenyloxy groups, etc.) containing groups that can have a cyclic structure (alkyl groups, alkenyl groups, alkynyl groups, etc.), and compounds that contain groups that can have a cyclic structure. be. When a group can form a cyclic skeleton, the lower limit of the number of atoms of the group forming the cyclic skeleton is 3 or more, regardless of the lower limit of the number of atoms specifically described below for the group that can adopt this structure, 5 or more is preferable.
In the description of the substituent group T below, for example, in order to clarify a group with a linear or branched structure and a group with a cyclic structure, such as an alkyl group and a cycloalkyl group, these are described separately. There is also

The groups included in the substituent group T include the following groups.
Alkyl group (the number of carbon atoms is preferably 1 to 20, more preferably 1 to 12, more preferably 1 to 8, particularly preferably 1 to 6, and most preferably 1 to 3. For example, methyl, ethyl, isopropyl, t- butyl, pentyl, heptyl, 1-ethylpentyl, benzyl, 2-ethoxyethyl, 1-carboxymethyl, trifluoromethyl, etc.), alkenyl groups (the number of carbon atoms is preferably 2 to 20, more preferably 2 to 12, 2 to 10 is more preferable, and 2 to 8 is particularly preferable.For example, vinyl, allyl, oleyl, etc.), alkynyl group (the number of carbon atoms is preferably 2 to 20, more preferably 2 to 12, more preferably 2 to 10, 2 to 8 are particularly preferred, such as ethynyl, butadiynyl, phenylethynyl, etc.), cycloalkyl groups (preferably having 3 to 20 carbon atoms, such as cyclopropyl, cyclopentyl, cyclohexyl, 4-methylcyclohexyl, etc.), cycloalkenyl groups (preferably having 5 to 20 carbon atoms, for example, cyclopentenyl, cyclohexenyl, etc.), an aryl group (which may be a monocyclic group, a condensed ring group (preferably a condensed ring group having 2 to 6 rings), The number of carbon atoms is preferably 6 to 26, more preferably 6 to 18, still more preferably 6 to 12. For example, phenyl, 1-naphthyl, 4-methoxyphenyl, 2-chlorophenyl, 3-methylphenyl etc.), a heterocyclic group (the number of carbon atoms is preferably 2 to 20. It has at least one oxygen atom, sulfur atom or nitrogen atom as a ring-constituting atom. It may be a monocyclic group, a condensed ring group ( (preferably a condensed ring group having 2 to 6 rings).In the case of a monocyclic group, the number of ring members is preferably 5 to 7, more preferably 5 or 6. The heterocyclic group is Aromatic heterocyclic groups (heteroaryl groups) and aliphatic heterocyclic groups (aliphatic heterocyclic groups) are included, such as 2-pyridyl, 4-pyridyl, 2-imidazolyl, 2-benzimidazolyl, 2-thiazolyl, 2-oxazolyl, 2-tetrahydropyranyl, etc.), alkoxy groups (the number of carbon atoms is preferably 1 to 20, more preferably 1 to 12. For example, methoxy, ethoxy, isopropyloxy, benzyloxy, etc.), alkenyloxy groups ( The number of carbon atoms is preferably 2-20, more preferably 1-12. propynyloxy, 4-butynyloxy, etc.), cycloalkyloxy groups (preferably having 3 to 20 carbon atoms, such as cyclopropyloxy, cyclopentyloxy, cyclohexyloxy, 4-methylcyclohexyloxy, etc.), aryloxy groups (having a carbon number of , 6 to 26 are preferred, and 6 to 14 are more preferred. For example, phenoxy, 1-naphthyloxy, 3-methylphenoxy, 4-methoxyphenoxy, etc.), heterocyclic oxy group (for the heterocyclic group portion in the heterocyclic oxy group, the description of the above heterocyclic group is preferably applied. For example, imidazolyloxy, benzimidazolyloxy, thiazolyloxy, benzothiazolyloxy, triazinyloxy, purinyloxy, tetrahydropyranyloxy), poly(alkyleneoxy) groups (having preferably 2 to 40 carbon atoms, 2 to 20 is more preferred, e.g., poly(ethyleneoxy) group),

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

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

A silyl group (preferably a silyl group having 1 to 20 carbon atoms and substituted with alkyl, aryl, alkoxy and aryloxy, such as triethylsilyl, triphenylsilyl, diethylbenzylsilyl, dimethylphenylsilyl, etc.), a silyloxy group ( Silyloxy groups preferably having 1 to 20 carbon atoms and substituted with alkyl, aryl, alkoxy and aryloxy, such as triethylsilyloxy, triphenylsilyloxy, diethylbenzylsilyloxy, dimethylphenylsilyloxy, etc.), hydroxy group , a cyano group, a nitro group, a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, preferably a fluorine atom), an oxygen atom (specifically, > _CH2 constituting a ring is >C =O), carboxy group (-CO ₂ H), phosphono group [-PO(OH) ₂ ], phosphonooxy group [-O-PO(OH) ₂ ], sulfo group (-SO ₃ H), boric acid group [-B(OH) ₂ ], onio group (ammonio group including cyclic ammonio group, sulfonio group (-SH ₂ ⁺ ), phosphonio group (-PH ₃ ⁺ ), preferably having 0 to 30 carbon atoms, more preferably 1 to 20), a sulfanyl group (—SH), an amino acid residue, or a polyamino acid residue. In addition, the above alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, and aryl group having a carboxy group, phosphono group, sulfo group, onio group, amino acid residue, or polyamino acid residue as a substituent. , heterocyclic group, alkoxy group, alkenyloxy group, alkynyloxy group, cycloalkyloxy group, aryloxy group, heterocyclicoxy group, alkoxycarbonyl group, cycloalkoxycarbonyl group, aryloxycarbonyl group, amino group, sulfamoyl group, Acyl group, acyloxy group, carbamoyl group, acylamino group, alkylthio group, cycloalkylthio group, arylthio group, heterocyclic thio group, alkyl, cycloalkyl or arylsulfonyl group.
A substituent selected from the substituent group T is more preferably an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an alkoxycarbonyl group, or a cycloalkoxycarbonyl group. , the above amino group, acylamino group, cyano group or halogen atom, and particularly preferably alkyl group, alkenyl group, heterocyclic group, alkoxy group, alkoxycarbonyl group, amino group, acylamino group or cyano group.

A substituent selected from the substituent group T includes a group formed by combining a plurality of the above groups unless otherwise specified. For example, when compounds or substituents contain alkyl groups, alkenyl groups, etc., these may be linear or branched, and may be substituted or unsubstituted. In addition, when containing an aryl group, a heterocyclic group, or the like, they may be monocyclic or condensed, and may be substituted or unsubstituted.

- Group represented by formula (A) -

In the formula, L ³ is a single bond, or an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and any of the following formulas (1-1) to (1-8). A linking group consisting of one or a combination of two or more of the groups is shown. That is, L ³ may be a single bond; an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, a group represented by formula (1-1) described below, a group represented by formula (1-2) ), a group represented by the formula (1-3), a group represented by the formula (1-4), a group represented by the formula (1-5), in the formula (1-6) a group represented by the formula (1-7), or a group represented by the formula (1-8); a group represented by the formula (1-1) described below, a group represented by the formula (1-2), a group represented by the formula (1-3), a group represented by the formula (1-4), selected from a group represented by formula (1-5), a group represented by formula (1-6), a group represented by formula (1-7), and a group represented by formula (1-8); It may be a group formed by combining two or more kinds of groups.
R ¹¹¹ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or a monovalent aliphatic heterocyclic group.
However, when the hydrogen atom in R ¹¹¹ is a dissociative hydrogen atom, this dissociative hydrogen atom may be dissociated.
Each group in L ³ and R ¹¹¹ may further have a substituent.
* indicates a joint.

Regarding the group represented by formula (A), L ³ and R ¹¹¹ shall be interpreted based on the following rules (i) and (ii).
(i) when the group represented by formula (A) has a group represented by any one of general formulas (1-1) to (1-8) * (bond) to general formula (1-1 ) to (1-8) are interpreted as ^L3 .
For example, when the group represented by the formula (A) is a carboxyphenyl group, the group represented by the formula (A) is L ³ : a phenylene group, the group represented by the formula (1-4) and the formula ( A group represented by a linking group obtained by combining the groups represented by 1-1) and R ¹¹¹ : a hydrogen atom. Further, when the group represented by the formula (A) is a sulfoalkyl group, the group represented by the formula (A) is L ³ : an alkylene group, a group represented by the formula (1-7) and a group represented by the formula ( A group represented by a linking group obtained by combining the groups represented by 1-1) and R ¹¹¹ : a hydrogen atom.
However, the group represented by any one of general formulas (1-1) to (1-8) does not form a ring as a ring-constituting atom.
Incidentally, when the group represented by the formula (A) contains two or more groups represented by any of the general formulas (1-1) to (1-8), the group represented by the formula (A) In general formula (1-1), where the number of shortest bond atoms from * (bond) to the group represented by any one of general formulas (1-1) to (1-8) is maximized Up to the group represented by any one of to (1-8) is interpreted as ^L3 .
(ii) when the group represented by formula (A) does not have a group represented by any of general formulas (1-1) to (1-8): the terminal of the group represented by formula (A) An alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or a monovalent aliphatic heterocyclic group located in the structure shall be interpreted as R ¹¹¹ .
For example, when the group represented by formula (A) is a phenyl group, the group represented by formula (A) is a group represented by L ³ : single bond and R ¹¹¹ : phenyl group. Further, when the group represented by formula (A) is —CH ₂ ═CHPh, the group represented by formula (A) is represented by L ³ : —CH ₂ ═CH— and R ¹¹¹ : Ph It is a group that is Here, Ph means a phenyl group.
However, the "terminal structure in the group represented by the formula (A)" refers to the group represented by the formula (A), which is located at the most terminal counting from * (bonding part) of the longest binding chain means structure.
In the above (i) and (ii), when the group represented by the formula (A) is a group having a substituent capable of binding to the biological substance described later, the biological substance described later in the group represented by the formula (A) Read about groups in which substituents that can bind to substances are replaced with hydrogen atoms. That is, substituents capable of binding to biological substances, which will be described later, are interpreted as substituents that each group in L ³ or R ¹¹¹ may further have.
In addition, each group defined by R ¹¹¹ or L ³ may further have a substituent within the scope conforming to the definitions of (i) and (ii) above.

( ^L3 )
The alkylene group that can be taken as L ³ has the same definition as the group obtained by removing one hydrogen atom from the alkyl group selected from the above-described substituent group T, and the preferred ones are also the same.
The alkenylene group that can be taken as ^L3 has the same definition as the group obtained by removing one hydrogen atom from the alkenyl group selected from the substituent group T described above, and the preferred ones are also the same.
The alkynylene group that can be taken as L ³ has the same definition as the group obtained by removing one hydrogen atom from the alkynyl group selected from the substituent group T described above, and the preferred ones are also the same.
The arylene group that can be taken as L ³ has the same definition as the group obtained by removing one hydrogen atom from the aryl group selected from the above-described substituent group T, and the preferred groups are also the same.
The heteroarylene group that can be taken as ^L3 has the same definition as the group obtained by removing one hydrogen atom from the heteroaryl group selected from the substituent group T described above, and the preferred ones are also the same.
An alkylene group, an alkenylene group, an alkynylene group, an arylene group and a heteroarylene group that can be taken as L ³ may be an unsubstituted group or a group having a substituent.
The substituents that the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group that can be taken as L ³ may have are not particularly limited, and are preferably selected from the substituent group T described above. , and more preferably a halogen atom, an alkyl group or an alkoxy group.
A halogen atom includes a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom or a chlorine atom.
In addition, the number of substituents that the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group that can be taken as L ³ may have is not particularly limited as long as it can be taken as a structure, and at least one or more The upper limit is not particularly limited, and for example, all hydrogen atoms in the alkylene group, alkenylene group, alkynylene group, arylene group and heteroarylene group may be substituted with a substituent.

One of the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and each group represented by any one of the following formulas (1-1) to (1-8), which can be taken as L ³ Alternatively, in the linking group in which two or more types are combined, the types of groups to be combined are not particularly limited as long as they have an appropriate chemical structure. For example, L ³ does not include a group in which two or more groups represented by any one of the following formulas (1-1) to (1-3) are consecutive.
One of the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and each group represented by any one of the following formulas (1-1) to (1-8), which can be taken as L ³ Alternatively, in the linking group in which two or more types are combined, the types of groups to be combined are not particularly limited, but, for example, 1 to 6 types are preferable, and 1 to 4 types are more preferable.
One of the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and each group represented by any one of the following formulas (1-1) to (1-8), which can be taken as L ³ Alternatively, in the linking group in which two or more types are combined, the number of groups to be combined is not particularly limited.

Groups represented by any of formulas (1-1) to (1-8) that can be used as L and ^L3 are as follows.

In the formula, R ³¹ and R ³² represent hydrogen atoms or substituents.
* indicates a joint.

The substituent that can be taken as R ³¹ is not particularly limited, and is preferably selected from the substituent group T described above. R ³¹ is preferably a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an acyl group or a sulfonyl group, more preferably a hydrogen atom or an alkyl group, still more preferably a hydrogen atom.
Any of the alkyl group, aryl group, heteroaryl group, acyl group and sulfonyl group that can be used as R ³¹ may be an unsubstituted group or a group having a substituent.
The substituent that can be taken as R ³² is not particularly limited, and is preferably selected from the substituent group T described above. R ³² is preferably a hydrogen atom, a hydroxy group, an alkoxy group, an aryloxy group, an alkyl group, an aryl group or a heteroaryl group, more preferably a hydroxy group, an alkoxy group or an aryloxy group, and still more preferably a hydroxy group.

The group in which the groups represented by any of the above formulas (1-1) to (1-8) are combined is represented by any of the following formulas (1A-1) to (1A-9) groups are preferred.

R ³¹ and R ³² are synonymous with R ³¹ and R ³² above.
* and ** indicate joints. In addition, ** indicates a bond with the R ¹¹¹ side in the case of a group that can be used as L ³ . Formula (1A-2) may be attached to R ¹¹¹ on any * side in L ³ .

As the linking group obtained by combining each group represented by any one of the above formulas (1-1) to (1-8), which can be used as the above L, the above formulas (1A-1) and (1A-2) or (1A-4), and more preferably a group represented by the above formula (1A-1) or (1A-2).
Examples of the linking group obtained by combining the groups represented by any of the above formulas (1-1) to (1-8) that can be used as L ³ include the above formulas (1A-1) and (1A-2 ) or (1A-4) is preferable, more preferably a group represented by the above formula (1A-1) or (1A-2), represented by the above formula (1A-1) is more preferred.
For example, in the group represented by formula (A), the group represented by the above formula (1A-1) and the hydrogen atom as R ¹¹¹ corresponds to a carboxy group, and the above formula (1A -4) and a hydrogen atom as R ¹¹¹ correspond to a sulfo group. Further, a group in which a hydrogen atom is dissociated from the carboxy group as a dissociative hydrogen atom corresponds to a carboxy group having an ionic structure or a salt structure, and a group in which a hydrogen atom is dissociated from the sulfo group as a dissociative hydrogen atom is , corresponds to a sulfo group having an ionic structure or a salt structure.

Further, L ³ includes each group represented by any one of formulas (1-1) to (1-8) or a linking group obtained by combining these groups, an alkylene group, an alkenylene group, an alkynylene group, and an arylene group. may be a linking group combining at least one or more of a group and a heteroarylene group, and through one or a combination of two or more of an alkylene group, an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group It may be a linking group in which two or three or more of the groups represented by any one of formulas (1-1) to (1-8) or a linking group combining these groups are linked.
Specific examples of the linking group obtained by combining two or more types that can be taken as L ³ include, for example, combining at least two types (preferably two to four types) of an alkylene group, an alkenylene group, an alkynylene group, an arylene group and a heteroarylene group. group, and at least one (preferably 1 to 4) of an alkylene group, an arylene group and a heteroarylene group, and at least one represented by any one of formulas (1-1) to (1-8) (preferably 1 to 4 types).

( ^R111 )
When the hydrogen atom that can be taken as R ¹¹¹ is a dissociative hydrogen atom, the dissociative hydrogen atom may be dissociated in the group represented by formula (A). That is, it may form an ionic structure or a salt structure. This is the same for the description of R ¹¹¹ below.
That the hydrogen atom is dissociable means, for example, that the acid dissociation constant (pKa) is 10 or less, preferably 7 or less, more preferably 5 or less. The above acid dissociation constant means the value at 25°C in water.

The alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group and monovalent aliphatic heterocyclic group that can be taken as R ¹¹¹ are each synonymous with the corresponding group in the substituent group T described above, The same applies to what is preferable.
Any of the above alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups and monovalent aliphatic heterocyclic groups that can be used as R ¹¹¹ may be unsubstituted groups or groups having substituents. may be
The substituent that each of the above groups that can be taken as R ¹¹¹ may have is not particularly limited, and includes groups selected from the aforementioned substituent group T, preferably a halogen atom. A halogen atom includes a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom. In addition, it may have a substituent capable of binding to a biological substance, which will be described later.
The number of substituents that each of the above groups that can be used as R ¹¹¹ may have is not particularly limited as long as it can be taken in the structure, and can be at least one. The upper limit is not particularly limited, for example, even if all hydrogen atoms in alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups and monovalent aliphatic heterocyclic groups are substituted with substituents good.
Of the alkyl groups that can be used as R ¹¹¹ , the alkyl group having a substituent is preferably a halogenoalkyl group. The halogenoalkyl group that can be taken as R ¹¹¹ has the same definition as the alkyl group in the substituent group T described above, except that at least one hydrogen atom in the alkyl group in the substituent group T described above is substituted with a halogen atom, The same applies to what is preferable.
A halogen atom includes a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom.
The number of halogen atoms constituting the halogenoalkyl group is not particularly limited, and may be, for example, a perhalogenoalkyl group.

Specific examples of the group represented by formula (A) include the following groups (1) and (2) as preferred forms. However, in these examples, the form in which the substituents in L ³ and R ¹¹¹ have substituents is not excluded, and they may be unsubstituted or have substituents. As the substituents that the substituents in L ³ and R ¹¹¹ may have, the description of the substituents that the substituents may have in L ³ and R ¹¹¹ can be preferably applied.
(1) When having a group represented by any ^one of general formulas (1-1) to (1-8) An alkynylene group, an arylene group, a heteroarylene group, and a linking group in which one or more of the groups represented by any of the above formulas (1-1) to (1-8) are combined, and R ¹¹¹ is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or a monovalent aliphatic heterocyclic group.
However, it has at least one group represented by any one of formulas (1-1) to (1-8). This is the same for the description related to (1) below.
In this case, L ³ is a single bond, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and the above formulas (1-1), (1-3), (1-4) and (1). -7) is preferably a linking group in which one or more of the groups represented by any one of -7) is combined, and a single bond, an alkylene group, an arylene group, a heteroarylene group, the above formula (1-1), A linking group obtained by combining one or more of the groups represented by the formulas (1-3), (1-4) and (1-7) is more preferable, and an alkylene group, an arylene group, heteroarylene group, one or more of the groups represented by the above formulas (1-1), (1-3), (1-4) and (1-7) A linking group is more preferable, and one or two of the groups represented by an alkylene group, an arylene group, a heteroarylene group, and the above formulas (1-1), (1-3) and (1-4) Linking groups that combine more than one species are particularly preferred. In these cases, R ¹¹¹ is preferably a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group or a monovalent aliphatic heterocyclic group, more preferably a hydrogen atom or an alkyl group, still more preferably a hydrogen atom.
(2) When not having a group represented by any of general formulas (1-1) to (1-8) In the group represented by formula (A), L ³ is a single bond or an alkylene group, A linking group consisting of one or more of an alkenylene group, an alkynylene group, an arylene group, and a heteroarylene group, wherein R ¹¹¹ is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, or an aryl group , a heteroaryl group or a monovalent aliphatic heterocyclic group.
In this case, L ³ is a single bond, and R ¹¹¹ is a hydrogen atom, an alkyl group, an alkenyl group, an aryl group, or a heteroaryl group, or L ³ is an alkenylene group or an alkynylene group, and R ¹¹¹ is preferably an alkyl group or an aryl group, more preferably L ³ is a single bond and R ¹¹¹ is a hydrogen atom, an alkyl group, an alkenyl group or an aryl group.

EXAMPLES The present invention will be described in more detail based on examples below, but the present invention is not limited thereto. In addition, room temperature means 25 degreeC.

[Example]
Compounds (2) to (4) and comparative compound (1) used in Examples are shown below.

Methods for synthesizing compounds and labeled antibodies are described in detail below, but starting materials, dye intermediates and synthetic routes are not limited to these.

Unless otherwise specified, SNAP KP-Sil Cartridge (trade name, manufactured by Biotage) and high flash column W001, W002, W003, W004 or W005 (trade name, manufactured by Yamazen) are used as carriers in silica gel column chromatography. bottom. SNAP KP-NH Cartridge (manufactured by Biotage) was used as NH silica. Mixing ratios in the eluent are volume ratios. For example, "ethyl acetate: n-hexane = 0: 100 → 100: 0" changes the eluent of "ethyl acetate: n-hexane = 0: 100" to the eluent of "ethyl acetate: n-hexane = 100: 0". It means that
MS spectrum, ACQUITY SQD LC / MS System [trade name, manufactured by Waters, ionization method: ESI (ElectroSpray Ionization)] or LCMS-2010EV [trade name, manufactured by Shimadzu Corporation, ionization methods: ESI and APCI (Atmospheric Pressure Chemical Ionization, atmospheric pressure chemical ionization) simultaneously].
Unless otherwise specified, the synthesized compounds and labeled antibodies were stored under light-shielding conditions when not used immediately after preparation. In addition, commercially available compounds and labeled antibodies were also used after being stored under light-shielding conditions until use.

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

[Synthesis Example 1: Synthesis of Compound (2) and Labeled Antibody (2)]
Compound (2) and labeled antibody (2) were synthesized according to the scheme below.

<Synthesis of Compound (2-A)>
3-bromo-aniline (13.1 g, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and acetonitrile (76 mL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 300 mL three-necked flask, and allyl bromide (19.8 mL, Fujifilm Wako Pure Chemical Industries, Ltd.) was added. Yakusha) and potassium carbonate (21.0 g, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were mixed and stirred at 80°C. Completion of the reaction was confirmed by LC/MS, and unnecessary substances were removed by filtration. After evaporating the solvent from the filtrate under reduced pressure, the resulting residue was dissolved in methylene chloride and purified by silica gel column chromatography (ethyl acetate:n-hexane) to collect the desired product and evaporate the solvent under reduced pressure. , to obtain compound (2-A) (17.2 g, yield 90%).
[M+H ⁺ ] ⁺ : 253

<Synthesis of compound (2-B)>
Compound (2-A) (2.55 g) and tetrahydrofuran (30 mL, super dehydrated, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 100 mL three-necked flask, cooled at −78° C. after purging with nitrogen, and n-butyllithium ( 7.23 mL, 1.6 M, n-hexane solution, manufactured by Kanto Kagaku) was added dropwise and stirred for 30 minutes. Subsequently, dichloromethylvinylsilane (0.700 mL, manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred for 1 hour. After confirming the completion of the reaction by LC/MS, a saturated aqueous ammonium chloride solution was added under ice-cooling, the mixture was extracted with ethyl acetate, dried over Glauber's salt, and the solvent was distilled off under reduced pressure. The resulting residue was dissolved in methylene chloride and purified by silica gel column chromatography (ethyl acetate:n-hexane=0:100→10:90) to collect the desired product and evaporate the solvent under reduced pressure to give a colorless oil. Compound (2-B) (1.98 g, yield 49%) was obtained.
[M+H ⁺ ] ⁺ : 415

<Synthesis of compound (2-C)>
Compound (2-B) (250 mg), 3,5-difluoro-4-formylbenzoic acid (112 mg, manufactured by COMBI-BLOCKS), zinc chloride (II) (247 mg, FUJIFILM Wako Pure) in a 5 mL test tube reaction vessel Yakusha) and ethanol (2.1 mL, ultra-dehydrated, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added, irradiated with microwaves, and heated at 140° C. for 30 minutes. After confirming the completion of the reaction by LC/MS, water was added to the reaction solution, the mixture was extracted with ethyl acetate, dried over Glauber's salt, and the solvent was distilled off under reduced pressure. The resulting residue was dissolved in methylene chloride and purified by silica gel column chromatography (ethyl acetate:n-hexane=0:100→30:70) to collect the desired product and evaporate the solvent under reduced pressure. A gum-like compound (2-C) (150 mg, yield 38%) was obtained.
[M+H ⁺ ] ⁺ : 583

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

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

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

<Synthesis of compound (2-NHS)>
Compound (2) (2.3 mg), N-hydroxysuccinimide (6.4 mg, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 1-ethyl-3-(3-dimethylaminopropyl hydrochloride) were placed in a 5 mL test tube reaction vessel. ) Carbodiimide (10.8 mg, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) and DMSO (330 μL, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were added and stirred at room temperature for 1 hour. After confirming the completion of the reaction by LC/MS, the reaction solution was directly purified by reversed-phase silica gel column chromatography (C18, Biotage, acetonitrile: aqueous solution containing 0.1% trifluoroacetic acid = 0:100 → 80:20). Then, the desired product was collected and the solvent was distilled off by freeze-drying to obtain compound (2-NHS) (1.4 mg, yield 53%) as dark blue powder.
[M+H ⁺ ] ⁺ : 846

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

[Synthesis Example 2: Synthesis of Compound (3) and Labeled Antibody (3)]
<Synthesis of compound (3)>
Compound (2) was synthesized in the same manner except that N-acetyl-D-galactosamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used in place of 4,6-O-ethylidene-α-D-glucopyranose. Compound (3) was obtained as dark blue powder.
[M+H ⁺ ] ⁺ : 831
<Synthesis of compound (3-NHS)>
Compound (3-NHS) was obtained as dark blue powder in the same manner as in the method for synthesizing compound (2-NHS), except that compound (3) was used instead of compound (2).
[M+H ⁺ ] ⁺ : 928
<Synthesis of labeled antibody (3)>
Labeled antibody (3) was obtained in the same manner as in the method for synthesizing labeled antibody (2), except that compound (3-NHS) was used instead of compound (2-NHS).

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

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

〔evaluation〕
The water solubility, maximum molar extinction coefficient and fluorescence quantum yield of each compound shown above, and the decrease rate of fluorescence quantum yield with increasing fluorescence labeling rate for each labeled antibody obtained were evaluated. Table 1 summarizes the results.

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

[2] Evaluation of Maximum Molar Extinction Coefficient A PBS solution (pH 7.4) of the above compound was evaluated by the method described in the reference materials below. The unit of maximum molar extinction coefficient in the evaluation criteria below is mol ⁻¹ Lcm ⁻¹ .
“TECH TIP #6 Extinction Coefficients” (Thermo Scientific document, available from https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0006-Extinction-coefficients.pdf)
Regarding the maximum molar extinction coefficient, a high evaluation rank is preferable because the maximum molar extinction coefficient is excellent. In this test, it is preferable that the maximum molar extinction coefficient satisfies the evaluation rank of "C" or higher from the viewpoint of practicality as a fluorescent compound.

- Evaluation criteria for maximum molar extinction coefficient -
A: 100000 or more B: 50000 or more and less than 100000 C: 10000 or more and less than 50000 D: Less than 10000 E: Not evaluated due to low water solubility

[3] Evaluation of Fluorescence Quantum Yield A PBS solution (pH 7.4) of the above compound was evaluated by the method described in the reference materials below.
“A Guide to Recording Fluorescence Quantum Yields” (Document from HORIBA Scientific, available from https://www.horiba.com/fileadmin/uploads/Scientific/Documents/Fluorescence/quantumyieldstrad.pdf)
Regarding the fluorescence quantum yield, a high evaluation rank is preferable because the fluorescence quantum yield is excellent. In this test, it is preferable that the fluorescence quantum yield satisfies an evaluation rank of "C" or higher from the viewpoint of practicality as a fluorescent compound.

- Evaluation criteria for fluorescence quantum yield -
A: 0.5 or more B: 0.3 or more and less than 0.5 C: 0.1 or more and less than 0.3 D: Less than 0.1 E: Not evaluated due to low water solubility

[4] Evaluation of decrease rate of fluorescence quantum yield with increase in fluorescence labeling rate of labeled antibody Approximate straight line obtained by plotting relationship between fluorescence labeling rate of labeled antibody and fluorescence quantum yield calculated by the following method (ie, a in linear approximation represented by Y=-aX+b) was applied to the following evaluation criteria, and the rate of decrease in fluorescence quantum yield accompanying an increase in the fluorescence labeling rate of the labeled antibody was evaluated.
A high evaluation rank is very preferable because the rate of decrease in fluorescence quantum yield accompanying an increase in the fluorescent labeling rate is small.

-Evaluation criteria for the rate of decrease in fluorescence quantum yield of the labeled antibody-
A: a is less than 0.05.
B: a is 0.05 or more and less than 0.10.
C: a is 0.10 or more and less than 0.20.
D: a is 0.20 or more.

The fluorescence labeling rate of the labeled antibody was evaluated by the following method.
Put 217 μL of anti-rabbit IgG antibody (2.3 mg/ml) and 21.7 μL of carbonate buffer in a microtube, shake and stir, and then add a dimethyl sulfoxide solution of the above compound in a molar equivalent amount to the antibody. Added so that the ratio would be 10, 20, 40 or 80, and further shaken and stirred. After allowing to stand at room temperature for 1 hour, the reaction solution was purified using gel filtration column chromatography PD10 (manufactured by GE Healthcare Life Sciences) and a PBS solution (phosphate buffered saline) to obtain a labeled antibody. For each labeled antibody obtained, the fluorescence labeling rate was calculated by the method shown below.
(Calculation of fluorescence labeling rate)
As a method for calculating the fluorescence labeling rate, a general method as shown below was used. The description in [ ] indicates a unit, and [-] means that there is no unit. In this study, protein means anti-rabbit IgG antibody.
Fluorescent labeling rate = Concentration of fluorescent dye/Concentration of protein The concentration of fluorescent dye means the total molar concentration [M] of the labeled fluorescent dye, and the concentration of protein is the molar concentration of the fluorescently labeled protein [ M], each of which is calculated by the following formula.
Concentration of fluorescent dye = Dye _max /ε _dye
Concentration of protein = (IgG ₂₈₀ - (Dye _max x CF))/ε _protein
Each symbol in the above formula is as follows.
Dye _max ; Absorption at maximum absorption wavelength of fluorescent dye [-]
ε _dye ; Molar extinction coefficient of fluorescent dye [M ^-1 cm ^-1 ]
IgG ₂₈₀ ; absorption of fluorescently labeled protein at 280 nm [-]
Dye ₂₈₀ ; absorption of fluorescent dye at 280 nm [-]
ε _protein ; molar extinction coefficient of protein [M ^-1 cm ^-1 ]
CF (Correction Factor); Dye ₂₈₀ /Dye _max [-]
(Evaluation of fluorescence quantum yield)
For the labeled antibody whose fluorescence labeling rate was calculated as described above, in the above [3] Evaluation of fluorescence quantum yield, the fluorescence quantum yield was evaluated in the same manner except that the labeled antibody was used instead of the compound. bottom.
Regarding the fluorescence labeling rate and the fluorescence quantum yield of the four samples obtained in this way, with the molar equivalent ratio of the compound to the antibody being 10, 20, 40 or 80, the vertical axis Y is the fluorescence quantum yield, and the horizontal axis is The axis X was plotted as the fluorescence labeling rate, and the slope a of the approximation straight line (ie, a in linear approximation represented by Y=−aX+b) was calculated.

(Note to table)
Compounds (2) to (4) and comparative compound (1) are each as described above.
The column of fluorescence quantum yield decrease rate (labeled antibody) describes the evaluation results of the fluorescence quantum yield decrease rate associated with an increase in the fluorescence labeling rate for the corresponding labeled antibody.

The results in Table 1 above show the following.
In the compound represented by the general formula (2), which has a structural portion composed of a Si-rhodamine compound as FL, the comparative compound (1) which does not have the group represented by the general formula (B) is the corresponding labeled antibody. However, the rate of decrease in fluorescence quantum yield with an increase in the fluorescent labeling rate was large and inferior. On the other hand, all of the compounds (2) to (4) having a group represented by the general formula (B) show an increase in the fluorescence labeling rate of the corresponding labeled antibody with respect to the comparative compound (1). The rate of decrease in fluorescence quantum yield associated with the method was suppressed to a level exceeding 2 times, indicating excellent suppression of the decrease in fluorescence quantum yield associated with an increase in the fluorescence labeling rate.

Claims (8)

A fluorescent compound represented by the following general formula (1).

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

In the above formula, R ¹ to R ³ represent a hydrogen atom, an alkyl group, an aryl group or a heteroaryl group.
L is a single bond, or an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and any of the groups represented by the following formulas (1-1) to (1-8) It represents a linking group of one type or a combination of two or more types.
* indicates a joint.
However, L and any one of R ¹ to R ³ do not combine to form a glycosyl structure.

In the above formula, R ³¹ and R ³² represent hydrogen atoms or substituents.
* indicates a joint. 2. The fluorescent compound according to claim 1, wherein said FL is a structural moiety comprising a dipyrromethene compound, a rhodamine compound, a xanthene compound, a rhodol compound, a pyrrolopyrrole compound, a pyrene compound, a coumarin compound or a cyanine compound. 3. The fluorescent compound according to claim 2, wherein said FL is a structural part comprising a rhodamine compound, a xanthene compound or a rhodol compound. 4. The fluorescent compound according to claim 3, represented by the following general formula (2).

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

In the formula, L ³ is a single bond, or an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and any of the above formulas (1-1) to (1-8). A linking group consisting of one or a combination of two or more of the groups is shown.
R ¹¹¹ represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or a monovalent aliphatic heterocyclic group.
However, when the hydrogen atom in R ¹¹¹ is a dissociative hydrogen atom, this dissociative hydrogen atom may be dissociated.
* indicates a joint. 5. The fluorescent compound according to claim 4, represented by the following general formula (3) or (4).

In the above formula, X represents —O— or >SiR ¹⁰ R ¹¹ .
R ¹ to R ¹¹ , L and Z have the same definitions as R ¹ to R ¹¹ , L and Z above. A fluorescence-labeled biological material obtained by binding the compound according to any one of claims 1 to 5 to a biological material. 7. The fluorescence-labeled biological material according to claim 6, wherein said biological material is any one of proteins, amino acids, nucleic acids, nucleotides, sugar chains and lipids. 7. The fluorescence-labeled biological material according to claim 6, wherein the binding between said compound and said biological material is by any one of the following i) to v).
i) non-covalent or covalent bonding between peptides,
ii) van der Waals interactions between long-chain alkyl groups in compounds and lipid bilayer membranes or lipids in biomaterials;
iii) an amide bond formed by reacting an N-hydroxysuccinimide ester in the compound with an amino group in the biological substance;
iv) a thioether bond formed by reacting a maleimide group in the compound with a sulfanyl group in the biological substance;
v) Bonding accompanied by formation of a triazole ring by Click reaction between an azide group in a compound and an acetylene group in a biological substance, or an acetylene group in a compound and an azide group in a biological substance