JP2001272350A - Fluorescent labeling reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a specified cyanine dye as a labeling compound, having an effective structure for inhibiting the association of cyanine dyes in water. SOLUTION: This fluorescent labeling reagent contains a compound, represented by the shown formula (where Z is nonmetal atom group; R is an alkyl or an aryl; Q is methine group or polymethine group substituted with a heterocyclic group or an aromatic group; L is a connecting group consisting of a nonmetal group; s is 1-3; t is 1-4; Y is an oxygen atom, a sulfur atom, or the like; V is a substituent group excluding a sulfonic group; n is 0-4; M is a counter ion; m is a number required for keeping the balance of electrical changes; and at least one of a functional group reacting with an amino group, a hydroxyl group, or a mercapto group for forming a double bond and/or a carboxyl group is contained in the molecule).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescent labeling reagent for labeling a natural or artificial chemical substance by attaching a fluorescent compound to the substance to facilitate the detection of the substance, and a method of using the reagent. It relates to the fluorescent labeling method used.

[0002]

2. Description of the Related Art A compound that emits fluorescence to a chemical substance (hereinafter referred to as a compound).
In this specification, it may be referred to as “fluorescent compound”. ) Is widely covalently labeled to detect the presence of the substance. Examples of compounds to be labeled include antibodies, proteins, peptides, enzyme substrates, hormones,
Lymphokine, metabolite, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin, carbohydrate, polysaccharide, nucleic acid, deoxynucleic acid, nucleic acid derivative, derived deoxynucleic acid, DNA fragment, RNA fragment, derived DNA fragment, derived RNA fragment , Natural drugs, virus particles, bacterial particles,
Virus components, yeast components, blood cells, blood cell components, bacteria, bacterial components, natural or synthetic resins, synthetic drugs, poisons, environmental pollutants, polymers, polymer particles, glass particles, plastic particles, polymer films, etc. No. Various compounds are used as the fluorescent compound bound to these, and an organic dye is particularly useful.

[0003] Among organic dyes, cyanine dyes, which have strong fluorescence and whose fluorescence wavelength can be easily selected, are particularly useful. Cyanine dyes have the drawback that an aggregate is easily formed in water and the fluorescence intensity is weakened. In order to overcome this problem, cyanine dyes having an arylsulfonic acid group have been developed, as described in, for example, Japanese Patent No. 2757965. That is, the presence of a sulfonic acid group directly bonded to an aromatic ring condensed to a heterocyclic ring called a basic nucleus, which constitutes a terminal group of a cyanine dye, prevents the arrangement of overlapping dyes due to electrostatic repulsion between anions. , The association is described as being inhibited.

[0004]

However, even with such a cyanine dye having an arylsulfonic acid group, it has been found that fluorescence quenching due to association is not necessarily escaped, but rather the association may be promoted. Was. This is probably because the bond between the sulfonic acid group and the aryl group to which the sulfonic acid group is bonded is within the plane of the aryl group, so that it is difficult to sterically hinder the overlap between the dyes. Accordingly, an object of the present invention is to provide a specific cyanine dye having a structure effective for inhibiting the association of a cyanine dye in water as a labeling compound, and to provide a method for labeling various compounds using the same. It is to be.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, the compound represented by the following general formula (I) or (II) was dissolved in water. They found that they did not associate and that they had particularly excellent properties as fluorescent labeling reagents, and completed the present invention.
That is, the present invention provides the following general formula (I):

Embedded image (Wherein, Z represents a nonmetallic atom group necessary for forming an aromatic ring; R represents an alkyl group which may have a substituent or an aryl group which may have a substituent. Q represents a methine group substituted with a heterocyclic group or an aromatic group or a polymethine group substituted with a heterocyclic group or an aromatic group; L represents a linking group consisting of a group of non-metallic atoms; T represents an integer of 1 to 4; Y represents an oxygen atom, a sulfur atom, NR ¹ , or C (R ² )
(R ³ ) (wherein R ¹ , R ² , or R ³ each independently represents an alkyl group which may have a substituent or an aryl group which may have a substituent); V represents a substituent (excluding a sulfonic acid group); n represents an integer of 0 to 4; M represents a counter ion; m represents a number necessary to maintain charge balance; A compound having at least one functional group and / or carboxyl group capable of forming a covalent bond by reacting with one or more groups selected from the group consisting of a group, a hydroxyl group, and a mercapto group) And a reagent for fluorescent labeling.

Further, according to the present invention, the following general formula (I)
I):

Embedded image (Wherein, Z ¹ and Z ² each independently represent a nonmetallic atom group necessary for forming an aromatic ring; R ⁴ and R ⁵ each independently represent an alkyl group which may have a substituent. Or an aryl group which may have a substituent; L ¹ and L ²
Represents a linking group comprising a group of non-metallic atoms; s1 and s2 each independently represent an integer of 1 to 3; t1 and t2
Each independently represents an integer of 1 to 4; p, q, and r each independently represent 0 or 1; Y ¹ and Y ² each independently represent an oxygen atom, a sulfur atom, NR ¹ , or C (R ² )
(R ³ ) (wherein, R ¹ , R ² and R ³ each independently represent an alkyl group which may have a substituent or an aryl group which may have a substituent); L ³ , L ⁴ , L ⁵ ,
L ⁶ , L ⁷ , L ⁸ , and L ⁹ each represent a methine group which may have a substituent; V represents a substituent (excluding a sulfonic acid group); M represents a counter ion; m represents a number necessary to maintain charge balance; provided that one or more groups selected from the group consisting of amino groups, hydroxyl groups, and mercapto groups Which has at least one functional group and / or carboxyl group in the molecule capable of forming a covalent bond by reacting with the compound).

[0007] From another viewpoint, the present invention provides a method for labeling a substance using the above-mentioned fluorescent labeling reagent, preferably a method for labeling a substance in an aqueous liquid. This method includes a step of reacting a compound represented by the above general formula (I) or the general formula (II) with a substance in an aqueous liquid to form a covalent bond. Further, according to the present invention,
Use of the compound represented by the general formula (I) or (II) as a fluorescent labeling reagent; the compound represented by the general formula (I) or (II) is bound as a fluorescent label Substance; A method for performing a fluorescence analysis on a substance to which a compound represented by the above general formula (I) or (II) is bound as a fluorescent label is provided.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the general formula (I), Q represents a methine group substituted with a heterocyclic group or an aromatic group or a polymethine group substituted with a heterocyclic group or an aromatic group. It shows a methine group or a polymethine group consisting of a partial structure of a methine dye. For example, when the methine dye is a cyanine dye, a preferable Q is a partial structure containing a part or all of the methine structure of the cyanine dye and one ring structure. Preferred examples of the methine dye include a cyanine dye, a merocyanine dye, a rhodocyanine dye, a trinuclear merocyanine dye, an allopolar dye, a hemicyanine dye, and a styryl dye.

The details of these methine dyes are described in FM Harmer, "Heterocyclic Compounds-Cyanin Soybean and Related Compounds".
e Dyes and Related Compounds), John Wiley & Sons, New York, London, 1964, D.M.
(DM Sturmer), Heterocyclic Compounds-Special Topics in Heterocyclic Compounds-Special to
pics in heterocyclic chemistry), Chapter 18, Chapter 1
Verses 4, 482-515. The general formulas of cyanine dyes, merocyanine dyes and rhodacyanine dyes are described, for example, in (XI), (XII), and (XIII) of Nos. 21 and 22 of US Pat. No. 5,340,694.
Is shown in The compound represented by the general formula (I) is
It is preferably a compound selected from the general formula (II).

In the general formula (II), the optionally substituted alkyl group represented by R ⁴ or R ⁵ is preferably a straight-chain, branched-chain, cyclic or C1-C12 alkyl group, or An alkyl group composed of a combination thereof (the same applies to the alkyl portion of another alkyl group or a substituent having an alkyl portion unless otherwise specified);
Particularly preferred is a linear or branched alkyl group having 1 to 8 carbon atoms. These alkyl groups may have a substituent. When the alkyl group has a substituent, the type, number, and substitution position of the substituent are not particularly limited, but the substituent may be, for example, a sulfonic acid group or the following V
Can be selected from the groups represented by It is preferable that the alkyl group has at least one acidic group or a salt thereof that becomes an anionic group such as a sulfonic acid group, a phosphonic acid group, and a carboxyl group as a substituent of the alkyl group, and an amino group and a hydroxyl group. And a group capable of forming a covalent bond by reacting with a group selected from the group consisting of a mercapto group.

The optionally substituted aryl group represented by R ⁴ or R ⁵ is preferably a phenyl group optionally having 6 to 12 carbon atoms. When the aryl group has a substituent, the type, number, and substitution position of the substituent are not particularly limited, but the substituent can be selected from, for example, a sulfonic acid group or a group represented by V below.
The aryl group preferably has at least one group of an acidic group such as a sulfonic acid group, a phosphonic acid group, and a carboxyl group, which is an anionic group or a salt thereof, such as a sulfonic acid group, a phosphonic acid group, and a carboxyl group. It is also preferable to have a group capable of reacting with a group selected from the group consisting of a group and a mercapto group to form a covalent bond. R is the same as above R ⁴ or R ⁵ in the general formula (I).

In the general formula (II), Y ¹ or Y ² represents an oxygen atom, a sulfur atom, NR ¹ or C (R ² ) (R ³ ).
The optionally substituted alkyl group represented by R ¹ , R ² , or R ³ is preferably an alkyl group having 1 to 12 carbon atoms which is linear, branched, cyclic, or a combination thereof. And particularly preferably a linear or branched alkyl group having 1 to 8 carbon atoms. These alkyl groups may have a substituent. When the alkyl group has a substituent, the type, number, and substitution position of the substituent are not particularly limited, but the substituent can be selected from, for example, a sulfonic acid group or a group represented by V below. R
² and R ³ may be linked to each other to form a cycloalkyl ring such as a cyclohexane ring or a hetero ring such as a morpholine ring together with one or more hetero atoms (eg, an oxygen atom, a sulfur atom, a nitrogen atom, etc.), One or more substituents may be present on the ring.

The optionally substituted aryl group represented by R ¹ , R ² or R ³ is preferably a phenyl group optionally having 6 to 12 carbon atoms. When the aryl group has a substituent, the type, number, and substitution position of the substituent are not particularly limited, and the substituent can be selected from, for example, a sulfonic acid group or a group represented by the following V. Further, as the substituent, a group which is an acidic group or a salt thereof which is ionized to be an anionic group such as a sulfonic acid group, a phosphonic acid group, and a carboxyl group is also preferable. Y ¹ and Y ² are preferably an oxygen atom or C (R ² ) (R ³ ), and more preferably C (R ² ) (R ³ ). R ² and R ³
Is preferably a methyl group. The same applies to Y in the general formula (I).

In the general formulas (I) and (II),
M can be present as a cation or an anion when needed to neutralize the ionic charge of the compound. M may be either a monovalent ion or a polyvalent ion. Typical cations include hydrogen ions (H ⁺ ), inorganic cations such as alkali metal ions (eg, sodium ion, potassium ion, lithium ion), alkaline earth metal ions (eg, calcium ion), and ammonium ions (eg, Organic ions such as ammonium ion, tetraalkylammonium ion, pyridinium ion, and ethylpyridinium ion. The anion may be either an inorganic anion or an organic anion, such as a halogen anion (for example, a fluorine ion, a chloride ion, or an iodine ion), or a substituted arylsulfonate ion (for example, p-toluenesulfonic acid ion, p-toluene ion). Chlorobenzenesulfonic acid ion), aryldisulfonic acid ion (for example, 1,3-benzenesulfonic acid ion, 1,5-naphthalenedisulfonic acid ion,
2,6-naphthalenedisulfonic acid ion), alkyl sulfate ion (eg, methyl sulfate ion), sulfate ion, thiocyanate ion, perchlorate ion, tetrafluoroborate ion, picrate ion, acetate ion, trifluoromethanesulfonic acid ion And the like. Further, as M, an ionic polymer or another dye having a charge opposite to that of the dye may be used. Moreover, CO ₂ ^- or SO ₃ ^-
Is CO ₂ H when it has a hydrogen ion as a counter ion,
It can also be expressed as SO ₃ H. m represents the number necessary to balance the charges, and is 0 when a salt is formed in the molecule.

In the general formula (I) or the general formula (II),
As the substituent represented by V, a substituent other than a sulfonic acid group can be used, but the type is not particularly limited. For example, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom may be used), a mercapto group, a cyano group, a carboxyl group, a phosphate group, a sulfo group,
Hydroxy group, having 1 to 10 carbon atoms, preferably 2 carbon atoms
To 8, more preferably a carbamoyl group having 2 to 5 carbon atoms (for example, a methylcarbamoyl group, an ethylcarbamoyl group, a morpholinocarbonyl group, etc.);
0, preferably 2 to 8, more preferably 2 to 5 sulfamoyl groups (eg, methylsulfamoyl group, ethylsulfamoyl group, piperidinosulfonyl group), nitro group, 1 to 20 carbon atoms Preferably an alkoxy group having 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms (for example, methoxy group, ethoxy group, 2-methoxyethoxy group, 2-phenylethoxy group);
To 20, preferably 6 to 12, more preferably 6 to 10 aryloxy groups (for example, phenoxy, p-methylphenoxy, p-chlorophenoxy, naphthoxy, etc.),

An acyl group having 1 to 20 carbon atoms, preferably 2 to 12 carbon atoms, more preferably 2 to 8 carbon atoms (eg, acetyl, benzoyl, trichloroacetyl, etc.), 1 to 20 carbon atoms, preferably An acyloxy group having 2 to 12 carbon atoms, more preferably 2 to 8 carbon atoms (eg, acetyloxy group, benzoyloxy group, etc.), 1 to 20 carbon atoms, preferably 2 to 1 carbon atoms
2, more preferably an acylamino group having 2 to 8 carbon atoms (such as an acetylamino group), 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 carbon atom.
To 8 sulfonyl groups (for example, methanesulfonyl group, ethanesulfonyl group, benzenesulfonyl group, etc.), sulfinyl groups having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 8 carbon atoms (eg, methanesulfinyl group) Benzenesulfinyl group), a sulfonylamino group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms (for example, methanesulfonylamino group, ethanesulfonylamino group,
Benzenesulfonylamino group),

An amino group, a substituted amino group having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 8 carbon atoms (for example, methylamino group, dimethylamino group, benzylamino group, anilino group, diphenylamino An ammonium group having 0 to 15 carbon atoms, preferably 3 to 10 carbon atoms, more preferably 3 to 6 carbon atoms (for example, trimethylammonium group, triethylammonium group, etc.), and 0 to 15 carbon atoms, preferably A hydrazino group having 1 to 10 carbon atoms, more preferably a hydrazino group having 1 to 6 carbon atoms (eg, a trimethylhydrazino group), a ureido group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms (E.g., ureido group, N, N-dimethylureido group, etc.), having 1 to 15 carbon atoms, preferably Number 1 to 10, more preferably imido group having 1 to 6 carbon atoms (e.g., succinimido group), 1 to 20 carbon atoms, preferably a carbon number of 1 1
2, more preferably an alkyl or arylthio group having 1 to 8 carbon atoms (eg, methylthio, ethylthio group, carboxyethylthio group, sulfobutylthio group, phenylthio group, etc.), 2 to 20 carbon atoms, preferably 2 to 12 carbon atoms. More preferably, it is an alkoxycarbonyl group having 2 to 8 carbon atoms (eg, methoxycarbonyl group, ethoxycarbonyl group, benzyloxycarbonyl group, etc.), 6 to 20 carbon atoms, preferably 6 to 12 carbon atoms, more preferably 6 to 8 carbon atoms. Aryloxycarbonyl group (such as a phenoxycarbonyl group),

An unsubstituted alkyl group having 1 to 18 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms (eg, a methyl group, an ethyl group, a propyl group, a butyl group, etc.); To 18, preferably 1 carbon atom
To 10, more preferably a substituted alkyl group having 1 to 5 carbon atoms (hydroxymethyl group, trifluoromethyl group,
Benzyl group, carboxyethyl group, ethoxycarbonylmethyl group, acetylaminomethyl group and the like, preferably an unsaturated hydrocarbon having 2 to 18 carbon atoms, more preferably 3 to 10 carbon atoms, and particularly preferably 3 to 5 carbon atoms. (For example, a vinyl group, an ethynyl group, a 1-cyclohexenyl group, a benzylidine group, a benzylidene group, etc.), having 6 to 20 carbon atoms, preferably 6 to 1 carbon atoms.
5, and more preferably a substituted or unsubstituted aryl group having 6 to 10 carbon atoms (for example, phenyl group, naphthyl group, p
-Carboxyphenyl group, p-nitrophenyl group, 3,
5-dichlorophenyl group, p-cyanophenyl group, m-
Fluorophenyl group, p-tolyl group),

A substituted or unsubstituted heterocyclic group having 1 to 20, preferably 2 to 10, more preferably 4 to 6 carbon atoms (for example, pyridyl group, 5-methylpyridyl group, thienyl group, furyl group) Morpholino group, tetrahydrofurfuryl group, etc.). Further, one or two or more of the substituents exemplified as V above may be present on these substituents.

In the general formula (II), V is the above-mentioned alkyl group, aryl group, alkoxy group, halogen atom, acyl group, cyano group or sulfonyl group, more preferably, alkyl group, aryl group, halogen atom, An acyl group or a sulfonyl group, particularly preferably a methyl group, a phenyl group, a methoxy group, a chlorine atom, a bromine atom,
Or it is an iodine atom. Most preferably, a phenyl group,
They are a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
The same applies to V in the general formula (I).

In the general formula (II), the linking group represented by L ¹ or L ² comprises a nonmetallic atom, and is preferably a divalent to tetravalent linking group. The linking group is preferably a group formed by a combination of atoms selected from the group consisting of a carbon atom, a hydrogen atom, a nitrogen atom, an oxygen atom, and a sulfur atom. For example, the general formula: -TG- More preferably, it is a group represented by Wherein T is an aliphatic group,
It preferably represents an alkylene group, alkenylene group or alkynylene group, and G represents an oxygen atom, a sulfur atom, -NH-
(The nitrogen atom may be substituted by an alkyl group),-
N (C = O) -, - O (C = O) -, - NHSO 2 -
(A nitrogen atom may be substituted with an alkyl group), or -NHCONH- (a nitrogen atom may be substituted with an alkyl group). The number of carbon atoms of the linking group is preferably 1 to 12, particularly preferably 1 to 6.
Examples of the case where L ¹ or L ² is trivalent or tetravalent are -CH
^{_{2 - (CH 2 -SO 3 -}} ) 2, -CH 2 - (CO-CH 2 -CH 2
_{^{-SO 3 -) 2, -CH2-}} C (CH 2 -SO 3 -) (SO 3 -) -
CH ₂ —SO ₃ ^{— and the} like can be mentioned. L in the general formula (I) is the same as L ¹ or L ² .

In the general formula (II), Z¹And Z^TwoIs aromatic
A group of ring constituting atoms necessary for forming an aromatic ring is shown.
Aromatic ring is aromatic hydrocarbon ring (aryl ring) or aromatic
Includes heterocycles (heteroaryl rings). Preferably
Is Z¹And Z^TwoIs from 3 to 8 atoms, preferably 3 or 4
Group of atoms including carbon atoms, nitrogen atoms, acids
In addition to elemental and sulfur atoms, metal sources such as selenium
May include children. Z ¹And Z^TwoFormed by
An aromatic ring is a condensed aromatic ring containing two or more rings.
You may. Preferably, the aromatic ring is an aromatic hydrocarbon ring
And more preferably a benzene ring or a naphthalene ring
is there. As the aromatic hetero ring, pyrazine ring, thiophene ring
Ring and the like. In the general formula (I)
The same applies to Z.

[0023] In formula (II), an aromatic ring containing a nitrogen-containing 5-membered ring and Z ¹ containing, or Y ¹ (nitrogen-containing 5-membered ring containing or Y ²⁾ nitrogen-containing 5-membered ring containing Y ¹ (Or a fused ring system formed by a 5-membered nitrogen-containing ring containing Y ² and an aromatic ring containing Z ² ) include, for example, a thiazoline nucleus, a thiazole nucleus, and a benzothiazole nucleus An oxazoline nucleus, an oxazole nucleus, a benzoxazole nucleus, a selenazoline nucleus, a selenazole nucleus, a benzoselenazole nucleus, a 3,3-dialkylindolenine nucleus (for example, 3,3-dimethylindolenine), an imidazoline nucleus,
Imidazole nucleus, benzimidazole nucleus, 2-pyridine nucleus, 4-pyridine nucleus, 2-quinoline nucleus, 4-quinoline nucleus, 1-isoquinoline nucleus, 3-isoquinoline nucleus, imidazo [4,5-b] quinoxalin nucleus, oxadi Examples thereof include an azole nucleus, a thiadiazole nucleus, a tetrazole nucleus, and a pyrimidine nucleus. Preferred are 3,3-dialkylindolenine nucleus, benzoxazole nucleus, benzothiazole nucleus, benzimidazole nucleus and quinoline nucleus, and more preferred are 3,3-dialkylindolenine nucleus, benzoxazole nucleus and benzothiazole nucleus. In the general formula (I), the same applies to a 5-membered nitrogen-containing ring containing Y or a condensed ring system formed by a 5-membered nitrogen-containing ring containing Y and an aromatic ring formed by Z.

[0024] The 1 to 4 atoms of the atoms contained in Z ¹ each when sulfonic acid groups are present and linked three to no 1 (L ¹ is 2 or more via L ¹ L ¹ is present the same or different or may be), the 1 to 4 atoms of the atoms contained in Z ² which has from 1 to sulfonic acid 3 bound via L ² (L ² is 2 or more In this case, each L ² may be the same or different). Among the atoms contained in Z ¹ , one to four substituents represented by V may be bonded to the atoms to which L ¹ and L ² are not bonded, but V may not be present. When a plurality of V exist, they may be the same or different. L ¹
And L ² can be bonded to any atom contained in Z ¹ or Z ² . When one or more V exists,
V can be bonded to any atom contained in Z ¹ or Z ² . When Z ¹ or Z ² forms a condensed aromatic ring containing two or more rings, one or two of those rings are
L ¹ or L ² , or one or more Vs may be present on any of the above rings. The same applies to L and V in the general formula (I).

In the general formula (II), the methine groups represented by L ^{3 to} L ⁹ may have a substituent. The substituent is, for example, a substituted or unsubstituted C 1 to C 15, preferably Represents 1 to 10 carbon atoms, more preferably 1 carbon atom
To 5 alkyl groups (for example, methyl group, ethyl group, 2-
A substituted or unsubstituted aryl group having 6 to 20 carbon atoms, preferably 6 to 15 carbon atoms, more preferably 6 to 10 carbon atoms (eg, phenyl group, o-carboxyphenyl group, etc.); Alternatively, unsubstituted C3 to C20, preferably C4 to C1
5, more preferably a heterocyclic group having 6 to 10 carbon atoms (such as an N, N-diethylbarbituric acid group), a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom); 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 carbon atom
To 5 alkoxy groups (for example, methoxy group, ethoxy group, etc.), having 1 to 15 carbon atoms, preferably 1 to 1 carbon atoms.
0, more preferably an alkylthio group having 1 to 5 carbon atoms (eg, a methylthio group, an ethylthio group, etc.);
To 20, preferably an arylthio group having 6 to 15 carbon atoms, more preferably 6 to 10 carbon atoms (such as a phenylthio group), 0 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 4 to 10 carbon atoms. 10 amino groups (for example, N, N-diphenylamino group, N-methyl-N-phenylamino group, N-methylpiperazino group)
And the like. It may form a ring with another methine group, or may form a ring with Z ¹ and / or Z ² .

The group capable of reacting with an amino group, a hydroxyl group or a mercapto group to form a covalent bond includes:
For example, isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, Examples include groups containing imide ester, hydrazine, azidonitrophenyl, azide, 3- (2-pyridylthio) propionamide, glyoxal, or aldehyde. Specific examples of these groups include Japanese Patent No. 275796.
The substituents described in claim 2 of JP-A No. 5 or the following groups are mentioned. These groups can be present at any position of the compound represented by the general formula (I) or the general formula (II), and the type, the position, and the number are not particularly limited.

[0027]

Embedded image

Specific examples of the compound of the formula (I) of the present invention are shown below, but the components of the fluorescent labeling reagent of the present invention are not limited to these. The following specific examples also include specific examples of the compound represented by the general formula (II).

[0029]

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[0030]

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[0031]

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[0032]

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[0033]

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[0034]

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[0035]

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The compounds represented by the above general formulas (I) and (II) are described, for example, by FM Harmer (FM Harr).
`` Heterocyclic Compounds-Cyanine Soybeans and Relied Compounds (Heteroc
yclic Compounds-Cyanine Dyesand Related Compound
s) ", John Wiley and Sons
& Sons), New York, London, 1964, DMSturmer, Heterocyclic Compounds-Special Topics in Heterocyclic Chemistry.
mpounds--Specialtopics in heterocyclic chemistr
y) ", Chapter 18, Section 14, 482-515 Tribute, John Wiley & Sons
-New York, London, 1977, "Rod's Chemistry of Carbon Compounds (Rodd '
s Chemistry of Carbon Compounds) '' 2nd.Ed.vol.IV, pa
rtB, 1977, Chapter 15, Chapters 369-422, published by Elsevier Science Publishing Company Inc.,
It can be synthesized based on the method described in New York, JP-B-46-3700 and the like.

A substance can be labeled using the fluorescent labeling reagent of the present invention, and preferably a substance in an aqueous liquid can be labeled. This method includes a step of reacting a compound represented by the above general formula (I) or the general formula (II) with a substance in an aqueous liquid to form a covalent bond. Generally, when a compound represented by the general formula (I) or (II) having a group capable of reacting with an amino group, a hydroxy group or a mercapto group is used, the above compound and the substance to be labeled are used. May be mixed in an aqueous medium. At this time, when an acid or a base is generated, it is preferable to add a base or an acid so that the acid or the base can be neutralized, or to allow the two to react in an aqueous medium having a buffering capacity such as a pH buffer. When the compound represented by the general formula (I) or the general formula (II) has a carboxyl group, the carboxyl group is converted into a corresponding mixed acid anhydride active ester, and then the above-mentioned reactive group is used. Or a reaction with the substance to be labeled in the presence of a condensation accelerator such as a dicyclohexylcarbodiimide derivative. Examples of the reaction conditions are described in, for example, Examples 1 to 5 of Japanese Patent No. 2757965.

The labelable substance is not particularly limited. Examples of the substance include antibodies, proteins, peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, antigens, haptens, lectins, avidins, streptavidins, toxins, carbohydrates, polysaccharides, Nucleic acids, deoxynucleic acids, derived nucleic acids, derived deoxynucleic acids, DNA fragments, RNA fragments, derived DNA fragments, derived RNA fragments, natural drugs, virus particles, bacterial particles, virus components, yeast components, blood cells, blood cell components, bacteria, Bacterial components, natural or synthetic lipids, synthetic drugs, poisons, environmental pollutants, polymers, polymer particles, glass particles,
Examples include plastic particles and polymer films.

[0039]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited by these Examples. Example 1 [Preparation of slide glass] Slide glass (25 mm × 75
mm) in a solution of 50 g of sodium hydroxide in 150 mL of distilled water and 200 mL of ethanol for 1 hour, and after washing, 10% by volume of poly-L-lysine (manufactured by Sigma).
It was immersed in the aqueous solution for 1 hour. This was centrifuged with a plate centrifuge and dried at room temperature. [Preparation of DNA fragment] G cloned in advance
P (Human α2HS Glycoprotein) was amplified by PCR using cDNA as a template and the following primers to obtain a target DNA fragment. Primer 1: (5'-NH2-ACC CCC GGA AAA CAC GCA CAG
T-3 ') Primer 2: (5'-TCA GGC ACT TTC ATT AAC AGG CAT
AT-3 ') The PCR product was purified by a column method and an ethanol precipitation method and dried. Furthermore, it was dissolved in TE buffer so as to have a final concentration of 0.5 mg / mL to form a spotting solution, and spotted on the surface of the slide glass using a spotter (Cartesian Pin Array System). Thereafter, the post-treatment liquid (1-
The solution was immersed in 31.5 mL of methyl-2-pyrrolidone and 35 mL of a 1M aqueous boric acid solution in which 5 g of succinic anhydride was dissolved) for 10 minutes to block nonspecific adsorption sites. Thereafter, the post-treatment liquid was removed with distilled water and ethanol, and then dried at room temperature. The slide glass was treated at 95 ° C. for 2 minutes while immersed in water, and used for the following hybridization.

[Preparation of Fluorescently Labeled DNA Probe] 0.5 μg of human liver mRNA (Clontech) and 0.5 μg of oligo dT primer (dT18-21, Gibco BRL) were mixed, heated at 70 ° C. for 10 minutes, and then placed on ice. And quenched. To this mixture, RnaseOUT (Gibco BRL) 40U, d
ATP (500 micromol), dGTP (500 micromol), dCT
P (500 micromolar), dTTP (200 micromolar), and a conjugate (100
Micromolar), SuperScript II reverse transcriptase (Gibco BR
L) 40 U, DEPC-treated water (total volume of 20 microliters) was added and reacted at 42 ° C. for 2 hours. After the reaction,
EDTA and NaOH were added and incubated at 65 ° C. for 1 hour to stop the reaction and degrade mRNA. Centr reaction solution
It was passed through an iSep column (PRINCETON SEPARATION, INC) to remove and purify unreacted compound 6 dUTP conjugate and the like.

[Hybridization of DNA Array] A solution (final concentration: 5 × SSC, 0.5%) of the fluorescent-labeled probe prepared by the method described above on the DNA spotted slide glass
(SDS) 50 microliters was overlaid, covered with a cover glass, placed in a moisturizing chamber, and hybridized overnight in a thermostat at 60 ° C. After the reaction, slide glass is washed with washing solution 1 (mixture of 0.1% by weight SDS and 2 × SSC)
Was washed three times for 10 minutes. Further, the washing liquid 2 (0.1% by weight SDS
And 0.2 × SSC) by rinsing once for 10 minutes and twice with a washing solution 2 kept at 37 ° C. to remove SDS.
Centrifuged for 20 seconds and dried at room temperature. The fluorescence intensity on the surface of the slide glass was measured with a fluorescence scanning device using a 532 nm SHG laser beam as a light source. Table 1 shows the results.

Example 2 Hybridization was carried out in the same manner as in Example 1 except that Compound 7 was used in place of Compound 6, and measurement was carried out using a fluorescent scanning apparatus.

Example 3 (Comparative Example) Hybridization was carried out in the same manner as in Example 1 except that a dUTP conjugate prepared using Comparative Compound A was used instead of the dUTP conjugate of Compound 6 in Example 1, and a fluorescence scan was performed. It was measured with a thinning device. Comparative compound A has a sulfonic acid group directly bonded to an aromatic nucleus, and has a structure that has been conventionally considered to be excellent as a fluorescent labeling compound.
Table 1 shows the results.

[0044]

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[0045]

[Table 1]

From the results shown in Table 1, it can be seen that the fluorescent labeling reagent of the present invention has higher hybridization detection sensitivity than the compound of the comparative example. Although the fluorescent labeling reagent of the present invention does not have a sulfonic acid group directly bonded to an aromatic nucleus,
The sensitivity was superior to that of the comparative compound.

[0047]

The fluorescent labeling reagent of the present invention gives higher detection sensitivity than conventional labeling reagents, and is particularly useful for detection in DNA hybridization and the like.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G054 CA22 CE01 EA03 GA04 4B024 AA11 CA01 CA04 CA09 CA11 HA13 4B029 AA07 AA23 BB15 BB20 FA01 FA11 FA12 FA15 4B063 QA01 QA13 QA17 QA18 QA19 QQ42 QQ52 QR56 QR84QS

Claims (4)

[Claims] 1. The following general formula (I): (Wherein, Z represents a nonmetallic atom group necessary for forming an aromatic ring; R represents an alkyl group which may have a substituent or an aryl group which may have a substituent. Q represents a methine group substituted with a heterocyclic group or an aromatic group or a polymethine group substituted with a heterocyclic group or an aromatic group; L represents a linking group consisting of a group of non-metallic atoms; T represents an integer of 1 to 4; Y represents an oxygen atom, a sulfur atom, NR ¹ , or C (R ² )
(R ³ ) (wherein R ¹ , R ² , or R ³ each independently represents an alkyl group which may have a substituent or an aryl group which may have a substituent); V represents a substituent (excluding a sulfonic acid group); n represents an integer of 0 to 4; M represents a counter ion; m represents a number necessary to maintain charge balance; A compound having at least one functional group and / or carboxyl group capable of forming a covalent bond by reacting with one or more groups selected from the group consisting of a group, a hydroxyl group, and a mercapto group) Includes fluorescent labeling reagents. 2. The following general formula (II): (Wherein, Z ¹ and Z ² each independently represent a nonmetallic atom group necessary for forming an aromatic ring; R ⁴ and R ⁵ each independently represent an alkyl group which may have a substituent. Or an aryl group which may have a substituent; L ¹ and L ²
Represents a linking group comprising a group of non-metallic atoms; s1 and s2 each independently represent an integer of 1 to 3; t1 and t2
Each independently represents an integer of 1 to 4; p, q, and r each independently represent 0 or 1; Y ¹ and Y ² each independently represent an oxygen atom, a sulfur atom, NR ¹ , or C (R ² )
(R ³ ) (wherein, R ¹ , R ² and R ³ each independently represent an alkyl group which may have a substituent or an aryl group which may have a substituent); L ³ , L ⁴ , L ⁵ ,
L ⁶ , L ⁷ , L ⁸ , and L ⁹ each represent a methine group which may have a substituent; V represents a substituent (excluding a sulfonic acid group); M represents a counter ion; m represents a number necessary to maintain charge balance; provided that one or more groups selected from the group consisting of amino groups, hydroxyl groups, and mercapto groups Having at least one functional group and / or carboxyl group in the molecule capable of forming a covalent bond by reacting with). 3. A method for fluorescently labeling a substance using the compound represented by the general formula (I) according to claim 1 or the compound represented by the general formula (II) according to claim 2. 4. The method according to claim 3, wherein the substance in the aqueous medium is labeled.