JP2003034697A - Fluorescent nucleotide and labeling method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new fluorescent nucleotide which is useful for labeling a nucleic acid. SOLUTION: The fluorescent nucleotide represented by formula X-Y-Z [wherein, X is the residue of a natural or unnatural nucleotide, oligonucleotide, polynucleotide or its derivative and combines with Y at the base part of the residue; Y is a bivalent linkage group or single bond; and Z is a monovalent group induced from a compound represented by formula (I) (wherein, R<1> to R<4> are each an alkyl group, provided that at least either R<1> or R<2> is a reactive group combining with Y, L<1> to L<7> are each methine group; A is a nonmetallic group which forms a 5- or 6-membered nitrogen-containing heterocyclic ring; M is pair ion; m is a number necessary for neutralizing the electric charge of the molecule; s, t, and u are each 0 or 1), a reactive group existing in R<1> or R<2> , and combines with Y].

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an azamethine compound useful as a fluorescent labeling reagent, a fluorescent nucleotide containing the same, and use thereof.

[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 hybridisation. 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 a probe and the nucleic acid to be detected, each complementary single-stranded nucleic acid is hybridized 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 method of using a radioisotope for labeling a gene probe is excellent in that it is particularly sensitive, but the handling of the radioisotope is accompanied by the complexity of ensuring the safety of the laboratory and the disposal of radioactive compounds. Existed. Further, since the radioisotope has a half-life, it has a problem that it can be used only for a certain period.

For the above reasons, a non-radioactive labeling method has been developed as a simpler method. For example, a biotin molecule as a gene probe (European Patent No. 63879).
Or the digoxigenin molecule (European Patent Publication No. 324474).
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 to them a (strept) avidin-marker enzyme complex or an anti-digoxigenin antibody-marker enzyme complex to digoxigenin. However, the detection method using such an enzyme is not sufficient in terms of sensitivity and specificity.

In addition to the above method, various methods for labeling a target substance with a fluorescent dye have been studied. As the performance required for the fluorescent labeling reagent, (1) it has a high fluorescence quantum yield, (2) it has a high molecular extinction coefficient,
(3) It is water-soluble and does not cause self-quenching by aggregating in an aqueous solvent, (4) It is less susceptible to hydrolysis,
(5) Photodegradation is less likely to occur, (6) Background fluorescence is less likely to be affected, and (7) Reactive substituents that cause a covalent bond with the target substance are introduced. Fluorescein isothiocyanate (FIT) has long been known as a fluorescent labeling reagent.
Although C) and rhodamine isothiocyanate have a high fluorescence quantum yield, they have a low molecular extinction coefficient and have excitation and emission wavelengths of 500 nm to 600 nm, and thus are easily affected by background fluorescence of a membrane used for blotting, for example. There are drawbacks.

Examples of dyes having a high molecular extinction coefficient include, for example, US Pat. No. 5,486,616 and JP-A-2-191.
No. 674, No. 5-287209, and No. 5-28.
No. 7266, No. 8-47400, No. 9-12
No. 7115, No. 7-145148, No. 6-2
Cyanine dye described in JP 22059, Journalo
Polymethine dyes such as oxonol barbituric acid described in f Fluorescence, 5, p. 231 (1995) are known, but they are difficult to dissolve in water, and even if they are dissolved, hydrolysis occurs. is there. In addition, due to strong intermolecular interaction between dyes, aggregates are generated in an aqueous medium,
Therefore, self-quenching of fluorescence is often observed. Further, the cyanine dyes described in JP-A-2-191674 and the like 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, it cannot be said that the fluorescence quantum yield is sufficiently high, and there is a problem that the synthesis of the dye becomes difficult due to the introduction of the sulfonic acid group. Under such circumstances, there has been a demand for the development of a fluorescent dye which is highly soluble in water, stable, and does not cause fluorescence quenching due to aggregation in addition to its strong fluorescence.

As another dye skeleton having strong fluorescence, there is an example of an azaindolenine cyanine dye described in British Patent No. 870,753, which is water-soluble,
Since there is no description about the essential properties of the fluorescent labeling reagent such as aggregating property and aqueous solution stability, and no example of introducing a reactive substituent that causes a covalent bond with the target substance is described, this azaindolenin The suitability of cyanine dyes as fluorescent labeling reagents was completely unknown. In addition, JP-A-4
No. 3,358,143, No. 3,135,553, No. 1-280750, and European Patent No. 341958 disclose examples in which azaindorenine cyanine is applied for photographic use. The absorption characteristics of renin cyanine were used, and the emission characteristics were not actively used.

[0008]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention and Problems to be Solved by the Invention An object of the present invention is to provide a novel fluorescent nucleotide useful for labeling nucleic acids, and more specifically, the above-mentioned fluorescent nucleotides. It is an object to provide a fluorescent nucleotide that can avoid the problems of the conventional techniques. As a result of repeated intensive investigations by the present inventors to solve the above-mentioned problems,
When a complex with a nucleotide is formed using a recently developed azaametin compound as a fluorescent labeling reagent and a nucleic acid is labeled and detected using the complex, the incorporation rate into the nucleic acid and the fluorescence intensity at the time of detection are excellent. Find out that
The present invention has been completed.

That is, the present invention provides a compound of the formula: XYZ [wherein X represents a residue of a natural or unnatural nucleotide, an oligonucleotide or a polynucleotide or a derivative thereof, and the base in the residue is a base. Bound to Y at a part;
Y represents a divalent linking group or a single bond; Z is the following general formula (I):

[Chemical 5] (In the formula, R ¹ , R ² , R ³ , and R ⁴ each independently represent a substituted or unsubstituted alkyl group, and R ³ and R ⁴ are linked to each other to form a saturated or unsaturated ring. However, at least one of R ¹ and R ² represents a reactive group which is bonded to Y; L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ and L ⁷ are each independently substituted. Or an unsubstituted methine group; A is 5
A non-metal atom group forming a 6-membered or 6-membered nitrogen-containing heterocycle; M is a counterion; m is the number necessary to neutralize the charge of the molecule; s is 0 or 1 ; T is 0 or 1
And u is a monovalent group derived from the compound represented by 0 or 1 and is bonded to Y by a reactive group present in R ¹ or R ² ] It provides nucleotides.

According to a preferred embodiment of the present invention, the compound represented by the general formula (I) has the following general formula (II):

[Chemical 6] (In the formula, R ¹ , R ² , R ³ , R ⁴ , L ¹ , L ² , L ³ , L ⁴ ,
L ⁵ , L ⁶ , L ⁷ , M, m, s, t, and u have the same meanings as defined in formula (I); V ¹ , V ² and V ³ are each independently a hydrogen atom or a monovalent group. Represents a substituent, V ¹
And V ² or V ² and V ³ may be linked to each other to form a saturated or unsaturated ring; W is an oxygen atom, a sulfur atom, —C
^{(R 3) (R 4)} - or ^{-N (R 5) -, (} R 3, R 4, and R ⁵ each independently represent a substituted or unsubstituted alkyl group) indicates; Q is a nitrogen atom or -C (V ⁴ )-(V ⁴ represents a hydrogen atom or a monovalent substituent, and may be bonded to V ³ to form a saturated or unsaturated ring)); (III):

[Chemical 7] (In the formula, V ¹ , R ¹ , R ² , R ³ , R ⁴ , L ¹ , L ² , L ³ ,
^{L 4, L 5, L 6} , L 7, W, M, m, s, t, and u have the same meanings as defined above, W is -N (R ⁵⁾ - or -C (R ³⁾
In the case of (R ⁴ )-, it may combine with a substituent on W to form a saturated or unsaturated ring); or the following general formula (I
V):

[Chemical 8] (In the formula, V ¹ , R ¹ , R ² , R ³ , R ⁴ , L ¹ , L ² , L ³ ,
L ⁴ , L ⁵ , L ⁶ , L ⁷ , W, M, m, s, t, and u are as defined above; E is a nitrogen atom or -C (R ⁶ ) =
(R ⁶ represents a hydrogen atom or a monovalent substituent, and R ⁶ may combine with V ¹ to form a saturated or unsaturated ring). Nucleotides are provided.

According to a further preferred embodiment, according to the invention, at least one of R ¹ and R ² is an active ester group in Y, which active group may be covalently bonded to an amino group, a hydroxyl group or a thiol group. ) The above-mentioned fluorescent nucleotide which is an alkyl group substituted with; the above-mentioned fluorescent nucleotide wherein at least one of R ¹ and R ² is an alkyl group substituted with a carboxyl group in Y; and X is a nucleotide or a derivative thereof. The fluorescent nucleotides above are provided which are residues of

In a more preferred embodiment, X is (1) A
MP, 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
P, 5-MeO-CDP, 5-MeO-CTP (Me represents a methyl group, MeO represents a methoxy group, Me or MeO
The number before the is a substitution position), a nucleotide selected from the group consisting of (2) a deoxynucleotide corresponding to the nucleotide described in (1) above and a dideoxynucleotide; and (3) the above (1) and (2) fluorescence of nucleotides or derivatives thereof selected from the group consisting of nucleotide derivatives derived from nucleotide residues which is described above according to the nucleotide; Y is -CH ₂ -, - CH = CH -, -C
A linking group consisting of ≡C-, -CO-, -O-, -S-, -NH-, or any combination of these groups (a hydrogen atom on the linking group is substituted with another substituent). And optionally Y), and Y is an aminoallyl group.

From another point of view, the present invention provides a method for producing a fluorescently labeled nucleic acid, which comprises a step of carrying out a nucleic acid synthesis reaction using a nucleic acid synthase, a template nucleic acid, and the above-mentioned fluorescent nucleotide. According to a preferred embodiment of this method, the nucleic acid synthesis reaction is one or more reactions selected from the group consisting of reverse transcription reaction, terminal transferase reaction, random prime method, PCR method, and nick translation method. Will be provided.

From still another aspect, a nucleic acid probe or primer labeled with the above-mentioned fluorescent nucleotide; a nucleic acid detection reagent comprising the above-mentioned nucleic acid probe or primer; the above-mentioned nucleic acid detection reagent for use in diagnosing a disease;
And a kit for detecting a nucleic acid, which comprises (1) the fluorescent nucleotide, (2) a nucleic acid synthase, and (3) a buffer solution.

According to the present invention, the above general formula (I)
In represented by azamethine dyes; the general formula (II) azamethine dyes (preferably W which is expressed by ^{-C (R 3) (R 4} ) -
Or an azamethine dye which is an oxygen atom, and W is-
C (R ³ ) (R ⁴ )-, wherein Y is a nitrogen atom, and the azamethine dye represented by the general formula (III) (preferably W is -C (R ³ ) (R). ⁴ ) -or a sulfur atom, wherein the azamethine dye); and the general formula (IV)
An azamethine dye represented by the formula (preferably the above azamethine dye in which W is an oxygen atom) is provided. In the azamethine dyes represented by the general formulas (II), (III) and (IV), at least one of R ¹ and R ² is an alkyl group substituted with a reactive group capable of covalently bonding to the labeled compound. An azamethine dye which is; general formulas (II), (III) and (I
In the azamethine dye represented by V), at least one of R ¹ and R ² is substituted with an active ester group (the active ester group can be covalently bonded to an amino group, a hydroxyl group or a thiol group in the compound to be labeled) The above-mentioned azamethine dye which is an alkyl group; and general formulas (II) and (II
In the azamethine dye represented by I) and (IV), R ¹
And at least one of R ² is an alkyl group substituted with a carboxyl group.

Furthermore, the above-mentioned general formula (I), general formula (II) and general formula (II) for the production of the above-mentioned fluorescent nucleotides.
I) or use of a compound represented by the general formula (IV); the above general formula (I), general formula (II), general formula (III), or general formula for producing the above-mentioned nucleic acid probe or primer. Use of a compound represented by the formula (IV); represented by the general formula (I), the general formula (II), the general formula (III), or the general formula (IV) for producing the reagent for detecting a nucleic acid. The present invention provides a method for diagnosing a disease, comprising the step of contacting a biological sample collected from a mammal such as a human with a reagent for detecting a nucleic acid as described above.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the natural or non-natural nucleotide represented by X include (1) AMP, ADP, AT
P, GMP, GDP, GTP, CMP, CDP, CT
P, UMP, UDP UTP, TMP, TDP, TT
P, 2-Me-AMP, 2-Me-ADP, 2-Me-
ATP, 1-Me-GMP, 1-Me-GDP, 1-M
e-GTP, 5-Me-CMP, 5-Me-CDP, 5
-Me-CTP, 5-MeO-CMP, 5-MeO-C
DP, a nucleotide represented by 5-MeO-CTP (Me represents a methyl group, MeO represents a methoxy group, and the number before Me or MeO represents a substitution position), (2) a deoxynucleotide corresponding to the nucleotide, And a dideoxynucleotide corresponding to the nucleotide, and (3) a derivative further derived from the nucleotide described in (1) and (2) above.
It is not limited to these. Specific examples of natural or non-natural nucleotides include, for example, ATP, CTP,
GTP, TTP, UTP, dATP, dCTP, dGT
P, dTTP, dUTP, ddATP, ddCTP, d
Examples thereof include, but are not limited to, dGTP, ddTTP, ddUTP, and derivatives thereof.

As the oligonucleotide, for example, about 1 to 50 of the above nucleotide or its derivative,
It is preferable to list about 1 to 30 and more preferably about 1 to 20 polymerized oligonucleotides, 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, but may be, for example, a number of bases ranging from several bp to several kb. The term “fluorescent nucleotide” as used herein includes all cases where the nucleic acid component is a nucleotide, an oligonucleotide and a polynucleotide as described above, and should be construed in the broadest sense.

X is bonded to Y at the base moiety in the 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 Y 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 can be bonded at the 2-position or 8-position, or can be bonded to the linking group Y by the amino group existing at the 6-position. When the purine base is guanine, it can be bonded at the 1-position or 8-position, or can be bonded to the linking group Y by the amino group existing at the 2-position. The binding site of the linking group Y in the pyrimidine base is not particularly limited as long as it is other than the 1-position that binds to the sugar component. For example, when the pyrimidine base is cytosine, it can be bonded at the 5-position or 6-position, or the amino group existing at the 4-position can be bonded to the linking group Y, and when the pyrimidine base is thymine, the 3-position Alternatively, it can be bonded at the 6-position, or it can be bonded to the linking group Y by a methyl group present at the 5-position. When the pyrimidine base is uracil, it can be bonded to the linking group Y at the 3-position, 5-position or 6-position.

In the above formula, Y represents a divalent linking group or a single bond. The type of the linking group is the property of the fluorescent nucleotide of the present invention, for example, the stability of the fluorescent nucleotide as a compound,
It is not particularly limited as long as it does not significantly affect the water solubility, the nucleic acid uptake rate, the fluorescence intensity, or the like. Those skilled in the art can appropriately select a divalent linking group suitable for linking the nucleotide moiety represented by X and the fluorescent compound moiety represented by Z. As the linking group Y, for example, -C
_{H 2 -, - CH = CH} -, - C≡C -, - CO -, - O
Examples of the linking group include-, -S-, -NH- or a combination thereof, and one or more hydrogen atoms on these linking groups may be substituted with other substituents. . 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.

Z is a monovalent group derived from the azamethine dye represented by the general formula (I), and is a reactive group present in R ¹ or R ² and is bonded to Y. The azamethine dye represented by the general formula (I) is preferably the above general formula (I
Examples include azamethine dyes represented by I), general formula (III), or general formula (IV).

In the general formula (I), A represents a non-metallic atomic group forming a 5- or 6-membered nitrogen-containing heterocyclic ring. Specifically, it is described by FM Harmer in "Heterocyclic." Compounds-Cyanine soybean and related compounds (Heterocyclic Compounds-
Cyanine Dyes and Related Compounds, John Wiley and Sons-New York, London, 1964, DMSturmer, Heterocyclic Compounds-Special Topics In Heterocyclic Compounds-Specia
l Topics in Heterocyclic Chemistry) ", Chapter 18, Chapter 1
Section 4, pp. 482-515, John Wiley and Sons-New York, London, 1977, etc.
More specifically, in the general formula (I), it is preferable that A forms an azamethine dye skeleton represented by the general formulas (II), (III) and (IV).

In the formulas (I), (II), (III) and (IV), R ¹ and R ² may be the same or different from each other, preferably an alkyl group having 1 to 20 carbon atoms. . The alkyl group may be linear, branched, cyclic, or a combination thereof (herein,
The same applies to other alkyl groups or alkyl moieties of other substituents having an alkyl moiety). For example, a methyl group, an ethyl group, an n-propyl group, a butyl group, a cyclohexyl group and the like can be mentioned. The above alkyl group may have a substituent at any position. The substituent on the alkyl group is not particularly limited, but the dye may be an antibody, protein, peptide, enzyme substrate, hormone, lymphokine,
Metabolite, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin, carbohydrate, polysaccharide, nucleic acid, deoxynucleic acid, derivative nucleic acid, derivative deoxynucleic acid, DNA fragment, RNA fragment, derivative DNA fragment, derivative RNA fragment, natural drug , Virus particles, bacterial particles, virus components, yeast components,
Covalent bond on substances including blood cells, blood cell components, bacteria, bacterial components, natural to synthetic lipids, synthetic drugs, poisons, environmental pollutants, polymers, polymer particles, glass particles, plastic particles, polymer membranes, etc. , Ionic bond,
It is preferable to introduce a reactive substituent for labeling by hydrogen bond or the like.

The type of the reactive substituent is not particularly limited, but examples thereof include a succinimidyl ester group, a halogen-substituted triazinyl group, a halogen-substituted pyrimidinyl group, a sulfonyl halide group, an α-haloacetyl group, a maleimidyl group, and an aziridinyl group. Examples of other substituents include a halogen atom (eg, fluorine, chlorine, bromine, iodine), a mercapto group, a cyano group, a nitro group, a carboxyl group, a phosphoric acid group, a sulfo group, a hydroxy group, an amino group, and isothiocyanate. Nato group, isocyanate group, alkoxy group having about 1 to 8 carbon atoms (eg, methoxy group, ethoxy group), aryloxy group having about 6 to 20 carbon atoms (eg, phenoxy group, naphthoxy group, etc.) ), An alkoxycarbonyl group having about 2 to 10 carbon atoms (eg, methoxycarbonyl group, ethoxycarbonyl group), an aryloxycarbonyl group having about 6 to 20 carbon atoms (eg, phenoxycarbonyl group), the number of carbon atoms Has about 2 to 10 acyl groups (eg, acetyl group, pivaloyl group, etc.), carbon atoms Having about 2 to 8 acyloxy groups (eg, acetyloxy group, benzoyloxy group, etc.), acylamino groups having about 2 to 8 carbon atoms (eg, acetylamino group), sulfonyl having about 1 to 8 carbon atoms A group (for example, a methanesulfonyl group, an ethanesulfonyl group, a benzenesulfonyl group, etc.), a sulfinyl group having about 1 to 20 carbon atoms (for example, a methanesulfinyl group, an ethanesulfinyl group, a benzenesulfinyl group), a carbon atom number A sulfonylamino group having about 1 to 8 (eg, methanesulfonylamino group, ethanesulfonylamino group, benzenesulfonylamino group, etc.), a carbamoyl group having about 1 to 10 carbon atoms (eg, carbamoyl group, methylcarbamoyl group, molybdenum group). Holinocarbamoyl group, etc.), carbon Child number from 1 20 about a substituted amino group (e.g., methylamino group, dimethylamino group, benzylamino group, anilino group, such as a diphenylamino group),

A sulfamoyl group having about 2 to 10 carbon atoms (eg, methylsulfamoyl group, ethylsulfamoyl group, piperidinosulfamoyl group, etc.), an ammonium group having about 0 to 15 carbon atoms (eg, Trimethylammonium group, triethylammonium group, etc.), hydrazino group having 0 to 15 carbon atoms (eg, trimethylhydrazino group), 1 carbon atom
To about 15 ureido groups (eg, ureido groups, N, N-
Dimethylureido group, etc.), imide group having 1 to 15 carbon atoms (eg, succinimide group), alkylthio group having 1 to 20 carbon atoms (eg, methylthio group, ethylthio group, etc.), carbon atom number 6 to 2
0 arylthio group (eg, phenylthio group, p-methylphenylthio group, p-chlorophenylthio group, 2-pyridylthio group, naphthylthio group, etc.), substituted or unsubstituted heterocyclic group having about 1 to 20 carbon atoms (For example, pyridyl group, 5-methylpyridyl group, thienyl group, furyl group, morpholino group, tetrahydrofuryl group, 2-pyrazyl group, etc.), unsaturated hydrocarbon group having 2 to 18 carbon atoms (for example, vinyl Group, ethynyl group, 1-cyclohexenyl group, benzylidine group, benzylidene group, etc.),
An aryl group having about 6 to 20 carbon atoms (eg, phenyl group, 4-sulfophenyl group, 2,5-disulfophenyl group, 4-carboxyphenyl group, naphthyl group, etc.), having 1 to 20 carbon atoms. Some alkyl groups (eg, methyl, ethyl, propyl, etc.) are included.

As R ¹ and R ² , a carboxyl group, an isothiocyanate group, a succinimidyl ester group, a sulfonyl halide group, an α-haloacetyl group, a maleimidyl group, a sulfonic acid group or a salt thereof is substituted with 1 to 10 carbon atoms. Is more preferable, and a carboxyl group, an isothiocyanate group, a succinimidyl ester group, an alkyl group having 1 to 6 carbon atoms substituted with a sulfonic acid group or a salt thereof, or a sulfo group, a carboxyl group or isothiocyanate 7 to 2 carbon atoms substituted with a nato group, a succinimidyl ester group, a sulfonyl halide group, an α-haloacetyl group, or a maleimidyl group.
It is an arylalkyl group of 0.

General formulas (I), (II), (III) and (I
In V), R ³ , R ⁴ and R ⁵ are preferably alkyl groups having 1 to 20 carbon atoms and have the substituents exemplified for R ¹ and R ² at arbitrary positions on the alkyl group. May be. R ³ and R ⁴ may be linked to each other to form a saturated carbocycle. As R ³ , R ⁴ and R ⁵ , an alkyl group having 1 to 6 carbon atoms is preferable, and an alkyl group having 1 to 3 carbon atoms is more preferable.

V ¹ , V ² and V ³ in the general formulas (II), (III) and (IV) are the same or different and each represents a hydrogen atom or a substituent. The substituents represented by V ¹ , V ² and V ³ are not particularly limited, and examples thereof include those exemplified as the substituents on the alkyl group for R ¹ and R ² . In the general formula (II), V ¹ and V ² , or V ² and V ³ may be connected to each other to form, for example, a 5-membered, 6-membered or 7-membered saturated or unsaturated ring. The unsaturated ring may contain one or more arbitrary heteroatoms such as oxygen atom, nitrogen atom, or sulfur atom. One or two substituents exemplified as the substituents on the alkyl group for R ¹ and R ² (hereinafter, these substituents are referred to as “exemplary substituents”) are formed at arbitrary positions of the ring formed. The above may be replaced.

As V ¹ , V ² and V ³ , an alkyl group having 1 to 6 carbon atoms (one or more exemplary substituents may be present at any position on the alkyl group), An aryl group having 6 to 20 carbon atoms (one or more exemplary substituents may be present at any position on the aryl group),
Halogen atom, thioalkyl group having 1 to 10 carbon atoms, alkylsulfone group having 1 to 10 carbon atoms, sulfonic acid group,
Phosphonic acid group, carboxyl group, alkoxy group having 1 to 10 carbon atoms, cyano group, substituted amino group, isothiocyanate group, isocyanate group, succinimidyl ester group, halogen-substituted triazinyl group, halogen-substituted pyrimidinyl group, sulfonyl halide group , Α-haloacetyl group, maleimidyl group, and aziridinyl group. As V ¹ , V ² and V ³ , more preferably a halogen atom, a carboxyl group, a sulfonic acid group or a phosphonic acid group, and a carbon atom having 6 substituted with a halogen atom, a carboxyl group, a sulfonic acid group or a phosphonic acid group. To 20 aryl groups, 1 to 10 carbon atoms substituted with a carboxyl group or a sulfo group, 1 to 1 carbon atoms substituted with a carboxyl group, a sulfonic acid group, or a phosphonic acid group
There may be mentioned 0 alkylthio groups.

General formulas (I), (II), (III) and (I
L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ and L ⁷ in V) each independently represent a substituted or unsubstituted methine group. Examples of the substituent include a substituted or unsubstituted C1 to C1
5, preferably 1 to 10 carbon atoms, particularly preferably 1 to 5 carbon alkyl groups (eg methyl groups,
Ethyl group, carboxyethyl group, etc.), a substituted or unsubstituted aryl group having 6 to 30 carbon atoms, preferably 6 to 20 carbon atoms, and more preferably 6 to 15 carbon atoms (eg, phenyl group, o- A carboxyphenyl group), a substituted or unsubstituted heterocyclic group having 3 to 20 carbon atoms, preferably 4 to 15 carbon atoms, and more preferably 6 to 10 carbon atoms (eg, N, N-dimethylbarbitur). Acid groups, etc.), halogen atoms (eg chlorine, bromine, iodine, fluorine etc.), alkoxy groups having 1 to 20 carbon atoms, preferably 1 to 15 carbon atoms, more preferably 1 to 10 carbon atoms (eg methoxy group, ethoxy group). Such),
0 to 20 carbon atoms, preferably 2 to 1 carbon atoms
5, more preferably an amino group having 4 to 15 carbon atoms (eg, methylamino group, dimethylamino group, N-methyl-N-phenylamino group, N-methylpiperazino group, etc.), 1 to 15 carbon atoms, preferably Has 1 carbon atom
To 10, more preferably an alkylthio group having 1 to 8 carbon atoms (eg, methylthio group, ethylthio group, etc.),
6 to 20 carbon atoms, preferably 6 to 1 carbon atoms
8, more preferably an arylthio group having 6 to 15 carbon atoms (eg, phenylthio group, p-methylthio group, etc.) and the like. s, t, and u each independently represent 0 or 1. Preferred is when s is 0 and t and u are 1, or when s and t are 0 and u is 1.

M represents a counter ion. M may be a cation or an anion, and examples of the cation 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, fluorine ion, chlorine ion, bromide ion, iodide ion, etc.), a substituted aryl sulfonate ion (eg, p-toluene sulfone). Acid ion, p-chlorobenzenesulfonate ion, etc.), aryldisulfonate ion (eg, 1,3-benzenedisulfonate ion, 1,5-naphthalenedisulfonate ion, etc.), alkylsulfate ion (eg, methylsulfate ion, etc.) ), Sulfate ion, thiocyanate ion, perchlorate ion, tetrafluoroborate ion, picrate ion, acetate ion, trifluoromethanesulfonate ion and the like. Further, M may be hydrogen ion. Examples of the cation include ammonium ion, alkali metal ion, halogen anion, and substituted aryl sulfonate ion, and more preferable examples include alkali metal ion, halogen anion, and substituted aryl sulfonate ion. m represents the number required to neutralize the charge of the molecule.

In the general formula (II), W is oxygen source.
Child, sulfur atom, or -C (R³) (R^Four)-Is preferred, oxygen
Atom and -C (R³) (R^Four)-Is more preferred. General formula
In (III), W is a sulfur atom or -C (R³)
(R^Four)-Is preferred, and -C (R³) (R ^Four)-Is more preferred
Yes. In formula (IV), W is an oxygen atom or-
C (R³) (R^Four)-Is preferable, and an oxygen atom is more preferable.
E in the general formula (IV) is a nitrogen atom or -C (R⁶) =
Represent, R⁶Represents a hydrogen atom or a substituent. R⁶Represented by
Examples of the substituent include the above-mentioned "exemplary substituents". R
⁶Is V¹When connected to form a saturated or unsaturated ring
Is preferred. More preferably, the following general formula (V)
As shown, when a benzisoxazole ring is formed,
It is the case.

[0033]

[Chemical 9] (R ¹ , R ² , R ³ , R ⁴ , L ¹ , L ² , L ³ , L ⁴ , L ⁵ ,
L ⁶ , L ⁷ , M, m, s, t, and u have the same meanings as defined above, and examples of V ¹¹ , V ¹² , V ¹³ , and V ¹⁴ include those exemplified for V ¹ to V ³ . The preferred embodiment is also the same as V ¹ to V ³ . )

The compound represented by the general formula (I) may have one or more asymmetric carbon atoms depending on the kind of the substituent,
Any of stereoisomers such as optical isomers and diastereomers, mixtures of stereoisomers, racemates and the like are included in the scope of the present invention. In addition, the compound represented by the general formula (I) may form a hydrate or a solvate,
All of these substances are included in the scope of the present invention. The same applies to the compounds represented by the general formulas (II), (III), (IV), and (V).

The above general formulas (I), (II), (III) and (I
Preferred examples of the compound represented by V) are shown below, but the scope of the present invention is not limited by the following specific compounds.

[0036]

[Chemical 10]

[0037]

[Chemical 11]

[0038]

[Chemical 12]

[0039]

[Chemical 13]

[0040]

[Chemical 14]

[0041]

[Chemical 15]

[0042]

[Chemical 16]

[0043]

[Chemical 17]

Since the method for producing a representative compound is specifically shown in the examples of the present specification, those skilled in the art can appropriately refer to the starting compounds, reaction conditions, reagents and the like while referring to the detailed description of the examples below. Any compound included in the above general formulas (I), (II), (III) and (IV) is produced by selecting and, if necessary, modifying or altering the method described in Examples. It is possible. However, it goes without saying that the method for producing the compounds represented by the above-mentioned general formulas I), (II), (III) and (IV) is not particularly limited, and those produced by any method can be used in the present invention.

The compounds represented by the above general formulas (I), (II), (III) and (IV) are used as a fluorescent labeling component in the fluorescent nucleotide of the present invention. There are various known methods for introducing a compound represented by the general formula (I), (II), (III) or (IV) into a nucleotide as a fluorescent label, and any means available to those skilled in the art can be appropriately selected. It is possible to use. For example, functional groups such as amino groups and hydroxyl groups in nucleotides and general formulas (I), (II),
Reactive substituents such as carboxyl group and active ester group in the compounds represented by (III) and (IV) are directly bonded by ionic bond or covalent bond, or a linking group is introduced into a part of nucleotide. After the chemical modification such as the reaction, the compounds of the general formulas (I), (II), (III) and (IV) may be reacted. The fluorescent nucleotide produced after the reaction can be purified by a general-purpose separation technique such as chromatography, electrophoresis, recrystallization and the like.

The present invention further relates to the use of the fluorescent nucleotides of the present invention. That is, the fluorescent nucleotide of the present invention can be used for detecting nucleic acid. When the fluorescent nucleotide of the present invention is used for DNA analysis such as detection of nucleic acid, it can be prepared, for example, by Ruth (Jerry L. Ruth, DN
The fluorescent nucleotide of the present invention can be incorporated into a probe or primer by the method described in A, 3, 123 (1984)). That is, the present invention provides a method for preparing a fluorescently labeled nucleic acid, which comprises a step of performing a nucleic acid synthesis reaction using a nucleic acid synthase, a template nucleic acid, and the fluorescent nucleotide of the present invention.

Examples of the nucleic acid synthase used in the method of the present invention include DNA polymerase (including any DNA polymerase such as Klenow enzyme and Taq DNA polymerase), RNA polymerase, reverse transcriptase or terminal transferase. It is not limited to these. The type of template nucleic acid is not particularly limited and may be DNA or RNA. In addition to naturally occurring DNA or RNA, recombinant DNA or RN
A or non-natural D such as chemically synthesized DNA or RNA
It may be NA or RNA (the same applies when referring to “nucleic acid” herein). The nucleic acid synthesis reaction uses, for example, a template DNA, a non-fluorescent nucleotide mixture, the fluorescent nucleotide of the present invention, and a nucleic acid synthetase under conditions suitable for the enzymatic reaction (including conditions such as salt concentration, pH, and temperature). Can be done in. Such nucleic acid synthesis method is well known to those skilled in the art, and depending on the purpose of labeling, etc.,
Those skilled in the art can appropriately select materials and reagents to be used.

Nucleic acids can be labeled by various means using the fluorescent nucleotides of the present invention. The random prime method is one method for labeling DNA, and includes a step of using a mixture of hexanucleotide sequences in any combination as a primer (random primer), and hybridizing the random primer to a nucleic acid to be labeled. . 3'-O of this random primer
Starting from the H-terminus, a strand complementary to the single strand is synthesized using a DNA polymerase such as Klenow enzyme or another DNA polymerase. At that time, four types of deoxyribonucleotides, which are substrates for the DNA polymerase, are contained in the complementary strand. Inserted in. By using the fluorescent nucleotide of the present invention as at least one of these deoxyribonucleotides, complementary DNA labeled with a fluorescent nucleotide is synthesized.

Instead of the random primer, 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 DNA complementary to the template DNA is initiated from the 3'-OH end of the specific primer. As in the case of the random prime method, when the complementary DNA is synthesized, the fluorescent nucleotide of the present invention is incorporated to synthesize the fluorescently labeled complementary DNA.

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 where the template double-stranded DNA is cleaved into a single strand is generated. At the same time, Escherichia coli DNA polymerase I, four types of deoxyribonucleotides that are substrates for this 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 deoxyribonucleotides or ribonucleotides to the 3'-OH end can be used. Terminal transferases require at least one deoxyribonucleotide or ribonucleotide as a substrate. By using the fluorescent nucleotide of the present invention as a substrate for terminal transferase, it is possible to synthesize a fluorescently labeled nucleic acid extended at the 3'-OH end.

Reverse transcription is performed by converting a single-stranded RNA to a complementary D
It is a reaction for synthesizing NA. First, an oligodeoxyribonucleotide as a primer is annealed to a complementary portion of RNA, and then an extension reaction is carried out using a reverse transcriptase, whereby a DNA strand complementary to the RNA strand starts from the 3'-OH end of the primer. And then synthesized. This D
Also in NA synthesis, four kinds of deoxyribonucleotides are used as enzyme substrates, and by adding the fluorescent nucleotide of the present invention thereto, the fluorescent nucleotide is inserted into the DNA chain which is being extended during the reverse transcription reaction, so that Labeled DNA is synthesized.

An enzyme that synthesizes RNA from DNA can also be used to synthesize RNA labeled with the fluorescent nucleotide of the present invention. Examples of enzymes that synthesize RNA from DNA include RNA polymerases encoded by phages such as SP6, T3 or T7 RNA polymerases. These enzymes are enzymes for synthesizing double-stranded DNA and RNA containing SP6, T3 or T7 promoters, and four kinds of ribonucleotides are used as substrates. Fluorescently labeled RNA can be synthesized by using the fluorescent nucleotide of the present invention as one of the substrates.

The polymerase chain reaction (PCR) can also be used to synthesize nucleic acids labeled with the fluorescent nucleotides of the present invention. In PCR, the nucleic acid to be detected in a biological sample is denatured into a single strand and two primers anneal to this single stranded nucleic acid. After annealing, an extension reaction is carried out with a polymerase (preferably Taq DNA polymerase) and deoxyribonucleotide as an enzyme substrate. Complementary DNA starting from the 3'-OH end of the primer
Are synthesized to form double-stranded DNA. By repeating this process, the DNA to be detected contained in the sample
Can be amplified. By using the fluorescent nucleotide of the present invention as one of the substrates in the extension reaction with Taq DNA 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 fluorescent nucleotide hybridized with the target nucleic acid can be easily detected by measuring the fluorescence intensity with a fluorescence intensity meter.

The fluorescent nucleotide of the present invention can be used for labeling a gene probe, and the probe is useful as a reagent for nucleic acid detection, especially as a diagnostic agent for diagnosing diseases of mammals including humans. When the probe labeled with the fluorescent nucleotide of the present invention is used as a reagent or a diagnostic agent for nucleic acid detection, one or more additives can be mixed and supplied in the form of a reagent composition. For example, a reagent in a desired form such as a solution can be prepared by using an appropriate additive such as a buffering agent, a solubilizing agent, a pH adjusting agent and a preservative. Those skilled in the art can appropriately select the form of the reagent and the method for producing the reagent. Although the above-mentioned diagnostic agent can be orally or parenterally administered to mammals including humans, biological samples such as blood, urine and saliva separated and collected from mammalian animals including humans can be used as the diagnostic agent. By bringing them into contact with each other, it becomes possible to diagnose a disease associated with a genetic abnormality.

The fluorescent nucleotide of the present invention can also be supplied in the form of a nucleic acid detection kit together with the enzyme used for the nucleic acid synthesis reaction, the buffer solution and the like.
The types of reagents to be included in the kit can be appropriately selected according to the purpose of the kit. In addition to the fluorescent nucleotide, the nucleic acid synthase, and the buffer, one or more (preferably four) Fluorescent nucleotide mixture,
Purified water and the like may be included in the kit. Primers such as random primers, oligo dT primers, or specific primers depending on the purpose can be included in the kit.

[0058]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. Synthesis Example 1: Synthesis of Compound I-1 Compound I-1 was synthesized by the following synthetic route.

[0059]

[Chemical 18]

(Synthesis of Compound I-1c) Compound I-1d
(2.4 g, 10 mmol) was dissolved in DMF (5 ml), ethyl 6-bromohexanoate (2.4 g, 10.8 mmol) was added, and the mixture was reacted at 130 ° C. for 3 hours. The oil component produced by adding ethyl acetate (50 ml) to the reaction solution was purified by silica gel column chromatography to obtain Compound I-1c. Yield: 2.6g, Yield: 65% mass (posi): 397

(Synthesis of Compound I-1b (anil Form)) Compound I-1e (1.6 g, 10 mmol) was dissolved in dimethylformamide (5 ml), and propane sultone (1.2 g, 10 mmo).
l) was added and reacted at 100 ° C. for 2 hours. Ethyl acetate (50 ml) was added to the reaction solution, and the obtained solid (2.8 g) was taken out by filtration. Acetic anhydride (10 ml) and N,
N'-diphenylformamidine (3.9 g, 20 mmol) was added and reacted at 100 ° C for 30 minutes. Ethyl acetate (50 ml) and hexane (50 ml) were added to the reaction solution after the reaction to separate the resulting oil component by decantation, and compound I-1b (anil derivative) was isolated by silica gel column chromatography. Yield: 1.5g, Yield: 40% mass (posi): 385

(Synthesis of Compound I-1a) Compound I-1b
(0.39 g, 1 mmol) and compound I-1c (0.40 g, 1 mmol)
It was dissolved in DMF (5 ml), triethylamine (0.5 ml) and acetic anhydride (0.5 ml) were added, and the mixture was reacted at 60 ° C for 1 hr. When ethyl acetate (50 ml) was added to the reaction solution, crystals were precipitated. The crude crystals were purified by gel filtration (SEPHADEX LH-20) to obtain the compound compound I-1a. mass (nega): 688

(Synthesis of Compound I-1) The Compound I-1a
The whole amount was dissolved in methanol (10 ml), 5% aqueous lithium hydroxide solution (5 ml) was added, and the mixture was reacted at room temperature for 2 hours. Aqueous potassium acetate solution (10 ml) was added to the reaction solution, and then methanol was concentrated under reduced pressure to precipitate crude crystals of compound I-1. The crude crystals were desalted by gel filtration (SEPHADEX LH-20) to obtain compound I-1. Yield: 0.17 g, yield: 25% (from compound I-1b) mass (nega): 661

Synthesis Example 2: Synthesis of compound I-2 Compound I-2 was synthesized according to the method of synthesizing compound I-1.

Synthesis Example 3: Synthesis of compound I-3 Compound I-1 (0.70 g, 1 mmol) was dissolved in DMF (5 ml), and pyridine (0.5 ml) and N, N'-disuccinimidyl carbonate were dissolved. (1.0 g) was added and the mixture was reacted at 40 ° C for 4 hours. Ethyl acetate (50 ml) was added to the reaction solution, and the resulting oil was separated by decantation. The oil was crystallized from diethyl ether to give compound I-3. Yield: 0.52g, Yield: 65% mass (nega): 758

Synthesis Example 4: Synthesis of compound I-4 and compound I-5 Synthesis was carried out according to the method of compound I-3. Table 1 shows the absorption characteristics in methanol.

[0067]

[Table 1]

Synthesis Example 5: Synthesis of Compounds I-6 to I-9 Anyl derivative derived from benzoxazole, indolenine, benzothiazole and benzimidazole by a known method and compounds I-1c to I-1a, Compound I
-1 and compound I-6 were synthesized according to the method of synthesizing compound I-6.

[0069]

[Chemical 19]

Since the optimum conditions for synthesizing an anil compound were different in the synthesis of compound I-6 to compound I-9, Table 2 shows the details of the conditions for synthesizing an anil compound. Further, in Table 3, compound I-
6 shows the absorption characteristics of compound I-9 in methanol.

[0071]

[Table 2]

[0072]

[Table 3]

Synthesis Example 6: Compound I-10 to Compound I-
Synthesis of 14: N, N′-diphenylformamidine used in the synthesis of Compound I-1b was replaced with 3-anilino-acrylaldehyde phenylimine, and Compound I-1 to Compound I-9 was used under the reaction conditions of Compound I-1 to Compound I-9. Compound I-14 was synthesized.
The dye structure was determined by mass spectrum and absorption spectrum.

Synthesis Example 7: Synthesis of Compound I-15 A compound was prepared under the same conditions as for Compound I-1, except that 3-anilino-acrylaldehyde phenylimine used in the synthesis of Compound I-1 was replaced with a glutaconaldehyde dianyl derivative. I-15 was synthesized. Table 4 shows the absorption characteristics of compound I-10 to compound I-15 in methanol.

[0075]

[Table 4]

Synthesis Example 8: Synthesis of compound II-1 Compound II-1 was synthesized by the following route.

[Chemical 20]

Compound II-1d (12.4 g, 0.1 mol) and propane sultone (12.2 g, 0.1 mol) were dissolved in 30 ml of toluene and heated under reflux for 4 hours to precipitate compound II in the reaction solution.
-1c was collected by filtration (quantitative). Acetonitrile (100 ml), N, N'-diphenylformamidine (19.6 g,
0.1 mol) and acetic anhydride (10.2 g, 0.1 mol) were added, and the mixture was heated under reflux for 4 hours to obtain compound II-1b (yield 96%). Compound II-1b (0.35 g, 1 mmol) and Compound I-1
c (0.40 g, 1 mmol) was dissolved in DMF (5 ml), triethylamine (0.5 ml) and acetic anhydride (0.5 ml) were added, and the mixture was reacted at 60 ° C for 1 hr. When ethyl acetate (50 ml) was added to the reaction solution, crude crystals of compound II-1a were precipitated. Compound II-1a was recrystallized from a mixed solvent of chloroform and methanol. Next, methanol (10 ml) and 5% lithium hydroxide aqueous solution (5 ml)
By ester hydrolysis (room temperature, 2 hours), salt exchange with potassium acetate in methanol, and recrystallization from methanol to give compound II-1. mass (posi): 625

Synthesis Example 9: Compound II-2 to Compound II-13
Compound II-2 to Compound II-13 were synthesized according to the methods exemplified so far. The dye structure was confirmed by mass spectrum and absorption spectrum. Table 5 shows the absorption characteristics of each compound in methanol.

[0079]

[Table 5]

Synthesis Example 10: Synthesis of Compound III-1 In the synthesis of Compound II-1 shown in 24, Compound II-1a was replaced by 3-methyl-benzo [d] isoxazole as a starting material. Compound III-1 was obtained under the same reaction conditions as above. mass (nega): 773 Absorption maximum (methanol): 549 nm Molecular extinction coefficient: 145000

Synthesis Example 11: Synthesis of compound III-3 According to the method described in Can. J. Chem., 66, 1405-1409 (1988), 3-methyl-benzo [d] isothiazole was synthesized to give compound III-1. Compound III-3 was synthesized in the same manner as. mass (nega): 695 Absorption maximum (methanol): 555 nm Molecular extinction coefficient: 145000

Synthesis Example 12: Synthesis of Compound III-4 In the synthesis of Compound II-1 above, the starting compound
Compound III-4 was obtained by replacing II-1a with 2-methyl-1-pyrroline manufactured by Aldrich under the same reaction conditions as for compound II-1. mass (posi): 681 Absorption maximum (methanol): 520 nm Molecular absorption coefficient: 110000

(Comparison of Fluorescence Intensity) The excitation wavelength and the fluorescence intensity of the dye of the present invention were compared with the conventional dyes shown below. Methanol was used as the solvent, and the dye concentration was 1.0 × 10 ⁻⁶ M. A spectrophotofluorometer RF-5300PC manufactured by Shimadzu Corporation was used for the measurement. The results are shown in Tables 6 to 8. As is apparent from the table, the dyes of the present invention have strong fluorescence intensity, and particularly, Table 6 and Table 7
It can be seen that the dyes shown in (1) have a remarkable difference from the conventional dyes.

[0084]

[Chemical 21]

[0085]

[Table 6]

[0086]

[Table 7]

[0087]

[Table 8]

Example 1 Synthesis of Compound I-1-dUTP Conjugate 5 mg (2.5 parts) of compound I-1 was dissolved in 2 ml of 0.1 M MES buffer to give WSC hydrochloride 3.66 mg (5 parts). And Sulfo-NHS 4.20 mg (5
Part) was added and the mixture was stirred at room temperature for 15 minutes. To this, 2.2 mg of aminoallyl-dUTP (Sigma) was dissolved in 200 μl of 0.1 M MES and added, and the mixture was reacted at room temperature for 2 hours. 1M Tris buffer (pH 7.
5) After adding 100 μl to stop the reaction, add 8 g of ODS silica.
Adsorb to a column packed with (YMC-ODS-AQ 120A), 30%
Elute with aqueous methanol. After concentration of the eluent, medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-4
The product was purified by 0B) to obtain a target product having a purity of 98% (yield 75%). MS analysis: M-1150

Example 2 Synthesis of Compound I-10-dUTP Conjugate 5.5 mg (2.5 parts) of Compound I-1 was dissolved in 200 μl of DMSO and WS
C hydrochloride 3.1 mg (5 parts) and Sulfo-NHS 3.55 mg (5 parts) 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 at room temperature.
The reaction was carried out 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 120
It was adsorbed on a column packed with A) and eluted with a 45% aqueous methanol solution. The eluate was concentrated and further purified by medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-40B) to obtain the target product with a purity of 95% (yield 69%). MS analysis: M-1176

Example 3: Synthesis of compound II-1-dUTP conjugate 6.00 mg (2.5 parts) of compound II-1 was dissolved in 200 μl of DMSO,
WSC hydrochloride 3.1 mg (5 parts) and Sulfo-NHS 3.55 mg (5 parts) were added, and the mixture was stirred at room temperature for 30 minutes. To this was added 2.2 mg of aminoallyl-dUTP (Sigma) dissolved in 2 ml of 0.1MMES,
The reaction was carried out at room temperature for 2 hours. 1M Tris buffer (pH 7.5) 100μ
After adding l to stop the reaction, 8 g of ODS silica (YMC-ODS-A
It was adsorbed on a column packed with Q 120A) and eluted with a 50% aqueous methanol solution. The eluate was concentrated and further purified by medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-40B) to obtain the desired product with a purity of 96% (yield 78%). MS analysis: M-1114

Example 4: Synthesis of compound II-10-dUTP conjugate 3.27 mg (1.0 part) of compound II-12 was dissolved in 200 μl of DMSO and 2 ml of aminoallyl-dUT dissolved in 0.1 M MES.
2.2 mg of P (Sigma) was added and reacted at room temperature for 2 hours. 1M
After adding 100 μl of Tris buffer (pH 7.5) to stop the reaction,
A column packed with g of ODS silica (YMC-ODS-AQ 120A) was adsorbed and eluted with 40% aqueous methanol solution. After concentration of the eluent, medium pressure preparative chromatography (YAMAZEN Ultr
apack ODS-S-40B) to obtain the target product with a purity of 95%.
(Yield 71%). MS analysis: M-1140

Example 5: Synthesis of compound III-1-dUTP conjugate 1.00 mg (1.0 part) of compound III-1 was dissolved in 300 μl of 0.1 M MES and 0.25 mg (0.4 part) of aminoallyl-dUTP was added thereto. (Si
gma), 300 μl of 1M carbonate buffer (pH 9.0) was further added, and the mixture was reacted overnight at room temperature. 1M Tris buffer (pH 7.5) 1
After the reaction was stopped by adding 00 μl, the product was purified by medium pressure preparative chromatography (YAMAZEN Ultrapack ODS-S-40B) to obtain a target product with a purity of 95% (yield 65%). MS analysis: M-1123

Use Example 1: Fluorescently labeled DN using transcription reaction
Preparation of A probe Human liver mRNA (Clontech) (0.5 μg) and oligo dT primer (dT18-21, Gibco BRL) (0.
5 μg) were mixed, heated at 70 ° C. for 10 minutes, and then rapidly cooled on ice. Add RNaseOUT (Gibco
BRL) (40U), dATP (500 μM), dGTP
(500 μM), dCTP (500 μM), dTTP
(200 μM), the compound I-1-dUTP conjugate obtained in Example 1 (100 μM), SuperScriptII reverse transcriptase (Gibco BRL) (400 U), DEPC-treated water (amount of 20 ul in total) was added, and the temperature was 42 ° C. And reacted for 2 hours. After completion of the reaction, EDTA and NaOH were added and incubated at 65 ° C. for 1 hour to stop the reaction and decompose the mRNA. The reaction solution is CentriSe
p column (PRINCETON SEPARATIO
N, INC) to remove unreacted compound I-1-dUTP conjugate and the like for purification.

For comparison, a fluorescent nucleotide for comparison (Cy3-AP3-dUTP; Amersham pharmacia biote) labeled with a dye (Cy3) instead of the compound I-1-dUTP conjugate was used.
ch) was used to carry out a reverse transcription reaction in the same manner as above to purify the reaction product. The purified reaction solutions were subjected to agarose gel electrophoresis, and SYBR GreenII (Molecu
Staining with Lar Probes) and scanning with FLA2000 (Fuji Photo Film). As a result, it was found that the compound I-1-dUTP conjugate of the present invention had a stronger fluorescence intensity and could be detected more clearly. Further, the fluorescence intensity was measured with a fluorometer, the amount of DNA was measured from the absorbance at 260 nm, and the incorporation rate calculated from these results and the fluorescence intensity per 1 μM probe were calculated. The results are shown in Table 8. As can be seen from Table 8, the incorporation rate and the fluorescence intensity were higher when the compound I-1-dUTP conjugate of the present invention was used.

[0095]

[Table 9]

Use Example 2: Preparation of fluorescent dye-labeled DNA probe 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 II-1-dUTP conjugate or Cy3-AP3-dUTP: 50 μM Pyrobest DNA polymerase (TAKARA): 0.5 u
nit Sterilized water: 20 μl in total (Note: template DNA is PC
R-ScriptTM SK (+) (STRATAGENE) with α-2-HS-glycoprotein gene incorporated is used, and the sequences of Primers 1 and 2 are shown in Sequence Listings 1 and 2, respectively.)

Using the above composition as a PCR reaction solution,
A PCR reaction was performed by performing 30 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The reaction solution was sent to CentriSep column (PRINCETON).
(SEPARATION, INC) to remove unreacted fluorescent nucleotides and the like to 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 Co., Ltd.). As a result, it was found that the compound II-1-dUTP conjugate of the present invention had a stronger fluorescence intensity and could be detected more clearly. In addition, the fluorescence intensity was measured with a fluorometer and
The amount of DNA was measured from the absorbance at 0 nm, and the uptake rate calculated from those results and the fluorescence intensity per 1 μM probe were calculated. The results are shown in Table 9. As can be seen from Table 9, the incorporation rate and fluorescence intensity were higher when the compound II-1-dUTP conjugate of the present invention was used.

[0098]

[Table 10]

[0099]

INDUSTRIAL APPLICABILITY The fluorescent nucleotide of the present invention is excellent in the incorporation rate during DNA synthesis and the fluorescence intensity after incorporation, and is useful as a labeling substance for nucleic acids.

[Sequence list] SEQUENCE LISTING <110> Fuji Photo Film Co. Ltd. <120> Fluorescent nucleotide and method of labeling by using the same <130> A11319M <160> 2 <210> 1 <211> 20 <212> DNA <213> Artificial <400> 1 tggccgcctt caacgctcag 20 <210> 2 <211> 26 <212> DNA <213> Artificial <400> 2 tcaggcactt tcattaacag gcacat 26

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 21/78 G01N 33/58 A 33/58 C12N 15/00 ZNAA (72) Inventor Hiroko Inomata 3 Izumisui, Asaka City, Saitama Prefecture 11-46 chome Fuji Shashin Film Co., Ltd. Asaka Research Institute (72) Inventor Masayoshi Kojima 3-chome 11-46 Izumi, Asaka-shi, Saitama F-Term (A reference) 2G045 AA25 DA12 DA13 DA14 FB02 FB07 FB12 GC15 2G054 AA06 AB05 CA22 CE02 EA03 GA04 4B024 AA11 CA01 CA09 HA14 HA19 4B063 QA01 QA18 QQ42 QQ52 QR56 QR62 QS34 QX02 4C057 KK21 LL44 MM05

Claims (18)

[Claims] 1. A formula: XYZ [wherein, X represents a residue of a natural or unnatural nucleotide, an oligonucleotide or a polynucleotide or a derivative thereof, and Y is a base moiety in the above residue. Combine;
Y represents a divalent linking group or a single bond; Z represents the following general formula (I): (In the formula, R ¹ , R ² , R ³ , and R ⁴ each independently represent a substituted or unsubstituted alkyl group, and R ³ and R ⁴ are linked to each other to form a saturated or unsaturated ring. However, at least one of R ¹ and R ² represents a reactive group which is bonded to Y; L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ and L ⁷ are each independently substituted. Or an unsubstituted methine group; A is 5
A non-metal atom group forming a 6-membered or 6-membered nitrogen-containing heterocycle; M is a counterion; m is the number necessary to neutralize the charge of the molecule; s is 0 or 1 ; T is 0 or 1
U is a monovalent group derived from the compound represented by 0 or 1 and is bonded to Y by a reactive group present in R ¹ or R ² ] nucleotide. 2. A compound represented by the general formula (I) has the following general formula (II): (In the formula, R ¹ , R ² , R ³ , R ⁴ , L ¹ , L ² , L ³ , L ⁴ ,
L ⁵ , L ⁶ , L ⁷ , M, m, s, t, and u have the same meanings as defined in formula (I); V ¹ , V ² and V ³ are each independently a hydrogen atom or a monovalent group. Represents a substituent, V ¹
And V ² or V ² and V ³ may be linked to each other to form a saturated or unsaturated ring; W is an oxygen atom, a sulfur atom, —C
^{(R 3) (R 4)} - or ^{-N (R 5) -, (} R 3, R 4, and R ⁵ each independently represent a substituted or unsubstituted alkyl group) indicates; Q is a nitrogen atom or -C (V ⁴ )-(V ⁴ represents a hydrogen atom or a monovalent substituent, and may be bonded to V ³ to form a saturated or unsaturated ring)); (III): (In the formula, V ¹ , R ¹ , R ² , R ³ , R ⁴ , L ¹ , L ² , L ³ ,
^{L 4, L 5, L 6} , L 7, W, M, m, s, t, and u have the same meanings as defined above, W is -N (R ⁵⁾ - or -C (R ³⁾
In the case of (R ⁴ )-, V ¹ may combine with a substituent on W to form a saturated or unsaturated ring); or the following general formula (IV): (In the formula, V ¹ , R ¹ , R ² , R ³ , R ⁴ , L ¹ , L ² , L ³ ,
L ⁴ , L ⁵ , L ⁶ , L ⁷ , W, M, m, s, t, and u are as defined above; E is a nitrogen atom or -C (R ⁶ ) =
(R ⁶ represents a hydrogen atom or a monovalent substituent, and R ⁶ may combine with V ¹ to form a saturated or unsaturated ring). The fluorescent nucleotide according to. 3. At least one of R ¹ and R ² is an alkyl group substituted with an active ester group in Y (the active ester group can be covalently bonded to an amino group, a hydroxyl group, or a thiol group). The fluorescent nucleotide according to claim 1 or 2. 4. 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 in Y. 5. The fluorescent nucleotide according to claim 1, wherein X is a residue of a nucleotide or a derivative thereof. 6. X 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
-MeO-CTP (Me represents a methyl group, MeO represents a methoxy group, and the number before Me or MeO represents a substitution position), and a nucleotide selected from the group consisting of (2) the nucleotide according to (1) above. A nucleotide selected from the group consisting of deoxynucleotides and dideoxynucleotides corresponding to the above; and (3) a nucleotide selected from the group consisting of nucleotide derivatives derived from the nucleotides described in (1) and (2) above, or a derivative thereof. The fluorescent nucleotide according to any one of claims 1 to 4, which is a residue. 7. Y is —CH ₂ —, —CH═CH—, —C
A linking group consisting of ≡C-, -CO-, -O-, -S-, -NH-, or any combination of these groups (a hydrogen atom on the linking group is substituted with another substituent). The fluorescent nucleotide according to any one of claims 1 to 6. 8. The fluorescent nucleotide according to claim 1, wherein Y is an aminoallyl group. 9. A nucleic acid synthase, a template nucleic acid, and claim 1.
9. A method for producing a fluorescently labeled nucleic acid, which comprises the step of performing a nucleic acid synthesis reaction using the fluorescent nucleotide according to any one of 1 to 8. 10. A nucleic acid synthesis reaction is a reverse transcription reaction, a terminal transferase reaction, a random prime method, or a PC.
The method according to claim 9, which is one or more reactions selected from the group consisting of R method and nick translation method. 11. A nucleic acid probe or primer labeled with the fluorescent nucleotide according to any one of claims 1 to 8. 12. A nucleic acid detection reagent comprising the nucleic acid probe or primer according to claim 11. 13. A method according to claim 12 for use in diagnosing a disease.
The reagent according to. 14. A kit for detecting a nucleic acid, which comprises (1) the fluorescent nucleotide according to any one of claims 1 to 8, (2) a nucleic acid synthase, and (3) a buffer solution. 15. An azamethine dye represented by the general formula (I) according to claim 1. 16. An azamethine dye represented by the general formula (II) according to claim 2. 17. An azamethine dye represented by the general formula (III) according to claim 2. 18. An azamethine dye represented by the general formula (IV) according to claim 2.