JPH10158293A - 3'-deoxyribonucleotide derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a 3′-deoxyribonucleotide derivative which is safe and highly sensitive and is useful in, for example, a method for determining a nucleotide sequence of a nucleic acid utilizing an RNA polymerase action. A rhodamine dye-labeled 3'-deoxyribonucleotide derivative represented by the following structural formula. Embedded image (Wherein Q represents a 3′-deoxyribonucleotide residue;
R represents a lower alkylene group, W represents a carboxyl group,
R1 and R2 each independently represent a hydrogen atom or a lower alkyl group. )

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a 3'-deoxyribonucleotide derivative which is useful, for example, for analyzing the base sequence of nucleic acids.

[0002]

2. Description of the Related Art DNA base sequence analysis is one of the fundamental techniques in molecular biology. As the DNA sequencing method, at present, the Maxam-Gilbert method (chemical decomposition method) [Methods of Enzymology, 65 , 499-560 (1980)]
And Sanger method (dideoxy chain termination method) [Proc. Nat
l. Acad. Sci., USA, 74 , 5463-5467 (1977)]. Among them, the dideoxy chain termination method has become the mainstream DNA sequencing method because the sequence can be determined more easily and in a shorter time than the chemical decomposition method.

[0003] The basic principle of the dideoxy chain termination method is as follows. First, the DN whose base sequence is to be determined
A single-stranded DNA containing the A fragment is prepared and used as a template for the replication process. Next, a primer that binds to the vicinity of the insertion site of the DNA fragment is bound thereto, and DNase complementary to the single-stranded DNA is digested with an enzyme called Klenow fragment.
A is synthesized. This synthesis involves four natural 2'-deoxyribonucleotides and a chain terminator.
The method is performed in the presence of labeled 2 ′, 3′-dideoxynucleotides corresponding to each of the four bases labeled with a radioisotope, a fluorescent dye, or the like. That is, 2 ′, 3′-dideoxynucleotide is a 2′-deoxyribonucleotide in which the OH group at the 3′-position is replaced with an H group,
Like a deoxyribonucleotide, it serves as a substrate for the Klenow fragment, but when 2 ', 3'-dideoxynucleotides bind, chain extension of the DNA stops there. As a result, various DNA strands having a common 5 'end but different chain lengths are synthesized. That is, for each base of adenine (A), guanine (G), cytosine (C), and thymine (T), the above operation was performed in combination with a 2 ′, 3′-dideoxynucleotide, followed by electrophoresis. , The sequence of the base sequence can be decoded based on the length of the DNA fragment.

[0004] However, in the current state of the so-called genome project, in which the determination of the total DNA base sequence of humans and animals and plants and the analysis of all genes have been advanced, the above-mentioned dideoxy chain termination method has been used. Problems such as complicated operation procedures (preparation of single-stranded DNA template, etc.) and difficulty in shortening the processing time.

[0005] As one of means for solving these problems, a method of determining a DNA base sequence by a chain extension reaction utilizing the properties of RNA polymerase has been considered. Among such base sequence determination methods using RNA polymerase, the chain termination method is a radioisotope as four types of natural ribonucleotides and a terminator.
^A method using labeled 3'-deoxynucleotides corresponding to each of four bases using a radioisotope such as ³² P as a label is known [Biochemistry, 24 , 5716-572.
3 (1985)]. However, since this label terminator uses a radioisotope as a label, there are problems such as adverse effects on human bodies and waste disposal.

As for the labeled terminator used in the dideoxy chain termination method as described above, for example, a report by Sanger et al. [J. Mol. Biol., 143 , 161-178 (198)
0)], Smith et al. [Nucleic Acids Res., 13 , 2399
-2412 (1985)] and a report by Prober [Science, 238 ,
336-341 (1987)], Connel et al.'S report [BioTechnique
s, 5 , 342-348 (1987)] and a report by Lee et al. [Nucleic Aci.
ds Res., 20 , 2471-2483 (1992)]
And various reports such as Japanese Patent Publication No. 7-121239, but none of the reports mention labeled 3'-deoxyribonucleotides.

[0007] On the other hand, in fields requiring advanced techniques such as DNA base sequence determination, fluorescently labeled compounds and fluorescently labeled 3'-deoxyribonucleotides are used in RNs.
Because it is used in the dideoxy chain termination method, which has a severe restriction that it must not interfere with the activity of A polymerase,
It is extremely difficult to estimate a fluorescently labeled terminator useful for the chain termination method using RNA polymerase from the known labeled terminators as described above, based on the structural correlation between DNA polymerase and RNA polymerase. In addition, Japanese Patent Publication No. 8-5908 discloses labeling 2 ',
Various 3'-dideoxyribonucleotides, labeled 2'-deoxyribonucleotides, labeled 3'-deoxyribonucleotides and labeled ribonucleotides have been disclosed, and among these various nucleotide derivatives, DNA
There is no suggestion as to which is particularly sensitive and which is particularly useful when used for sequencing.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a 3'-deoxyribonucleotide derivative which is safe and highly sensitive and is useful, for example, in a method for determining the nucleotide sequence of a nucleic acid utilizing RNA polymerase action. It is assumed that.

[0009]

The present invention is directed to a 3'-deoxyribonucleotide derivative represented by the following general formula [I].

[0010]

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[0011] wherein, Q is a 3'-deoxyribonucleotide residue, R represents a lower alkylene group, W represents a carboxyl group, X is -O -, - S -, - NR'- and -CH ₂
(Wherein, R ′ represents a hydrogen atom, a lower alkyl group, an aralkyl group, or an aryl group). In addition, one of ring A and ring B is

[0012]

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And the other is

[0014]

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(Where Z is ＝ O or ＮN ⁺ R ¹ R ² , Y is —OH or —NR ¹ R ² , and R ¹ and R ² are each independently Represents a hydrogen atom or a lower alkyl group, R ¹ and R ² both represent a trimethylene group, and the other end represents —NR ¹ R ² or Ａ on ring A or ring B.
N ⁺ R ¹ R ² may be bonded to both adjacent carbon atoms to the bonded carbon atom. ), Ring C

[0016]

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A broken line in the above formula means a bond at a position corresponding to the structure of the ring A and the ring B. Further, each of the rings and the benzene ring may further have a substituent. ]

That is, the present inventors have made intensive studies to achieve the above object, and as a result, the fluorescently labeled 3′-deoxyribonucleotide derivative represented by the above general formula [I]
It is highly safe to the human body and environment, and can be detected with high sensitivity, and can be a substrate for RNA polymerase.
The present inventors have found that when used as a terminator in a method for determining the nucleotide sequence of a nucleic acid utilizing the A polymerase action, it is possible to perform high-speed analysis of the nucleotide sequence, and have completed the present invention.

In the general formula [I], 3 ′ represented by Q
As the deoxyribonucleotide residue, a purine nucleotide residue represented by the following general formulas (II) and (III), and a 3′-deoxyribonucleotide residue of a pyrimidine nucleotide represented by the following general formulas (IV) and (V): No.

[0020]

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[0021]

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[0022]

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[0023]

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In the above general formulas [II] to [V],
R ³ is _{-PO 3 H 2, -P 2 O} 6 H 3, -P 3 O 9 H 4 or an salt thereof, specific examples of such salts include, for example, sodium salts, potassium salts, lithium salts, etc. Preferable examples include alkali metal salts such as alkaline earth metal salts such as barium salts, and organic amine salts such as ammonium salts, triethylammonium salts and pyridine salts.

In formula [I], the lower alkylene group represented by R may be linear, branched or cyclic. For example, an alkylene group having 1 to 10 carbon atoms is preferable. And specifically, a methylene group, an ethylene group,
Propylene group, butylene group, 2-methylpropylene group, pentylene group, 2,2-dimethylpropylene group, 2-ethylpropylene group, hexylene group, heptylene group, octylene group, 2-ethylhexylene group, nonylene, decylene group, Examples thereof include a cyclopropylene group, a cyclopentylene group, and a cyclohexylene group, and preferably a straight-chain lower alkylene group having 1 to 6 carbon atoms.

In the general formula [I], the lower alkyl group represented by R 'in -NR'- represented by X may be any of linear, branched or cyclic. Examples thereof include an alkyl group having 1 to 6 carbon atoms, specifically, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-
Examples thereof include a butyl group, a pentyl group, an isopentyl group, a tert-pentyl group, a 1-methylpentyl group, an n-hexyl group, an isohexyl group, a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group. Examples of the aralkyl group represented by R ′ include an aralkyl group having 7 to 20 carbon atoms, and specific examples thereof include benzyl, phenethyl, phenylpropyl, methylbenzyl, methylphenethyl, ethylbenzyl, and naphthyl. Examples of the group include a methyl group and a naphthylethyl group. Examples of the aryl group include a phenyl group, a tolyl group, a xylyl group, and a naphthyl group.

In the general formula [I], a lower alkyl group represented by R ¹ and R ² in = N ⁺ R ¹ R ² represented by Z or -NR ¹ R ² represented by Y May be linear, branched or cyclic, and includes, for example, an alkyl group having 1 to 6 carbon atoms, specifically, a methyl group,
Ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, tert-pentyl group, 1-methylpentyl group, n-hexyl group , An isohexyl group, a cyclopropyl group, a cyclopentyl group, a cyclohexyl group and the like.

The 3'-deoxyribonucleotide derivative represented by the general formula [I] of the present invention comprises a 3'-deoxyribonucleotide residue: Q, a linker: a general formula [VI]

[0029]

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Wherein R is as defined above. ] And a fluorescent dye group: General formula [VII]

[0031]

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Wherein W and X are as defined above. ] Can be divided into three components.

The component represented by the general formula [VI] is
Q is a linker for bonding the 3'-deoxyribonucleotide residue represented by Q and the fluorescent dye group represented by the general formula [VII], and one end of the triple bond has a lower alkylene group R as described above. An alkynylamino group bonded to the amino group through That is, the other end of the triple bond of the linker binds to the 5-position of pyrimidine and the 7-position of 7-deazapurine among the 3′-deoxyribonucleotide residues Q as described above, Furthermore, the amino group of the linker forms an amide bond with the carboxyl group at the 5-position on the fluorescent dye group, thereby forming a compound having a 3′-deoxyribonucleotide residue and a fluorescent dye group. As such a linker, in the general formula [VI], R is preferably a linear lower alkylene group having 1 to 6 carbon atoms.

The fluorescent dye groups of the general formula [VII] are, in particular, fluorescent dyes which produce a detectable luminescent emission following stimulation by energy absorption from a suitable source such as an argon laser.

In the above general formula [VII], when the carboxyl group represented by W is bonded to the position shown in the following general formula [VIII],

[0036]

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The part of the fluorescent dye group in the 3′-deoxyribonucleotide derivative of the present invention can take any of the following formulas.

[0038]

(Equation 1)

The carboxyl group may be an alkali metal salt such as a sodium salt, a potassium salt, a lithium salt, etc.
For example, alkaline earth metal salts such as barium salts may be formed, and salts such as organic amine salts such as ammonium salts, triethylammonium salts and pyridine salts may be formed.

In the above general formula [VII], the substituent Z in ring A or ring B is ＮN ⁺ R ¹ R ² and Y is -NR ¹
When R ² , R ¹ and R ² both represent a trimethylene group, and the other end is ＝ N ⁺ R ¹ R ² or-on ring A or ring B.
Examples of the case where NR ¹ R ² is bonded to each of the carbon atoms on both sides of the bonded carbon atom include those represented by the following formula.

[0041]

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In the above general formula [VII], the ring C

[0043]

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The broken line in the above-mentioned means a bond at a position corresponding to the structure of the ring A and the ring B. This is specifically shown as follows, for example. Become. That is,
Ring A is

[0045]

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In this case, the position of the bond at ring C is as follows:

[0047]

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Also, when ring B is

[0049]

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In this case, the position of the bond at ring C is as shown below.

[0051]

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Further, when X is -NH-, the ring A (or ring B) and the ring C can take any of the following states.

[0053]

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Each ring and the benzene ring in the above general formula [VII] may further have a substituent. Examples of such a substituent include an alkyl group, an alkoxy group and a halogen atom. Can be As the alkyl group, linear,
It may be branched or cyclic, and may have a double bond. Examples thereof include an alkyl group having 1 to 20 carbon atoms, and preferably a lower alkyl group having 1 to 6 carbon atoms. Is mentioned. Specifically, methyl group, ethyl group, n-
Propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, 1-methylpentyl, n-hexyl, isohexyl, cyclohexyl Preferred examples include a propyl group, a cyclopentyl group, and a cyclohexyl group. As the alkoxy group, a lower alkoxy group, for example, an alkoxy group having 1 to 6 carbon atoms is preferable, and specifically, a methoxy group, an ethoxy group, an n-propoxy group,
Isopropoxy group, n-butoxy group, amyloxy group,
Isoamyloxy group, tert-amyloxy group, 1-
Examples thereof include a methylpentyloxy group, an n-hexyloxy group, and an isohexyloxy group. Examples of the halogen atom include fluorine, chlorine, bromine, and iodine.

More specifically, examples of the fluorescent dye group represented by the general formula [VII] include, for example, 5 (or 6) carboxytetramethyl rhodamine, 5 (or 6) carboxy rhodamine X, and 5 (or 6) carboxy. Rhodamine 6G, 5 (or 6) carboxy rhodamine 110,
5 (or 6) carboxyfluorescein, 5 (or 6) carboxy-2 ', 7'-dichlorofluorescein, 5 (or 6) carboxy-2', 4 ', 5', 7'-tetrachlorofluorescein Olesesin, 5 (or 6) carboxy 4,7-dichloro-2 ', 7'-dimethoxyfluorescein, 5 (or 6) carboxy-4,7,4', 5'-tetrachloro-2 ', 7 '-Dimethoxyfluorescein,
5 (or 6) carboxy-4 ', 5'-dichloro-2',
7′-dimethoxyfluorescein, 5 (or 6) carboxy-4,7-dichloro-1 ′, 2 ′, 7 ′, 8′-dibenzofluorescein, 5 (or 6) carboxy-4,
Preferred are those derived from fluorescent dyes such as 7-dichloro-1 ′, 2 ′, 7 ′, 8′-dibenzofluorescein.

The 3′-deoxyribonucleotide derivative of the present invention represented by the general formula [I] can be easily synthesized, for example, according to the following synthesis scheme. still,
In the following synthesis scheme, R ⁴ represents a fluorescent dye group as described above. The formal names used in the following synthesis schemes are as follows.

CAN: diammonium cerium (IV) nitrate AcOH: acetic acid NPTFA: N-propargyl trifluoroacetamide NPETFA: 5-trifluoroacetamide-1-pentyne Et ₃ N: triethylamine (Ph ₃ P) ₄ Pd: tetrakis (triphenylphosphene) palladium (0) DMF: N, N- dimethylformamide NHTfa: trifluoroacetamide (EtO) ₃ PO: triethyl phosphate tris (TBA) PP: tris (tri -n- butylammonium) pyro Phosphate TEAB: Triethylammonium hydrogen carbonate buffer TBDMSCl: tert-butyldimethylsilyl chloride THF: Tetrahydrofuran TCDI: 1,1′-thiocarbonyldiimidazole n-Bu ₃ SnH: hydrogenated-n-tributyltin AIBN: 2,2'-azobis (isobutyronitrile) pyr. : Pyridine n-Bu ₄ NF: tetrabutylammonium fluoride Ac ₂ O: acetic anhydride MeOH: methanol NIS: N-iodosuccinimide STC: 4-thiocresol HMPA: hexamethylphosphoramide MCPBA: m-chloroperbenzoic acid R ⁴ -OSu: imidyl succinate of fluorescent dye group

(1) Fluorescently labeled 3'-deoxyuridine-
Synthesis of 5'-triphosphate (compound in which R in general formula [I] is a methylene group).

[0059]

(Equation 2)

(2) Fluorescently labeled 3'-deoxycytidine-
Synthesis of 5'-triphosphate (compound in which R in general formula [I] is a methylene group).

[0061]

(Equation 3)

(3) Fluorescent labeling 7-deaza-3'-deoxyadenosine-5'-triphosphate (general formula [I]
Wherein R is a methylene group.

[0063]

(Equation 4)

(4) Fluorescently labeled 7-deaza-3'-deoxyguanosine-5'-triphosphate (general formula [I]
Wherein R is a methylene group.

[0065]

(Equation 5)

(5) Fluorescently labeled 3'-deoxycytidine-
5'-triphosphate (R in the general formula [I] is n
-Compound which is a propylene group).

[0067]

(Equation 6)

The 3'-deoxyribonucleotide derivative of the present invention is very effective as an RNA elongation reaction terminator in a DNA sequence determination method by a chain termination method using RNA polymerase. The sequence can be determined simply and in a short time. That is, the template DNA whose base sequence is to be determined
It is connected downstream of the A polymerase promoter and modified with four types of ribonucleotides, adenine (A), guanine (G), cytosine (C), and uracil (U), and different fluorescent dyes of the present invention corresponding to the ribonucleotides. In the presence of the four types of 3′-deoxyribonucleotide derivatives, an elongation stop reaction of RNA polymerase is performed at each base site, the products are mixed and electrophoresed in one lane. Can be sequentially determined by spectroscopy.

The RNA polymerase used in the above-described chain termination method using an RNA polymerase is not particularly limited, but an RNA polymerase derived from a promoter-dependent phage having a high processivity of the extension reaction is preferably exemplified. Specific examples of these RNA polymerases, T ₇ RNA polymerase, T ₃ RNA
Polymerase, SP6 RNA polymerase and the like. Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited thereto.

[0070]

【Example】

Reference Example 1. 3'-Deoxy-5-iodouridine (3'-Deoxy-5
-iodouridine) (corresponding to compound 2 in the above-mentioned synthesis scheme, and is hereinafter abbreviated as compound 2. In addition, the compounds in the synthesis scheme are similarly shown below.)
Synthesis of After 3'-deoxyuridine (compound 1: 2.08 g, 9.11 mmol) was dissolved in acetic acid (75 ml), diammonium cerium (IV) nitrate (2.50 g, 4.56 mmol) and iodine (1.39 g, 2.73 mmol) were dissolved.
l) was added and stirred at 80 ° C. for 30 minutes. After the completion of the reaction, the acetic acid was concentrated under reduced pressure, and then three times with an ethanol-toluene mixture (1: 2 v / v; 30 ml) and a water-ethanol mixture (1: 2 v / v; 3).
(0 ml) three times to obtain an oily substance. The resulting residue was purified by silica gel column chromatography (eluent; methylene chloride-methanol mixed solvent) to give 3'-deoxy-5.
1.78 g of -iodouridine (compound 2) was obtained (yield 55.08).
%). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.92-2.01 (m, 1H,
3'-Ha), 2.17-2.23 (m, 1H, 3'-Hb), 3.52, 3.71 (2dd,
2H, J = 3.2, 12.2; 2.7, 11.9, 5'-Ha, b), 4.20-4.40
(m, 2H, 2'-H, 4'-H), 5.10 (brs, 1H, 5'-OH), 5.50
(d, 1H, J = 4.5Hz, 2'-OH), 5.78 (d, 1H, J = 2.7Hz, 1'-
H), 8.30 (s, 6H), 11.32 (brs, 1H, NH)

Reference Example 2 Synthesis of N-propargyltrifluoroacetamide Methyl trifluoroacetate cooled to 0 ° C. (manufactured by Tokyo Chemical Industry;
69.2 g, 0.54 mol) with propargylamine (manufactured by Altrich Co.);
(25 g, 0.45 mol) was added dropwise. After reacting at 0 ° C for 2 hours, purification was performed by distillation under reduced pressure (23 mmHg; boiling point: 77 ° C) to obtain 43.8 g (86.0%) of N-propargyl trifluoroacetamide.

Reference Example 3. 3'-Deoxy-5- (3 "-trifluoroacetamido-1" -propynyl) uridine [3'-Deoxy-5-
Synthesis of (3 "-trifluoroacetamido-1" -propynyl) uridine] (compound 3). 3'-deoxy-5-iodouridine (Compound 2: 1.56 g, 4.4 m
mol) of D-propargyl trifluoroacetamide (1.54 ml, 1
3.2 mmol), copper (I) iodide (168 mg, 0.88 mmol), tetrakis (triphenylphosphine) palladium (0)
(508 mg, 0.44 mmol) and triethylamine (1.23 ml, 8.8
mmol) and reacted at room temperature for 4 hours. The residue obtained by concentrating the reaction solution under reduced pressure was mixed with a methylene chloride-methanol mixture (40 m
l) and dissolved in the ion exchange resin AG1X8 (Bio-Rad, HCO
₃ ^- type; 4g) was stirred for 30 minutes added. After filtration, the filtrate was concentrated and the resulting residue was purified by silica gel column chromatography (eluent; methylene chloride-methanol mixed solution) to give a novel substance, 3'-deoxy-5- (3 "-trifluoroacetamide- 918 mg of 1 "-propynyl) uridine (compound 3) was obtained (55.3%). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.89-1.92 (m, 1H,
3'-Ha), 2.17-2.26 (m, 1H, 3'-Hb), 3.53, 3.76 (2d
d, 2H, J = 3.1, 11.9; 2.7, 12.1Hz, 5'-Ha, b), 4.22-4.
30 (m, 4H, 2'-H, 4'-H, -CH _2- ), 5.20 (brs, 1H, 5'-O
H), 5.52 (d, 1H, J = 4.0, 2'-OH), 5.75 (d, 1H, J = 1.9H
z, 1'-H), 8.34 (s, 1H, 6-H), 10.06 (t, 1H, J = 5.4Hz,
NHTfa), 11.67 (s, 1H, NH)

Reference Example 4. Tris (tri-n-butylammoniu) pyrophosphate (Tris (tri-n-butylammoniu)
m) Synthesis of pyrophosphate). Tetrasodium pyrophosphate decahydrate (2.23 g) in water (5
0W) and passed through a column of Dowex 50Wx8 (manufactured by Dowex; H ⁺ type, 45ml). The column was further eluted with water and collected until the eluate pH was nearly neutral. bird
-N-butylamine (3.55 ml) was added to the eluate, and the mixture was stirred well. The mixture was concentrated under reduced pressure, and the residue was ethanol, pyridine,
The mixture was further azeotropically concentrated to dryness with dimethylformamide (DMF). The obtained residue was dissolved in dry DMF, and the volume was increased to 10 ml to obtain 0.5 M concentration of tris (tri-n-butylammonium) pyrophosphate.

Reference Example 5.5 5- (3 "-amino-1" -propynyl) -3'-deoxyuridine-5'-triphosphate [5-
(3 "-Amino-1" -propynyl) -3'-deoxyuridine 5'-triphos
phate] (compound 4). 3'-Deoxy-5- (3 "-trifluoroacetamido-1" -propynyl) uridine (compound 3: 42 mg, 0.11 mmol) was dissolved in triethyl phosphate (1.13 ml) and cooled to -20 ° C. Phosphorus oxychloride (25 μl) was added and the mixture was stirred at −20 ° C.
After 30 minutes, phosphorus oxychloride (22 µl) was added, and the mixture was further stirred for 5 hours. This reaction solution was cooled to −20 ° C., and 0.5M-tris (tri-n-butylammonium) obtained in Reference Example 4 was obtained.
The solution was added to a solution of pyrophosphate in DMF (3.6 ml) and stirred at room temperature for 2 hours. After the reaction was completed, a mixed solution of triethylamine and water (4 ml) was added, and the mixture was allowed to stand overnight.
l) was added and allowed to stand for 4 hours. The reaction mixture was washed with ether and concentrated to dryness. The resulting residue was subjected to DEAE-Toyopearl ion exchange column chromatography (Tosoh Corporation; 1.2 × 3).
0 cm; eluent: triethylammonium bicarbonate buffer
(pH 7.5) 0M → 0.3M linear concentration gradient (total volume 1L))
115 mg of a novel substance 5- (3 "-amino-1" -propynyl) -3'-deoxyuridine-5'-triphosphate (compound 4) was obtained (yield 41.3%).

Embodiment 1 TMR-labeled 3'-deoxyuridin-5'-triphosphate
e Synthesis of 5'-triphosphate). 5- (3 "-amino-1" -propynyl) -3'-deoxyuridine-
5M-triphosphate (compound 4: 10.5 μmol) in 1 M triethylammonium bicarbonate buffer (pH 9.05; 1.2 ml)
Of 5-carboxytetramethylrhodamine succinimide ester (Molecular Probes) (15 mg) in DMF
A solution (0.9 ml) was added and the mixture was stirred at room temperature overnight. The reaction solution was diluted by adding water (30 ml), and then subjected to DEAE-Toyopearl ion exchange column chromatography (1.2 × 30 cm; eluent: triethylammonium hydrogen carbonate buffer (pH 7.5) 0.0
Purified by 5M → 0.7M linear concentration gradient (total volume 2L)), and abbreviated as TMR-labeled 3′-deoxyuridine-5′-triphosphate (hereinafter, TMR-3′dUTP (n1)) represented by the following formula: .) 7.38 μmol (70.2% yield).

[0076]

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[Wherein, Me represents a methyl group. Further, the obtained TMR-3′dUTP (n1) was converted to DU640
FIG. 1 shows the results of measuring an ultraviolet-visible absorption spectrum (measurement wavelength: 700 nm to 200 nm, symmetry: distilled water) using an ultraviolet-visible spectroscopic analysis system (manufactured by Beckman Co., Ltd.).

Reference Example 6. Synthesis of 3'-Deoxy-5-iodocytidine (Compound 6) 3′-deoxycytidine (compound 5: 3.0 g, 13.2 mmol) was added to 1,
After suspending in a mixed solution of 4-dioxane (300 ml) and ethanol (30 ml) and cooling to 10 ° C., silver trifluoroacetate (7.0 g, 31.7 mmol) and iodine (8.04 g, 31.7 mmol)
Was added and stirred at room temperature for 2 hours. After completion of the reaction, the precipitate was filtered off through celite, washed with 1,4-dioxane,
The filtrate and the washing solution were combined and concentrated to obtain 3.84 g of 3'-deoxy-5-iodocytidine (compound 6) (yield: 82.4%). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.64-1.70 (m, 1H, 3 '
-Ha), 1.88-1.99 (m, 1H, 3'-Hb), 3.60-3.89 (m, 2H,
5'-Ha, b), 4.20-4.21 (m, 1H, 4'-H), 4.37-4.39 (m, 1
H, 2'-H), 5.59 (s, 1H, 1'-H), 7.58 (brs, 1H, NH
₂ a), 8.41 (brs, 1H, NH ₂ b), 8.79 (brs, 1H, 6-H)

Reference Example 7. 3'-Deoxy-5- (3 "-trifluoroacetamido-1" -propynyl) cytidine [3'-Deoxy-5-
Synthesis of (3 "-trifluoroacetamido-1" -propynyl) cytidine] (compound 7). 3'-deoxy-5-iodocytidine (Compound 6: 1.0 g, 2.83 m
mol) in DMF (14 ml) under nitrogen atmosphere under N-propargyl trifluoroacetamide (0.99 ml, 8.
50 mmol), copper (I) iodide (108 mg, 0.566 mmol), tetrakis (triphenylphosphine) palladium (0)
(327 mg, 0.283 mmol) and triethylamine (0.8 ml, 5.6
6 mmol) and reacted at room temperature for 30 minutes. The reaction solution was diluted with a methylene chloride-methanol mixture (25 ml), ion-exchange resin AG1X8 (HCO ₃ ^- type; 2.6 g) was added, and the mixture was stirred for 30 minutes. After filtration, the filtrate was concentrated and the resulting residue was purified by silica gel column chromatography (eluent; methylene chloride-methanol mixed solution) to give a novel substance, 3'-deoxy-5- (3 "-trifluoroacetamide- 879 mg of 1 "-propynyl) cytidine (compound 7) was obtained (82.5% yield). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.64-1.71 (m, 1H,
3'-Ha), 1.86-1.96 (m, 1H, 3'-Hb), 3.52-3.57 (m, 1H,
5'-Ha), 3.79-3.83 (m, 1H, 5'-Hb), 4.14-4.28 (m, 4
H, 2'-H, 4'-H, -CH _2- ), 5.18 (t, 1H, J = 5.0Hz, 5'-O
H), 5.53 (d, 1H, J = 3.8Hz, 2'-OH), 5.63 (s, 1H,
1'-H), 6.76 (brs, 1H, NH ₂ a), 7.74 (brs, 1H, NH ₂ b),
8.38 (s, 1H, 6-H), 9.93 (brs, 1H, NHTfa)

Reference Example 8.5- (3 "-amino-1" -propynyl) -3'-deoxycytidine-5'-triphosphate [5- (3 "
-Amino-1 "-propynyl) -3'-deoxycytidine-5'-triphospha
te] (compound 8). 3'-Deoxy-5- (3 "-trifluoroacetamido-1" -propynyl) cytidine (compound 7: 113 mg, 0.3 mmol) was dissolved in triethyl phosphate (1.13 ml) and cooled to -20 ° C. Phosphorus oxychloride (25 μl) was added and the mixture was stirred at −20 ° C.
After 30 minutes, phosphorus oxychloride (22 µl) was added, and the mixture was further stirred for 5 hours. This reaction solution was cooled to −20 ° C., and 0.5M-tris (tri-n-butylammonium) obtained in Reference Example 4 was obtained.
The solution was added to a solution of pyrophosphate in DMF (3.6 ml) and stirred at room temperature for 2 hours. After the reaction was completed, a mixed solution of triethylamine and water (4 ml) was added, and the mixture was allowed to stand overnight.
l) was added and allowed to stand for 4 hours. The reaction mixture was washed with ether and concentrated to dryness. The residue obtained was subjected to DEAE-Toyopearl ion exchange column chromatography (1.2 × 30 cm; eluent: triethylammonium hydrogen carbonate buffer (pH 7.5) 0 M)
→ 0.3M linear concentration gradient (total volume 1L))
(3 "-amino-1" -propynyl) -3'-deoxycytidine-5 '
-Triphosphate (compound 8) 115 mg was obtained (yield 41.3).
%).

Embodiment 2 FIG. XR-labeled 3'-deoxycytidine
-5'-triphosphate (XR-labeled 3'-deoxycytidine
Synthesis of 5′-triphosphate) (compound of the general formula [I] wherein R is a methylene group). 5- (3 "-amino-1" -propynyl) -3'-deoxycytidine-
1 in which 5′-triphosphate (compound 8: 8 μmol) was dissolved
M-triethylammonium bicarbonate buffer (pH 9.05; 0.4
To this solution, a DMF solution (0.9 ml) of 5-carboxy-X-rhodamine succinimide ester (manufactured by Molecular Probes) (15 mg) was added, and the mixture was stirred at room temperature overnight. Water (3
0 ml) for dilution, and then DEAE-Toyopearl ion exchange column chromatography (1.2 × 30 cm; eluent: triethylammonium hydrogen carbonate buffer (pH 7.5) 0.1 M → 0.7)
An XR-labeled 3'-deoxycytidine-5'-triphosphate represented by the following formula (a compound in which R is a methylene group in the general formula [I]), purified by an M linear concentration gradient (total volume: 2 L).
Hereinafter, it is abbreviated as XR-3'dCTP (n1). ) 2.93 μmol was obtained (yield 36.6%).

[0082]

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Further, the obtained XR-3′dCTP (n1) was
FIG. 2 shows the results of measuring an ultraviolet-visible absorption spectrum (measurement wavelength: 700 nm to 200 nm, symmetry: distilled water) using a DU640 ultraviolet-visible spectral analysis system (manufactured by Beckman Co., Ltd.).

Reference Example 9.6 6-Chloro-9- {2,5-bis (O-ter
t-butyldimethylsilyl) -3-O-[(imidazol-1-yl) thiocarbonyl] -β-D-ribofuranosyl｝ -7-deazapurine (6-Chloro-9- {2,5-bis (O-tert -butyldimethyl
silyl) -3-O-[(imidazol-1-yl) thiocarbonyl] -β-D-ribo
Synthesis of furanosyl} -7-deazapurine) (compound 11). After dissolving 6-chloro-9- (β-D-ribofuranosyl) -7-deazapurine (compound 9: 3.58 g, 12.5 mmol) in THF (160 ml), pyridine (5.1 ml, 62.7 mmol), silver nitrate (4.68 g, 27.6
mmol) and tert-butyldimethylsilyl chloride (4.1
6g, 27.6mmol) and stirred overnight at room temperature. After completion of the reaction, the precipitate was filtered off through Celite, and the filtrate was concentrated.
After dissolving in chloroform, it was washed with 0.2N hydrochloric acid and saturated saline. After the chloroform layer was dried over anhydrous magnesium sulfate, chloroform was distilled off, and a new substance, 6-chloro-9-
[2,5-bis (O-tert-butyldimethylsilyl) -β-D-ribofuranosyl] -7-deazapurine (compound 10) 6.42 g
(Quantitative) was obtained. DM without further purification
After dissolving in F (120 ml), 1,1'-thiocarbonyldiimidazole (13.36 g, 75.0 mmol) was added, and the mixture was stirred at room temperature overnight. After completion of the reaction, ethyl acetate and water were added, and the organic layer was washed with saturated saline. The ethyl acetate layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent; ethyl acetate).
-n-hexane mixed solvent) to purify the new substance 6-chloro-
9- ｛2,5-bis (O-tert-butyldimethylsilyl) -3-O-
[(Imidazol-1-yl) thiocarbonyl] -β-D-ribofuranosyl フ ラ -7-deazapurine (compound 11) (4.03 g) was obtained (yield: 51.5%).

Reference Example 10. 6-Chloro-9- [2,5-bis (Ot
ert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurine (6-Chloro-9- [2,5-bis (Ot
ert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosy
l] -7-deazapurine) (Compound 12). 6-chloro-9- {2,5-bis (O-tert-butyldimethylsilyl) -3-O-[(imidazol-1-yl) thiocarbonyl]
-β-D-ribofuranosyl｝ -7-deazapurine (Compound 1
1: 4.0 g, 6.41 mmol) in toluene (200 ml),
2'-Azobis (isobutyronitrile) (0.21 g, 1.3 mmol)
And n-tributyltin hydride (3.45 ml, 13 mmol) were added, and the mixture was stirred at 80 ° C. for 30 minutes under a nitrogen atmosphere. After completion of the reaction, toluene was distilled off, and the residue was purified by silica gel column chromatography (eluent; ethyl acetate-n-hexane mixed solvent) to give a new substance, 6-chloro-9- [2,5-bis (O-tert- 2.60 g of (butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurine (compound 12) was obtained (yield).
81.5%).

Reference Example 11. 6-chloro-9- (3-deoxy-β
-D-ribofuranosyl) -7-deazapurine [6-Chloro-9- (3-d
eoxy-β-D-ribofuranosyl) -7-deazapurine] (Compound 1
Synthesis of 3). 6-Chloro-9- [2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurine (Compound 12: 2.6 g, 5.2 mmol) was added to THF (30 ml). ) And 1M tetrabutylammonium fluoride (12.5m
1,12.5 mmol) and stirred at room temperature overnight. After the completion of the reaction, the reaction solution is concentrated under reduced pressure, and silica gel column chromatography (eluent; chloroform-methanol mixed solvent)
To purify the new substance 6-chloro-9- (3-deoxy-β-D-
1.47 g of ribofuranosyl) -7-deazapurine (compound 13)
Was obtained (quantitative). ¹ H-NMR (270MHz, DMSO-d6) δppm: 1.93, 2.23 (m, 2H,
3'-Ha, b), 3.55, 3.71 (2dd, 2H, J = 4.1, 11.9; 3.
2, 11.6Hz, 5'-Ha, b), 4.36 (m, 1H, 2'-H), 4.45 (m,
1H, 4'-H), 5.02 (brs, 1H, 5'-OH), 5.66 (brs, 1H,
2'-OH), 6.19 (d, 1H, J = 2.4Hz, 1'-H), 6.70 (d, 1H, J
= 3.8Hz, 7-H), 8.02 (d, 1H, J = 4.1Hz, 8-H), 8.66 (s,
(1H, 2-H)

Reference Example 12. 6-Chloro-9- (3-deoxy-2,
5-di-O-acetyl-β-D-ribofuranosyl) -7-deazapurine [6-Chloro-9- (3-deoxy-2,5-di-O-acetyl-β-D-ribofu
ranosyl) -7-deazapurine] (compound 14). 6-Chloro-9- (3-deoxy-β-D-ribofuranosyl) -7-deazapurine (Compound 13: 1.47 g, 5.45 mmol) was dissolved in pyridine (15 ml), and acetic anhydride (5 ml) was added. And stirred overnight. After completion of the reaction, the mixture was cooled to 0 ° C and methanol (5
ml), concentrated under reduced pressure, dissolved in chloroform, and washed with 0.5N hydrochloric acid and saturated saline. After the chloroform layer was dried over anhydrous magnesium sulfate, chloroform was distilled off and a new substance, 6-chloro-9- (3-deoxy-2,5-di-O-acetyl-β
-D-ribofuranosyl) -7-deazapurine (compound 14) 2.
00 g was obtained (quantitative).

Reference Example 13. 6-Chloro-7-iodo-9- (3-deoxy-2,5-di-O-acetyl-β-D-ribofuranosyl) -7-
Deazapurine (6-Chloro-7-iodo-9- (3-deoxy-2,5-di-O-
Synthesis of acetyl-β-D-ribofuranosyl) -7-deazapurine) (compound 15). 6-chloro-9- (3-deoxy-2,5-di-O-acetyl-β-D-ribofuranosyl) -7-deazapurine (Compound 14: 1.44 g,
4.07 mmol) was dissolved in acetonitrile (67 ml), cerium (IV) nitrate (IV) (0.62 g, 2.44 mmol) and iodine (1.12 g, 2.03 mmol) were added, and the mixture was stirred at 80 ° C. for 30 minutes. After completion of the reaction, acetonitrile was concentrated under reduced pressure, dissolved in ethyl acetate, and washed with a 5% sodium hydrogen sulfite solution, a saturated sodium hydrogen carbonate solution, and a saturated saline solution. After drying the ethyl acetate layer over anhydrous magnesium sulfate, the ethyl acetate was distilled off and crystallized from methylene chloride-ether to give a new substance 6
-Chloro-7-iodo-9- (3-deoxy-2,5-di-O-acetyl-
β-D-ribofuranosyl) -7-deazapurine (compound 15)
1.46 g was obtained (75.0% yield). Melting point: 149-150 ° C ¹ H-NMR (270 MHz, DMSO-d6) δppm: 2.14, 2.17 (2s, 6
H, 2Ac), 2.23-2.49 (m, 2H, 3'-Ha, b), 4.25- 4.48 (m,
2H, 5'-Ha, b), 4.58-4.68 (m, 1H, 4'-H), 5.52-5.54
(m, 1H, 2'-H), 6.33 (d, 1H, J = 1.4Hz, 1'-H), 7.64
(s, 1H, 8-H), 8.63 (s, 1H, 2-H)

Reference Example 14. 7-Iodo-3'-deoxy-7-deazaadenosine
Synthesis of (Compound 16). 6-chloro-7-iodo-9- (3-deoxy-2,5-di-O-acetyl
-β-D-ribofuranosyl) -7-deazapurine (Compound 1)
5: 1.63 g, 3.40 mmol) and ammonia-methanol (70 m
l) was reacted at 110 ° C. for 20 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, crystallized with methanol, and a new substance, 7-iodo-3′-deoxy-7-deazaadenosine (compound 16), was added.
0 g was obtained (78.8% yield). Melting point: 223-225 ° C (decomposition)

Reference Example 15. 7-Deaza-7- (3 "-trifluoroacetamido-1" -propynyl) -3'-deoxyadenosine
[7-Deaza-7- (3 "-trifluoroacetamido-1" -propynyl) -3'-
deoxyadenosine] (compound 17). 7-Iodo-3'-deoxy-7-deazaadenosine (Compound 1
6: 0.5 g, 1.33 mmol) in a DMF (6.7 ml) solution under a nitrogen atmosphere, N-propargyl trifluoroacetamide (0.47 ml, 3.99 mmol) obtained in Reference Example 2, copper (I) iodide (51 mg, 0.
266 mmol), tetrakis (triphenylphosphine) palladium (0) (154 mg, 0.133 mmol) and triethylamine (0.37 ml, 2.66 mmol) were added and reacted at room temperature for 30 minutes. The reaction mixture was diluted with a methylene chloride-methanol mixture (12 ml), and ion-exchange resin AG1X8 (HCO ₃ ^- type; 1.22 g) was added.
Stirred for 0 minutes. After filtration, the filtrate was concentrated and the resulting residue was purified by silica gel column chromatography (eluent; a mixed solution of methylene chloride and methanol) to give a novel substance 7-deaza-7- (3 "-trifluoroacetamide-1 "-Propynyl)
436 mg of -3'-deoxyadenosine (compound 17) was obtained (82.3% yield). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.83-1.91 (m, 1H,
3'-Ha), 2.14-2.24 (m, 1H, 3'-Hb), 3.48-3.56 (m, 1H,
5'-Ha), 3.66-3.73 (m, 1H, 5'-Hb), 4.30-4.35 (m, 4
H, 2'-H, 4'-H, -CH _2- ), 5.08 (t, 1H, J = 5.5Hz, 5'-O
H), 5.59 (d, 1H, J = 3.8Hz, 2'-OH), 6.02 (d, 1H, J = 2.
2Hz, 1'-H), 6.80 (brs, 2H, NH 2), 7.79 (s, 1H, 8-H),
8.12 (s, 1H, 2-H), 10.08 (brs, 1H, NHTfa)

Reference Example 16.7-Deaza-7- (3 "-amino-1"-
Propinyl) -3'-deoxyadenosine-5'-triphosphate [7-deaza-7- (3 "-amino-1" -propynyl) -3'-deoxyaden
Synthesis of [osine5'-triphosphate] (compound 18). 7-deaza-7- (3 "-trifluoroacetamide-1" -propynyl) -3'-deoxyadenosine (Compound 17: 120 mg, 0.3 m
mol) was dissolved in triethyl phosphate (1.2 ml), cooled to −20 ° C., phosphorus oxychloride (25 μl) was added, and the mixture was stirred at −20 ° C. After 30 minutes, phosphorus oxychloride (22 µl) was added, and the mixture was further stirred for 4 hours. This reaction solution was added to a DMF solution (3.6 ml) of 0.5 M-tris (tri-n-butylammonium) pyrophosphate obtained in Reference Example 4 cooled to −20 ° C., and stirred at room temperature for 3 hours. After completion of the reaction, a mixed solution of triethylamine and water (4 ml) was added, and the mixture was allowed to stand overnight. After that, 25% aqueous ammonia (20 ml) was added, and the mixture was allowed to stand for 4 hours. After the reaction solution was washed with ether, the residue obtained by concentration to dryness was subjected to DEAE-Toyopearl ion exchange column chromatography (1.2 × 30 c.
m; Eluate: triethylammonium bicarbonate buffer (pH
7.5) Purify with 0M → 0.3M linear concentration gradient (1L in total)) and purify the new substance 7-deaza-7- (3 "-amino-1" -propynyl) -3'-deoxyadenosine-5'-triphosphate (Compound 18) 11
4 mg was obtained (39.9% yield).

Embodiment 3 FIG. R6G-labeled 7-deaza-3'-deoxyadenosine-5'-triphosphate (R6G-labeled 7-de
Synthesis of aza-3'-deoxyadenosine 5'-triphosphate). 7-deaza-7- (3 "-amino-1" -propynyl) -3'-deoxyadenosine-5'-triphosphate (compound 18: 8 μmo
l) is dissolved in 1M-triethylammonium hydrogen carbonate buffer solution (pH 9.05; 0.4 ml), and 5-carboxyrhodamine 6G succinimide ester (manufactured by Molecular Probes)
(14.5 mg) in DMF (0.8 ml) was added and stirred at room temperature overnight. Water (30 ml) was added to the reaction solution to dilute it, and then DEAE-
Toyopearl ion exchange column chromatography (1.2
× 30 cm; eluent: purified with triethylammonium hydrogen carbonate buffer (pH 7.5) 0.1 M → 0.6 M linear concentration gradient (total volume 2 L)) and R6G-labeled 7-deaza-3′- represented by the following formula Deoxyadenosine-5'-triphosphate (hereinafter referred to as R6G-3'dA
Abbreviated as TP (n1). 2.54 μmol was obtained (yield 31.7).
%).

[0093]

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[In the formula, Et represents an ethyl group, and Me represents a methyl group. The obtained R6G-3'dATP (n1) was measured for its UV-visible absorption spectrum using a DU640 UV-visible spectroscopic analysis system (manufactured by Beckman Co., Ltd.) (measurement wavelength: 700 nm to 200 nm, symmetric : Distilled water) is shown in FIG.

Reference Example 17.6-Methoxy-2-methylthio-9-
｛2,5-bis (O-tert-butyldimethylsilyl) -3-O-
[(Imidazol-1-yl) thiocarbonyl] -β-D-ribofuranosyl｝ -7-deazapurine (6-methoxy-2-methylt
hio-9- {2,5-bis (O-tert-butyldimethylsilyl) -3-O-[(im
idazol-1-yl) thiocarbonyl] -β-D-ribofuranosyl} -7-de
Synthesis of azapurine) (compound 21). 6-methoxy-2-methylthio-9- (β-D-ribofuranosyl)-
7-Deazapurine (Compound 19: 12.86 g, 39.28 mmol) was added to TH
After dissolving in F (580 ml), pyridine (16.8 ml, 208 mmol), silver nitrate (15.55 g, 91.5 mmol) and tert-butyldimethylsilyl chloride (13.80 g, 91.5 mmol) were added, and the mixture was stirred at room temperature overnight. After completion of the reaction, the precipitate was separated by filtration through Celite, and the filtrate was concentrated, dissolved in chloroform, and washed with 0.2N hydrochloric acid and saturated saline. After the chloroform layer was dried over anhydrous magnesium sulfate, chloroform was distilled off to obtain crude 6-chloroform.
24.0 g of methoxy-2-methylthio-9- [2,5-bis (O-tert-butyldimethylsilyl) -β-D-ribofuranosyl] -7-deazapurine (compound 20) was obtained. (Compound 20) in DMF
(400 ml), 1,1′-thiocarbonyldiimidazole (35.0 g, 196.5 mmol) was added, and the mixture was stirred at room temperature overnight. After completion of the reaction, ethyl acetate and water were added, and the organic layer was washed with saturated saline. The ethyl acetate layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent; ethyl acetate).
-Chloroform mixed solvent) to give a new substance 6-methoxy-2-methylthio-9- {2,5-bis (O-tert-butyldimethylsilyl) -3-O-[(imidazol-1-yl) Thiocarbonyl] -β-D-ribofuranosyl｝ -7-deazapurine (Compound 21) (20.45 g, yield 78.2%).

Reference Example 18.6-Methoxy-2-methylthio-9-
[2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurine (6-methoxy
-2-methylthio-9- [2,5-bis (O-tert-butyldimethylsily
l) Synthesis of 3-deoxy-β-D-ribofuranosyl] -7-deazapurine) (compound 22). 6-methoxy-2-methylthio-9- {2,5-bis (O-tert-butyldimethylsilyl) -3-O-[(imidazol-1-yl) thiocarbonyl] -β-D-ribofuranosyl} -7 -Deazapurine (Compound 21: 20.45 g, 30.7 mmol) in toluene (1 L)
Dissolved in 2,2'-azobis (isobutyronitrile) (1.0
1 g, 6.1 mmol) and hydrogenated n-tributyltin (16.5 ml, 6
1.4 mmol) and stirred at 80 ° C. for 30 minutes under a nitrogen atmosphere. After completion of the reaction, the toluene was distilled off, and the residue was purified by silica gel column chromatography (eluent; a mixed solvent of ethyl acetate-n-hexane) to give a novel substance 6-methoxy-2-methylthio-9- [2,5-bis ( O-tert-butyldimethylsilyl) -3
-Deoxy-β-D-ribofuranosyl] -7-deazapurine (Compound 22) (14.57 g) was obtained (yield: 87.9%).

Reference Example 19.7-Iodo-6-methoxy-2-methylthio-9- [2,5-bis (O-tert-butyldimethylsilyl)
-3-deoxy-β-D-ribofuranosyl] -7-deazapurine (7-iodo-6-methoxy-2-methylthio-9- [2,5-bis (O-tert-
butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-
deazapurine) (compound 23). 6-methoxy-2-methylthio-9- [2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurine (Compound 22: 15.57 g, 28.84 mmo)
l) was dissolved in DMF, and N-iodosuccinimide (7.79 g, 3
4.61 mmol), and the mixture was stirred at room temperature for 5 hours while shielding light under a nitrogen atmosphere. After completion of the reaction, the reaction solution was cooled to 0 ° C., ethyl acetate and water were added, and the organic layer was washed with a 5% sodium thiosulfate solution, a saturated sodium hydrogen carbonate solution, and a saturated saline solution. After drying the ethyl acetate layer over anhydrous magnesium sulfate,
Ethyl acetate was distilled off, and the residue was purified by silica gel column chromatography (eluent; mixed solvent of chloroform and n-hexane) to give a novel substance, 7-iodo-6-methoxy-2-methylthio-9.
19.06 g of-[2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurine (compound 23) was obtained (99.3% yield). ¹ H-NMR (270MHz, DMSO-d6) δppm: -0.10, -0.06, 0.1
2, 0.13 (4s, 12H, 4MeSi), 0.80, 0.93 (2s, 18H, 2t-
Bu), 1.90-2.10 (m, 1H, 3'-Ha), 2.18-2.28 (m, 1H, 3 '
-Hb), 2.54 (s, 3H, SMe), 3.72 (dd, 1H, J = 2.7, 11.6
Hz, 5'-Ha), 3.94 (dd, 1H, J = 2.2, 11.6Hz, 5'-Hb), 4.
03 (s, 3H, OMe), 4.32-4.48 (m, 2H, 2'-H, 4'-H), 6.
07 (d, 1H, J = 3.0Hz, 1'-H), 7.59 (s, 1H, 8-H)

Reference Example 20.7 7-Iodo-2-methylthio-9-
[2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurin-6-one (7-
iodo-2-methylthio-9- [2,5-bis (O-tert-butyldimethyls
ilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurine-6-
one) Synthesis of (compound 24). After dissolving 4-thiocresol (3.36 g, 27.0 mmol) in methanol, sodium methoxide (1.61 g, 29.7 mmol) was added, and the mixture was stirred for 5 minutes, and then methanol was distilled off. To this, 7-iodo-6-methoxy-2-methylthio-9- [2,5-bis (O-tert-butyldimethylsilyl) dissolved in toluene (150 ml)
-3-deoxy-β-D-ribofuranosyl] -7-deazapurine (compound 23: 4.00 g, 6 mmol) and hexamethylphosphoramide (10 ml, 57.1 mmol) were added, and under a nitrogen atmosphere,
Refluxed for 4.5 hours. After completion of the reaction, ethyl acetate and water were added, and the organic layer was washed with saturated saline. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (eluent: methylene chloride).
-Methanol mixed solvent) to purify the new substance 7-iodo-
2.35 g of 2-methylthio-9- [2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurin-6-one (compound 24) was obtained ( Yield 59.9
%). ¹ H-NMR (270MHz, DMSO-d6) δppm: 0.06, 0.08, 0.20,
0.24 (4s, 12H, 4MeSi), 0.92, 1.03 (2s, 18H, 2t-Bu),
2.07-2.13 (m, 1H, 3'-Ha), 2.34-2.38 (m, 1H, 3'-H
b), 2.63 (s, 3H, SMe), 3.81-4.05 (m, 2H, 5'-Ha, b),
4.46-4.57 (m, 2H, 2'-H, 4'-H), 6.12 (d, 1H, J = 3.0H
z, 1'-H), 7.47 (s, 1H, 8-H), 12.44 (s, 1H, 1-H)

Reference Example 21.7-Iodo-2 ', 5'-bis (O-ter
t-butyldimethylsilyl) -3'-deoxy-7-deazaguanosine (7-iodo-2 ', 5'-bis (O-tert-butyldimethylsily
l) Synthesis of -3'-deoxy-7-deazaguanosine) (compound 25). 7-iodo-2-methylthio-9- [2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl]-
7-deazapurin-6-one (Compound 24: 2.30 g, 3.53 mmo
l) was dissolved in methylene chloride (90 ml), and cooled to 0 ° C.
Add chloroperbenzoic acid (0.96 g, 3.88 mmol) and add
The mixture was stirred for 1 minute and further at room temperature for 1 hour. After the reaction,
The reaction solution was washed with a saturated sodium hydrogen carbonate solution and a saturated saline solution, dried over anhydrous magnesium sulfate, and then methylene chloride was distilled off. The obtained residue is subjected to silica gel column chromatography (eluent; mixed solvent of chloroform and methanol).
2.02 g of 7-iodo-2-methylsulfonyl-9- [2,5-bis (O-tert-butyldimethylsilyl) -3-deoxy-β-D-ribofuranosyl] -7-deazapurin-4-one (Yield 83.
9%). This is dissolved in 1,4-dioxane (20 ml),
After cooling to -78 ° C, add liquid ammonia (60ml) and add
For 6 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure,
The obtained residue was purified by silica gel column chromatography (eluent; methylene chloride-methanol mixed solvent) to give a novel substance, 7-iodo-2 ', 5'-bis (O-tert-butyldimethylsilyl) -3'-. 1.60 g of deoxy-7-deazaguanosine (compound 25) was obtained (yield: 73.1%).

Reference Example 22.3-Deoxy-7-iodo-7-deazaguanosine
Synthesis of (Compound 26). 7-iodo-2 ', 5'-bis (O-tert-butyldimethylsilyl)
-3'-deoxy-7-deazaguanosine (Compound 25: 1.6 g,
2.58 mmol) was dissolved in THF (50 ml), and 1M-tetrabutylammonium fluoride (6.2 ml, 6.17 mmol) was added.
Stirred overnight at room temperature. After completion of the reaction, the reaction solution is concentrated under reduced pressure, purified by silica gel column chromatography (eluent; chloroform-methanol mixed solvent), crystallized with methanol, and a novel substance 3′-deoxy-7-iodo-7-deazaguanosine is obtained. (Compound 26) 0.80 g was obtained (yield 79.1)
%). Melting point: 176-178 ° C (decomposition) ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.81-1.89 (m, 1H,
3'-Ha), 2.10-2.20 (m, 1H, 3'-Hb), 3.43-3.64 (m, 2
H, 5'-Ha, b), 4.18-4.29 (m, 2H, 2'-H, 4'-H), 4.91
(t, 1H, J = 5.5Hz, 5'-OH), 5.46 (d, 1H, J = 4.3Hz, 2'-OH)
OH), 5.80 (d, 1H, J = 2.4Hz, 1'-H), 6.30 (brs, 2H, 2-
NH ₂ ), 7.10 (s, 1H, 8-H), 10.45 (brs, 1H, 1-H)

Reference Example 23.3 3'-deoxy-7- (3 "-trifluoroacetamido-1" -propynyl) -7-deazaguanosine (3'-deoxy-7- (3 "-trifluoroacetamido-1") -propynyl)
Synthesis of -7-deazaguanosine) (compound 27). 3'-deoxy-7-iodo-7-deazaguanosine (compound 2
6: 0.65 g, 1.7 mmol) in a DMF (8.5 ml) solution under a nitrogen atmosphere, N-propargyltrifluoroacetamide (0.58 ml, 5.0 mmol) obtained in Reference Example 2, copper (I) iodide (63 mg, 0.3
3 mmol), tetrakis (triphenylphosphine) palladium (0) (192 mg, 0.17 mmol) and triethylamine (0.46 ml, 3.31 mmol) were added and reacted at room temperature for 1 hour. The reaction solution was diluted with a methylene chloride-methanol mixture (16 ml), ion-exchange resin AG1X8 (HCO ₃ ^- type; 1.6 g) was added, and the mixture was stirred for 30 minutes. After filtration, the filtrate was concentrated and the resulting residue was purified by silica gel column chromatography (eluent; mixed solution of chloroform and methanol) to give a novel substance 3'-deoxy-
485 mg of 7- (3 "-trifluoroacetamide-1" -propynyl) -7-deazaguanosine (compound 27) was obtained (yield 70.5).
%). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.81-1.91 (m, 1H,
3'-Ha), 2.10-2.21 (m, 1H, 3'-Hb), 3.42-3.68 (m, 2H,
5'-Ha, b), 4.22-4.30 (m, 4H, 2'-H, 4'-H, -CH _2- ),
4.94 (t, 1H, J = 5.5Hz, 5'-OH), 5.49 (d, 1H, J = 4.3H
z, 2'-OH), 5.81 (d, 1H, J = 2.4Hz, 1'-H), 6.32 (brs,
2H, 2-NH ₂ ), 7.27 (s, 1H, 8-H), 10.05 (brs, 1H, NH
Tfa), 10.50 (brs, 1H, 1-H)

Reference Example 24.7-Deaza-7- (3 "-amino-1"-
Propinyl) -3'-deoxyguanosine-5'-triphosphate (7-deaza-7- (3 "-amino-1" -propynyl) -3'-deoxygua
Synthesis of nosine-5'-triphosphate (compound 28). 3'-deoxy-7- (3 "-trifluoroacetamido-1" -propynyl) -7-deazaguanosine (Compound 27: 125 mg, 0.3
mmol) in triethyl phosphate (1.25 ml),
After cooling to -20 ° C, add phosphorus oxychloride (25 µl)
With stirring. After a lapse of 30 minutes, phosphorus oxychloride (22 µl) was added, and the mixture was further stirred overnight. This reaction solution was added to a DMF solution (3.6 ml) of 0.5 M-tris (tri-n-butylammonium) pyrophosphate obtained in Reference Example 4 cooled to −20 ° C., and stirred at room temperature for 3 hours. After the completion of the reaction, a mixed solution of triethylamine and water (4 ml) was added, and the reaction solution was washed with ether, and then subjected to DEAE-Toyopearl ion exchange column chromatography (1.2 × 30 cm; eluent: triethylammonium hydrogen carbonate buffer (pH 7.5)). ) 0M → 0.6M linear concentration gradient (total amount 2
L)). To this was added 25% aqueous ammonia (15 ml), and the mixture was allowed to stand for 1 hour.
Purification by AE-Toyopearl ion exchange column chromatography (1.2 × 30 cm; eluent: triethylammonium bicarbonate buffer (pH 7.5) 0M → 0.3M linear concentration gradient (total volume 1 L)), a new substance 7-deaza 75 mg of -7- (3 "-amino-1" -propynyl) -3'-deoxyguanosine-5'-triphosphate (compound 28) was obtained (yield 25.9%).

Embodiment 4 FIG. R110-labeled 7-deaza-3′-deoxyguanosine-5′-triphosphate (R110-labeled 7-d
Synthesis of eaza-3'-deoxyguanosine-5'-triphosphate). 7-deaza-7- (3 "-amino-1" -propynyl) -3'-deoxyguanosine-5'-triphosphate (compound 28: 10.5μ)
mol) and 5-carboxyrhodamine 110-bis-trifluoroacetate succinimide ester (Molecular probe (31.5 mg) dissolved in DMF (0.49 ml) -water (0.23 ml), triethylamine (0.16 ml) and pyridine (0.29
ml) and stirred overnight. The reaction mixture was diluted with water (30 ml) and diluted with DEAE-Toyopearl ion exchange column chromatography (1.2 × 30 cm; eluent: triethylammonium hydrogen carbonate buffer (pH 7.5) 0.1 M → 0.8 M linear concentration gradient ( Purified in a total volume of 1 L)) to give R110-
Labeled 7-deaza-3'-deoxyguanosine-5'-triphosphate (hereinafter abbreviated as R110-3'dGTP (n1)) 3.67
μmol was obtained (34.9% yield).

[0104]

Embedded image

Further, the obtained R110-3′dGTP (n1) was
FIG. 4 shows the results of measuring an ultraviolet-visible absorption spectrum (measurement wavelength: 700 nm to 200 nm, symmetry: distilled water) using a DU640 ultraviolet-visible spectral analysis system (manufactured by Beckman Co., Ltd.).

Reference Example 25.5-trifluoroacetamide-1
Synthesis of -pentyne (5-trifluoroacetamido-1-pentyne) DM of sodium hydride (60% oily; 5.99 g, 0.15 mol)
Trifluoroacetamide (19,2 g, 0.17 m
ol) was added in 10 portions under ice-cooling. Then sodium iodide (20.4 g, 0.136 mol) was added, followed by 5-chloro-1
A solution of -pentyne (13.97 g, 0.136 mol) in DMF (50 ml) was added, and the mixture was stirred and reacted at room temperature for 4.5 hours and then at 60 ° C for 21 hours. After cooling the reaction solution, potassium dihydrogen phosphate (59.2
g) Aqueous solution (500 ml) was added and the solution was extracted with ether (500 ml). The ether layer was washed with water, dried over magnesium sulfate and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent; hexane-ethyl acetate mixed solvent) to give 5-trifluoroacetamide -1
-17.6 g of pentyne was obtained (67% yield). ^{1 H-NMR (270MHz, CDCl} 3) δppm: 1.83 (m, 2H, -CH 2 C H 2 CH 2
-), 2.04 (t, 1H, J = 2.7Hz, H-CC-), 2.31 (dt, 2H, J = 2.
7, 6.6Hz, -CCCH _2- ), 3.52 (q, 2H, J = 6.7Hz, CH ₂ N), 6.
88 (brs, 1H, NHTfa)

Reference Example 26.3'-Deoxy-5- (5 "-trifluoroacetamido-1" -pentynyl) cytidine (3'-deoxy
-5- (5 "-trifluoroacetamido-1" -pentynyl) cytidine)
Synthesis of (Compound 29) 3′-Deoxy-5-iodocytidine (Compound 6: 777 mg, 2.20 mmol) obtained in the same manner as in Reference Example 3 was obtained in Reference Example 2 under a nitrogen atmosphere in a DMF (11 ml) solution. 5-trifluoroacetamide-1-pentyne (1.18 g, 6.60 mmol), copper (I) iodide
(83.8 mg, 0.44 mmol), tetrakis (triphenylphosphine) palladium (0) (254 mg, 0.22 mmol) and triethylamine (0.613 ml, 4.4 mmol) were added and reacted at room temperature for 30 minutes. The reaction mixture methylene chloride - diluted with methanol mixture (20ml) ion exchange resin AG1X8 (Biorad Corporation; HCO ₃ ^-; 2.
02g) and stirred for 30 minutes. After filtration, the filtrate was concentrated and the resulting residue was subjected to silica gel chromatography (eluent;
Purified with a mixed solution of chloroform and methanol) and crystallized from a mixed solution of methanol and ether to give a novel substance, 3'-deoxy-5- (5 "-trifluoroacetamide-1" -pentynyl)
409 mg of cytidine was obtained (46.0% yield). Melting point 191-193 ° C ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.63-1.78 (m, 3H, 3 '
-Ha and CH ₂ C H ₂ CH ₂ N), 3.51-3.82 (m, 2H, 5'-Ha, b), 4.1
2-4.31 (m, 2H, 2'-H and 4'-H), 5.15 (t, 1H, J = 5.1Hz,
5'-OH), 5.51 (d, 1H, J = 4.1Hz, 2'-OH), 5.62 (s, 1H,
1'-H), 6.69 (brs, 1H, 4-NHa), 7.64 (brs, 1H, 4-NHb),
8.30 (s, 1H, 6-H), 9.50 (brs, 1H, NHTfa)

Reference Example 27.5- (5 "-amino-1" -pentynyl) -3'-deoxycytidine-5'-triphosphate (5-
(5 "-amino-1" -pentynyl) -3'-deoxycitydine-5'-triphos
Synthesis of 3'-deoxy-5- (5 "-trifluoroacetamido-1" -pentynyl) cytidine (121 mg, 0.3 mmol) in triethyl phosphate
(1.21 ml), cooled to -20 ° C, added phosphorus oxychloride (25 µl), and stirred at -20 ° C. After a lapse of 30 minutes, phosphorus oxychloride (22 µl) was added, and the mixture was further stirred for 5 hours.
This reaction solution was cooled to −20 ° C. and tris (tri-n-butylammonium) pyrophosphate obtained in Reference Example 27 was cooled to
It was added to a 0.5 M DMF solution (3.6 ml) and stirred at room temperature for 2 hours. After the reaction, triethylamine-water mixture (5 ml)
And then left overnight, add 25% aqueous ammonia (20 ml) and add
Let stand for hours. The reaction mixture was washed with ether and concentrated to dryness. The residue obtained was subjected to DEAE-Toyopearl ion exchange column chromatography (manufactured by Tosoh Corporation; 1.7 × 30 cm; eluent:
Triethylammonium hydrogen carbonate buffer (pH 7.5) 0M → 0.3
Purification by M linear concentration gradient (total volume 1L)), new substance 5- (5 "-
105 mg of amino-1 "-pentynyl) -3'-deoxycytidine-5'-triphosphate was obtained (yield 36.6%).

Embodiment 5 FIG. XR-labeled 3'-deoxycytidine
-5'-triphosphate (XR-labeled 3'-deoxycitydine-
Synthesis of 5'-triphosphate) (the compound in which R is an n-propylene group in the general formula [I]) 5- (5 "-amino-1" -pentynyl) -3'-deoxycytidine-
DMF (300 μl) in which 5′-triphosphate (8 μmol) was dissolved
l)-water (300 μl) mixed with triethylamine (10 μl) and 5-
Carboxy-X-rhodamine succinimide ester (manufactured by Molecular Probes) (26.9 μmol) was added, and the mixture was stirred at room temperature overnight. Water (30 ml) was added to the reaction solution to dilute it.
AE-Toyopearl ion exchange column chromatography
(1.7 × 15 cm; eluent: Triethylammonium hydrogen carbonate buffer (pH 7.5) 0.1 → 0.7 M linear concentration gradient (total volume 1 L)), and XR-labeled 3′-deoxycytidine represented by the following formula:
-5'-triphosphate (a compound in which R is a straight-chain alkylene group having 3 carbon atoms in the general formula [I]; hereinafter, XR-3 '
Abbreviated as dCTP (n3). 6.28 μmol was obtained (78.5 yield).
%).

[0110]

Embedded image

Further, the obtained XR-3′dCTP (n3) was
DU640 UV-visible spectroscopy system (Beckman Co., Ltd.)
FIG. 5 shows the results of measuring the ultraviolet-visible absorption spectrum of the sample (measured wavelength: 700 nm to 200 nm, target: distilled water).

Experimental Example 1 Evaluation of the incorporation rate of various fluorescently labeled 3'-deoxy terminators This evaluation was performed according to Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4076.
-4080, June 1989. [Template DNA] pBSKS (+) / pvuII (Stratagene) [Terminator]-TMR-3'dUTP (n1) (compound of Example 1)-XR-3'dCTP (n1) (compound of Example 2) R6G-3'dATP (n1) (compound of Example 3) R110-3'dGTP (n1) (compound of Example 4) [Operation method] 1 μg of template DNA is used as each terminator, guanosine-5'-triphosphate. (RGTP), uridine
5'-triphosphate (rUTP), adenosine-5'-triphosphate (rATP), cytidine-5'-triphosphate (rCTP), MgCl _2, spermidine - (HC
l) ₃ , dithiothreitol (DTT), Tris / HCl (pH
7.5) and T7 RNA polymerase were added to a sample tube containing the final concentration under the following conditions, and the total volume was adjusted to 10 μl with distilled water to prepare a sample.
<Conditions> Terminator TMR-3'dUTP (n1): 10 µM R6G-3'dATP (n1): 20 µM R110-3'dGTP (n1): 1 µM XR-3'dCTP (n1): 1 µM rUTP: 500 µM rATP: 250 μM rGTP: 500 μM rCTP: 250 μM MgCl ₂ : 5 to 10 mM Spermidine- (HCl) ₃ : 2 mM DTT: 10 mM Tris / HCl (pH 7.5): 40 mM T7 RNA polymerase: 25 units / 10 μl

After the sample was heated at 37 ° C. for 1 hour, the terminator which was not incorporated by gel filtration was removed.
The precipitate was collected by centrifugation. The precipitate is then
μl formamide dye (95% formamide containing 10 mM
DTA) and heated at 80 ° C. for 5 minutes. After cooling, 1.5 μl was applied to a 4% sequence gel, electrophoresis was performed for 5 hours using an ABI377 fluorescence automatic sequencer (manufactured by ABI), and the final fluorescence signal was detected on the gel image of the sequencer.

[Results] As a result of the detection, it was found that a fluorescent signal capable of identifying the target nucleic acid sequence was obtained for each of the terminators. In other words, it was found that any of the fluorescently labeled terminators of the present invention had excellent properties as a terminator for a chain termination method using RNA polymerase. Also, XR-3'dCT
Instead of 1 μM of P (n1), 1 μM of XR-3′dCTP (n3)
When the final fluorescent signal was detected in the same manner as described above, except that was used, the same fluorescent signal as that of XR-3'dCTP (n1) was obtained for XR-3'dCTP (n3). From this, XR-3'dCTP (n3) is also XR-3'dCTP (n3).
Like TP (n1), it was found to have excellent properties as a terminator for the chain termination method using RNA polymerase.

[0115]

Industrial Applicability The present invention provides a fluorescently labeled 3'-deoxyribonucleotide derivative which is useful, for example, for safely and sensitively determining a target nucleotide sequence in a nucleic acid utilizing an RNA polymerase action. When the chain termination method using RNA polymerase is performed using the fluorescently labeled 3′-deoxyribonucleotide derivative of the present invention, an excellent effect is obtained in that the nucleotide sequence of a nucleic acid can be determined easily and in a short time. This is an extremely large invention that contributes to the industry.

[0116]

[Brief description of the drawings]

FIG. 1 shows the results of measuring the ultraviolet-visible absorption spectrum of TMR-labeled 3′-deoxyuridine-5′-triphosphate (n1) obtained in Example 1.

FIG. 2 shows the results of measuring an ultraviolet-visible absorption spectrum of XR-labeled 3′-deoxycytidine-5′-triphosphate (n1) obtained in Example 2.

FIG. 3 shows R6G-labeled 7-deaza-3′- obtained in Example 3.
3 shows the results of measuring the ultraviolet-visible absorption spectrum of deoxyadenosine-5′-triphosphate (n1).

FIG. 4 shows R110-labeled 7-deaza-3′- obtained in Example 4.
3 shows the results of measuring the ultraviolet-visible absorption spectrum of deoxyguanosine-5′-triphosphate (n1).

FIG. 5 shows the results of measuring an ultraviolet-visible absorption spectrum of XR-labeled 3′-deoxycytidine-5′-triphosphate (n3) obtained in Example 5.

 ──────────────────────────────────────────────────の Continued on the front page (72) Kaori Ozawa 6-1 Takada-cho, Amagasaki-shi, Hyogo Wako Pure Chemical Industries, Ltd. Osaka Research Laboratory Co., Ltd. No. 1 Wako Pure Chemical Industries, Ltd. Osaka Research Laboratories (72) Inventor Takumi Takumi 6-1, Takadacho, Amagasaki City, Hyogo Prefecture Wako Pure Chemical Industries Co., Ltd. Osaka Research Laboratory

Claims (1)

[Claims] 1. A 3′-deoxyribonucleotide derivative represented by the following general formula [I]. Embedded image [Wherein Q represents a 3′-deoxyribonucleotide residue;
R is a lower alkylene group, W represents a carboxyl group, X is -O -, - S -, - NR'- and -CH ₂ -, and each represents (wherein, R 'is a hydrogen atom, a lower alkyl group , An aralkyl group or an aryl group.). In addition, one of ring A and ring B is And the other is (Where Z is ＯO or ＮN ⁺ R ¹ R
² , Y represents —OH or —NR ¹ R ² , respectively, and R ¹ and R
² each independently represents a hydrogen atom or a lower alkyl group. Further, R ¹ and R ² both represent a trimethylene group, and the other end is —NR ¹ R ² or ＮN ⁺ R ¹ R ² on ring A or ring B.
May be bonded to carbon atoms on both sides of the carbon atom to which is bonded. ), Ring C In the above, a broken line means a bond at a position corresponding to the structure of Ring A and Ring B. Further, each of the rings and the benzene ring may further have a substituent. ]