JP2001163900A - Fluorescent avidin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new fluorescent avidin useful for the assay of a body composition such as a nucleic acid, a protein or a saccharide. SOLUTION: This fluorescent avidin is represented by the formula X-C, wherein, X is a residue of the avidin or streptavidin; and C is a monovalent group derived from the general formula (I), wherein, R1 and R2 are each independently an alkyl (substituted with a reactive group capable of covalently bonding to X); R3, R4, R5 and R6 are each independently an alkyl, or R3 and R4, and/or R5 and R6 may form a saturated carbon ring by bonding to each other through carbon atoms bonded thereto; V1 to V10 are each independently H or a monovalent substituent, and neighboring two groups thereof may form a ring buy bonding to each other; L1, L2 and L3 are each a (substituted) methine; m, n, s and t are each 0 or 1 with the proviso that m+n=1 and s+t=1; p is 1, 2 or 3; M is a counter ion; and q is the number required for neutralizing the electric charge of the molecule, and bonded to the B by the reactive group present in the R1 and R2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a fluorescent avidin using an azamethine compound useful as a fluorescent labeling reagent and its use.

[0002]

BACKGROUND OF THE INVENTION One of the most commonly used molecular biology methods for the detection of homologous nucleic acid sequences is DNA / DNA, R
NA / RNA or RNA / DNA hybridization. In this method, a nucleic acid (DNA or RNA) used as a probe is labeled, and the labeled nucleic acid is hybridized with a nucleic acid (DNA or RNA) to be detected. When there is homology between the nucleic acid used as the probe and the nucleic acid to be detected, each complementary single-stranded nucleic acid hybridizes to form a double strand,
The double strand is detected by labeling the probe.

Conventionally, when a nucleic acid is used as a probe, a method of labeling the probe with a radioisotope has been used, and the presence or absence of hybridization between the probe and the target nucleic acid has been detected by autoradiography. The use of radioisotopes for labeling gene probes is particularly advantageous in terms of high sensitivity, but the complexity of handling radioisotopes involves ensuring laboratory safety and disposing of radioactive compounds. Existed. In addition, since radioisotopes have a half-life, there is also a problem that they can be used only for a certain period.

[0004] For the above reasons, a non-radioactive labeling method has been developed as a simpler method. For example, a gene probe is used as a biotin molecule (EP 063 879) or a digoxigenin molecule (EP 0 324).
474 A1) is known. After hybridization between the labeled nucleic acid probe and the nucleic acid sequence to be detected, a biotin molecule or a digoxigenin molecule is present in the formed double-stranded nucleic acid. After hybridization, the nucleic acid hybridized with the probe can be detected by binding the (strept) avidin-marker enzyme complex or the anti-digoxigenin antibody-marker enzyme complex to digoxigenin. However, the detection method using such an enzyme has not been sufficient in terms of sensitivity and specificity.

[0005] In addition to the above methods, various methods for labeling a target substance with a fluorescent dye have been studied. The performance required of the fluorescent labeling reagent is (1) having a high fluorescence quantum yield, (2) having a high molecular extinction coefficient,
(3) water-soluble and does not undergo self-quenching by aggregating in an aqueous solvent; (4) being less susceptible to hydrolysis;
(5) It is difficult for photodecomposition to occur, (6) It is hard to be affected by background fluorescence, and (7) A reactive substituent which causes a covalent bond with a target substance is introduced. Fluorescein isothiocyanate (FIT) which has long been known as a fluorescent labeling reagent
C) Although rhodamine isothiocyanate has a high fluorescence quantum yield, it has a low molecular extinction coefficient and has an excitation and emission wavelength of 500 nm to 600 nm, and thus is susceptible to the background fluorescence of a membrane used for blotting, for example. There are drawbacks.

As dyes having a high molecular extinction coefficient, for example, US Pat. No. 5,486,616, JP-A-2-191.
Nos. 674, 5-287209, 5-28
No. 7266, No. 8-47400, No. 9-12
Nos. 7115, 7-145148 and 6-2
No. 22059, cyanine dyes, Journal
Polymethine dyes such as oxonol barbiturates described in of Fluorescence, 5, page 231 (1995) are known. However, these are difficult to dissolve in water and have problems such as hydrolysis when dissolved. is there. In addition, since the intermolecular interaction between the dyes is strong, aggregates are formed in the aqueous medium, so that self-quenching of fluorescence is often observed.
Further, the cyanine dyes described in JP-A-2-191677 are excellent dyes which impart water solubility by introducing a sulfonic acid group into a relatively stable chromophore and suppress the formation of aggregates. However, there are problems that the fluorescence quantum yield cannot be said to be sufficiently high, and that the introduction of a sulfonic acid group makes it difficult to synthesize a dye. Under such circumstances, in addition to the property of strong fluorescence, there has been a demand for the development of a fluorescent dye which has high water solubility, is stable and does not cause fluorescence quenching due to aggregation.

[0007] As another dye skeleton having strong fluorescence, there is an example of an azaindolenine cyanine dye described in British Patent No. 870,753.
This azaindolenine is not described because there is no description about the essential properties of the fluorescent labeling reagent, such as aggregability and aqueous solution stability, and further, there is no description of an example of introducing a reactive substituent that causes a covalent bond with a target substance. The suitability of cyanine dyes as fluorescent labeling reagents was unknown at all. In addition, Japanese Unexamined Patent Application Publication No.
JP-A-358143, JP-A-3-135553, JP-A-1-280750, and EP 341958 show examples in which azaindolenine cyanine is applied to photographic applications. It was based on the absorption characteristics of renin cyanine, and was not focused on the emission characteristics and was not actively utilized.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the prior art. That is, an object of the present invention is to provide a novel fluorescent avidin which is useful for analyzing biological components such as nucleic acids, proteins or sugars.

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have formed a conjugate with streptavidin using a recently developed azaamethine compound as a fluorescent labeling reagent, As a result of detecting a biotin-labeled nucleic acid using the conjugate, it was found that fluorescence having an intensity depending on the amount of the nucleic acid was detected, thereby completing the present invention.

That is, according to the present invention, there is provided a fluorescent avidin represented by the formula: XC. [Wherein, X represents a residue of avidin or streptavidin, C is a monovalent group derived from the following general formula (I), and R ¹ or R ²
It is bonded to B by a reactive group present therein.

Embedded image(Where R¹And R^TwoAre independently covalently linked to X
Represents an alkyl group which may be substituted with a reactive group
R^Three, R^Four, R^Five, And R⁶Are each independently alkyl
Represents a group, but R^ThreeAnd R^FourAnd / or R^FiveAnd R⁶Are connected to each other
A saturated carbocycle with the carbon atoms to which they are attached
May be formed; V¹, V^Two, V^Three, V^Four, V ^Five, V⁶, V
⁷, V⁸, V⁹, And V^TenAre each independently a hydrogen atom or
Represents a monovalent substituent, of which two adjacent groups are
L may combine with each other to form a ring;¹, L^Two, And L
^ThreeRepresents a substituted or unsubstituted methine group; m, n, s, and
And t represent 0 or 1, provided that m + n = 1 and s + t =
P is 1, 2, or 3; M is a counter ion
Represents the number required to neutralize the molecular charge
)]

Preferably, at least one of R ¹ and R ² is an alkyl group substituted with an active ester group capable of covalently bonding to an amino group, a hydroxyl group, or a thiol group in the compound to be labeled. More preferably, at least one of R ¹ and R ² is an alkyl group substituted with a carboxyl group. Preferably, X is a residue of streptavidin.

According to another aspect of the present invention, there is provided an analytical reagent or a diagnostic agent comprising the fluorescent avidin of the present invention. According to still another aspect of the present invention, there is provided an analysis kit comprising (1) the fluorescent avidin of the present invention, and (2) biotin or a biotin-labeled substance.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments and a method for implementing the present invention will be described in detail. The present invention relates to a fluorescent avidin represented by the formula: XC. In the above formula, X represents a residue of avidin or streptavidin. Avidin or streptavidin is a proteinaceous molecule with a molecular weight of about 60 kDa. The position at which X binds to C is not limited, but may be a free amino group or free carboxyl group at the end of a protein molecule or an amino group, carboxyl group, hydroxyl group, etc. existing on a constituent amino acid of a protein.
Can be combined with Alternatively, a linking group having an appropriate reactive group may be introduced in advance into avidin or streptavidin, and this may be bound to a compound of the general formula (I) described below. The term fluorescent avidin used in the present specification is used to include both cases where the component represented by X is avidin or streptavidin.

In the above formula, C is a monovalent group derived from the following general formula (I), and is bonded to B by a reactive group present in R ¹ or R ² .

[0015]

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(Wherein R¹And R^TwoAre each independently X
Optionally substituted with a reactive group capable of covalently bonding with
A kill group; R^Three, R^Four, R^Five, And R⁶Are independent
Represents an alkyl group, but R^ThreeAnd R^FourAnd / or R^FiveAnd R⁶
Are bound together and saturated with the carbon atoms to which they are attached.
V may form a sum carbocycle; V¹, V^Two, V^Three, V^Four, V
^Five, V⁶, V⁷, V⁸, V⁹, And V^TenAre each independently water
Represents an atom or a monovalent substituent;
The two groups may be joined together to form a ring;¹,
L^Two, And L^ThreeRepresents a substituted or unsubstituted methine group; m,
n, s, and t represent 0 or 1, provided that m + n = 1
S + t = 1; p represents 1, 2, or 3;
Indicates a counter ion; q is required to neutralize the molecular charge
Indicate number)

In the present specification, the alkyl moiety of the alkyl group or a substituent containing an alkyl moiety (for example, an alkoxy group) may be linear, branched, cyclic, or a combination thereof. Unless otherwise specified, the number of carbon atoms is about 1 to 20. The alkyl groups represented by R ¹ and R ² may be the same or different, for example,
Methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl, sec-butyl group, tert-butyl group, cyclopropylmethyl group, n
-Pentyl, n-hexyl, cyclohexyl and the like. The alkyl group represented by R ¹ and R ² may have one or more substituents at any position on the alkyl chain. When it has two or more substituents, they may be the same or different.

The type of the substituent on the alkyl group represented by R ¹ and R ² is not particularly limited. In order to introduce the compound of the general formula (I) as a fluorescent label into avidin or streptavidin to be labeled, It is preferable that a reactive substituent capable of forming a bond such as a covalent bond, an ionic bond, or a hydrogen bond with a substance to be labeled is introduced (in the present specification, the "reactive substituent" Means a substituent having).

Examples of the reactive substituent that can be introduced on the alkyl group represented by R ¹ and R ² include a succinimidyl ester group, a halogen-substituted triazinyl group, a halogen-substituted pyrimidinyl group, a sulfonyl halide group, an α-haloacetyl group, Maleimidyl group, aziridinyl group and the like can be mentioned. In addition to these reactive substituents, for example,
A halogen atom (a “halogen atom” in the present specification may be a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), a mercapto group, a cyano group, a nitro group, a carboxyl group, a phosphate group, Sulfo group,
A hydroxy group, an amino group, an isothiocyanate group, an isocyanate group, an alkoxy group having 1 to 8 carbon atoms (for example, methoxy group, ethoxy group, etc.),
To 20 aryloxy groups (for example, phenoxy group, naphthoxy group, etc.), an alkoxycarbonyl group having 2 to 10 carbon atoms (for example, methoxycarbonyl group, ethoxycarbonyl group), and an aryloxy group having 6 to 20 carbon atoms. A carbonyl group (eg, phenoxycarbonyl group), an acyl group having 2 to 10 carbon atoms (eg, acetyl group, pivaloyl group), an acyloxy group having 2 to 8 carbon atoms (eg, acetyloxy group, benzoyl) An oxy group), an acylamino group having 2 to 8 carbon atoms (for example, an acetylamino group, etc.),
8 sulfonyl groups (eg, methanesulfonyl group, ethanesulfonyl group, benzenesulfonyl group, etc.), sulfinyl groups having 1 to 20 carbon atoms (eg, methanesulfinyl group, ethanesulfinyl group, benzenesulfinyl group, etc.), carbon atom A sulfonylamino group having 1 to 8 carbon atoms (for example, methanesulfonylamino group, ethanesulfonylamino group, benzenesulfonylamino group, etc.); a carbamoyl group having 1 to 10 carbon atoms (for example, carbamoyl group, methylcarbamoyl group, A substituted amino group having 1 to 20 carbon atoms (eg, a methylamino group, a dimethylamino group, a benzylamino group, an anilino group, a diphenylamino group, etc.);
A sulfamoyl group having 2 to 10 carbon atoms (for example, a methylsulfamoyl group, an ethylsulfamoyl group, a piperidinosulfamoyl group, etc.), and a carbon atom number of 0 to 15
(E.g., trimethylammonium group, triethylammonium group, etc.) having 0 carbon atoms
To 15 hydrazino groups (e.g., trimethylhydrazino group, etc.), ureido groups having 1 to 15 carbon atoms (e.g., ureido groups, N, N-dimethylureido groups, etc.), and imides having 1 to 15 carbon atoms. Group (e.g., succinimide group), alkylthio group having 1 to 20 carbon atoms (e.g., methylthio group, ethylthio group, etc.), arylthio group having 6 to 20 carbon atoms (e.g., phenylthio group, p-methylphenyl) A thio group, a p-chlorophenylthio group, a 2-pyridylthio group, a naphthylthio group, etc., a substituted or unsubstituted heterocyclic group having 1 to 20 carbon atoms (for example, a pyridyl group, a 5-methylpyridyl group, a thienyl group,
Furyl, morpholino, tetrahydrofuryl, 2-
A pyrazyl group), an unsaturated hydrocarbon group having 2 to 18 carbon atoms (for example, a vinyl group, an ethynyl group, a 1-cyclohexenyl group, a benzylidine group, a benzylidene group, etc.), a substituted or unsubstituted group having 6 to 20 carbon atoms. Unsubstituted aryl group (for example, phenyl group, 4-sulfophenyl group, 2,5
-Disulfophenyl group, 4-carboxyphenyl group, naphthyl group, etc.) and an alkyl group having 1 to 20 carbon atoms (eg, methyl group, ethyl group, propyl group, etc.).

Preferred examples of R ¹ and R ² include a carboxyl group, an isothiocyanate group, a succinimidyl ester group, a sulfonyl halide group, an α-haloacetyl group,
Or an alkyl group having 1 to 15 carbon atoms substituted by a maleimidyl group, and a carboxyl group, an isothiocyanate group, a succinimidyl ester group, a sulfonyl halide group, an α-haloacetyl group, or a carbon atom having 7 to 15 carbon atoms substituted by a maleimidyl group. And 20 arylalkyl groups. More preferred examples include an alkyl group having 1 to 10 carbon atoms substituted by a carboxyl group, an isothiocyanate group, or a succinimidyl ester group.

As R ³ , R ⁴ , R ⁵ and R ⁶ , for example, an alkyl group having 1 to 20 carbon atoms can be used, and R ¹ and R ² are exemplified at arbitrary positions on the alkyl group. (Preferably excluding a reactive substituent). R ³ and R ⁴ may be connected to each other to form a saturated carbocyclic ring, or R ⁵ and R ⁶ may be connected to each other to form a saturated carbocyclic ring. As a saturated carbocyclic ring, 3
And an 8-membered ring, preferably a 5- or 6-membered ring. One or more substituents such as an alkyl group may be present on the carbocyclic ring. R ³ , R ⁴ , R ⁵ , and R ⁶
As an example, an alkyl group having preferably 1 to 6 carbon atoms can be mentioned, and more preferably 1 to 6 carbon atoms.
3 alkyl groups.

V ¹ , V ² , V ³ , V ⁴ , V ⁵ , V ⁶ , V ⁷ ,
V ⁸ , V ⁹ and V ¹⁰ each independently represent a hydrogen atom or a monovalent substituent. ^{V 1, V 2, V 3} , V 4, V 5, V 6,
The types of the substituents represented by V ⁷ , V ⁸ , V ⁹ , and V ¹⁰ are not particularly limited, and they may be the same or different. As the substituents represented by these groups, for example, those exemplified as the substituents on the alkyl group represented by R ¹ and R ² (including reactive substituents) can be used.

V ¹ , V ² , V ³ , V ⁴ , V ⁵ , V ⁶ , V ⁷ ,
Two adjacent groups among V ⁸ , V ⁹ and V ¹⁰ may be linked to each other to form a saturated or unsaturated ring. Examples of the ring formed include a 5- to 7-membered ring. The unsaturated ring may form a condensed aromatic ring. The unsaturated ring may contain a hetero atom such as an oxygen atom, a nitrogen atom and a sulfur atom. R ¹ may be located at any position on the ring to be formed.
And one or more of the substituents exemplified as the substituent on the alkyl group for R ² and R ² may be substituted.

V ¹ , V ² , V ³ , V ⁴ , V ⁵ , V ⁶ , V ⁷ ,
Preferred examples of V ⁸ , V ⁹ and V ¹⁰ include, for example, a hydrogen atom and an alkyl group having 1 to 6 carbon atoms (R ¹ and R ²
May be substituted at any position as a substituent on the alkyl group represented by (including a reactive substituent), and an aryl group having 6 to 20 carbon atoms (R ¹ and R ²
A substituent (including a reactive substituent) on the alkyl group represented by may be substituted at any position), a halogen atom, a thioalkyl group having 1 to 10 carbon atoms, a carbon atom An alkylsulfone group having 1 to 10 atoms,
Phosphoric acid group, carboxyl group, sulfo group, having 1 carbon atom
To 10 alkoxy groups, substituted amino groups, isothiocyanate groups, isocyanate groups, succinimidyl ester groups, halogen-substituted triazinyl groups, halogen-substituted pyrimidinyl groups, sulfonyl halide groups, α-haloacetyl groups, maleimidyl groups, aziridinyl groups and the like. Can be mentioned.

More preferably, a hydrogen atom, a halogen atom, or an aryl group having 6 to 20 carbon atoms (even when substituted with an isothiocyanate group, an isocyanate group, a succinimidyl ester group, a carboxyl group, or a sulfo group) Good), a sulfo group, an alkoxy group having 1 to 10 carbon atoms (which may be substituted with an isothiocyanate group, an isocyanate group, a succinimidyl ester group, a carboxyl group, or a sulfo group), 1 to 10 alkylthio groups (which may be substituted with an isothiocyanate group, an isocyanate group, a succinimidyl ester group, a carboxyl group, or a sulfo group), an isothiocyanate group, a succinimidyl ester group, a sulfonyl halide group, It is an α-haloacetyl group or a maleimidyl group.

L ¹ , L ² and L ³ each independently represent a substituted or unsubstituted methine group. The number, position and type of substituents on the methine group are not particularly limited, and when two or more substituents are present, they may be the same or different. Examples of the substituent include a substituted or unsubstituted carbon atom having 1 to 15, preferably 1 carbon atom.
Alkyl groups having 1 to 5 carbon atoms (e.g., methyl group, ethyl group, carboxyethyl group, etc.), substituted or unsubstituted carbon atoms having 6 to 30, preferably carbon atoms having 1 to 5 carbon atoms. An aryl group having 6 to 20, more preferably 6 to 15 carbon atoms (for example, phenyl group, o
-Carboxyphenyl group), substituted or unsubstituted carbon atoms having 3 to 20, preferably 4 to 15 carbon atoms,
More preferably, a heterocyclic group having 6 to 10 carbon atoms (eg, N, N-dimethylbarbituric acid group and the like),
A halogen atom having 1 to 20 carbon atoms, preferably 1 to
15, more preferably 1 to 10 alkoxy groups (eg, methoxy group, ethoxy group, etc.), 0 to 0 carbon atoms
20, preferably an amino group having 2 to 15 carbon atoms, more preferably 4 to 15 carbon atoms (eg, a methylamino group, a dimethylamino group, an N-methyl-N-phenylamino group, an N-methylpiperazino group) Etc.), an alkylthio group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms (for example, methylthio group, ethylthio group, etc.), and 6 carbon atoms. To 20, preferably 6 to 18 carbon atoms, more preferably 6 to 15 carbon atoms such as an arylthio group (e.g., phenylthio group, p-methylthio group). ^Two substituents on L ¹ , L ² and L ³ may be bonded to each other to form a ring. For example, ^two substituents on L ² and L ³ may be bonded to each other to form an alkylene chain having 2 or 3 carbon atoms to form a ring.

P represents 1, 2, or 3, and preferably represents 1 or 2. In particular, when p is 1, the semiconductor laser or helium-neon laser excitation (excitation wavelength 6
Since the excitation efficiency at 30 to 650 nm) is very high, p is most preferably 1. m, n, s, or t
Represents 0 or 1, but satisfies the conditions of m + n = 1 and s + t = 1.

M represents a counter ion, and may be either a cation or an anion. The cation may be either an inorganic cation or an organic cation, and examples thereof include alkali metal ions such as sodium ion, potassium ion and lithium ion, and organic ions such as tetraalkylammonium ion and pyridinium ion.
The anion may be either an inorganic anion or an organic anion, a halogen anion (eg, a fluorine ion, a chloride ion, a bromine ion, an iodine ion, etc.), a substituted aryl sulfonate ion (eg, a p-toluene sulfonate ion, p-chlorobenzenesulfonate ion),
Aryl disulfonate ions (e.g., 1,3-benzenedisulfonate ion, 1,5-naphthalenedisulfonic acid ion, etc.), alkyl sulfate ions (e.g., methyl sulfate ion, etc.), sulfate ion, thiocyanate ion,
Examples include perchlorate ion, tetrafluoroborate ion, picrate ion, acetate ion, and trifluoromethanesulfonic acid ion. M may be a hydrogen ion. Examples of preferred counter ions include, for example, ammonium ion, alkali metal ion, halogen anion, substituted aryl sulfonate ion, and more preferably, alkali metal ion,
Examples include a halogen anion and a substituted aryl sulfonate ion. q represents a number necessary to neutralize the charge of the molecule, but q may be 0 when the compound represented by the general formula (I) forms an intramolecular counter ion.

The compound of the general formula (I) may have one or more asymmetric carbon atoms depending on the type of the substituent, but it may be a stereoisomer such as an optical isomer or a diastereoisomer, or a stereoisomer. And racemic forms are included in the scope of the present invention. Preferred examples of the compound represented by the general formula (I) are shown below, but the scope of the present invention is not limited by the following specific compounds.

[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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[0036]

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[0037]

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[0038]

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[0039]

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[0040]

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[0041]

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Since the methods for producing the representative compounds are specifically shown in the examples of the present specification, those skilled in the art can appropriately refer to the following specific description of the examples to determine the raw material compounds, reaction conditions, reagents and the like. The above formula (I) can be selected and optionally modified or modified in the methods described in the examples.
It is possible to produce any compound encompassed by However, the method for producing the compound represented by the general formula (I) is not particularly limited, and it goes without saying that compounds produced by any method are included in the scope of the present invention. The compound of the general formula (I) is disclosed in Japanese Patent Application No. 11-264.
No. 845, which is also described in Japanese Patent Application No. 11-26.
All the contents described in the specification of No. 4845 are incorporated herein. The compound represented by the above general formula (I) is used as a fluorescent labeling component in the fluorescent avidin of the present invention.

Various methods are known for introducing the compound represented by the general formula (I) into avidin or streptavidin as a fluorescent label, and it is possible to appropriately select and use means available to those skilled in the art. It is possible. For example, a functional group such as an amino group, a carboxyl group or a hydroxyl group in avidin is directly bonded to a reactive substituent such as a carboxyl group or an active ester group in the compound of the general formula (I) by an ionic bond or a covalent bond. Alternatively, the compound of general formula (I) may be reacted after chemical modification such as introduction of a linker into a part of avidin or streptavidin. Fluorescent avidin generated after the reaction can be purified by a general-purpose separation technique such as chromatography, electrophoresis, and recrystallization.

The present invention further relates to the use of the fluorescent avidin of the present invention. That is, the fluorescent avidin of the present invention is a nucleic acid,
It can be used for detection of biological components such as proteins or sugars. When the fluorescent avidin of the present invention is used for DNA analysis such as nucleic acid detection, for example, it is preferable that biotin is previously incorporated into a probe or a primer to be detected with the fluorescent avidin of the present invention. When the fluorescent avidin of the present invention is added to a probe or primer having biotin, the two bind to each other due to the affinity between biotin and (strept) avidin to form a complex. Alternatively, primers can be detected.

The method for preparing a probe or primer labeled with biotin is well known to those skilled in the art. Specifically, a biotin-labeled nucleotide (for example, biotinylated d
UTP, etc.), random prime method, nick translation method, method using terminal transferase, reverse transcription reaction, polymerase chain reaction (PC
R) or the like, a biotin-labeled nucleic acid can be synthesized. Such a biotin-labeled nucleic acid can be used as a probe or primer to be detected with the fluorescent avidin of the present invention.

Similarly, the fluorescent avidins of the present invention can be used for protein analysis. For example, by reacting a biotin-labeled antibody with a biological sample and then reacting with the fluorescent avidin of the present invention, a complex is formed between the biotin-labeled antibody and the fluorescent avidin of the present invention. By measuring the fluorescence of the complex, the presence of the biotin-labeled antibody, that is, the presence of the target protein in the biological sample can be detected. For example, the presence of a cancer cell or cancer tissue can be proved by labeling an anti-tumor antibody with biotin, contacting the labeled antibody with a tissue or organ, and then reacting with the fluorescent avidin of the present invention. For diagnosis,
Tissue sections and the like may be immobilized by an appropriate method such as a paraffin method and observed under a microscope. Alternatively, a biological tissue may be immunochemically stained using an endoscope and observed. Recently, various fluorescent imaging methods using near-infrared fluorescent materials have been proposed (for example, see Japanese Patent Application Laid-Open No. 9-3098).
No. 45; J. Neurosurg., 87, pp. 738-745, 1997;
Medical electronics and biotechnology, 34, pp. 316-322, 1996, etc.), and the fluorescent avidin of the present invention can also be used as a diagnostic agent using a fluorescent imaging technique.

The fluorescence of the fluorescent avidin of the present invention bound to the biotin component in the substance to be detected (nucleic acid, protein or sugar, etc.) can be easily detected by measuring the fluorescence intensity with a fluorescence intensity meter. it can.

As described in detail above, the fluorescent avidin of the present invention is useful as a reagent for analyzing a biological component. When the fluorescent avidin of the present invention is used as an analytical reagent or a diagnostic agent, one or more additives can be blended and supplied in the form of a reagent composition. For example, a reagent in a desired form such as a solution can be prepared using appropriate additives such as a buffer, a solubilizer, a pH adjuster, and a preservative. The form of the reagent and the method for producing the reagent can be appropriately selected by those skilled in the art.

[0049] The fluorescent avidin of the present invention can also be supplied in the form of an analysis kit together with biotin or a biotin-labeled substance. The type of reagent to be included in the kit can be appropriately selected depending on the purpose of the kit,
In addition to the fluorescent avidin, biotin or biotin-labeled substance (such as biotin-labeled nucleotide or biotin-labeled antibody) of the present invention, primers (random primers, oligo dT primers, specific primers, etc.), nucleotide mixtures, buffers, purified water, etc. Can be combined according to the purpose of use of the kit. The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the examples. It will be apparent to those skilled in the art that the materials and methods described in the examples can be modified, improved, or substituted without departing from the spirit of the invention.

[0050]

Examples Compounds 1, 4, 6, and 6 synthesized in the following synthesis examples
7, 9, 10, 11, 12, 13, 14, and 17 are each compounds X shown as preferred compounds herein
-1, X-4, X-6, X-7, X-9, X-10, X
-11, X-12, X-13, X-14 and X-17
Corresponding to Synthesis Example 1: Synthesis of Compound 1 A synthesis scheme of Compound 1 is shown below.

[0051]

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(Synthesis of Compound 1b) Compound 1a (2.3
6 g, 10 mmol) in acetone (10 ml),
Ethyl 6-bromohexanoate (3.3 g, 15 mmol)
Was added and the mixture was heated under reflux for 5 hours. In this reaction, alkylation to a heterocyclic nitrogen atom (indole ring portion) other than the target site also proceeds, but after the reaction mixture is reacted with ethyl acetate (50 m
l) and hexane (50 ml) are added to separate the oil component produced by decantation. Ethyl acetate (10 ml) and isopropyl alcohol (5 ml) are added to the oil, and the mixture is heated under reflux for 30 minutes to obtain the desired oil. Only compound 1b, which is a pyridine ring nitrogen quaternization product, could be crystallized. Yield: 2.8 g, yield: 60% mass (posi): 379

(Synthesis of Compound 1c) Compound 1b (2.3 g,
5 mmol) was dissolved in a mixed solvent of pyridine (5 ml) and acetic acid (2 ml), and triethyl orthoformate (1 ml) and triethylamine (1 ml) were added, followed by a reaction at 140 ° C. for 1 hour. When ethyl acetate (50 ml) is added to the reaction solution, a solid containing compound 1c precipitates. This was filtered out and used for the next reaction without purification. mass (posi): 768

(Synthesis of Compound 1) The above-mentioned unpurified compound 1c
The whole amount was methanol (10 ml) and tetrahydrofuran (5
ml) and water (10 ml), 10% aqueous sodium hydroxide solution (5 ml) was added, and the mixture was reacted at room temperature for 30 minutes. Saturated aqueous sodium hydrogen carbonate solution (10 ml)
, And concentrated under reduced pressure to precipitate a crude crystal of Compound 1. Gel filtration of crude crystals (SEPHADEX LH-20)
To give Compound 1. Yield: 0.7 g, Yield: 40% (from compound 1b) mass (nega): 709 (M-Na) Maximum absorption (methanol): 634 nm Molecular absorption coefficient: 190,000

Synthesis Example 2: Synthesis of Compound 4 Compound 4 was synthesized in exactly the same manner as the synthesis method shown in Compound 1 except that Compound 1a was changed to 5- (3-methoxyphenyl) -7-azaindolenin. mass (nega): 769 (M-Na) Absorption maximum (methanol): 636 nm Molecular absorption coefficient: 190000

Synthesis Example 3: Synthesis of Compound 9 Compound 9 was synthesized in exactly the same manner as the synthesis method shown in Compound 1 except that Compound 1a was changed to 5-benzofuranyl-7-azaindolenin. mass (nega): 790 (M-Na) Absorption maximum (methanol): 658 nm Molecular absorption coefficient: 200000

Synthesis Example 4: Synthesis of Compound 6 A synthesis scheme of Compound 6 is shown below.

[0058]

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(Synthesis of Compound 6b) Compound 6a (2.7)
g, 10 mmol) was dissolved in acetone (10 ml), methyl iodide (4.3 g, 30 mmol) was added, and the mixture was heated under reflux for 2 hours. One hour after the start of the reaction, pale yellow crystals of the compound 6b which started to precipitate were added with ethyl acetate (20 ml) and collected by filtration to obtain a compound 6b. Yield: 3.2 g, yield: 80% mass (posi): 281

(Synthesis of Compound 6c) Compound 6b (4.1
g, 10 mmol), N, N'-diphenylformamidine (9.8 g, 50 mmol) and acetic anhydride (5 ml).
And reacted at 60 ° C. for 1 hour and at 100 ° C. for 3 hours. Compound 6c was precipitated when ethyl acetate (50 ml) was added to the reaction solution. Yield: 3.3 g, yield: 60% mass (posi): 426

(Synthesis of Compound 6d) Compound 6a (2.66)
g, 10 mml) in dimethylformamide (10 ml) and ethyl 6-bromohexanoate (5.9 g, 30 ml).
(mmol) was added and reacted at 120 ° C. for 1.5 hours. In this reaction, alkylation to the heterocyclic nitrogen atom (indole ring portion) other than the target site also proceeds, but after the reaction, ethyl acetate (50 ml) is added, and the resulting oil component is separated by decantation to give diethyl ether. As a result, only compound 6d, which is the target pyridine ring nitrogen quaternization product, was obtained. Yield: 2.3 g, yield: 60% mass (posi): 380

(Synthesis of Compound 6) Compound 6c (2.76)
g, 5 mmol) and compound 6d (1.9 g, 5 mmol)
l) was dissolved in dimethylformamide (20 ml), triethylamine (2 ml) was added, and the mixture was reacted at room temperature for 30 minutes. When methyl acetate (30 ml) was added to the reaction solution, crude crystals of compound 6 precipitated. The obtained crude crystals were purified by methanol recrystallization to obtain Compound 6. Yield: 1.8 g, Yield: 55% mass (posi): 671 Absorption maximum (methanol): 637 nm Molecular absorption coefficient: 190000

Synthesis Example 5: Synthesis of Compound 7 Compound 6 (0.67 g, 1 mmol) was dissolved in dimethylformamide (2 ml), and sulfoloxide oxide dimethylformamide complex (0.76 g, 5 m
mol) was added and reacted at 90 ° C. for 5 hours. When acetone (30 ml) was added to the reaction solution, a crude crystal of Compound 7 precipitated, and was collected by filtration. The crude crystals were dissolved in methanol, and potassium acetate (0.5 g) was added to carry out a salt formation reaction, whereby Compound 7 was precipitated. Yield: 0.59 g, Yield: 65% mass (nega): 868 Maximum absorption (methanol): 640 nm Molecular absorption coefficient: 190000

Synthesis Example 6: Synthesis of Compound 10 A synthesis scheme of Compound 10 is shown below.

[0065]

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(Synthesis of Compound 10b) Compound 10a (2.
76 g, 10 mml) was dissolved in fusetone (10 ml), butanesultone (2.0 g, 15 mmol) was added, and the mixture was heated under reflux for 5 hours to precipitate compound 10b. Ethyl acetate (20 ml) was added, and this was filtered. did. Yield: 3.3 g, yield: 80% mass (posi): 412

(Synthesis of Compound 10c) Compound 10b (4.
1 g, 10 mmol), N, N'-diphenylformamidine (9.8 g, 50 mmol) and acetic anhydride (5 m
1) were mixed and reacted at 60 ° C. for 1 hour and at 100 ° C. for 3 hours. When ethyl acetate (50 ml) was added to the reaction solution, compound 10c was precipitated. Yield: 2.8 g, yield: 50% mass (posi): 557

(Synthesis of Compound 10e) Compound 10e was synthesized from compound 10d according to the synthesis method of compound 6d. mass (posi): 380

(Synthesis of Compound 10) Compound 10c (2.7)
9 g, 5 mmol) and compound 6d (1.9 g, 5 mmol)
l) was dissolved in dimethylformamide (20 ml), triethylamine (2 ml) was added, and the mixture was reacted at room temperature for 30 minutes. When methyl acetate (30 ml) was added to the reaction solution, crude crystals of Compound 10 were precipitated. The obtained crude crystals were purified by recrystallization from a mixed solvent of methanol and chloroform to obtain Compound 10. Yield: 1.8 g, Yield: 45% mass (posi): 803 Absorption maximum (methanol): 646 nm Molecular absorption coefficient: 190000

Synthesis Example 7: Synthesis of Compound 11 Compound 10 (0.80 g, 1 mmol) was dissolved in dimethylformamide (2 ml), and sulfuryl oxide dimethylformamide complex (0.46 g, 3 mmol) was dissolved.
mmol) and reacted at 90 ° C. for 5 hours. When acetone (30 ml) was added to the reaction solution, crude crystals of Compound 11 were precipitated, and were collected by filtration. The crude crystals were dissolved in methanol, and potassium acetate (0.5 g) was added to perform a salt-forming reaction, whereby Compound 11 was precipitated. Yield: 0.46 g, Yield: 50% mass (nega): 882 Maximum absorption (methanol): 648 nm Molecular absorption coefficient: 190000

Synthesis Example 8: Synthesis of Compound 12 Compound 12 was synthesized according to the synthesis method of Compound 10. mass (posi): 803 Absorption maximum (methanol): 646 nm Molecular absorption coefficient: 190000

Synthesis Example 9: Synthesis of Compound 13 Compound 13 was synthesized according to the synthesis method of Compound 11. mass (posi): 882 Absorption maximum (methanol): 648 nm Molecular absorption coefficient: 190000

Synthesis Example 10: Synthesis of Compound 14 A synthesis scheme of Compound 14 is shown below.

[0074]

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(Synthesis of Compound 14b) Compound 14a (1.
9 g, 10 mmol) in acetone (10 ml),
Methyl iodide (4.3 g, 30 mmol) was added, and the mixture was heated under reflux for 2 hours. When methyl acetate (40 ml) was added to the reaction solution, the precipitated crystals were collected by filtration to obtain Compound 14b. Yield: 4.0 g, yield: 85% mass (posi): 349

(Synthesis of Chemical Compound 14c) Compound 14b (2.
4 g, 5 mmol) in pyridine (5 ml), acetic acid (2 ml).
In a mixed solvent of triethyl orthoformate (1 ml)
And triethylamine (1 ml) were added and reacted at 140 ° C. for 1 hour. When ethyl acetate (50 ml) and hexane (50 ml) are added to the reaction solution, a solid containing compound 14c precipitates. This was filtered out and used for the next reaction without purification. mass (posi): 708

(Synthesis of Compound 14) Unpurified Compound 1
4c to methanol (10 ml), tetrahydrofuran
(5 ml) and water (10 ml), 10% aqueous sodium hydroxide solution (5 ml) was added, and the mixture was reacted at room temperature for 2 hours. Saturated aqueous sodium hydrogen carbonate solution (10 ml)
, And concentrated under reduced pressure to precipitate a crude crystal of compound 14. The crude crystals were subjected to gel filtration (SEPHADEX LH-2).
Purification according to 0) gave compound 14. Yield: 0.59 g, Yield: 35% (from compound 14b) mass (nega): 650 (M-Na) Maximum absorption (methanol): 632 nm Molecular absorption coefficient: 170,000

Synthesis Example 11: Synthesis of Compound 17 A synthesis scheme of Compound 17 is shown below.

[0079]

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(Synthesis of Compound 17b) Compound 17a
(2.6 g, 16.2 mmol) in acetone (10 ml)
And dissolved in ethyl 6-bromohexanoate (3.6 g, 1
(6.2 mmol) and heated under reflux for 5 hours. In this reaction, alkylation to the heterocyclic nitrogen atom (indole ring portion) other than the target site also proceeds, but after the reaction, the oil component generated by adding ethyl acetate (50 ml) and hexane (50 ml) is decanted. Separation, ethyl acetate (10 ml) and isopropyl alcohol (5 ml) were added to the oil, and the mixture was heated under reflux for 30 minutes to crystallize only the desired pyridine ring nitrogen quaternary product, compound 17b. did it. Yield: 4.0 g, yield: 65% mass (posi): 337

(Synthesis of Compound 17c) Compound 17b (4.0
g, 10.4 mmol) in pyridine (5 ml) and acetic acid (2 m
l) in a mixed solvent, and triethyl orthoformate (2 m
l) and triethylamine (0.5 ml).
For 1 hour. Ethyl acetate (50
ml) and hexane (50 ml) precipitate a solid containing compound 17c. This was filtered out and used for the next reaction without purification. mass (posi): 615

(Synthesis of Compound 17) Unpurified Compound 1
The whole amount of 7c was dissolved in methanol (10 ml) and water (10 ml), a 10% aqueous sodium hydroxide solution (5 ml) was added, and the mixture was reacted at room temperature for 30 minutes. A saturated aqueous solution of sodium hydrogencarbonate (10 ml) was added to the reaction solution, and then methanol was concentrated under reduced pressure, whereby crude crystals of compound 17 were precipitated. The crude crystals were purified by gel filtration (SEPHADEX LH-20) to obtain Compound 17. Yield: 1.5 g, Yield: 50% (from compound 17b) H-NMR (DMSO-d6) δ; 8.90 (t, 1)
H), 8.08 (d, 2H), 7.80 (d, 2
H), 6.98 (t, 2H), 6.00 (d, 2H),
4.42 (t, 4H), 2.13 (t, 4H), 1.7
8 (m, 4H), 1.58 (m, 4H), 1.37
(S, 12H), 1.12 (m, 4H) Absorption maximum (methanol): 609 nm Molecular absorption coefficient: 120,000

Synthesis Example 12: Synthesis of Compound 18 The same operation as the synthesis method shown for Compound 1 was carried out in place of 5- (4-methylphenyl) -7-azaindolenine for Compound 1a used in the synthesis of Compound 1. Compound 18 was synthesized. mass (nega): 738 (M-Na) Absorption maximum (methanol): 636 nm Molecular absorption coefficient: 190,000

Synthesis Example 13 Synthesis of Compound 19 Compound 18 was dissolved in concentrated sulfuric acid and reacted at 100 ° C. for 30 minutes. The reaction solution was ice-cooled, adjusted to pH 8 with a 10% aqueous sodium hydroxide solution, and then concentrated under reduced pressure. The obtained residue was purified three times using Sephadex LH20 to obtain Compound 19. mass (nega): 942 (M-Na) Absorption maximum (methanol): 636 nm Molecular absorption coefficient: 190,000

Synthesis Example 14: Synthesis of Compound 20 The same operation as in the synthesis method shown for Compound 6 was carried out in place of 5- (4-methylphenyl) -7-azaindolenine for Compound 6a used in the synthesis of Compound 6. Compound 20 was synthesized. mass (nega): 639 (M-Na) Absorption maximum (methanol): 636 nm Molecular absorption coefficient: 190,000

Synthesis Example 15: Synthesis of Compound 21 Compound 20 was synthesized in the same manner as in the synthesis method of Compound 19, except that Compound 18 used in the synthesis of Compound 19 was replaced with Compound 20. mass (nega): 798 (MK) Absorption maximum (methanol): 638 nm Molecular extinction coefficient: 195000

Synthesis Example 16: Synthesis of Compound 22 A synthesis scheme of Compound 22 is shown below.

[0088]

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(Synthesis of Compound 22b) Compound 22a
(5.0 g, 19 mmol) in 47% aqueous hydrogen bromide (50
ml) and heated under reflux for 3 hours. After cooling the reaction solution to room temperature, water was added to crystallize Compound 22b. Yield: 3.8 g, yield: 80% mass (posi): 253 (M + H)

(Synthesis of Compound 22c) Compound 22b
(3.0 g, 12 mmol) was dissolved in dimethylformamide (30 ml), and 62.5% sodium hydride (0.46 g, 12 mmol) and propane sultone (1.5 g, 12 mmol) were added under ice cooling. The reaction was performed for 1 hour. Ethyl acetate and isopropyl alcohol were added to the reaction solution to crystallize compound 22c. Yield: 4.4 g, yield: 93% mass (nega): 373 (M-Na)

(Synthesis of Compound 22d) Compound 22c
(3.0 g, 7.6 mmol) was dissolved in dimethylformamide (5 ml), and ethyl 6-bromohexanoate (3.3 g, 15 mmol) was added, followed by a reaction at 120 ° C. for 6 hours. In this reaction, alkylation to a heterocyclic nitrogen atom (indole ring portion) other than the target site also proceeds,
Isopropyl alcohol (5 ml) was added to the reaction
By heating and refluxing for one minute, only the target pyridine ring nitrogen quaternization product, compound 22d, could be crystallized. Yield: 2.8 g, yield: 72% mass (nega): 488 (M-Na)

(Synthesis of Compound 22) Compound 10c (2.7)
9 g, 5 mmol) and compound 22d (2.6 g, 5 mm
ol) was dissolved in dimethylformamide (100 ml), triethylamine (2 ml) was added, and the mixture was reacted at 40 ° C for 1 hour. When methyl acetate (30 ml) was added to the reaction solution, crude crystals of compound 22 were precipitated. The obtained crude crystals were purified by recrystallization from a mixed solvent of methanol and chloroform to obtain Compound 22. Yield: 1.8 g, Yield: 40% mass (posi): 910 (M-Na) Absorption maximum (methanol): 648 nm Molecular absorption coefficient: 195000

Example 1 Streptavidin-Compound 1
Synthesis of Conjugate of Activation of Fluorescent Dye 100 μl of 24 mg / ml DMSO solution of Compound 1
31 μl of a 48 mg / ml DMSO solution of WSC (manufactured by Wako Pure Chemical Industries) and 48 mg / ml of sufoNHS (manufactured by Dojindo Chemical)
36 ml of DMSO solution was successively stirred at room temperature for 1 hour. 2. Synthesis of Streptavidin-Compound 1 Conjugate 4 mg / ml of streptavidin (manufactured by Wako Pure Chemical Industries)
83 μl of the activated form of the fluorescent dye (compound 1) prepared above was added to 300 μl of a 0 nM carbonate buffer (pH 8.3) solution, and the mixture was stirred at room temperature for 1 hour. This reaction solution was used for NAP2
5 (manufactured by Pharmacia) in 100 mM phosphate buffer (pH
In 7.5), unreacted fluorescent dye was removed by gel filtration.

Example 2 Streptavidin-Compound 1
Synthesis of Conjugate 0 Activation of Fluorescent Dye 100 μl of 24 mg / ml DMSO solution of Compound 10
29 μl of a 48 mg / ml DMSO solution of WSC (manufactured by Wako Pure Chemical Industries) and 48 mg / ml of sufoNHS (manufactured by Dojindo Chemical)
Then, 33 μl of DMSO solution was sequentially added and stirred at room temperature for 1 hour. 2. Streptavidin-Synthesis of fluorescent dye 4 mg / ml of streptavidin (manufactured by Wako Pure Chemical Industries)
83 μl of the activated form of the fluorescent dye (compound 10) prepared above was added to 300 μl of a 0 nM carbonate buffer (pH 8.3) solution.
Was added and stirred at room temperature for 1 hour. This reaction solution is
25 (manufactured by Pharmacia) in 100 mM phosphate buffer (pH
In 7.5), unreacted fluorescent dye was removed by gel filtration.

Example 3 Streptavidin-Compound 1
Synthesis of Conjugate 4 Activation of fluorescent dye 100 μl of 12 mg / ml DMSO solution of compound 14
In 16 μl of a 48 mg / ml DMSO solution of WSC (manufactured by Wako Pure Chemical), 48 mg / ml of sufoNHS (manufactured by Dojindo)
17 μl of a DMSO solution was sequentially added thereto, followed by stirring at room temperature for 1 hour. 2. Streptavidin-Synthesis of fluorescent dye 4 mg / ml of streptavidin (manufactured by Wako Pure Chemical Industries)
40 μl of the activated form of the fluorescent dye (compound 14) prepared above was added to 150 μl of a 0 nM carbonate buffer (pH 8.3) solution.
Was added and stirred at room temperature for 1 hour. This reaction solution is
25 (manufactured by Pharmacia) in 100 mM phosphate buffer (pH
In 7.5), unreacted fluorescent dye was removed by gel filtration.

Example 4 Confirmation of Streptavidin-Fluorescent Dye Conjugate Using the streptavidin-compound 14 conjugate synthesized in Example 1, the bond between streptavidin and compound 14 was confirmed as follows. . Streptavidin (2.5 μg), streptavidin-compound 14 conjugate (3.6 μg) and molecular weight marker (1.7 μg)
g) was electrophoresed by SDS-PAGE in a conventional manner. The gel was scanned with FLA2000 (Fuji Photo Film) to measure the fluorescence. As a result, about 15 kDa was observed in the lane in which the streptavidin-compound 14 conjugate was migrated.
And a fluorescent band was detected at a position of about 30 kDa. Streptavidin is a tetrameric protein composed of four subunits and is known to have a molecular weight of about 60 kDa. About 15k detected above
The Da and approximately 30 kDa bands are the monomer and 2 bands, respectively.
It is thought to correspond to the subunit of the monomer. The same gel was protein stained with Cypro Orange to detect protein. As a result, fluorescent bands were detected at about 15 kDa and about 30 kDa in the lanes of the conjugate of streptavidin and streptavidin-compound 14. From the above results, it can be seen that the conjugate of streptavidin-compound 14 having fluorescence was certainly synthesized.

Use Example 1: Various biotin-labeled PCR products encoding α-2-HS-glycoprotein (20, 200, 500, 1000, 1500 bp / 40 ng / ml each) were diluted 2- to 512-fold. 1 μl of the solution was spotted on a nylon membrane (Hybond / Amersham Pharmacia), air-dried, and
V Cross Link, DIGWash and Blo
The membrane was washed and blocked with ck Set (manufactured by Roche), impregnated with the avidin-fluorescent dye (protein concentration: 3 μg / ml) prepared in Example 1, and incubated at room temperature for 3 hours.
Left for 0 minutes. After washing with the above-mentioned washing solution, FLA20
Fluorescence was measured at 00 (manufactured by Fuji Photo Film). Figure 1 shows the results
Shown in As can be seen from the results of FIG. 1, the target nucleic acid (PC
R product) was observed. Use Example 1 confirmed that the fluorescent substance streptavidin of the present invention can detect and quantify a target substance.

[0098]

The fluorescent avidin of the present invention is a novel substance and is useful as a reagent for analyzing biological components such as nucleic acids, proteins or sugars.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of detecting biotinylated DNA using the fluorescent streptavidin of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C07D 471/04 104 C07D 471/04 104Z (72) Inventor Masayoshi Kojima 3-11 Izumi, Asaka-shi, Saitama No. 46 Fujisha Shin Film Co., Ltd., Asaka Research Laboratory (72) Inventor Yukio Sudo 3--11 Izumi, Asaka City, Saitama Prefecture Fujisha Shin Film Co., Ltd., Asaka Research Laboratory (72) Inventor Junji Nishigaki Minami Ashigara, Kanagawa Prefecture 210 Nakanaka, Fuji Photo Film, Ashigara Research Laboratories (72) Inventor Osamu Shishimoto 3-11-46, Izumi, Asaka-shi, Saitama Fujisha Shin-Film Co., Ltd. Asaka Research Laboratory F-term (reference) FB07 FB12 4B063 QA01 QA18 QQ42 QR41 QR48 QR66 QS03 QX02 4C065 AA04 BB04 CC01 DD02 EE02 HH09 JJ02 JJ09 KK02 KK06 LL01 PP10 PP11 4H045 AA10 AA30 BA51 BA70 CA40 EA50 EA65 4H056 CA01 CC02 CC08 CE03 DD03 FA05

Claims (6)

[Claims] 1. A fluorescent avidin represented by the formula: XC. Wherein X is avidin or
Represents a residue of streptavidin, and C represents the following general formula:
A monovalent group derived from (I),¹Or R^TwoDuring ~
Is bonded to B by a reactive group existing in B. Embedded image(Where R¹And R^TwoAre independently covalently linked to X
Represents an alkyl group which may be substituted with a reactive group
R^Three, R^Four, R^Five, And R⁶Are each independently alkyl
Represents a group, but R^ThreeAnd R^FourAnd / or R^FiveAnd R⁶Are connected to each other
A saturated carbocycle with the carbon atoms to which they are attached
May be formed; V¹, V^Two, V^Three, V^Four, V ^Five, V⁶, V
⁷, V⁸, V⁹, And V^TenAre each independently a hydrogen atom or
Represents a monovalent substituent, of which two adjacent groups are
L may combine with each other to form a ring;¹, L^Two, And L
^ThreeRepresents a substituted or unsubstituted methine group; m, n, s, and
And t represent 0 or 1, provided that m + n = 1 and s + t =
P is 1, 2, or 3; M is a counter ion
Represents the number required to neutralize the molecular charge
)] 2. The method according to claim ^1, wherein at least one of R ¹ and R ² is an alkyl group substituted with an active ester group capable of covalently bonding to an amino group, a hydroxyl group, or a thiol group in the compound to be labeled. The fluorescent avidin as described. 3. The fluorescent avidin according to claim 1, wherein at least one of R ¹ and R ² is an alkyl group substituted with a carboxyl group. 4. X is a residue of streptavidin,
The fluorescent avidin according to any one of claims 1 to 3. 5. An analytical reagent or diagnostic comprising the fluorescent avidin according to claim 1. Description: 6. An analysis kit comprising (1) the fluorescent avidin according to any one of claims 1 to 4, and (2) biotin or a biotin-labeled substance.