JP2022131357A - Fluorescent dye and labeled biological substance using the same - Google Patents

Abstract

To provide a fluorescent dye that can yield a labeled biological material that exhibits excellent fluorescence intensity in any of the states of solution, membrane and dot plot, and to provide a labeled biological substance.SOLUTION: Provided are a fluorescent dye and a labeled biological substance that are represented by the following general formula (I) or (II). In the formula, M represents a t-valent linking group, and t is an integer of 3 or more. L represents a divalent linking group formed by combining at least two of -C≡C-, an arylene group and a heteroarylene group. X represents an ether bond, thioether bond, alkylene group, amide bond, ester bond or amino bond. R indicates a fluophor part.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to fluorescent dyes and labeled biological substances using the same.

Fluorescence produced by labeling biomolecules (antibodies, etc.) that bind to the substance to be detected with a fluorescent compound (fluorochrome) 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, in vivo fluorescence 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, the luminance (fluorescence intensity) is generally increased by using a fluorescently labeled biological material to which a plurality of fluorescent dye molecules are bound. However, most fluorescent organic dyes, such as cyanine dyes and rhodamine dyes, have aromatic chromophores with high planarity, so they tend to interact with each other. Fluorescence intensity is likely to decrease due to interaction such as self-association between them. In particular, as the number of fluorescent dye molecules per biomolecule (fluorescence labeling rate: DOL) increases, the fluorescence intensity due to self-association tends to decrease.
As a technique for coping with this problem, for example, Patent Document 1 discloses that by polymerizing a fluorescent dye with a branched polyether skeleton having a core portion composed of a polyhydroxy compound, a polyamino compound, or a polythio compound, high fluorescence It is described that the strength is realized and the decrease in DOL due to self-quenching is suppressed.

JP 2017-2284 A

Dyes used for fluorescent labeling are required to exhibit excellent fluorescence intensity in various conditions such as solutions, membranes or dot plots. However, as a result of further studies on the fluorescence intensity by the present inventors, the fluorescence labeling using a fluorescent dye polymerized by a branched polyether skeleton disclosed in the above-mentioned Patent Document 1 has a solution, a membrane and a dot It has been found that sufficient levels of fluorescence intensity cannot be obtained in any state of the plot.
An object of the present invention is to provide a fluorescent dye that allows obtaining a labeled biological substance that exhibits excellent fluorescence intensity in any of the states of solution, membrane, and dot plot. Another object of the present invention is to provide a labeled biological substance obtained by binding this fluorescent dye to a biological substance.

式中、Ｍはｔ価の連結基を示し、ｔは３以上の整数である。
Ｌは、－Ｃ≡Ｃ－、アリーレン基及びヘテロアリーレン基のうちの少なくとも２つを組合わせてなる２価の連結基を示す。
Ｘは、エーテル結合、チオエーテル結合、アルキレン基、アミド結合、エステル結合又はアミノ結合を示す。
Ｒは、蛍光体部を示す。
〔２〕
上記Ｒが、キサンテン色素、ローダミン色素、クマリン色素、シアニン色素、ピレン色素、オキサジン色素、ピリジルオキサゾール色素及びピロメテン色素のうちの少なくとも１種の色素からなる構造部である、〔１〕に記載の蛍光色素。
〔３〕
上記蛍光色素が上記一般式（Ｉ）で表され、上記ｔが３であり、上記Ｍが下記いずれかの構造の連結基である、〔１〕又は〔２〕に記載の蛍光色素。

上記構造式中、＊はＬとの結合位置を示す。
〔４〕
上記蛍光色素が上記一般式（Ｉ）で表され、上記ｔが４であり、上記Ｍが下記構造の連結基である、〔１〕又は〔２〕に記載の蛍光色素。

上記構造式中、＊はＬとの結合位置を示す。
〔５〕
上記Ｒのうちの少なくとも１つがカルボキシ基又は生体物質に結合可能な置換基を有する、〔１〕～〔４〕のいずれか１つに記載の蛍光色素。
〔６〕
上記Ｌ及びＭのうちの少なくとも１つが水溶性置換基を有する、〔１〕～〔５〕のいずれか１つに記載の蛍光色素。
〔７〕
上記水溶性置換基が、カルボキシ基、スルホ基、ホスホノ基、ホスホノオキシ基、スルファモイル基及びポリオキシアルキレン基のうちの少なくとも１種である、〔６〕に記載の蛍光色素。
〔８〕
上記Ｒがシアニン色素からなる構造部である、〔１〕～〔７〕のいずれか１つに記載の蛍光色素。
〔９〕
上記シアニン色素が下記一般式（ＩＩＩ）で表される、〔８〕に記載の蛍光色素。

式中、Ｒ^１～Ｒ^４は、アルキル基又は－（ＣＨ_２－ＣＨ_２－Ｏ）_ｍ－Ｒ^２１を示す。ｍは１～５０であり、Ｒ^２１はアルキル基を示す。
Ｒ^１１～Ｒ^１３は、水素原子、アルキル基、アルコキシ基、アリールオキシ基、アルキルチオ基、アリールチオ基、アミノ基又はハロゲン原子を示し、隣接する基同士が互いに結合して５又は６員環を形成していてもよい。
Ｒ^２２～Ｒ^２５及びＲ^３２～Ｒ^３５は、水素原子、アルキル基、アルコキシ基、アリール基、スルホ基、スルファモイル基、カルボキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アシルオキシ基、カルバモイル基、アシルアミノ基、ニトロ基又はハロゲン原子を示す。
Ｒ^４１及びＲ^４２は、アルキル基又は－（ＣＨ_２－ＣＨ_２－Ｏ）_ｍ－Ｒ^２１を示す。Ｒ^２１及びｍは、上記のＲ^２１及びｍと同義である。Ｒ^４１及びＲ^４２は互いに結合して環を形成していてもよい。
ｎは、１～３の整数である。
上記のＲ^１～Ｒ^４、Ｒ^１１～Ｒ^１３、Ｒ^２２～Ｒ^２５、Ｒ^３２～Ｒ^３５、Ｒ^４１又はＲ^４２のうちのいずれか１つにより、上記Ｘと結合する。
上記蛍光色素中における少なくとも１つの式（ＩＩＩ）で表されるシアニン色素は、カルボキシ基又は生体物質に結合可能な置換基を有する。
ただし、式（ＩＩＩ）で表されるシアニン色素からなる構造部は、中性の構造部である。
〔１０〕
上記Ｒ^１１～Ｒ^１３のうちのいずれか１つにより上記Ｘと結合する、〔９〕に記載の蛍光色素。
〔１１〕
〔１〕～〔１０〕のいずれか１つに記載の蛍光色素と生体物質とが結合してなる標識生体物質。
〔１２〕
上記生体物質がタンパク質、アミノ酸、核酸、糖鎖及びリン脂質のいずれかである〔１１〕に記載の標識生体物質。 That is, the above problems of the present invention have been solved by the following means.
[1]
A fluorescent dye represented by the following general formula (I) or (II).

In the formula, M represents a t-valent linking group, and t is an integer of 3 or more.
L represents a divalent linking group formed by combining at least two of —C≡C—, an arylene group and a heteroarylene group.
X represents an ether bond, thioether bond, alkylene group, amide bond, ester bond or amino bond.
R indicates a phosphor portion.
[2]
The fluorescence according to [1], wherein the R is a structural moiety composed of at least one dye selected from xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, and pyrromethene dyes. pigment.
[3]
The fluorescent dye according to [1] or [2], wherein the fluorescent dye is represented by the general formula (I), t is 3, and M is a linking group having any one of the following structures.

In the above structural formula, * indicates the bonding position with L.
[4]
The fluorescent dye according to [1] or [2], wherein the fluorescent dye is represented by the general formula (I), t is 4, and M is a linking group having the following structure.

In the above structural formula, * indicates the bonding position with L.
[5]
The fluorescent dye according to any one of [1] to [4], wherein at least one of R has a carboxy group or a substituent capable of binding to a biological substance.
[6]
The fluorescent dye according to any one of [1] to [5], wherein at least one of L and M has a water-soluble substituent.
[7]
The fluorescent dye of [6], wherein the water-soluble substituent is at least one of a carboxy group, a sulfo group, a phosphono group, a phosphonooxy group, a sulfamoyl group and a polyoxyalkylene group.
[8]
The fluorescent dye according to any one of [1] to [7], wherein the R is a structural moiety composed of a cyanine dye.
[9]
The fluorescent dye according to [8], wherein the cyanine dye is represented by the following general formula (III).

In the formula, R ¹ to R ⁴ represent an alkyl group or —(CH ₂ —CH ₂ —O) _m —R ²¹ . m is 1 to 50, and R ²¹ represents an alkyl group.
R ¹¹ to R ¹³ each represent a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group or a halogen atom, and adjacent groups are bonded to each other to form a 5- or 6-membered ring; You may have
R ²² to R ²⁵ and R ³² to R ³⁵ are hydrogen atom, alkyl group, alkoxy group, aryl group, sulfo group, sulfamoyl group, carboxy group, alkoxycarbonyl group, aryloxycarbonyl group, acyloxy group, carbamoyl group, acylamino represents a radical, a nitro group or a halogen atom.
R ⁴¹ and R ⁴² represent an alkyl group or -(CH ₂ -CH ₂ -O) _m -R ²¹ . R ²¹ and m have the same definitions as R ²¹ and m above. R ⁴¹ and R ⁴² may combine with each other to form a ring.
n is an integer of 1-3.
Any one of R ¹ to R ⁴ , R ¹¹ to R ¹³ , R ²² to R ²⁵ , R ³² to R ³⁵ , R ⁴¹ or R ⁴² above binds to X above.
At least one cyanine dye represented by formula (III) in the fluorescent dye has a carboxy group or a substituent capable of binding to a biological substance.
However, the structural part composed of the cyanine dye represented by formula (III) is a neutral structural part.
[10]
The fluorescent dye according to [9], which binds to X through any one of R ¹¹ to R ¹³ .
[11]
A labeled biological substance obtained by binding the fluorescent dye according to any one of [1] to [10] to a biological substance.
[12]
The labeled biological material of [11], wherein the biological material is any one of proteins, amino acids, nucleic acids, sugar chains and phospholipids.

The fluorescent dye of the present invention can provide a labeled biological substance that exhibits excellent fluorescence intensity in any state of solution, membrane, and dot plot. In addition, the labeled biological material of the present invention exhibits excellent fluorescence intensity.

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. In addition, when a plurality of substituents and the like are close to each other (especially when they are adjacent), they may be linked together to form a ring unless otherwise specified. In addition, unless otherwise specified, rings such as alicyclic rings, aromatic rings and heterocyclic rings may be further condensed to form condensed rings.
In this specification, unless otherwise specified, the double bond may be either E-type or Z-type in the molecule, or a mixture thereof. In addition, unless otherwise specified, when a compound has diastereomers and enantiomers, it may be either one or a mixture thereof.

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 neutral compounds. For example, in the structural moiety composed of the cyanine dye represented by general formula (III), the formal charge of the nitrogen atom to which R ⁴² is bound is +1, and paired with this formal charge, general formula (III) By dissociating groups such as sulfo groups in the cyanine dye represented by having an ionic structure such as sulfonate ion, the compound of the present invention having a structural portion consisting of a cyanine dye represented by the general formula (III) is , the compound as a whole becomes a compound with an electric charge of 0.
In the cyanine dye represented by general formula (III) or the like defined in the present invention, the positive charge possessed by the compound is specifically indicated as a structure possessed by a specific nitrogen atom for convenience. However, since the cyanine dye represented by the general formula (III) or the like 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 generally Any compound that can have a structure represented by formula (III) or the like is included in the cyanine dye represented by general formula (III) or the like. This also applies to negative charges.
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, 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. In other words, 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 dye of the present invention is represented by the following general formula (I) or (II). Although the details of the reason why the fluorescent dye of the present invention can obtain a labeled biological substance exhibiting excellent fluorescence intensity in any of the states of solution, membrane and dot plot are not clear, it is considered as follows.
In the fluorescent dye of the present invention, two or more phosphor moieties R are -XLX- or -XLMLX-, as represented by general formula (I) or (II). wherein L is a rigid divalent linking group formed by combining at least two of -C≡C-, arylene and heteroarylene groups Therefore, it is considered that the interaction between the phosphor portions R is suppressed, and the decrease in fluorescence intensity due to the self-association of the fluorescent dye of the present invention can be suppressed. Further, the above L and the phosphor portion R are bonded by X having no π-conjugation, that is, an ether bond, a thioether bond, an alkylene group, an amide bond, an ester bond, or an amino bond, so that the phosphor portion R is It is possible to suppress a decrease in fluorescence intensity while maintaining the absorption emission wavelength within a desired range. In addition, as a result of effectively suppressing the self-association of the fluorescent dye of the present invention, it is thought that excellent fluorescence intensity can be exhibited even when a labeled biological substance exhibiting a high DOL is used.

The fluorescent dye represented by formula (I) or (II) of the present invention will be described in detail below.

<Fluorescent dye represented by general formula (I) or (II)>
The fluorescent dye represented by general formula (I) or (II) of the present invention (hereinafter also referred to as "the compound of the present invention") is as follows.

In the formula, M represents a t-valent linking group, and t is an integer of 3 or more.
L represents a divalent linking group formed by combining at least two of —C≡C—, an arylene group and a heteroarylene group.
X represents an ether bond, thioether bond, alkylene group, amide bond, ester bond or amino bond.
R indicates a phosphor portion.

In the compound represented by the general formula (I), L is a divalent linking group, M has a t-valent multi-branched structure, and the number of atoms constituting M is minimized , M.

1) M, n
M represents a t-valent linking group.
M is not particularly limited as long as the effects of the present invention are exhibited. For example, M is a t-valent aliphatic group having a chain or cyclic structure, a t-valent aromatic group, or a chain or cyclic structure and a t-valent group comprising a combination of an aliphatic group and an aromatic group. These groups may be substituted with a substituent, and possible substituents include groups in the group of substituents T described below, with water-soluble substituents described below being preferred. However, it is preferable not to include a phosphor portion. The chain-like t-valent aliphatic group includes a group composed of one atom having a t-valent bond.
As the aliphatic group constituting the t-valent group that can be taken as M, the description of the monovalent aliphatic group (alkyl group, alkenyl group or alkynyl group) in the substituent group T described later is replaced with the corresponding t-valent aliphatic group groups substituted for the group groups. Some or all of the carbon atoms and hydrogen atoms that constitute this aliphatic group may be replaced with heteroatoms such as oxygen atoms, nitrogen atoms or sulfur atoms, and the constituent atoms are carbon atoms and hydrogen atoms. is preferred.
The aromatic group constituting the t-valent group that can be taken as M may be either an aromatic hydrocarbon ring group or an aromatic heterocyclic group, and a monovalent aromatic hydrocarbon group in the substituent group T described later. Examples include groups in which the description of a hydrogen ring group or aromatic heterocyclic group is replaced with the corresponding t-valent aromatic hydrocarbon ring group or aromatic heterocyclic group.
In the aromatic heterocyclic group, the ring-constituting atoms are preferably carbon atoms and nitrogen atoms.
The aromatic group constituting the t-valent group that can be used as M may be either a monocyclic or condensed ring group, but is preferably a monocyclic group. Alternatively, a trivalent 6-membered ring aromatic group having bonds at the 2-, 4- and 6-positions is more preferable.
In addition, for an aliphatic group or an aromatic group that constitutes a t-valent group obtained by combining an aliphatic group having a chain or cyclic structure and an aromatic group that can be taken as M, M is a t-valent group and It is not particularly limited as long as it is used, and examples thereof include groups formed by combining the above-described aliphatic groups or aromatic groups.
M is preferably a t-valent aliphatic group having a chain or cyclic structure or a t-valent aromatic group, more preferably a t-valent chain aliphatic group or a t-valent aromatic group. , a group consisting of one atom having a t-valent bond, or a t-valent monocyclic aromatic group is more preferred.
t is an integer of 3 or more, preferably an integer of 3 to 6, more preferably an integer of 3 to 5, and even more preferably an integer of 3 or 4.
The number of atoms constituting M is preferably 1 to 24, more preferably 1 to 12, even more preferably 1 to 6. The number of atoms constituting M does not include the number of hydrogen atoms and the number of atoms constituting substituents of the t-valent group as M.
As M, for example, a trivalent group or a tetravalent group shown below, and a hexavalent group formed by combining an aliphatic group and an aromatic group having a chain or cyclic structure are preferable. can. Each aromatic group in the following structural formulas may be an unsubstituted group or may be substituted with a substituent. In the structures below, * means a bond.

2) L.
L represents a divalent linking group formed by combining at least two of —C≡C—, an arylene group and a heteroarylene group.

Examples of the arylene group constituting L include groups obtained by replacing the description of an aryl group in the substituent group T described later with a corresponding divalent group, preferably a phenylene group, more preferably a 1,4-phenylene group. .
The heteroarylene group constituting L includes a group in which the description of a heteroaryl group in the substituent group T described later is replaced with a corresponding divalent group, and the ring-constituting atoms are carbon atoms and nitrogen atoms. is preferred. Further, it is preferably a monocyclic group, more preferably a divalent 6-membered ring heteroarylene group having a bond at the para-position, 2,5-pyrazinediyl group, 1,4-triazinediyl group or 1 , 2,4,5-tetrazinediyl groups are more preferred.
The arylene group or heteroarylene group that may constitute L may be substituted with a substituent. Substituents that the above arylene group or heteroarylene group can take include groups in the group of substituents T described below, and water-soluble substituents described below are preferred. However, the above arylene group and heteroarylene group preferably do not contain a phosphor moiety.

The total number of -C≡C-, arylene groups and heteroarylene groups constituting L may be at least two, preferably from 2 to 12, more preferably from 2 to 10, and even more preferably from 2 to 7.
The types of -C≡C-, arylene group and heteroarylene group constituting L may be one or more, preferably two or more, and a combination of -C≡C- and an arylene group or a heteroarylene group. is more preferred. The upper limit is not particularly limited, and can be, for example, 4 or less, preferably 3 or less.
Regarding the types of arylene group and heteroarylene group, when the chemical structures including the binding sites are different, they are interpreted as different types of groups. For example, when L is composed of a phenylene group and a naphthalenediyl group, both are arylene groups, but the types of groups constituting L are counted as two types. A 1,4-phenylene group and a 1,3-phenylene group having different binding sites are also counted as different kinds of groups.
Examples of L include groups having skeleton structures shown below. However, it is not limited to groups having these skeleton structures. Moreover, the arylene group and the heteroarylene group in the following structural formulas may each be an unsubstituted group or may be substituted with a substituent. In the structures below, * means a bond, and in the compound represented by the general formula (I), it may be bonded to M on either side.

In the fluorescent dye of the present invention, at least one of L and M preferably has a water-soluble substituent. That is, in the fluorescent dye represented by general formula (I), at least one of M and t L preferably has a water-soluble substituent, and in the fluorescent dye represented by general formula (II) preferably L has a water-soluble substituent. In the fluorescent dye of the present invention, at least one of L and M has a water-soluble substituent, thereby suppressing the self-association of the fluorescent dye and further improving the fluorescence intensity in the state of solution, membrane and dot plot. is considered possible.
Water-soluble substituents include a carboxy group, a sulfo group (--SO ₂ (OH)), a phosphono group (phosphonic acid group, --PO(OH) ₂ ), and a phosphonooxy group (phosphoric acid group, --OPO(OH) ₂ ). , a sulfamoyl group (--SO ₂ NH ₂ ) or a polyoxyalkylene group (--(alkylene-O) _m --R ²¹ , m and R ²¹ preferably apply the description of m and R ²¹ in general formula (III) below. It is possible.) is preferably mentioned.
When L or M has a water-soluble substituent as a substituent, L or M may have the water-soluble substituent directly or via a linking group. When a linking group is interposed, the linking group is not particularly limited, but an alkylene group can be mentioned, for example.
In the present invention, from the viewpoint of water solubility, at least one L preferably has the water-soluble substituent, and at least one L is a carboxy group, a sulfo group, a phosphono group, a phosphonooxy group, a sulfamoyl group and a polyoxy More preferably, it has at least one alkylene group. Here, "at least one L" means at least one of t L in the fluorescent dye represented by the general formula (I), and the fluorescence represented by the general formula (II) In dyes, it means L.

When L has a water-soluble substituent, the number of water-soluble substituents in L preferably satisfies the following relationship.
The number of water-soluble substituents in L + the number of aromatic rings that constitute L ≥ 1 When L has a water-soluble substituent, the upper limit of the number of water-soluble substituents in L is not particularly limited, It is preferable to satisfy the following relationship.
Number of water-soluble substituents in L ≤ 2 × [number of aromatic rings constituting L]

3) X
X represents an ether bond (--O--), a thioether bond (--S--), an alkylene group, an amide bond (--NHCO--), an ester bond (--COO--) or an amino bond (--NH--).
As the alkylene group, an alkylene group obtained by removing one hydrogen atom from an alkyl group in the substituent group T described later can be applied.
X is preferably an ether bond or an amide bond, more preferably an ether bond.

4) R
R indicates a phosphor portion.
Multiple R's present in a compound may be the same or different.
The phosphor moiety that can be used as R is not particularly limited as long as it is a structural moiety composed of an organic compound exhibiting fluorescence and capable of bonding with L via X.
Examples of R include a structural moiety composed of at least one dye selected from xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes and pyrromethene dyes, and is preferred.
As the xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, and pyrromethene dyes, dyes commonly known as these formulas can be used without particular limitation.

From the viewpoint of versatility in bioimaging, at least one of R preferably has a carboxy group or a substituent capable of binding to a biosubstance described below.
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 a biological substance possessed by 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 above R is preferably a structural moiety composed of a cyanine dye, and more preferably a structural moiety composed of a cyanine dye represented by the following general formula (III).

In the formula, R ¹ to R ⁴ represent an alkyl group or —(CH ₂ —CH ₂ —O) _m —R ²¹ . m is 1 to 50, and R ²¹ represents an alkyl group.
R ¹¹ to R ¹³ each represent a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group or a halogen atom, and adjacent groups are bonded to each other to form a 5- or 6-membered ring; You may have
R ²² to R ²⁵ and R ³² to R ³⁵ are hydrogen atom, alkyl group, alkoxy group, aryl group, sulfo group, sulfamoyl group, carboxy group, alkoxycarbonyl group, aryloxycarbonyl group, acyloxy group, carbamoyl group, acylamino represents a radical, a nitro group or a halogen atom.
R ⁴¹ and R ⁴² represent an alkyl group or -(CH ₂ -CH ₂ -O) _m -R ²¹ . R ²¹ and m have the same definitions as R ²¹ and m above. R ⁴¹ and R ⁴² may combine with each other to form a ring.
n is an integer of 1-3.
Any one of R ¹ to R ⁴ , R ¹¹ to R ¹³ , R ²² to R ²⁵ , R ³² to R ³⁵ , R ⁴¹ or R ⁴² above binds to X above.
At least one cyanine dye represented by formula (III) in the fluorescent dye has a carboxy group or a substituent capable of binding to a biological substance.
However, the structural part composed of the cyanine dye represented by formula (III) is a neutral structural part.

The cyanine dye represented by the above general formula (III) has a length of 2n+3 repeating methine chains connected by conjugated double bonds, near 585 nm when n=1, and In the case of n=3, it has an excitation absorption wavelength near 685 nm, and near 785 nm in the case of n=3. Therefore, the compound of the present invention having a structural portion composed of a cyanine dye represented by the general formula (III) as a phosphor portion R is, respectively, fluorescent labels using excitation light sources of around 600 nm, 700 nm, and 800 nm, It can be used as a compound that exhibits excellent fluorescence intensity.

A multicolor WB detects a plurality of emission colors in the range from the visible region to the near-infrared region. Therefore, it is necessary to select the absorption and emission waveforms of a plurality of dyes so that they have an appropriate wavelength relationship so that crosstalk does not occur due to mutual interference when the dyes are excited to emit light. Ideally, the excitation light should be arranged so that only one dye glows and the others do not. From this point of view, for example, two types of excitation light sources, near 700 nm and near 800 nm, which are separated by a certain wavelength, are used for light emission in the near-infrared region of multicolor WB.
Fluorescence detection by near-infrared light excitation can suppress the autofluorescence of the membrane, that is, background fluorescence, compared to detection by visible light excitation, so it is easy to increase the signal-to-noise ratio (S/N ratio) and target proteins with high sensitivity. detection becomes possible. Therefore, in recent years, the need for fluorescence detection WB using light emission in the near-infrared region has increased in analysis research of trace proteins.
However, in the near-infrared region, the fluorescence quantum yield of fluorescent dyes is generally low, making it difficult to obtain a high amount of signal. Among the compounds of the present invention having a structural portion composed of a cyanine dye represented by the general formula (III) as a phosphor portion R, the compounds in which n = 2 or 3 are the two types near 700 nm and near 800 nm. It can be used as a compound that exhibits excellent fluorescence intensity even in multicolor WB having a can show excellent fluorescence intensity.

(i) R ¹ to R ⁴
R ¹ to R ⁴ each independently represent an alkyl group or —(CH ₂ —CH ₂ —O) _m —R ²¹ .

The alkyl groups that can be used as R ¹ to R ⁴ are synonymous with the alkyl groups in the substituent group T described later.
The unsubstituted alkyl group preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, and still more preferably 1 to 2 carbon atoms.
When the alkyl group has a substituent, the number of carbon atoms in the alkyl group portion of the alkyl group having a substituent is preferably 1 to 10, more preferably 1 to 8, still more preferably 2 to 6, particularly 2 to 5. preferable. Further, the number of atoms constituting the longest chain of the substituted alkyl group is preferably 3 to 35, more preferably 3 to 25, still more preferably 3 to 15, and particularly preferably 3 to 11.
In the present invention, "the number of carbon atoms in the alkyl group portion of an alkyl group having a substituent" means the number of carbon atoms in the alkyl group excluding the substituent portion.
In the present invention, "the number of atoms constituting the longest chain of an alkyl group having a substituent" refers to the number of atoms including the substituent moiety (that is, the total number of atoms minus the number of atoms of the molecular chain that does not constitute the longest chain. atomic number). When a substituent having a dissociable hydrogen atom such as a sulfo group or a carboxy group constitutes the longest chain, the hydrogen atom is included in the calculation regardless of the presence or absence of dissociation. In addition, the number of atoms in the substituent moiety capable of binding to the biological substance described later is not included.

Substituents that the alkyl group that can be taken as R ¹ to R ⁴ may have include an alkoxy group, a carboxy group, an alkoxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a sulfo group, a phosphono group and —(CH ₂ -CH ₂ -O) _m -R ²¹ , as well as groups consisting of combinations of these substituents. In addition, a substituent capable of binding to a biological substance, which will be described later, can be used. In addition, the above-mentioned alkoxy group, carboxy group, alkoxycarbonyl group, acyloxy group, carbamoyl group, acylamino group, sulfo group and phosphono group, and the alkyl group portion of the group consisting of a combination of these substituents binds to the biological substance described later. It may have possible substituents.
The alkyl group having a substituent that can be taken as R ¹ to R ⁴ is not particularly limited as long as it is an alkyl group having the above substituents, but an alkyl group having a terminal carboxy group or a substituent capable of binding to a biological substance. is preferably In this case, the alkyl group may be directly substituted with a carboxy group or a substituent capable of binding to a biological substance, or substituted as a group consisting of a combination of an alkoxy group and a carboxy group or a substituent capable of binding to a biological substance. may
An unsubstituted alkyl group is preferable as the alkyl group that can be used as R ¹ to R ⁴ .

(—(CH ₂ —CH ₂ —O) _m —R ²¹ )
In —(CH ₂ —CH ₂ —O) _m —R ²¹ that can be taken as R ¹ to R ⁴ , m is 1 to 50, and R ²¹ represents an alkyl group.
m means the average number of repetitions (also referred to simply as the number of repetitions), preferably 1 to 24, more preferably 1 to 12, even more preferably 1 to 10, particularly preferably 4 to 10, most preferably 4 to 8 preferable.
The above average number of repetitions can be calculated from the average integrated value obtained by subjecting the compound to ¹ H-NMR measurement. The average number of repetitions defined in the present invention means the number obtained by rounding off the average number of repetitions calculated by the above method to the first decimal place.
As for the alkyl group for R ²¹ , the description of the alkyl group that can be used as R ¹ to R ⁴ above can be applied.
—(CH ₂ —CH ₂ —O) _m that can be taken as R ¹ to R ⁴ —(CH ₂ —CH ₂ —O) _m that the alkyl group as R ²¹ and R ¹ to R ⁴ can have as a substituent —R ²¹ is preferably —(CH ₂ —CH ₂ —O) _m —unsubstituted alkyl group.

At least one of R ¹ to R ⁴ preferably contains a structure represented by —(CH ₂ —CH ₂ —O) _m —, and at least one of R ¹ and R ² and at least one of R ³ and R ⁴ It is more preferred that one of them contains a structure represented by -(CH ₂ -CH ₂ -O) _m -. At least one of R ¹ and R ² and at least one of R ³ and R ⁴ contain a structure represented by —(CH ₂ —CH ₂ —O) _m —, thereby suppressing self-association of the fluorescent dye. , solution, membrane, and dot-plot conditions, the fluorescence intensity can be further improved.
All R in the compound of the present invention is a structural moiety consisting of a cyanine dye represented by general formula (III), wherein at least one of R ¹ and R ² and at least one of R ³ and R ⁴ are - It is further preferred to include the structure represented by (CH ₂ —CH ₂ —O) _m —.
The structure represented by —(CH ₂ —CH ₂ —O) _m — is introduced by adopting —(CH ₂ —CH ₂ —O) _m —R ²¹ as R ¹ to R ⁴ . preferable.
m in —(CH ₂ —CH ₂ —O) _m — has the same meaning as m in —(CH ₂ —CH ₂ —O) _m —R ²¹ above.
Since the substituents of R ¹ to R ⁴ extend in the direction perpendicular to the cyanine dye skeleton (plane), the inclusion of the structure represented by —(CH ₂ —CH ₂ —O) _m — as this substituent gives It is presumed that the condensed ring portion becomes difficult to interact with π-π (the effect of suppressing association is strengthened), and the decrease in fluorescence intensity due to association can be suppressed.

(ii) R ¹¹ to R ¹³
R ¹¹ to R ¹³ each independently represent a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group or a halogen atom. Adjacent groups may combine with each other to form a 5- or 6-membered ring.
The alkyl group, alkoxy group, aryloxy group, alkylthio group, arylthio group, amino group and halogen atom that can be taken as R ¹¹ to R ¹³ are the alkyl group, alkoxy group, aryloxy group and alkylthio group in the substituent group T described later. , arylthio group, amino group and halogen atom, and the preferred ranges are also the same.
Examples of substituents that the alkyl group, alkoxy group, aryloxy group, alkylthio group, arylthio group and amino group in R ¹¹ to R ¹³ may have include substituents in the substituent group T described later.

Among R ¹¹ to R ¹³ , the 5- or 6-membered ring formed by bonding adjacent groups to each other may be either aromatic or aliphatic, preferably aliphatic. . Moreover, it is preferable to form a 6-membered ring. The number of 5- or 6-membered rings in the compound is not particularly limited, but is preferably 1 or 2, more preferably 1.
For example, when n=3, the structure having a ring formed by bonding adjacent groups among R ¹¹ to R ¹³ preferably includes the following structures. In the following examples, R ¹¹ to R ¹³ that do not form a ring structure are hydrogen atoms, and the ring structure has no substituents, but is not limited to these. Also, in the following description, the structure beyond the dashed line is omitted.

R ¹¹ and R ¹³ of the carbon atoms bonded to the indolenine ring are preferably hydrogen atoms.
R ¹² and R ¹³ other than the above are preferably a hydrogen atom or an alkyl group.
Of R ¹¹ to R ¹³ , adjacent groups in R ¹² to R ¹³ other than R ¹¹ and R ¹³ possessed by the carbon atom bonded to the indolenine ring are bonded to each other to form a 5- or 6-membered ring; preferably, and more preferably form a six-membered ring. Moreover, it is preferable to form the above 5- or 6-membered ring at the central portion of the bond connecting the indoline ring and the indolenine ring. The ring formed at the central portion of the bond connecting the indoline ring and the indolenine ring means a ring containing, as ring-constituting atoms, carbon atoms such that the number of bond atoms from the indoline ring and the indolenine ring is equal.

R above is bound to X through any one of R ¹ to R ⁴ , R ¹¹ to R ¹³ , R ²² to R ²⁵ , R ³² to R ³⁵ , R ⁴¹ or R ⁴² above.
Specifically, one hydrogen atom is removed from the substituents that can be taken as R ¹ to R ⁴ , R ¹¹ to R ¹³ , R ²² to R ²⁵ , R ³² to R ³⁵ , R ⁴¹ or R ⁴² . hydrogen atoms that are bonded to the above X or can be taken as R ¹ to R ⁴ , R ¹¹ to R ¹³ , R ²² to R ²⁵ , R ³² to R ³⁵ , R ⁴¹ or R ⁴² are removed, and R ¹¹ to R ¹³ , R ²² to R ²⁵ or R ³² to R ³⁵ are bonded to the carbon atom to which X is directly bonded.
Among them, R is preferably bonded to X by any one of R ¹¹ to R ¹³ , and any one of the hydrogen atoms that can be taken as R ¹¹ to R ¹³ is removed, and R ¹¹ It is more preferable that the carbon atom to which R ¹³ is bonded and the above X are directly bonded, and the number of hydrogen atoms that can be taken as R ¹³ other than R ¹³ possessed by R ¹² and the carbon atom bonded to the above indolenine ring Any one of them is removed, and the carbon atom to which R ¹³ other than R ¹³ possessed by R ¹² or the carbon atom bonded to the indolenine ring is more preferably directly bonded to X, It is particularly preferred that the carbon atoms having the same number of bond atoms from the indoline ring and the indolenine ring and the above X are directly bonded.

(iii) R ²² -R ²⁵ and R ³² -R ³⁵
R ²² to R ²⁵ and R ³² to R ³⁵ are hydrogen atom, alkyl group, alkoxy group, aryl group, sulfo group, sulfamoyl group, carboxy group, alkoxycarbonyl group, aryloxycarbonyl group, acyloxy group, carbamoyl group, acylamino represents a radical, a nitro group or a halogen atom. Adjacent groups of R ²² to R ²⁵ and R ³² to R ³⁵ may be bonded to each other to form a condensed ring.
Alkyl groups, alkoxy groups, aryl groups, sulfo groups, ^sulfamoyl groups, ^{alkoxycarbonyl} groups, ^{aryloxycarbonyl} groups, ^acyloxy groups, carbamoyl groups, acylamino groups, nitro groups and A halogen atom is an alkyl group, an alkoxy group, an aryl group, a sulfo group, a sulfamoyl group, an alkoxycarbonyl group, an aryloxycarbonyl group, an acyloxy group, a carbamoyl group, an acylamino group, a nitro group and a halogen in the substituent group T described later, respectively. Synonymous with atom.
The condensed ring formed by bonding adjacent groups of R ²² to R ²⁵ and R ³² to R ³⁵ to each other is not particularly limited, and examples thereof include naphthalene rings. From the viewpoint of suppressing association, it is preferable that adjacent groups among R ²² to R ²⁵ and R ³² to R ³⁵ are not bonded to each other to form a condensed ring.

From the viewpoint of improving water solubility and ^suppressing association, at least one of R ²² to R ²⁵ and at least one of R ³² to R ³⁵ preferably have a hydrophilic group ^. More preferably, each ring to which ³² to R ³⁵ are bonded has at least one hydrophilic group. For example, when adjacent groups among R ²² to R ²⁵ and R ³² to R ³⁵ are bonded to each other to form naphthalene rings as condensed rings, the number of rings to which R ²² to R ²⁵ are bonded is two. , the number of rings to which R ³² to R ³⁵ are bonded is two, and at least two of R ²² to R ²⁵ and at least two of R ³² to R ³⁵ preferably have a hydrophilic group. The upper limit is not particularly limited as long as the structure is possible, and can be adjusted as appropriate according to the number of hydrophilic groups in the compound as a whole, which will be described later.
Examples of hydrophilic groups include, but are not limited to, substituted alkoxy groups, carboxy groups, sulfo groups and phosphono groups, with sulfo groups being preferred.

R ²² to R ²⁵ and R ³² to R ³⁵ are preferably hydrogen atom, alkyl group, sulfo group, nitro group or halogen atom, more preferably hydrogen atom, alkyl group, sulfo group or halogen atom, hydrogen atom or alkyl group or a sulfo group is more preferred.

(iv) ^R41 and ^R42
R ⁴¹ and R ⁴² each independently represent an alkyl group or —(CH ₂ —CH ₂ —O) _m —R ²¹ . R ²¹ and m have the same definitions as R ²¹ and m above.
Examples of substituents that the alkyl groups in R ⁴¹ and R ⁴² may have include alkoxy groups, carboxy groups, alkoxycarbonyl groups, acyloxy groups, carbamoyl groups, acylamino groups, sulfo groups, phosphono groups, and substituents of these groups. Groups consisting of combinations are included. In addition, a substituent capable of binding to a biological substance, which will be described later, can be used. In addition, the above-mentioned alkoxy group, carboxy group, alkoxycarbonyl group, acyloxy group, carbamoyl group, acylamino group, sulfo group and phosphono group, and the alkyl group portion of the group consisting of a combination of these substituents binds to the biological substance described later. It may have possible substituents.

The alkyl group that can be used as R ⁴¹ and R ⁴² is synonymous with the alkyl group in the substituent group T described later.
The unsubstituted alkyl group preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, and still more preferably 1 to 3 carbon atoms.
The number of carbon atoms in the alkyl group portion of the alkyl group having a substituent is preferably 1 to 10, more preferably 1 to 8, still more preferably 1 to 7, still more preferably 1 to 6, and even more preferably 1 to 5. Further, the number of atoms constituting the longest chain of the substituted alkyl group is preferably 3-14, more preferably 3-12, and even more preferably 3-10.

As the alkyl group having a substituent that can be used as R ⁴¹ and R ⁴² , an alkyl group having at least one of an alkoxy group, a carboxy group, a sulfo group and a phosphono group as a substituent is preferred from the viewpoint of further improving water solubility. An alkyl group having at least one of a carboxy group and a sulfo group as a substituent is more preferred. An alkyl group having a substituent consisting of a combination of the preferred substituents (alkoxy group, carboxyl group, sulfo group and phosphono group) described above and groups other than these substituents may also be used.
In addition, the form of an alkyl group having a substituent which can be taken by the above R ¹ to R ⁴ can also be preferably applied.

—(CH _{2 —CH 2 —O) m —R 21 that can be taken as R 41 and R 42 preferably applies the description of —(CH 2 —CH 2 —O) m} ^{—R 21 in R} _{1 to R} ^{4 above .} can do.
— ⁽ CH ₂ —CH ₂ —O) _m —R ²¹ that can be used for R ⁴¹ and R ⁴² is an unsubstituted alkyl group, a carboxyl group at the terminal, or a An alkyl group having a substituent is preferred. In the case of an alkyl group having a terminal carboxy group or a substituent capable of binding to a biological substance, the alkyl group may be directly substituted with the carboxy group or a substituent capable of binding to a biological substance, and an alkoxycarbonyl group and a carboxy group Alternatively, it may be substituted as a group consisting of a combination with a substituent capable of binding to a biological substance.

R ⁴¹ and R ⁴² may combine with each other to form a ring.
Among the cyanine dyes represented by the general formula (III), a cyanine dye represented by the following general formula (IV) is preferable as the structure in which R ⁴¹ and R ⁴² are bonded to each other to form a ring.

In the formula, L ³ and L ⁴ represent an alkylene group or -(CH ₂ -CH ₂ -O) _m -alkylene-*. * indicates the bonding position with U.
The linking group U represents a divalent linking group having 1 to 100 atoms.
R ¹ to R ⁴ , R ¹¹ to R ¹³ , R ²² to R ²⁵ , R ³² to R ²⁵ , m and n are R ¹ to R ⁴ , R ¹¹ to R ¹³ and R ²² to It has the same meaning as R ²⁵ , R ³² to R ²⁵ , m and n, and the preferred ranges are also the same unless otherwise specified.
At least one of R ¹ to R ⁶ , L ¹ , R ³¹ and L ³ to L ⁶ includes a structure represented by -(CH ₂ -CH ₂ -O) _m -. m is synonymous with the above m.
However, the structural part composed of the cyanine dye represented by formula (III) is a neutral structural part.

The alkylene group that can be used as L ³ and L ⁴ corresponds to an alkylene group obtained by removing one hydrogen atom or one substituent from an alkyl group that has a substituent that can be used as R ⁴¹ and R ⁴² .
As for the number of carbon atoms in the alkylene group portion of the alkylene group that can be used as L ³ and L ⁴ , the description of the number of carbon atoms in the alkyl group portion of the substituted alkyl group for R ⁴¹ and R ⁴² can be preferably applied.

—(CH ₂ —CH ₂ —O) _m -alkylene-* which can be taken as L ³ and L ⁴ is —(CH ₂ —CH ₂ ^—O ) _m ^{—R 21} (R ²¹ represents an alkyl group having a substituent), which corresponds to —(CH ₂ —CH ₂ —O) _m -alkylene obtained by removing one hydrogen atom or substituent from the alkyl group as R ²¹ .
In —(CH ₂ —CH ₂ —O) _m -alkylene-* that can be used as L ³ and L ⁴ , m is preferably 1 to 10, more preferably 1 to 8, and the number of carbon atoms in the alkylene group portion is R The description of the number of carbon atoms in the alkyl group portion of the substituted alkyl group in ⁴¹ and R ⁴² can be preferably applied.

From the viewpoint of further improving fluorescence intensity, both L ³ and L ⁴ preferably contain a structure represented by -(CH ₂ -CH ₂ -O) _m -.

The total number of atoms constituting the linking group U is 1 to 100, preferably 10 to 90, more preferably 20 to 90, even more preferably 30 to 80.
The linking group U is a divalent linking group formed by combining three or more selected from an alkylene group, -O-, -NR ⁵⁰ -, -COO-, -CONR ⁵⁰ - and -SO ₂ NR ⁵⁰ -. is preferably ^R50 represents a hydrogen atom or an alkyl group.
The number of carbon atoms in the alkylene portion of the alkylene group that can be used as the linking group U is preferably 1 to 10, more preferably 1 to 8, still more preferably 1 to 7, particularly preferably 1 to 6, and most preferably 1 to 5.
The description of the alkyl group for R ¹ to R ⁴ above can be preferably applied to the alkyl group that can be used as R ⁵⁰ .
A hydrogen atom is preferred as R ⁵⁰ .
The number of the alkylene groups, —O—, —NR ⁵⁰ —, —COO—, —CONR ⁵⁰ — and —SO ₂ NR ⁵⁰ — constituting the linking group U is preferably 3 to 11, more preferably 3 to 7. 3 to 5 are more preferred, and 3 is particularly preferred.
The linking group U preferably has --O--, --NR ⁵⁰ --, --COO--, --CONR ⁵⁰ -- or --SO ₂ NR ⁵⁰ -- at the linking portion with L ³ and L ⁴ . That is, the linking group U is an alkylene group of L ³ and L ⁴ via —O—, —NR ⁵⁰ —, —COO—, —CONR ⁵⁰ — or —SO ₂ NR ⁵⁰ — constituting the linking group U. is preferably bound to. In the connecting group U, the portion connecting L ³ and L ⁴ is —O—, —NR ⁵⁰ —, —COO—, —CONR ⁵⁰ — or —SO ₂ NR ⁵⁰ —, and the connecting portions are alkylene It is more preferably a divalent linking group linked by a group.
The linking group U is preferably a carboxyl group or a divalent linking group having a substituent capable of binding to a biological substance, which will be described later. In the linking group U, the portion having a carboxy group or a substituent capable of binding to a biological substance includes an alkylene group or an alkyl group as R ⁵⁰ , and an alkylene group is preferred.
In the linking group U, the alkylene group or the alkyl group as R ⁵⁰ may be directly bonded to the carboxy group or a substituent capable of bonding to a biological substance, or may be bonded via a linking group ZZZ. .
Examples of the linking group ZZZ include an alkylene group, —NR ⁵⁰ —, —COO—, —CONR ⁵⁰ — and —(CH ₂ —CH ₂ —O) _p —, and a group consisting of a combination of these substituents. . The number to be combined is, for example, preferably 2-7, more preferably 2-5.
R ⁶⁰ has the same definition as R ⁶⁰ above, but R ⁶⁰ does not have a carboxy group or a substituent capable of binding to a biological substance.
p represents the number of repetitions, preferably from 1 to 10, more preferably from 1 to 8, even more preferably from 1 to 4.

(v)n
n is an integer of 1 to 3, preferably an integer of 2 or 3.

When the compound of the present invention has a structural moiety consisting of a cyanine dye represented by the general formula (III) as R, at least one cyanine dye represented by the general formula (III) in the compound is a carboxy group Alternatively, it has a substituent capable of binding to a biological substance, which will be described later.
In the cyanine dye represented by general formula (III), the position having a carboxyl group or a substituent capable of binding to a biological substance is not particularly limited, but any position of R ¹ to R ⁴ , R ⁴¹ or R ⁴² preferably has at least one in, more preferably has at least one in R ⁴¹ or R ⁴² , and more preferably has at least one on the ring formed by connecting R ⁴¹ and R ⁴² .
The number of groups having a carboxy group or a substituent capable of binding to a biological substance in the compound of the present invention is as described above.

In the cyanine dye represented by the general formula (III), at least one of R ¹ to R ⁴ , R ⁴¹ and R ⁴² contains a structure represented by —(CH ₂ —CH ₂ —O) _m — is preferred. m is synonymous with the above m. As a result, the compound of the present invention having a structural moiety composed of a cyanine dye represented by general formula (III) can have moderate hydrophilicity and moderate excluded volume effect, and the resulting labeled biological substance is excellent. It is thought that fluorescence intensity can be indicated.

In addition, the cyanine dye represented by the general formula (III), from the viewpoint of imparting sufficient hydrophilicity as the compound of the present invention, the number of hydrophilic groups per molecule of the cyanine dye represented by the general formula (III) is The number is preferably 2 or more, more preferably 2 to 8, even more preferably 2 to 6, and particularly preferably 3 to 6.
As the hydrophilic group, the description of the hydrophilic group that can be taken by R ²² to R ²⁵ and R ³² to R ³⁵ can be applied.
The position of the hydrophilic group is not particularly limited unless otherwise specified, and preferred examples of the group having the hydrophilic group include R ²² to R ²⁵ , R ³² to R ³⁵ , R ⁴¹ and R ⁴² . be done.
In addition, when the cyanine dye represented by the general formula (III) has the hydrophilic group as the carboxy group or a substituent capable of binding to the biological substance, the carboxy group or the substituent capable of binding to the biological substance It is practical and preferred to have one or more hydrophilic groups in addition to the groups. Specific examples include a carboxy group or a sulfo group.

Specific examples of the compounds of the present invention are shown below, but the present invention is not limited to these compounds. In the specific examples below, the sulfo group may have a salt structure by dissociating a hydrogen atom. In the specific examples below, EO _m , mPEG ₄ and PEG ₄ have the following structures, where _{m in EO m} is the average repeating number and Me represents a methyl group. However, _EOm and PEG ₄ are bonded to a nitrogen atom or an oxygen atom on the carbon atom side.

When the compound of the present invention has a substituent capable of binding to a biological substance, at least one substituent capable of binding to a biological substance possessed by the compound may bind to a biological substance such as a protein, peptide, amino acid, nucleic acid, sugar chain or lipid. It can be bound and used as a labeled biomaterial.
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 compounds of the present invention can be synthesized by known methods, except that the compound structure is defined by general formula (I) or (II). For example, for syntheses of M, L and X structures, see Acidochromicity of Bisarylethynylbenzenes: Hydroxyversus Dialkylamino Substituents, J. Am. Org. Chem. 2009, 74, 8909-8913 and Fourfold Suzuki-Miyaura and Sonogashira Cross-Coupling Reactions on Tetrahedral Methane and Adamantane Derivatives, Eur. J. Org. Chem. 2011, 1743-1754, etc., and for the synthesis of cyanine dyes, the methods described in WO 2005/000218, WO 2006/047452 and WO 2012/012595, etc. mentioned.
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 (I) or (II). See, for example, Bioconjugate Techniques (Third Edition, by Greg T. Hermanson).

<<Labeled biological substance>>
The labeled biological substance of the present invention 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 and exhibits an absorption wavelength peak suitable for color development in the near-infrared region and excellent fluorescence intensity, it can be preferably used as a labeled biological substance. The binding between the compound represented by general formula (I) or (II) and the biological substance may be in the form of direct binding between the compound represented by general formula (I) or (II) and the biological substance. It may be in the form of being linked via a group.

Proteins, peptides, amino acids, nucleic acids, sugar chains, and lipids are preferred examples of the biological substances. Antibodies are preferred as proteins, and phospholipids, fatty acids and sterols are preferred as lipids, and phospholipids are more preferred.
Among the biological substances mentioned above, clinically pathologically useful substances are not particularly limited, but examples include immunoglobulins such as IgG, IgM, IgE, IgA and IgD, complement, C-reactive protein (CRP ), ferritin, α1 _- microglobulin, β2 _- microglobulin and other plasma proteins and their antibodies, α-fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA19-9, CA-125, etc. tumor markers and their antibodies, luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotrobin (hCG), estrogen, hormones such as insulin 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), antibodies thereof, and the like.
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 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 digokinisin drugs, anti-asthma drugs such as theophylline, drugs such as antibiotics such as chloramphenicol and gentamicin, antibodies thereof, other enzymes, exotoxins (such as styrelidine O) and antibodies thereof. 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, lipids, etc. in biological materials;
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;
is 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, 2013, Vol. 1, p. 62-83. Also in the production of the labeled biological material of the present invention, the methods and the like described in the literature 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, is not particularly limited, and the form can be appropriately selected according to the purpose of use.
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. This 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"); The step of 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.

Examples of the target biological substances include proteins, so-called disease markers. Disease markers include, but are not limited to, α-fetoprotein (AFP), PIVKA-II, BCA225, basic fetoprotein (BFP), CA15-3, CA19-9, CA72-4, and 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 beta, 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-causing 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.
Among the compounds of the present invention, a labeled biological substance using a compound having a structural portion composed of a cyanine dye represented by the above-described general formula (III) as the phosphor portion R and having n equal to 1 is around 585 nm ( 560 to 620 nm), the wavelength range of the irradiated light is preferably 530 to 650 nm, more preferably 550 to 630 nm. A labeled biological material using this compound can be suitably used as a labeled biological material that exhibits excellent fluorescence intensity with respect to an excitation light source near 600 nm in the visible region.
Among the compounds of the present invention, a labeled biological substance using a compound having a structural portion composed of a cyanine dye represented by the above-described general formula (III) as the phosphor portion R and having n=2 has a wavelength around 685 nm ( 660 to 720 nm), the wavelength range of the irradiated light is preferably 630 to 750 nm, more preferably 650 to 730 nm. A labeled biological material using this compound can be suitably used as a labeled biological material that exhibits excellent fluorescence intensity against an excitation light source near 700 nm in the near-infrared region such as multicolor WB.
Among the compounds of the present invention, a labeled biological substance using a compound having a structural portion composed of a cyanine dye represented by the general formula (III) as the phosphor portion R and having n=3 is around 785 nm ( 760 to 820 nm), the wavelength range of the light to be irradiated is preferably 730 to 850 nm, more preferably 750 to 830 nm. A labeled biological material using this compound can be suitably used as a labeled biological material that exhibits excellent fluorescence intensity against an excitation light source near 800 nm in the near-infrared region such as multicolor WB.

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 matters 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, and devices that are commonly used can be appropriately selected in accordance with various methods using fluorescent labels.

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), the target biological substance can be 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 method 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), the target biological substance can be 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

Groups included in the substituent group T include the following groups.
Alkyl group (preferably 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms, still more preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, particularly preferably 1 to 3 carbon atoms), an alkenyl group (preferably 2 to 30 carbon atoms, more preferably 2 to 20 carbon atoms, still more preferably 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms, still more preferably 2 to 6 carbon atoms 2 to 4), an alkynyl group (preferably having 2 to 30 carbon atoms, more preferably 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms, still more preferably 2 to 6 carbon atoms, still more preferably 2 to 6 carbon atoms) 4), a cycloalkyl group (preferably having 3 to 20 carbon atoms), a cycloalkenyl group (preferably having 5 to 20 carbon atoms), an aryl group (which may be a monocyclic group, a condensed group (preferably 2 to 6-ring condensed ring group).When it is a condensed ring group, it consists of a 5- to 7-membered ring, etc. The aryl group preferably has 6 to 40 carbon atoms, more preferably 6 to 6 carbon atoms. 30, more preferably 6 to 26 carbon atoms, particularly preferably 6 to 10 carbon atoms), a heterocyclic group (having at least one nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom or selenium atom as a ring-constituting atom) may be a monocyclic group or a condensed ring group (preferably a condensed ring group with 2 to 6 rings).In the case of a monocyclic group, the number of ring members is 5 to It is preferably 7-membered, more preferably 5- or 6-membered, and the heterocyclic group preferably has 2 to 40 carbon atoms, more preferably 2 to 20. The heterocyclic group is an aromatic heterocyclic group (heteroaryl group). and aliphatic heterocyclic groups (aliphatic heterocyclic groups).), alkoxy groups (preferably 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms), alkenyloxy groups (preferably 2 carbon atoms to 20, more preferably 2 to 12 carbon atoms), alkynyloxy groups (preferably 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms), cycloalkyloxy groups (preferably 3 to 20 carbon atoms), aryl Oxy group (preferably 6 to 40 carbon atoms, more preferably 6 to 26 carbon atoms, more preferably 6 to 14 carbon atoms), heterocyclic oxy group (preferably 2 to 20 carbon atoms), polyalkyleneoxy group (preferably has 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms),

An alkoxycarbonyl group (preferably having 2 to 20 carbon atoms), a cycloalkoxycarbonyl group (preferably having 4 to 20 carbon atoms), an aryloxycarbonyl group (preferably having 6 to 20 carbon atoms), an amino group (preferably having 0 to 20, an unsubstituted amino group (—NH ₂ ), (mono- or di-)alkylamino group, (mono- or di-)alkenylamino group, (mono- or di-)alkynylamino group, (mono- or di-) cycloalkylamino group, (mono- or di-) cycloalkenylamino group, (mono- or di-) arylamino group, (mono- or di-) heterocyclic amino group. Each of the above substituting groups is synonymous with the corresponding group of the substituent group T.), a sulfamoyl group (preferably an alkyl, cycloalkyl or aryl sulfamoyl group having 0 to 20 carbon atoms.), an acyl group ( preferably 1 to 20 carbon atoms, more preferably 2 to 15 carbon atoms), acyloxy group (preferably 1 to 20 carbon atoms), carbamoyl group (preferably 1 to 20 carbon atoms, alkyl, cycloalkyl or aryl carbamoyl groups are preferred.),

acylamino group (preferably 1 to 20 carbon atoms), alkylthio group (preferably 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms), cycloalkylthio group (preferably 3 to 20 carbon atoms), arylthio group (preferably has 6 to 40 carbon atoms, more preferably 6 to 26 carbon atoms, more preferably 6 to 14 carbon atoms), a heterocyclic thio group (preferably 2 to 20 carbon atoms), an alkyl, cycloalkyl or arylsulfonyl group (preferably has 1 to 20 carbon atoms),

A silyl group (preferably having 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, and is preferably a silyl group substituted with alkyl, aryl, alkoxy or aryloxy.), a silyloxy group (preferably having 1 to 20 carbon atoms, , alkyl, aryl, alkoxy or aryloxy-substituted silyloxy groups are preferred.), hydroxy group, cyano group, nitro group, halogen atom (e.g. fluorine atom, chlorine atom, bromine atom or iodine atom), oxygen atom (specifically in which >CH ₂ constituting the ring is replaced with >C=O), carboxy group (-CO ₂ H), phosphono group [-PO(OH) ₂ ], phosphoryl group [-O-PO(OH) ₂ ], a sulfo group (-SO ₃ H), a boric acid group [-B(OH) ₂ ], an onio group (including an ammonio group, a sulfonio group, a phosphonio group including cyclic ammonium, preferably having 0 to 30 carbon atoms, more 1 to 20), a sulfanyl group (-SH), an amino acid residue, or a polyamino acid residue.
A carboxy group, a phosphono group, a sulfo group, an onio group, an amino acid residue, a polyamino acid residue, or -(CH ₂ -CH ₂ -O) _m -alkyl group (m is the same as m in R ¹ to R ⁶ ) as a substituent, the above alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, heterocyclic group, alkoxy group, alkenyloxy group, alkynyloxy group, cycloalkyloxy group, aryloxy group, 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 groups, alkyl, cycloalkyl or arylsulfonyl groups.

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, and a cycloalkoxycarbonyl group, amino group, acylamino group, cyano group or halogen atom, particularly preferably alkyl group, alkenyl group, aryl 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 and the like contain alkyl groups, alkenyl groups and the like, these may be substituted or unsubstituted. In addition, when containing an aryl group, a heterocyclic group, etc., they may be monocyclic or condensed, and may be substituted or unsubstituted.

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.

Compounds (1) to (5), which are carboxy forms of compounds (1-NHS) to (5-NHS) used in Examples, and comparative compounds (1-NHS) to (3-NHS) are shown below. show.
In the example compounds, the sulfo group may have a salt structure (for example, potassium salt, sodium salt, TEA (triethylamine) salt or DIPEA (N,N-diisopropylethylamine) salt) even if not otherwise specified. good. EO ₃ and EO ₄ are synonymous with EO ₃ and EO ₄ in the specific examples of the compound represented by the general formula (I) or (II) above. m means the average repetition number. All of the compounds were synthesized using, as starting materials, compounds in which the first decimal place of the average repetition number was 0.

The method for synthesizing each compound will be described in detail below, but the starting materials, dye intermediates and synthetic routes are not limited to these.
In the synthetic routes below, room temperature means 25°C.

The abbreviations used in synthesizing each component shown below are as follows.
DMF: N,N-dimethylformamide Pd/SPhos G3: SPhos Pd G3 (trade name), manufactured by Sigma-Aldrich, Pd catalyst for cross-coupling TBAF: Tetrabutylammonium fluoride TEA: Triethylamine THF: Tetrahydrofuran Ac: Acetyl group tBu: tert-butyl group Et: ethyl group TMS: trimethylsilyl group Ts: p-toluenesulfonyl group

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

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].

<Synthesis of compound (1-NHS)>
A compound (1-NHS) was synthesized based on the following scheme.

1) Synthesis of compound (1-B) 20 g of compound (1-A) and 150 ml of distilled water were placed in a 1 L three-necked flask, and 75 ml of a 30% hydrochloric acid aqueous solution was added dropwise while stirring. While cooling in a salt ice bath and maintaining the temperature below 3°C, a solution of 7 g of sodium nitrite dissolved in 80 ml of distilled water was slowly added dropwise, followed by stirring at 0 to 3°C for 45 minutes. Subsequently, a solution of 38 g of tin(II) chloride dissolved in 90 ml of distilled water and 30 ml of 30% HCl was slowly added dropwise, followed by stirring at 7° C. or less for 40 minutes. The solvent was concentrated and the residue was washed with isopropanol to obtain 14 g of compound (1-B).

2) Synthesis of compound (1-D) 1.9 g of compound (1-B), 20 ml of acetic acid (AcOH), 1.3 g of compound (1-C) and 0.98 g of potassium acetate (AcOK) are placed in a 200 ml eggplant flask. was added and stirred at 140° C. for 1 hour under a nitrogen atmosphere. The solvent was distilled off under reduced pressure, and the residue was purified by reverse phase column chromatography (eluent: acetonitrile/water=0/100→35/65) to obtain 0.99 g of compound (1-D).

3) Synthesis of compound (1-E) 478 mg of compound (1-D), 2 ml of sulfolane, 680 mg of butane sultone and 0.132 ml of triethylamine (Et ₃ N) were placed in a 50 ml eggplant-shaped flask and heated with stirring at 120°C for 6 hours. did. Ethyl acetate was added to the reaction solution to cause precipitation. The precipitate was purified by reverse phase column chromatography (eluent: acetonitrile/water=0/100→20/100) to obtain 344 mg of compound (1-E).

4) Synthesis of compound (1-F) 478 mg of compound (1-D), 2 ml of sulfolane, 772 mg of 6-bromohexanoic acid and 0.132 ml of triethylamine (Et ₃ N) were placed in a 50 ml eggplant flask and heated at 120°C. The mixture was heated and stirred for 6 hours. Ethyl acetate was added to the reaction solution to cause precipitation. The precipitate was purified by reverse phase column chromatography (eluent: acetonitrile/water=0/100→20/100) to obtain 296 mg of compound (1-F).

5) Synthesis of compound (1-H) 187 mg of compound (1-E), 43 mg of compound (1-G), 49 mg of potassium acetate (AcOK) and 2 mL of acetic anhydride (Ac ₂ O) were added to a test tube, and the mixture was stirred under a nitrogen atmosphere. and 60° C. for 2 hours. After the reaction converged, distilled water was added and the mixture was purified by reverse phase column chromatography (eluent: acetonitrile/water=0/100→25/75) to obtain 141 mg of compound (1-H).

6) Synthesis of compound (1-I) Compound (1-E) 94 mg, compound (1-F) 88 mg, compound (1-G) 43 mg, potassium acetate (AcOK) 49 mg and acetic anhydride (Ac ₂ O) in a test tube. ) was added, and the mixture was stirred at 60° C. for 2 hours under a nitrogen atmosphere. After the reaction converged, distilled water was added and the mixture was purified by reverse phase column chromatography (eluent: acetonitrile/water=0/100→25/75) to obtain 63 mg of compound (1-I).

7) Synthesis of compound (1-K) 10 g of compound (1-J), 50 mL of methylene chloride, and 6.32 mL of acetic anhydride (Ac ₂ O) were added to a 200 mL eggplant flask, and 9.3 mL of triethylamine was slowly added dropwise at room temperature. did. After stirring at room temperature for 1 hour, the solvent was distilled off under reduced pressure and purified by normal phase column chromatography (eluent: hexane/ethyl acetate = 100/0→25/75) to give compound (1-K)11. 5 g was obtained.

8) Synthesis of compound (1-L) Compound (1-K) 2.4 g, trimethylsilylacetylene 1.9 mL, tetrakis(triphenylphosphine) palladium (0) 500 mg, and copper iodide (I) are placed in a 100 mL eggplant flask. 86 mg, 30 mL of triethylamine was added. After vacuum degassing, the mixture was stirred at 80° C. for 3 hours under a nitrogen stream. The reaction solution was cooled to room temperature, filtered through celite, and the solvent was distilled off under reduced pressure. 20 mL of tetrahydrofuran (THF) was added to the residue, and the mixture was cooled to 0° C. with ice water. 10 mL of a THF solution of 1M tetrabutylammonium fluoride (TBAF) was slowly added dropwise, and the mixture was stirred at 0°C for 1 hour. The solvent was distilled off under reduced pressure, and the residue was purified by normal phase column chromatography (eluent: hexane/ethyl acetate=100/0→80/20) to obtain 1.1 g of compound (1-L).

9) Synthesis of compound (1-O) 7 g of compound (1-M), 8.3 g of compound (1-N) and 70 mL of THF were added to a 300 ml eggplant flask. 5.4 g of tBuOK was slowly added while cooling with ice water. The mixture was stirred at room temperature for 3 hours under nitrogen stream. After distilling off the solvent under reduced pressure, 50 mL of ethyl acetate was added, and after filtering, the solvent was distilled off from the filtrate under reduced pressure. Purification by normal phase column chromatography (eluent: hexane/ethyl acetate=100/0→0/100→ethyl acetate/methanol 50/50) gave 7.8 g of compound (1-O).

10) Synthesis of compound (1-P) Into a 50 ml round-bottom flask, 200 mg of compound (1-L), 1.2 g of compound (1-O), 100 mg of tetrakis(triphenylphosphine)palladium (0), copper iodide ( I) 12 mg, 10 mL triethylamine and 5 mL THF were added. After vacuum degassing, the mixture was stirred at 80° C. for 3 hours under a nitrogen stream. The reaction solution was cooled to room temperature, filtered through celite, and the solvent was distilled off under reduced pressure. Purification by normal phase column chromatography (eluent: methanol/ethyl acetate=0/100→50/50) gave 850 mg of compound (1-P).

11) Synthesis of compound (1-Q) Compound (1-P) 850 mg, trimethylsilylacetylene 0.48 mL, tetrakis(triphenylphosphine) palladium (0) 80 mg, copper (I) iodide 11 mg, 5 mL of triethylamine and 2.5 mL of THF were added. After vacuum degassing, the mixture was stirred at 80° C. for 3 hours under a nitrogen stream. The reaction solution was cooled to room temperature, filtered through celite, and the solvent was distilled off under reduced pressure. Tetrahydrofuran (THF) (10 mL) was added to the residue, and the mixture was cooled to 0°C with ice water. 1 mL of a 1 M tetrabutylammonium fluoride THF solution was slowly added dropwise, and the mixture was stirred at 0° C. for 1 hour. The solvent was distilled off under reduced pressure, and the residue was purified by normal phase column chromatography (eluent: methanol/ethyl acetate=0/100→50/50) to obtain 550 mg of compound (1-Q).

12) Synthesis of compound (1-R) Compound (1-P) 110 mg, compound (1-Q) 102 mg, tetrakis(triphenylphosphine)palladium (0) 20 mg, copper (I) iodide in a 50 ml eggplant flask. 2.8 mg, 5 mL triethylamine and 2.5 mL THF were added. After vacuum degassing, the mixture was stirred at 80° C. for 3 hours under a nitrogen stream. The reaction solution was cooled to room temperature, filtered through celite, and the solvent was distilled off under reduced pressure. 1 mL of tetrahydrofuran (THF), 1 mL of MeOH, and 440 μL of 3N aqueous sodium hydroxide solution were added to the residue, and the mixture was stirred at room temperature for 1 hour. After liquid separation with ethyl acetate and saturated brine, the organic layer was evaporated under reduced pressure and purified by normal phase column chromatography (eluent: methanol/ethyl acetate = 0/100 → 50/50) to give compound (1-R ) to give 28 mg.

13) Synthesis of Compound (1) Add 8.9 mg of compound (1-H), 8.6 mg of compound (1-I), 7.7 mg of compound (1-R), 300 μL of distilled water and 300 μL of acetonitrile to a test tube, Stirred at 95°C. A solution prepared by dissolving 1 mg of sodium hydroxide in 100 μL of distilled water was added dropwise to this solution, and the mixture was stirred at 95° C. for 30 minutes. The reaction solution was cooled to room temperature, purified by preparative HPLC, and lyophilized to obtain 7.1 mg of compound (1). The results of MS measurement of compound (1) were as follows.
MS (ESI m/z): (M+H ⁺ ) ⁺ =2970, (M−H ⁺ ) ⁻ =2968

14) Synthesis of compound (1-NHS) To 3.0 mg of compound (1), 0.30 ml of N,N-dimethylformamide (DMF), N,N,N',N'-tetramethyl-O-(N- An N,N-dimethylformamide solution in which 1 mg of succinimidyl)uronium hexafluorophosphate was dissolved and triethylamine (Et ₃ N) were added and stirred for 1 hour. Thereafter, the solvent was distilled off under reduced pressure, 1.5 mL of ethyl acetate was added, the supernatant was removed, and vacuum drying was performed to obtain compound (1-NHS).

<Synthesis of compound (2-NHS)>
Based on the scheme below, compound (2-NHS) was synthesized in the same manner as compound (1-NHS). The results of MS measurement of compound (2) were as follows: MS (ESI m/z): (M+H ⁺ ) ⁺ =3197, (M−H ⁺ ) ⁻ =3195

<Synthesis of compound (3-NHS)>
Based on the scheme below, compound (3-NHS) was synthesized in the same manner as compound (1-NHS). The results of MS measurement of compound (3) were as follows: MS (ESI m/z): (M+H ⁺ ) ⁺ =3610, (M−H ⁺ ) ⁻ =3608

<Synthesis of compound (4-NHS)>
Based on the scheme below, compound (4-NHS) was synthesized in the same manner as compound (1-NHS). The results of MS measurement of compound (4) were as follows: MS (ESI m/z): (M+H ⁺ ) ⁺ =4178, (M−H ⁺ ) ⁻ =4176

<Synthesis of compound (5-NHS)>
Based on the scheme below, compound (5-NHS) was synthesized in the same manner as compound (1-NHS). The results of MS measurement of compound (5) were as follows: MS (ESI m/z): (M+H ⁺ ) ⁺ =8728, (M−H ⁺ ) ⁻ =8726

The comparative compound (1-NHS) is IRDye800CW (trade name) manufactured by LI-COR Biotechnology.
Comparative compound (2-NHS) was obtained from N-(acid-PEG3)-N-bis(PEG3-amine) (manufactured by BROADPHARM, trade name: BP-23868) and comparative compound (1-NHS). , was synthesized in the same manner as in the synthesis of each NHS ester above using a compound that is the carboxy form of the comparative compound (2-NHS). The results of MS measurement of the carboxy compound of the comparative compound (2-NHS) were as follows.
MS (ESI m/z): (M+H ⁺ ) ⁺ =2541, (M−H ⁺ ) ⁻ =2539
Comparative compound (3-NHS) was synthesized in the same manner as in the synthesis of each NHS ester above, using comparative compound (3) synthesized according to the scheme below. The results of MS measurement of Comparative Compound (3) were as follows.
MS (ESI m/z): (M+H ⁺ ) ⁺ =2042, (M−H ⁺ ) ⁻ =2040

<Example 1>
The NHS ester (N-hydroxysuccinimide ester) form of each of the above compounds was evaluated for fluorescence labeling rate, fluorescence intensity on the membrane, and dot blot.
In addition, henceforth, the NHS ester form of each compound is simply called a "compound" for short. That is, the compound (1-NHS) is abbreviated as compound 1, and the compounds (2-NHS) to (5-NHS) and the comparative compounds (1-NHS) to (3-NHS) are similarly referred to as compounds. 2-5 and comparative compounds 1-3 for short.

[0] Preparation of fluorescence-labeled antibody In a microtube, put 217 µL of anti-rabbit IgG antibody (2.3 mg/ml) and 21.7 µL of carbonate buffer and shake and stir. It was added to the antibody so that the molar equivalent ratio shown in Table 1 was obtained, and then shaken and stirred. After allowing to stand at 4°C for 24 minutes, the reaction solution was purified using a centrifugal ultrafiltration filter Amicon Ultra UFC510096 (trade name, manufactured by Merck) and a PBS solution (phosphate buffered saline), and labeled antibody 1 was added. Obtained. The fluorescence labeling rate of the obtained labeled antibody was calculated by the method shown below. This fluorescence labeling rate means DOL (number of molecules of fluorescent dye bound per molecule of antibody).
Labeled antibodies for each compound and comparative compound were obtained in the same manner. Table A summarizes the results.

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 [-]

(Note to table)
In the labeled antibody column, the notation of compound Z-IgG or comparative compound Z-IgG means the IgG-labeled antibody of compound Z or the IgG-labeled antibody of comparative compound Z, respectively. Z means the number of each compound. The same applies to the following tables.

The results in Table A above show the following.
Compounds 1 to 5, which are the compounds of the present invention, were added in any molar equivalent amount of 3 equivalents, 5 equivalents, 10 equivalents, and 25 equivalents to the antibody, and comparative compound 1, which is a commercially available labeled compound, was used. The fluorescence labeling rate was at the same level as or higher than that obtained with the antibody, and the binding property to the antibody was at a level sufficient for practical use. This means that No. cA11 and No. It can be read from a comparison with A1 to A5.
Among them, in compounds 2 to 5, at least one of the cyanine dyes represented by the general formula (III) having the phosphor moiety R is a substituent on which R ⁴¹ and R ⁴² are bonded to each other has a substituent capable of binding to a biological substance and exhibits a higher fluorescence labeling rate (No. A2 to A5 compared to No. A1).

[1] Evaluation of solution fluorescence intensity The labeled antibody solution prepared above was adjusted to a protein concentration of 0.005 mg / mL, and a spectrofluorometer (trade name: RF-5300, manufactured by Shimadzu Corporation) was used to An excitation light of 785 nm was used, and the exposure conditions were standardized to calculate the integrated value of the fluorescence intensity in the fluorescence wavelength range of 810 to 840 nm. Using the DOL2.1 fluorescence intensity of comparative compound 1-IgG as a reference value, the ratio to this reference value was calculated and evaluated based on the following evaluation criteria. Table 1 summarizes the results.
In this test, an evaluation rank of "D" or higher for the fluorescence intensity is acceptable.

- Evaluation criteria for fluorescence intensity -
A: The ratio of fluorescence intensity to the reference value is 7.0 times or more B: The ratio of fluorescence intensity to the reference value is 4.0 times or more and less than 7.0 times C: The ratio of fluorescence intensity to the reference value is 2.5 times or more Less than 4.0 times D: The ratio of fluorescence intensity to the reference value is 1.5 times or more and less than 2.5 times E: The ratio of fluorescence intensity to the reference value is 1.2 times or more and less than 1.5 times F: Relative to the reference value Ratio of fluorescence intensity is 0.8 times or more and less than 1.2 times G: Ratio of fluorescence intensity to reference value is less than 0.8 times

The results in Table 1 above show the following.
Comparative Compound 2 has a structure different from that defined in the present invention in that the group connecting the phosphor portion is a polyoxyalkylene group. Comparative Compound 3 has a phenylene group and a polyoxyalkylene It is not a structure defined in the present invention in that it consists of a group. The fluorescence intensity in the labeled antibody solution using these comparative compounds 2 or 3 was low (No. c12 and c13).
In contrast, all of the labeled antibodies of Compounds 1 to 5 of the present invention have a fluorescence intensity 1.5 times or more that of the comparative labeled antibody 1, showing excellent fluorescence intensity. (No. 101-105 against No. c11). In addition, when the comparative compounds 1 to 3 were used, a decrease in fluorescence intensity due to self-association was observed with an increase in DOL. It was found that the decrease in fluorescence intensity due to self-association accompanying the increase was effectively suppressed (see the fluorescence intensity between ad in Nos. 101 to 105 and c11 to c13).
Among them, the structure portion having a hydrophilic group on the linking group connecting the phosphor portions R and further comprising a cyanine dye represented by the general formula (III) has at least one of R ¹ and R ² and R Compounds 3 to 5 of the present invention, in which at least one of ³ and R ⁴ has a structure containing a structure represented by —(CH ₂ —CH ₂ —O) _m —, exhibit superior fluorescence in the state of solution. strength (Nos. 103-105 versus Nos. 101 and 102). In particular, the labeled antibody using the compound 5 of the present invention, which is bound between the phosphor moieties R using a linking group M having a valence of 3 or more, can be obtained in a solution (No. 105 compared to Nos. 103 and 104).

[2] Evaluation of Fluorescence Intensity on Membrane An anti-rabbit IgG solution was adjusted to a protein concentration of 5.0 ng/mL, and 2 μL of the solution was carefully spotted on a nitrocellulose membrane. After drying the membrane, it was then blocked with Fish Gelatin blocking buffer in TBS-T. The membrane was incubated for 1 hour at room temperature with agitation. The blocking solution was removed, and the PBS solution of the labeled antibody was diluted 20,000 times with TBS buffer. The membrane was immersed in the diluted solution and incubated with agitation for 1 hour. The membrane was washed with TBS-T buffer three times for 10 minutes and finally with TBS buffer for 10 minutes. The resulting membrane was dried on a hot plate at 40° C. for 1 hour and imaged using an Amersham Typhoon scanner (manufactured by GEHC). The fluorescence intensity of was calculated. Using the DOL2.1 fluorescence intensity of comparative compound 1-IgG as a reference value, the ratio to this reference value was calculated and evaluated based on the following evaluation criteria. Table 2 summarizes the results.
In this test, an evaluation rank of "D" or higher for the fluorescence intensity is acceptable.

- Evaluation criteria for fluorescence intensity -
A: The ratio of fluorescence intensity to the reference value is 7.0 times or more B: The ratio of fluorescence intensity to the reference value is 4.0 times or more and less than 7.0 times C: The ratio of fluorescence intensity to the reference value is 2.5 times or more Less than 4.0 times D: The ratio of fluorescence intensity to the reference value is 1.5 times or more and less than 2.5 times E: The ratio of fluorescence intensity to the reference value is 1.2 times or more and less than 1.5 times F: Relative to the reference value Ratio of fluorescence intensity is 0.8 times or more and less than 1.2 times G: Ratio of fluorescence intensity to reference value is less than 0.8 times

The results in Table 2 above show the following.
As described above, the fluorescence intensity on the membrane of the labeled antibody using comparative compound 2 or 3, which does not have the structure defined by the present invention, was low (No. c22 and c23).
In contrast, all of the labeled antibodies of Compounds 1 to 5 of the present invention have a fluorescence intensity 1.5 times or more that of the comparative labeled antibody 1, showing excellent fluorescence intensity. (No. 201-205 against No. c21). In addition, when the comparative compounds 1 to 3 were used, a decrease in fluorescence intensity due to self-association was observed with an increase in DOL. It was found that the decrease in fluorescence intensity due to self-association accompanying the increase was effectively suppressed (see fluorescence intensities a to d in Nos. 201 to 205 and c21 to c23).
Among them, the structure portion having a hydrophilic group on the linking group connecting the phosphor portions R and further comprising a cyanine dye represented by the general formula (III) has at least one of R ¹ and R ² and R Compounds 3 to 5 of the present invention, in which at least one of ³ and R ⁴ has a structure containing a structure represented by —(CH ₂ —CH ₂ —O) _m —, exhibited superior fluorescence intensity on the membrane. (Nos. 203 to 205 for Nos. 201 and 202). In particular, the labeled antibody using the compound 5 of the present invention, which is bound between the phosphor moieties R using a linking group M having a valence of 3 or more, can Fluorescence intensity was better than above (No. 205 versus Nos. 203 and 204).

[3] Dot Blot Evaluation Transferrin (20 mg/mL) was adjusted to 50 ng/mL with TBS-T buffer and 2 μL was carefully spotted on a nitrocellulose membrane. After drying the membrane, it was then blocked with Fish Gelatin blocking buffer in TBS-T. Subsequently, 6 μL of rabbit anti-human transferrin polyclonal antibody was added to 30 mL of PBS-T buffer, and the membrane was soaked and shaken for 1 hour. After that, the membrane was taken out and washed 4 times with TBS-T buffer. After that, 15 μL of labeled antibody (anti-rabbit IgG) was added to 30 mL of TBS-T buffer, the membrane was immersed therein, and incubated at room temperature for 1 hour with stirring. The membrane was washed with TBS-T buffer three times for 10 minutes and finally with TBS buffer for 10 minutes. The resulting membrane was dried on a hot plate at 40° C. for 1 hour and imaged using an Amersham Typhoon scanner (manufactured by GEHC). The fluorescence intensity of was calculated. Using the DOL2.1 fluorescence intensity of comparative compound 1-IgG as a reference value, the ratio to this reference value was calculated and evaluated based on the following evaluation criteria. Table 3 summarizes the results.
In this test, an evaluation rank of "D" or higher for the fluorescence intensity is a pass.

- Evaluation criteria for fluorescence intensity -
A: The ratio of fluorescence intensity to the reference value is 7.0 times or more B: The ratio of fluorescence intensity to the reference value is 4.0 times or more and less than 7.0 times C: The ratio of fluorescence intensity to the reference value is 2.5 times or more Less than 4.0 times D: The ratio of fluorescence intensity to the reference value is 1.5 times or more and less than 2.5 times E: The ratio of fluorescence intensity to the reference value is 1.2 times or more and less than 1.5 times F: Relative to the reference value Ratio of fluorescence intensity is 0.8 times or more and less than 1.2 times G: Ratio of fluorescence intensity to reference value is less than 0.8 times

The results in Table 3 above show the following.
As described above, the fluorescence intensity on the dot blot of the labeled antibody using comparative compound 2 or 3, which does not have the structure defined by the present invention, was low (No. c32 and c33).
In contrast, all of the labeled antibodies of Compounds 1 to 5 of the present invention have a fluorescence intensity 1.5 times or more that of the comparative labeled antibody 1, showing excellent fluorescence intensity. (No. 301 to 305 against No. c31). In addition, when the comparative compounds 1 to 3 were used, a decrease in fluorescence intensity due to self-association was observed with an increase in DOL. It was found that the decrease in fluorescence intensity due to self-association accompanying the increase was effectively suppressed (see fluorescence intensity between a to d in Nos. 301 to 305 and c31 to c33).
Among them, the structure portion having a hydrophilic group on the linking group connecting the phosphor portions R and further comprising a cyanine dye represented by the general formula (III) has at least one of R ¹ and R ² and R Compounds 3 to 5 of the present invention, in which at least one of ³ and R ⁴ has a structure containing a structure represented by —(CH ₂ —CH ₂ —O) _m —, exhibited superior fluorescence on dot blots. strength (Nos. 303-305 versus Nos. 301 and 302). In particular, the labeled antibody using the compound 5 of the present invention, which is bound between the phosphor moieties R using a linking group M having a valence of 3 or more, is a dot It showed better fluorescence intensity on the blot (No. 305 versus No. 303 and 304).

Thus, in the compounds of the present invention represented by general formula (I) or (II), two or more phosphor moieties R are -X-L-X- or -X-L-M-L-X A compound linked by a linking group represented by -, wherein L is a divalent linking group formed by combining at least two of -C≡C-, arylene and heteroarylene groups, and X is an ether bond, a thioether bond, an alkylene group, an amide bond, an ester bond, or an amino bond, the resulting labeled biological substance exhibits excellent fluorescence intensity in any state of solution, membrane, or dot plot. be able to.

Claims (12)

A fluorescent dye represented by the following general formula (I) or (II).

In the formula, M represents a t-valent linking group, and t is an integer of 3 or more.
L represents a divalent linking group formed by combining at least two of —C≡C—, an arylene group and a heteroarylene group.
X represents an ether bond, thioether bond, alkylene group, amide bond, ester bond or amino bond.
R indicates a phosphor portion. The fluorescence according to claim 1, wherein the R is a structural moiety consisting of at least one of xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes and pyrromethene dyes. pigment. 3. The fluorescent dye according to claim 1, wherein said fluorescent dye is represented by said general formula (I), said t is 3, and said M is a linking group having any one of the following structures.

In the above structural formula, * indicates the bonding position with L. 3. The fluorescent dye according to claim 1, wherein said fluorescent dye is represented by said general formula (I), said t is 4, and said M is a linking group having the following structure.

In the above structural formula, * indicates the bonding position with L. The fluorescent dye according to any one of claims 1 to 4, wherein at least one of said R has a carboxy group or a substituent capable of binding to a biological substance. The fluorescent dye according to any one of claims 1 to 5, wherein at least one of said L and M has a water-soluble substituent. 7. The fluorescent dye according to claim 6, wherein said water-soluble substituent is at least one of a carboxy group, a sulfo group, a phosphono group, a phosphonooxy group, a sulfamoyl group and a polyoxyalkylene group. The fluorescent dye according to any one of claims 1 to 7, wherein said R is a structural moiety comprising a cyanine dye. 9. The fluorescent dye according to claim 8, wherein the cyanine dye is represented by the following general formula (III).

In the formula, R ¹ to R ⁴ represent an alkyl group or —(CH ₂ —CH ₂ —O) _m —R ²¹ . m is 1 to 50, and R ²¹ represents an alkyl group.
R ¹¹ to R ¹³ each represent a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an amino group or a halogen atom, and adjacent groups are bonded to each other to form a 5- or 6-membered ring; You may have
R ²² to R ²⁵ and R ³² to R ³⁵ are hydrogen atom, alkyl group, alkoxy group, aryl group, sulfo group, sulfamoyl group, carboxy group, alkoxycarbonyl group, aryloxycarbonyl group, acyloxy group, carbamoyl group, acylamino represents a radical, a nitro group or a halogen atom.
R ⁴¹ and R ⁴² represent an alkyl group or -(CH ₂ -CH ₂ -O) _m -R ²¹ . R ²¹ and m are synonymous with R ²¹ and m above. R ⁴¹ and R ⁴² may combine with each other to form a ring.
n is an integer of 1-3.
Any one of R ¹ to R ⁴ , R ¹¹ to R ¹³ , R ²² to R ²⁵ , R ³² to R ³⁵ , R ⁴¹ or R ⁴² described above binds to X.
At least one cyanine dye represented by formula (III) in the fluorescent dye has a carboxy group or a substituent capable of binding to a biological substance.
However, the structural part composed of the cyanine dye represented by formula (III) is a neutral structural part. 10. The fluorescent dye according to claim 9, which binds to said X through any one of said R ¹¹ to R ¹³ . A labeled biological substance obtained by binding the fluorescent dye according to any one of claims 1 to 10 to a biological substance. 12. The labeled biological material according to claim 11, wherein said biological material is any one of proteins, amino acids, nucleic acids, sugar chains and phospholipids.