JP2001163895A - Fluorescent nucleotide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new fluorescent nucleotide useful for labeling a nucleic acid. SOLUTION: This fluorescent nucleotide is represented by the formula A-B-C, wherein, A is a residue of a natural or synthetic nucleotide, an oligonucleotide, a polynucleotide or a derivative thereof, and linked to B with the base part in the residue; B is a divalent linking group or a single bond; and C is a monovalent group derived from the general formula (I) and linked to B with a reactive group present in R1 or R2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fluorescent nucleotide 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 nucleotide useful for labeling a nucleic acid.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, formed a complex with a nucleotide using an azamethine compound recently developed as a fluorescent labeling reagent. As a result of labeling and detecting the nucleic acid using the complex, it was found that the incorporation rate into the nucleic acid and the fluorescence intensity upon detection were excellent, and the present invention was completed.

That is, according to the present invention, there is provided a fluorescent nucleotide represented by the formula: ABC. [In the formula, A represents a residue of a natural or synthetic nucleotide, oligonucleotide, polynucleotide or a derivative thereof, and binds to B at a base moiety in the residue, and B represents a divalent linking group or a single bond. Wherein 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 .

Embedded image(Where R¹And R^TwoAre independently and covalently linked to B
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 B. More preferably, at least one of R ¹ and R ² is an alkyl group substituted with a carboxyl group.

Preferably, A is the residue of a nucleotide or a derivative thereof. More preferably, A is (1) AMP, ADP, ATP, GMP, GDP, GT
P, CMP, CDP, CTP, UMP, UDP UT
P, TMP, TDP, TTP, 2-Me-AMP, 2-
Me-ADP, 2-Me-ATP, 1-Me-GMP,
1-Me-GDP, 1-Me-GTP, 5-Me-CM
P, 5-Me-CDP, 5-Me-CTP, 5-MeO
A group consisting of nucleotides consisting of -CMP, 5-MeO-CDP and 5-MeO-CTP; (2) a group consisting of deoxynucleotides and dideoxynucleotides corresponding to the nucleotides; and (3) the above (1) and (2). And natural or synthetic nucleotides or derivatives thereof selected from the group consisting of derivatives further derived from the nucleotides described in 1. above.

Preferably, B is -CH _2- , -CH = C
H-, -C≡C-, -CO-, -O-, -S-, -NH
Or a linking group consisting of a combination thereof,
A hydrogen atom on the linking group is a linking group which may be further substituted with a substituent. More preferably, B is an aminoallyl group.

According to another aspect of the present invention, there is provided a method for preparing a fluorescently labeled nucleic acid, comprising performing a nucleic acid synthesis reaction using a nucleic acid synthase, a template nucleic acid and the fluorescent nucleotide of the present invention. . Preferably, the nucleic acid synthesis reaction is a reaction selected from the group consisting of a reverse transcription reaction, a terminal transferase reaction, a random prime method, a PCR method and a nick translation method.

According to still another aspect of the present invention, there is provided a nucleic acid probe or primer labeled with the fluorescent nucleotide of the present invention. According to still another aspect of the present invention, there is provided a nucleic acid detecting reagent or diagnostic agent comprising the fluorescent nucleotide of the present invention. According to still another aspect of the present invention, there is provided a kit for detecting a nucleic acid, comprising (1) the fluorescent nucleotide of the present invention, (2) a nucleic acid synthase, and (3) a buffer.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments and a method for implementing the present invention will be described in detail. The present invention has the formula: AB-
The fluorescent nucleotide represented by C. In the above formula, A
Represents a residue of a natural or synthetic nucleotide, oligonucleotide or polynucleotide or a derivative thereof. Examples of natural or synthetic nucleotides include (1) AM
P, ADP, ATP, GMP, GDP, GTP, CM
P, CDP, CTP, UMP, UDP UTP, TM
P, TDP, TTP, 2-Me-AMP, 2-Me-A
DP, 2-Me-ATP, 1-Me-GMP, 1-Me
-GDP, 1-Me-GTP, 5-Me-CMP, 5-
Me-CDP, 5-Me-CTP, 5-MeO-CM
A group consisting of nucleotides consisting of P, 5-MeO-CDP, 5-MeO-CTP; (2) a group consisting of deoxynucleotides and dideoxynucleotides corresponding to the nucleotides; and (3) the above (1) and (2)
And derivatives derived from the nucleotides described in (1), or natural or synthetic nucleotides or derivatives thereof selected from the group consisting of, but not limited to. More specific examples of natural or synthetic nucleotides include ATP, CTP, GTP, TTP, UTP, dATP, dCTP, dGT
P, dTTP, dUTP, ddATP, ddCTP, ddGTP, ddTTP, ddUTP,
Or a derivative thereof, but is not limited thereto.

Oligonucleotides include about 1 to 50 nucleotides or derivatives thereof, preferably 1 to 50 nucleotides or derivatives thereof.
It is formed by polymerizing about 30, more preferably about 1 to 20, and each nucleotide of the structural unit may be the same or different. The polynucleotide is a polymer obtained by polymerizing a large number of the above nucleotides or derivatives thereof, and the size (length) thereof is not particularly limited, and may be, for example, a number of bases ranging from several bp to several kb. As used herein, the term fluorescent nucleotide is used to include all cases where the nucleic acid component is a nucleotide, oligonucleotide and polynucleotide as described above.

A binds to B at a base moiety in a nucleotide residue. Examples of the base portion of the nucleotide residue include a purine derivative and a pyrimidine derivative. The binding site to the linking group B in the purine base is not particularly limited as long as it is other than the 9-position that binds to the sugar component. For example, when the purine base is adenine, it is present at the 2- or 8-position, or at the 6-position. When the purine base is guanine, it can be bonded to the linking group B at the 1-position or 8-position, or at the 2-position amino group. The binding site to the linking group B in the pyrimidine base is not particularly limited as long as it is other than position 1 which binds to the sugar component. For example, when the pyrimidine base is cytosine, it is located at position 5 or 6 or position 4 When the pyrimidine base is thymine, the amino group can bind to the linking group B, and when the pyrimidine base is thymine, the amino group can bind to the linking group B at a methyl group located at the 3-, 6-, or 5-position. In the case of uracil, it can bind to the linking group B at the 3-, 5- or 6-position.

In the above formula, B represents a divalent linking group or a single bond. The type of the linking group depends on the properties of the fluorescent nucleotide of the present invention (ie, the stability of the fluorescent nucleotide as a compound, water solubility, incorporation rate into nucleic acid, or fluorescence intensity)
There is no particular limitation as long as it does not significantly affect
Those skilled in the art can appropriately select a divalent linking group suitable for linking the nucleotide moiety represented by A and the fluorescent compound component represented by C. Linking group B is generally _{-CH 2 -, - CH = CH} -, - C≡C -, - CO
-, -O-, -S-, -NH- or a linking group thereof, wherein a hydrogen atom on the linking group may be further substituted with a substituent. The number of carbon atoms in the main chain of the linking group is not particularly limited, but is generally 1 to 50, preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5.

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 .

[0021]

Embedded image

Where R¹And R^TwoAre each independently B
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 any of straight-chain, branched-chain, 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. However, in order to introduce the compound of the general formula (I) as a fluorescent label into a nucleotide, the nucleotide (or a linkage bonded to the nucleotide) is used. 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 the group is introduced (in the present specification, the “reactive substituent” has the above-described characteristics. Having substituents).

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 (an isothiocyanate group, an isocyanate group, a succinimidyl ester group, a carboxyl group, or a sulfo group may be substituted). 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 diastereomer, 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.

[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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[0042]

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[0043]

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[0044]

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[0045]

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[0046]

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[0047]

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The method for producing a typical compound is specifically shown in the examples of the present specification, and 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 any compound produced by any method can be used in the present invention. The compound of the general formula (I) is disclosed in Japanese Patent Application No. 11-264845.
And Japanese Patent Application No. Hei 11-26484.
No. 5 is incorporated herein by reference in its entirety. The compound represented by the general formula (I) is used as a fluorescent label component in the fluorescent nucleotide of the present invention.

There are various known techniques for introducing the compound represented by the general formula (I) into a nucleotide as a fluorescent label, and a means available to those skilled in the art can be appropriately selected and used. . For example, a functional group such as an amino group or a hydroxyl group in a nucleotide 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 ionic bonding or covalent bonding, or The compound of general formula (I) may be reacted after chemical modification such as introduction of a linking group into a part of the nucleotide. The fluorescent nucleotide 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 nucleotide of the present invention. That is, the fluorescent nucleotide of the present invention can be used for nucleic acid detection. When the fluorescent nucleotide of the present invention is used for DNA analysis such as nucleic acid detection, for example, loose (Jerry L. Ruth, DNA,
3, 123 (1984)), the fluorescent nucleotide of the present invention can be incorporated into a probe or primer. That is, the present invention also provides a method for preparing a fluorescently labeled nucleic acid, comprising performing a nucleic acid synthesis reaction using a nucleic acid synthase, a template nucleic acid, and the fluorescent nucleotide of the present invention.

Specific examples of the nucleic acid synthase referred to in this specification include DNA polymerase (including all DNA polymerases such as Klenow enzyme and Taq DNA polymerase), R
Examples include, but are not limited to, NA polymerase, reverse transcriptase or terminal transferase. The type of template nucleic acid may be DNA or RNA, and DNA or RNA of natural origin, recombinant DNA or RN
A, Any of chemically synthesized DNA or RNA may be used. The nucleic acid synthesis reaction is carried out, for example, using a template DNA, a non-fluorescent nucleotide mixture, the fluorescent nucleotide of the present invention, and a nucleic acid synthase, under conditions suitable for the enzymatic reaction (salt concentration, pH,
Temperature, etc.). Such a nucleic acid synthesis method is well known to those skilled in the art, and the materials and reagents to be used can be appropriately selected by those skilled in the art according to the purpose of labeling and the like.

The nucleic acid (DNA or RNA) can be labeled with the fluorescent nucleotide of the present invention using various methods. The random prime method is one method for labeling DNA. A mixture of hexanucleotide sequences in any combination is used as a primer (random primer), and the random primer is hybridized to a nucleic acid to be labeled. Starting from the 3'-OH end of this random primer, a strand complementary to a single strand is
It is synthesized using a DNA polymerase such as an enow enzyme or another DNA polymerase, in which four deoxyribonucleotides which are substrates of the DNA polymerase are inserted into a complementary strand. By using the fluorescent nucleotide of the present invention as at least one of these deoxyribonucleotides, a complementary DNA labeled with the fluorescent nucleotide is synthesized.

In place of the random primer, an oligo DNA having a specific sequence (specific primer) can be used. The specific primer binds to a complementary region in the template DNA, and synthesis of a complementary DNA to the template DNA starts from the 3'-OH end of the specific primer. As with the random prime method, the complementary DNA
When the fluorescent nucleotide of the present invention is incorporated when is synthesized, a fluorescently labeled complementary DNA is synthesized.

The nick translation method is a method utilizing the action of DNase I on double-stranded DNA. D
Due to the action of Nase I, a site is cut into a single strand of the template double-stranded DNA. At the same time, Escherichia coli DNA polymerase I, four types of deoxyribonucleotides which are substrates of the enzyme, and the fluorescent nucleotide of the present invention are added to the reaction mixture. E. coli DNA polymerase I cleaves the cleaved single-stranded 5'-terminal deoxyribonucleoside and simultaneously inserts one substrate deoxyribonucleotide adjacent to the free 3'-OH terminus. By repeating this process, the cleavage site moves to the 3 'end. By including the fluorescent nucleotide of the present invention in the nucleotide of the substrate, fluorescent DNA can be synthesized using the nick translation method.

When labeling the 3 'end of double-stranded or single-stranded DNA, a terminal transferase which is an enzyme that binds deoxyribonucleotide or ribonucleotide to the 3'-OH end can be used. Terminal transferase requires at least one deoxyribonucleotide or ribonucleotide as a substrate. By using the fluorescent nucleotide of the present invention as a substrate for terminal transferase, 3′-
Fluorescently labeled nucleic acids extended at the OH terminus can be synthesized.

The reverse transcription method uses complementary D from single-stranded RNA.
This is a reaction for synthesizing NA. First, an oligodeoxyribonucleotide is annealed to a complementary portion of RNA as a primer, and then an extension reaction is performed using a reverse transcriptase, so that a DNA strand complementary to the RNA strand starts from the 3′-OH end of the primer. And synthesized. In this DNA synthesis, four types of deoxyribonucleotides are used as enzyme substrates, and the fluorescent nucleotides of the present invention are inserted into the growing DNA strand during the reverse transcription reaction by adding the fluorescent nucleotides therein,
Fluorescently labeled DNA is synthesized.

The RNA labeled with the fluorescent nucleotide of the present invention can be synthesized using an enzyme for synthesizing RNA from DNA. Enzymes for synthesizing RNA from DNA include phage-encoded RNA polymerases such as SP6, T3 or T7 RNA polymerase. These enzymes are enzymes for synthesizing double-stranded DNA and RNA containing SP6, T3 or T7 promoters, and use four types of ribonucleotides as substrates. By using the fluorescent nucleotide of the present invention as one of the substrates, a fluorescently labeled RNA can be synthesized.

Alternatively, the polymerase chain reaction (P
CR) can be used to synthesize a nucleic acid labeled with the fluorescent nucleotide of the present invention. In PCR, a nucleic acid to be detected in a biological sample is denatured into a single strand, and two kinds of primers anneal to the single-stranded nucleic acid. After annealing, a polymerase (preferably Taq DNA polymerase)
An extension reaction is performed with deoxyribonucleotides as enzyme substrates. Starting from the 3'-OH terminus of the primer, complementary DNA is synthesized to form double-stranded DNA. By repeating this step, the DNA to be detected contained in the sample can be amplified. TaqDNA
By using the fluorescent nucleotide of the present invention as one of the substrates during the extension reaction by the polymerase, a fluorescently labeled amplified nucleic acid can be obtained.

The fluorescent nucleic acid labeled with the fluorescent nucleotide of the present invention prepared as described above can be used as a gene probe for detecting a homologous nucleic acid sequence by hybridization. The hybridized fluorescent nucleotide of the target nucleic acid can be easily detected by measuring the fluorescence intensity with a fluorescence intensity meter.

As described in detail above, since the fluorescent nucleotide of the present invention can be used for labeling a gene probe, it is useful as a reagent for detecting nucleic acids or a diagnostic agent. When the fluorescent nucleotide of the present invention is used as a reagent for nucleic acid detection 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.

The fluorescent nucleotide of the present invention can also be supplied in the form of a kit for detecting a nucleic acid, together with the enzyme used in the above-described nucleic acid synthesis reaction and a buffer. The types of reagents to be included in the kit can be appropriately selected depending on the purpose of the kit. In addition to the fluorescent nucleotide, the nucleic acid synthetase, and the buffer, one or more (preferably 4
Kind), a non-fluorescent nucleotide mixture, purified water and the like. In addition, a primer such as a random primer, an oligo dT primer, or a specific primer depending on the purpose can be included in 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.

[0062]

Examples Compounds 1, 4, 6, and 6 synthesized in the following synthesis examples
7, 9, 10, 11, 12, 13, 14, 17, and 18 to 22, each of the compounds X-1, X-4, X-6, X-7, which are shown as preferred compounds herein. X-
9, X-10, X-11, X-12, X-13, X-1
4, X-17 and X-18 to X-22. Synthesis Example 1: Synthesis of Compound 1 A synthesis scheme of Compound 1 is shown below.

[0063]

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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) 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.

[0070]

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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 sulfuryl 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.

[0077]

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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 method for synthesizing 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 sulfololoxide 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.

[0086]

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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.

[0091]

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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 conducted, except that Compound 1a used in the synthesis of Compound 1 was replaced with 5- (4-methylphenyl) -7-azaindolenine. 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 procedure as the synthesis method shown for Compound 6 was used, except that Compound 6a used in the synthesis of Compound 6 was replaced with 5- (4-methylphenyl) -7-azaindolenine. 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.

[0100]

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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 Synthesis of Compound 17-dUTP Conjugate 5 mg (2.5 parts) of compound 17 was dissolved in 2 ml of 0.1 M MES buffer, and 3.66 mg (5 parts) of WSC hydrochloride and 4.20 mg of Sulfo-NHS were dissolved. (5 copies)
Was added and stirred at room temperature for 15 minutes. In addition, aminoallyl-d
2.2 mg of UTP (Sigma) was dissolved in 200 μl of 0.1 M MES and added, followed by reaction at room temperature for 2 hours. 1 M Tris buffer (pH 7.5) 10
After adding 0 μl to stop the reaction, 8 g of ODS silica (YMC-OD
It was adsorbed on a column packed with S-AQ 120A) and eluted with a 30% aqueous methanol solution. After the eluate was concentrated, it was further purified by medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-40B) to obtain the desired product with a purity of 95% (yield 71%). MS analysis value: M-1128

[0106]

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Example 2 Synthesis of Compound 14-dUTP Conjugate 5.5 mg (2.5 parts) of Compound 14 was dissolved in 200 μl of DMSO, and
3.1 mg (5 parts) of C hydrochloride and 3.55 mg (5 parts) of Sulfo-NHS were added, and the mixture was stirred at room temperature for 15 minutes. To this, 2.2 mg of aminoallyl-dUTP (Sigma) dissolved in 2 ml of 0.1 M MES was added and added at room temperature.
Allowed to react for hours. After stopping the reaction by adding 100 μl of 1 M Tris buffer (pH 7.5), 8 g of ODS silica (YMC-ODS-AQ120
The mixture was adsorbed on a column packed with A) and eluted with a 45% aqueous methanol solution. After the eluate was concentrated, it was further purified by medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-40B) to obtain the desired product with a purity of 94% (yield 66%). MS analysis value: M-1206

Example 3 Synthesis of Compound 1-dUTP Conjugate 6.00 mg (2.5 parts) of Compound 1 was dissolved in 200 μl of DMSO and
3.1 mg (5 parts) of hydrochloride and 3.55 mg (5 parts) of Sulfo-NHS were added, and the mixture was stirred at room temperature for 30 minutes. To this, 2.2 mg of aminoallyl-dUTP (Sigma) dissolved in 2 ml of 0.1 M MES was added and reacted at room temperature for 2 hours. After stopping the reaction by adding 100 μl of 1 M Tris buffer (pH 7.5), 8 g of ODS silica (YMC-ODS-AQ 120A)
Was eluted with a 50% aqueous methanol solution. After the eluate was concentrated, the eluate was further purified by medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-40B) to obtain the desired product with a purity of 92% (yield 74%). MS analysis value: M-1266

Example 4 Synthesis of Compound 10-dUTP Conjugate 3.27 mg (1.0 part) of Compound 10 was dissolved in 200 μl of DMSO, and
SC hydrochloride 0.76 mg (1.1 parts) and Sulfo-NHS 0.86 mg (1.1 parts)
Was added and stirred at room temperature for 30 minutes. To this, 2ml of 0.1M MES
2.2 mg of aminoallyl-dUTP (Sigma) dissolved in
The reaction was performed at room temperature for 2 hours. 100 μl of 1 M Tris buffer (pH 7.5)
Was added to stop the reaction, and then 8 g of ODS silica (YMC-ODS-AQ
The mixture was adsorbed on a column packed with 120A) and eluted with a 40% aqueous methanol solution. After the eluate was concentrated, it was further purified by medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-40B) to obtain the desired product with a purity of 95%. (Yield: 77%) MS analysis: M-1359

Example 5 Synthesis of Compound 19-dUTP Conjugate 1.00 mg (1.0 part) of compound 19 was dissolved in 300 μl of 0.1 M MES, and 0.44 mg (2.2 parts) of WSC hydrochloride and 0.50 mg of Sulfo-NHS ( 2.
2 parts) and stirred at room temperature for 60 minutes. Add 0.25 mg (0.
4 parts) of aminoallyl-dUTP (Sigma) was added, and 300 μl of 1M carbonate buffer (pH 9.0) was further added, followed by reaction at room temperature overnight. After stopping the reaction by adding 100 μl of 1 M Tris buffer (pH 7.5), medium pressure preparative chromatography (YAMAZ
The product was purified by EN Ultrapack ODS-S-40B) to obtain the desired product with a purity of 90%. (Yield: 62%) MS analysis: M-1513

Example 6 Synthesis of Compound 22-dUTP Conjugate 1.00 mg (1.0 parts) of compound 22 was dissolved in 300 μl of 0.1 M MES, and 0.23 mg (1.1 parts) of WSC hydrochloride and 0.26 mg of Sulfo-NHS ( 1.
1 part) and stirred at room temperature for 60 minutes. Add 0.66mg (1.
(0 parts) of aminoallyl-dUTP (Sigma), and 300 μl of 1M carbonate buffer (pH 9.0) was further added thereto, followed by reaction at room temperature overnight. After stopping the reaction by adding 100 μl of 1 M Tris buffer (pH 7.5), medium pressure preparative chromatography (YAMAZ
The product was purified by EN Ultrapack ODS-S-40B) to obtain the desired product with a purity of 96%. (Yield: 44%) MS analysis: M-1503

Use Example 1: Fluorescent Labeled DN Using Transcription Reaction
Preparation of probe A Human liver mRNA (Clontech) (0.5 μg) and oligo dT primer (dT _18-21 , Gibco BRL)
(0.5 μg), heated at 70 ° C. for 10 minutes, and quenched on ice. To this mixture, RNaseOUT
(Gibco BRL) (40 U), dATP (500 μM),
dGTP (500 μM), dCTP (500 μM), d
TTP (200 μM), compound 17-dU obtained in Example 1
TP conjugate (100 μM), SuperScript II
Reverse transcriptase (Gibco BRL) (400U), DEP
C-treated water (total amount of 20 ul) was added and reacted at 42 ° C. for 2 hours. After completion of the reaction, EDTA and NaOH
Was added and incubated at 65 ° C. for 1 hour to terminate the reaction and degrade mRNA. Centrifuge the reaction solution
riSep column (PRINCETON SEPARA
Unreacted compound 17-dUTP
The conjugate was removed and purified.

For comparison, a fluorescent nucleotide for comparison (Cy5-AP3-dUTP; Amershamp) labeled with a dye (Cy5) instead of the compound 17-dUTP conjugate was used.
reverse transcription reaction was carried out in the same manner as described above using harmacia biotech), and the reaction product was purified. Each of the purified reaction solutions was subjected to agarose gel electrophoresis, and SYBR Green II was used.
FLA after staining with (Molecular Probes)
Scanned with 2000 (Fuji Photo Film). As a result, it was found that when the compound 17-dUTP conjugate of the present invention was used, the fluorescence intensity was higher and the detection could be performed more clearly.
In addition, the fluorescence intensity was measured with a fluorometer, the amount of DNA was measured from the absorbance at 260 nm, and the incorporation rate and the fluorescence intensity per 1 μM of the probe were calculated from the results. Table 1 shows the results. As can be seen from Table 1, the uptake rate and the fluorescence intensity were higher when the compound 17-dUTP conjugate of the present invention was used.

[0114]

[Table 1]

Usage Example 2: Preparation of DNA Probe Labeled with Fluorescent Dye by PCR Method Composition of PCR Reaction Solution: Template DNA: 10 pg Primers 1 and 2: 0.5 μM each dATP, dGTP, dCTP: 200 μM each dTTP: 150 μM each compound 17-dUTP conjugate or Cy5-AP3-dUTP: 50 μM Pyrobest DNA polymerase (TAKARA): 0.5 u
nit sterilized water: 20 μl in total volume (Note: pCR-Script ^™ SK (+)
(STRATAGENE) into which α-2-HS-glycoprotein gene was incorporated, and primers 1 and 2 were used.
Are shown in Sequence Listings 1 and 2, respectively)

Using the above composition solution as a PCR reaction solution,
30 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds were performed 30 times to perform PCR reaction. The reaction solution is applied to a CentriSep column (PRINCETON).
(SEPARATION, INC) to remove unreacted fluorescent nucleotides and the like, and purify the product. The purified reaction solution was subjected to agarose gel electrophoresis, and SYBR Gr
After staining with eenII (Molecular Probes), scanning was performed with FLA2000 (Fuji Photo Film). As a result, it was found that when the compound 17-dUTP conjugate of the present invention was used, the fluorescence intensity was higher and the detection could be performed more clearly. The fluorescence intensity was measured with a fluorometer,
The DNA amount was measured from the absorbance at 0 nm, and the incorporation rate and the fluorescence intensity per 1 μM of the probe were calculated from the results. Table 2 shows the results. As can be seen from Table 2, the uptake rate and the fluorescence intensity were higher when the compound 17-dUTP conjugate of the present invention was used.

[0117]

[Table 2]

[0118]

Industrial Applicability The fluorescent nucleotide of the present invention is a novel substance, and is useful as a labeling substance for nucleic acids because of its good uptake rate during DNA synthesis and good fluorescence intensity after uptake.

[0119]

[Sequence list] SEQUENCE LISTING <110> Fuji Photo Film Co. Ltd., <120> Fluorescent Nucleotide <130> 99389M <160> 2 <210> 1 <211> 20 <212> DNA <213> Artificial DNA <400> 1 tggccgcctt caacgctcag 20 <210> 2 <211> 26 <212> DNA <213> Artificial DNA <400> 2 tcaggcactt tcattaacag gcacat 26

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masayoshi Kojima 3-11-46 Izumi, Asaka-shi, Saitama Prefecture Fujisha Shin Film Co., Ltd. Asaka Research Laboratory (72) Inventor Yukio Sudo 3--11 Izumi, Asaka-shi, Saitama No. 46 Fuji Photo Shin Film Co., Ltd., Asaka Research Laboratory (72) Inventor Junji Nishigaki 210 Nakanakanuma, Minamiashigara-shi, Kanagawa Prefecture Fuji Photo Film Co., Ltd. Ashigara Research Laboratory F-term (reference) 4B024 AA11 CA01 CA05 CA09 CA12 DA20 HA08 HA13 4B063 QA01 QQ08 QQ42 QQ52 QQ53 QR08 QR32 QR42 QR56 QR62 QR66 QS03 QS16 QS25 QS36 QX02 4C057 BB02 BB05 DD01 LL10 LL17 LL18 LL19 LL21 LL22 LL29 LL40 LL41 LL42 LL44 LL45 MM02 MM05

Claims (12)

[Claims] 1. A fluorescent nucleotide represented by the formula: ABC. Wherein A is natural or synthetic
Mature nucleotides, oligonucleotides or polynucleotides
Indicates the residue of otide or a derivative thereof;
Binds to B at a base moiety in B, and B is a divalent linking group or
C represents a monovalent group derived from the following general formula (I)
And R is¹Or R^TwoWith a reactive group present in
Are combined. Embedded image(Where R¹And R^TwoAre independently and covalently linked to B
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 B. Fluorescent nucleotide. 3. The fluorescent nucleotide according to claim 1, wherein at least one of R ¹ and R ² is an alkyl group substituted with a carboxyl group. 4. The fluorescent nucleotide according to claim 1, wherein A is a residue of a nucleotide or a derivative thereof. 5. A is (1) AMP, ADP, ATP, G
MP, GDP, GTP, CMP, CDP, CTP, UM
P, UDP UTP, TMP, TDP, TTP, 2-M
e-AMP, 2-Me-ADP, 2-Me-ATP, 1
-Me-GMP, 1-Me-GDP, 1-Me-GT
P, 5-Me-CMP, 5-Me-CDP, 5-Me-
CTP, 5-MeO-CMP, 5-MeO-CDP, 5
-A group consisting of nucleotides consisting of MeO-CTP;
(2) natural or synthetic selected from the group consisting of deoxynucleotides and dideoxynucleotides corresponding to the nucleotides, and (3) derivatives further derived from the nucleotides of (1) and (2). The fluorescent nucleotide according to any one of claims 1 to 4, which indicates a residue of the nucleotide or a derivative thereof. Wherein B is _{-CH 2 -, - CH = CH} -, - C
A linking group comprising C-, -CO-, -O-, -S-, -NH- or a combination thereof, wherein a hydrogen atom on the linking group may be further substituted with a substituent. The fluorescent nucleotide according to any one of claims 1 to 5, which is: 7. The method according to claim 1, wherein B is an aminoallyl group.
7. The fluorescent nucleotide according to any one of 6. 8. A method for preparing a fluorescently labeled nucleic acid, comprising performing a nucleic acid synthesis reaction using a nucleic acid synthase, a template nucleic acid and the fluorescent nucleotide according to any one of claims 1 to 7. 9. The nucleic acid synthesis reaction includes a reverse transcription reaction, a terminal transferase reaction, a random prime method, and a PC.
The method according to claim 8, which is a reaction selected from the group consisting of the R method and the nick translation method. A nucleic acid probe or primer labeled with the fluorescent nucleotide according to any one of claims 1 to 7. 11. A nucleic acid detecting reagent or a diagnostic agent comprising the fluorescent nucleotide according to any one of claims 1 to 7. 12. A kit for detecting a nucleic acid, comprising: (1) the fluorescent nucleotide according to any one of claims 1 to 7, (2) a nucleic acid synthase, and (3) a buffer.