JPH06345794A - Nucleoside or nucleotide derivative - Google Patents

Abstract

PURPOSE:To provide the novel compound capable of introducing the arbitrary number of nonradiative labels to the arbitrary positions of an oligomer and useful as a DNA-diagnosing medicine, etc., without deteriorating the original functions of a nucleic acid, etc. CONSTITUTION:A compound of formula I [B is purine, pyrimidine; R<1> is H, hydroxyl group-protecting group, phosphoric acid residue, diphosphoric acid residue, triphosphoric acid residue; either of R<2>, R<3> is H, a hydroxyl group- protecting group; the other is (CH2)nR<4> (n is >=3; R<4> is a nonradiative label having a phenolic hydroxyl group or carboxyl group bonded through 0)]. For example, a compound of formula II. The compound of formula I is obtained e.g. by reacting a compound of formula III [either of R<5>, R<6> is H, a hydroxyl group-protecting group; the other is (CH2)nX (X is halogen)] with a non-radiating label having a phenolic hydroxyl group or carboxyl group in the presence of a base such as sodium carbonate in a solvent such as THF.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a modified DNA in which an arbitrary number of non-radioactive labels are introduced at arbitrary positions in an oligomer, which are necessary for use in the development of, for example, DNA diagnostic agents.
The present invention provides nucleoside and nucleotide derivatives which are important in the synthesis of and is widely used in the pharmaceutical industry.

[0002]

2. Description of the Related Art In recent years, with the development of molecular biology,
Research on diagnostic agents using the so-called DNA probe method has been actively conducted. In these fields, regardless of organic chemical synthesis or biochemical synthesis, not only natural type DNA but also DNA with specific modification, that is, DNA having thiol or amino linker or non-isotopic label introduced is a research material. It is being used extensively as, and the demand for it is increasing rapidly. Correspondingly, in the field of synthetic organic chemistry,
Synthetic studies have been carried out on various nucleoside derivatives that can be used as raw materials for synthesizing modified DNA. That is, it is a synthetic study of a nucleoside in which a linker or a non-isotopic label is introduced into the nucleic acid base portion, and its representative results can be found in the following documents and the like. JP. Roduit, et al. Nucleosides , Nucleotides , 6 , 3
49 (1987) J. Haralambidis, et al. Nucleic Acids Res ., 15 , 48
57 (1987) Institut Pasteur, WO-88 / 00593 AF Cook, et al. Nucleic Acids Res ., 16 , 4077 (19
88) A. Roget, et al. Nucleic Acids Res ., 17 , 7643 (198
9) Co-Ori Ori Interstitial Society Anonym, Published Patent Publication No. 3-5
05209 These achievements have made a great contribution to the field of molecular biology by providing a novel modified DNA. However, since the nucleoside derivatives described in the above-mentioned documents require expensive halogenated or thiolated nucleosides or nucleobases in their synthetic routes, modified D synthesized with them is required.
NA is also expensive, and it cannot be denied that the introduction of a linker or a non-radioactive label into the nucleic acid base portion may cause a change in functions such as hydrogen bonding of the nucleic acid base.

Other researchers have developed a method for introducing a non-radioactive label or the like into a specific portion of the furanose ring constituting a nucleoside, instead of the nucleic acid base portion, and the contents thereof are disclosed in the following publication. There is. California Institute of Technology, Published Patent Publication No. 4-
60600 Hoechst Actel Sergeft, JP-A-4-29089
6 However, these derivatives are structurally characterized in that the hydroxyl group of the furanose ring is converted into an amino group or a thiol group and do not include a linker (spacer) portion. Therefore, these derivatives satisfy the problem that the modified DNA having a long-chain linker has a higher double-strand forming ability, as claimed by J Haralambidis et al. (Nucleic Acids Res., 15, 4857 (1987)). It does not give an answer.

In the nucleoside derivative which is required in order to provide a modified DNA whose demand is rapidly increasing in the field of molecular biology, the inventors of the present invention use a readily available natural nucleoside as a starting material and Without
Succeeded in developing a method for synthesizing a derivative in which a non-radioactive label is bound to a specific portion of a furanose ring constituting a nucleoside via a thiol linker or an amino linker, or either linker, and a synthetic intermediate thereof,
We previously filed two applications for the invention (filed on March 9, 1993).

[0005]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The inventors of the present invention have proposed a modified D for which the demand is rapidly increasing in the field of molecular biology.
The research was conducted with the object of synthesizing the nucleoside and nucleotide derivatives necessary for providing NA more easily and cheaply than those previously developed.

[0006]

[Means for Solving the Problems] The inventors of the present invention can further easily and inexpensively synthesize a nucleoside or a nucleotide derivative in which a non-radioactive label is bound to a specific portion of the furanose ring constituting the nucleoside, not to the nucleobase portion. And succeeded in completing the present invention. That is, the present invention relates to a nucleoside or nucleotide derivative represented by the following formula [I].

[0007]

[Chemical 2]

However, B in the formula is a purine or pyrimidine base, R ¹ is a hydrogen atom, a protective group for a hydroxyl group, a phosphoric acid residue, a diphosphoric acid residue or a triphosphoric acid residue, and R ² , R ³ One is a hydrogen atom or a hydroxyl-protecting group, and the other is (C
H ₂ ) _n R ⁴ and n represents any integer of 3 or more, and R ⁴
Represents a non-radioactive label having a phenolic hydroxyl group or a carboxyl group bonded via an oxygen atom.

Method for synthesizing the derivative represented by the formula [I] The nucleoside represented by the formula [I] or the nucleotide derivative is, for example, a nucleoside represented by the following formula [II]:
It can be synthesized by a nucleophilic substitution reaction by elimination of hydrogen halide with a non-radioactive label having a phenolic hydroxyl group or a carboxyl group via a nucleotide derivative.

[0010]

[Chemical 3]

However, B and R ¹ in the formula are the same as those in the formula [I], one of R ⁵ and R ⁶ is a hydrogen atom or a hydroxyl-protecting group, and the other is (CH ₂ ) _n X. And X represents a halogen atom.

That is, 3'- and 5'-hydroxyl-protected nucleoside derivatives (in the formula [II], R ¹ and R ⁵ are
A protective group for a hydroxyl group, the purine or pyrimidine residue of B is preferably an adenine residue, a guanine residue, a cytosine residue or a uracil residue, and the halogen atom of X is preferably a bromine atom) and phenolic With a non-radioactive label having a hydroxyl group or a carboxyl group, in a suitable solvent, by reacting a base (however, if the non-radioactive label having a phenolic hydroxyl group or a carboxyl group forms a salt with a base in advance No additional base is required), and the nucleoside of the formula [I] is synthesized by reacting for several hours to several days. The non-radioactive label having a phenolic hydroxyl group or a carboxyl group bonded via an oxygen atom is not particularly limited in its use as long as it can be used for detection of a DNA probe or the like, but is preferable for the present invention. Those are fluorescein, fluorescein methyl ester or ethyl ester. The solvent used in this reaction may be selected from those which do not interfere with the progress of this reaction. Among them, tetrahydrofuran, 1,5-dioxane, tetrahydrofuran / water, 1,4-dioxane / water, dimethoxyethane, Acetone, N, N-dimethylformamide, dimethylsulfoxide, N, N-dimethylformamide / water mixture and the like are preferred. Examples of the base include alkali metal carbonates or bicarbonates, metal hydroxides, metal hydrides, alkyl metals,
A Grignard reagent or the like can be used, and sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide or tetra-n-butylammonium hydroxide is preferable. Further, as the catalyst, tetra-n-butylammonium bromide or tetra-n-butylammonium hydrogensulfate, tris [2- (2-methoxyethoxy) ethyl] amine, potassium iodide, triphenylphosphine or tributylphosphine, etc. It is effective to add, these catalysts can be used alone or in combination,
The amount to be used is preferably 0.05 equivalent to 2 equivalents. The reaction temperature may be room temperature to the reflux temperature. Examples of the hydroxyl protecting group include a trityl protecting group, a benzyl and allyl protecting group, an acyl protecting group,
Examples of the silyl-type protecting group and the like are preferable for the present invention: 4,4′-dimethoxytrityl group, 4-methoxytrityl group, and tert-butyldimethylsilyl group. Removal of the hydroxyl-protecting group may be carried out by a conventional method,
By performing phosphorylation reaction or triphosphorylation reaction, various nucleosides and nucleotide derivatives represented by the formula [I] can be obtained. In addition, in these reactions, a protecting group can be introduced into the purine or pyrimidine residue by a known method, if necessary. N, which represents the chain length of the alkylene group in R ² and R ³ , represents an arbitrary integer of 3 or more. If n is less than 3, synthesis is almost impossible, and in view of difficulty in obtaining raw materials. N is 2 for the present invention
An integer of 0 or less is preferable.

Utilization method The nucleoside or nucleotide derivative having a non-radioactive label at the 2'- or 3'-hydroxyl group of the present invention has an arbitrary number of non-radioactive labels introduced at any position in an oligomer such as DNA or RNA. However, the known method can be used for introducing into oligomers such as DNA and RNA, and is used for detection of probes. Phosphate triester method, phosphoramidite method, H-phosphonate method, etc. Alternatively, an organic chemical method such as the thiophosphite method and a biochemical method utilizing the action of an enzyme can be exemplified.

[0014]

In the nucleoside or nucleotide derivative having a non-radioactive label on the 2'- or 3'-hydroxyl group of the present invention,
It is possible to introduce an arbitrary number of non-radioactive labels at any position in the oligomer, and a conventional method of introducing it into the nucleobase part, or directly introducing the non-radioactive label into a specific part of the furanose ring constituting the nucleoside. Compared with the method described above, it shows an effect that there is less possibility of impairing the original function of nucleic acid. Further, the derivative of the present invention can be produced using inexpensive fluorescein or its ester without using expensive raw materials such as fluorescein isothiocyanate.

[0015]

[Examples] 2'-provided in the present invention by the following examples
Or a nucleoside having a non-radioactive label on the 3'-hydroxyl group,
Examples of synthesizing nucleotide derivatives will be described, but the present invention is not limited to these examples. The following abbreviations are used in the structural formulas below. That is, Ad = adenine residue, Ur = uracil residue, M
MTr = 4-methoxytrityl group, TBDMS = t-butyldimethylsilyl group.

Example 1 Compound 1 and compound 2 represented by the following formulas were synthesized as follows.

[0017]

[Chemical 4]

[0018]

[Chemical 5]

5'-O-4-methoxytrityl-2'-O- (5-
Bromopentyl) uridine 4.07 g (6.11 mmol) of 1,4-
To a solution of 70 ml of dioxane, 4.59 g (12.2 mmol) of disodium fluorescein and 0.25 g of sodium hydroxide.
(6.25 mmol) and 1.02 g of potassium iodide (6.14 mmo)
A 25 ml solution of l) in water was added dropwise, and the mixture was heated under reflux for 48 hours. After allowing to cool, 5% aqueous citric acid solution was added, extracted with chloroform, and washed with saturated saline. The extract was dried over anhydrous magnesium sulfate, concentrated, and then purified by silica gel column chromatography to obtain two types of tan powder compounds. It was confirmed by NMR and IR analyzes that they were the above compounds 1 and 2, respectively. The yield of compound 1 is 1.
20 g (21%). The yield of compound 2 was 0.49 g (9%). The chemical shifts of ¹ H-NMR, the wave numbers of IR signals and the mobilities of silica gel thin layer chromatography are shown below, and IR charts are shown in FIGS. 1 and 2. Compound 1 ¹ H-NMR (CDCl ₃ ) δ: 0.97-2.03 (6H,
m) 3.23-4.55 (13H, m) 5.25-5.53 (1H, m) 5.73-6.00 (1H, m) 6.42-8.08 (25H, m) IR (KBr) cm ⁻¹ : 3410, 2940, 1770, ¹
710, 1660, 1610, 1460, 1250, 1
180. Silica gel thin layer chromatography Rf: 0.47 (chloroform: methanol = 10: 1) Compound 2 ¹ H-NMR (CDCl ₃ ) δ: 0.67-2.00 (6H,
m) 3.22-4.57 (13H, m) 5.20-5.53 (1H, m) 5.77-6.03 (1H, m) 6.50-8.40 (25H, m) IR (KBr) cm ⁻¹ : 3410, 2930, 1710, ¹
660, 1590, 1460, 1250, 1100. Silica gel thin layer chromatography Rf: 0.39 (chloroform: methanol = 10: 1)

Example 2 A compound 3 represented by the following formula was synthesized as follows.

[0021]

[Chemical 6]

2.80 g of compound 1 synthesized in Example 1
(3.05 mmol) was dissolved in 20 ml of chloroform and formic acid 2.
00 ml (53.0 mmol) was added, the mixture was stirred at room temperature for 2.5 hours, and then washed with water and saturated saline. After drying over anhydrous magnesium sulfate and concentrating, the product was purified by silica gel column chromatography to give a brownish brown compound 0.7.
1 g was obtained (yield 36%). It was confirmed to be the above compound 3 by ¹ H-NMR and IR analysis. ¹ H-NM
The chemical shift of the R signal, the wave number of the IR signal and the mobility of silica gel thin layer chromatography are shown below, and the ¹ H-NMR chart is shown in FIG. ¹ H-NMR (CDCl ₃ -CD ₃ OD) δ: 1.23-2.1
3 (6H, m) 3.50-4.33 (9H, m) 5.68 (1H, d) 5.83 (1H, d) 6.33-6.80 (6H, m) 7.07- 7.33 (1H, m) 7.43-8.07 (4H, m) IR (KBr) cm ^-1 : 3410,2940,1740,1
710, 1660, 1470, 1250, 1190, 1
100. Silica gel thin layer chromatography Rf: 0.51 (chloroform: methanol = 5: 1)

Example 3 A compound 4 represented by the following formula was synthesized as follows.

[0024]

[Chemical 7]

1.25 g of compound 2 synthesized in Example 1
(1.36 mmol) was dissolved in 10 ml of chloroform, and formic acid 1.
00 ml (26.5 mmol) was added, the mixture was stirred at room temperature for 2.5 hours, and then washed with water and saturated saline. After drying over anhydrous magnesium sulfate and concentrating, the product was purified by silica gel column chromatography to give a tan powdery compound 0.3.
3 g was obtained (yield 37%). It was confirmed to be the above compound 4 by ¹ H-NMR and IR analysis. ¹ H-NM
The chemical shift of the R signal, the wave number of the IR signal and the mobility of silica gel thin layer chromatography are shown below, and the ¹ H-NMR chart is shown in FIG. ¹ H-NMR (CDCl ₃ -CD ₃ OD) δ: 0.87-1.8
3 (6H, m) 3.37-4.33 (9H, m) 5.70 (1H, d) 5.87 (1H, d) 6.37-8.37 (11H, m) IR (KBr) cm ⁻¹ : 3390, 1710, 1660, ¹
590, 1460, 1260, 1210, 1100. Silica gel thin layer chromatography Rf: 0.19 (chloroform: methanol = 5: 1)

Example 4 Compound 5 represented by the following formula was synthesized as follows.

[0027]

[Chemical 8]

First, the compounds 6 and 7 represented by the following formulas were prepared as follows.

[0029]

[Chemical 9]

[0030]

[Chemical 10]

Adenosine 6.36 g (23.8 mmol), 1,10-
To 100 ml of a suspension of 35.7 g (119 mmol) of dibromodecane in 100 ml of N, N-dimethylformamide was added 1.00 g (25.0 mmol) of 60% oily sodium hydride at 0 ° C., and the mixture was added at room temperature to 3.5.
After stirring for an hour, the mixture was extracted with chloroform and washed with saturated saline. After drying over anhydrous magnesium sulfate and concentration, purification by silica gel column chromatography was performed.
Two colorless crystalline compounds were obtained. ¹ H-NMR and IR
According to the analysis, compound 6 (yield 31%) and compound 7 respectively
It was confirmed that the yield was 3%. The chemical shifts of ¹ H-NMR signals, the wave numbers of IR signals, and the mobilities of silica gel thin layer chromatography are shown below. Compound 6 ¹ H-NMR (CDCl ₃ -DMSO-d ⁶ ) δ: 0.90
-2.10 (16H, m) 3.17-3.90 (7H, m) 4.07-4.27 (1H, m) 4.33-4.77 (3H, m) 5.98 (1H , D) 6.83-7.20 (2H, br) 8.10 (1H, s) 8.15 (1H, s) IR (KBr) cm ^-1 : 3330, 3160, 2930, 2
850, 1670, 1600, 1330, 1300, 1
090. Silica gel thin layer chromatography Rf: 0.33 (chloroform: methanol = 10:
1) Compound 7 ¹ H-NMR (CDCl ₃ -DMSO-d ⁶ ) δ: 1.00
-2.10 (16H, m) 3.17-4.33 (9H, m) 4.67-5.03 (2H, m) 5.88 (1H, d) 6.73-7.17 (2H) , Br) 8.06 (1H, s) 8.13 (1H, s) IR (KBr) cm ⁻¹ : 3320, 3170, 2930, 2
850, 1690, 1610, 1340, 1300, 1
100. Silica gel thin layer chromatography Rf: 0.25 (chloroform: methanol = 10:
1)

Then, the compound 8 represented by the following formula was prepared as follows.

[0033]

[Chemical 11]

Fluorescein disodium 5.10 g (1
3.6 mmol) and tetra-n-butyl hydrogensulfate 4.60 g (1
3.5 mmol of isopropyl alcohol-water (1: 1) 6
Diethyl sulfate (3.60 ml, 27.5 mmol) was added to the 0 ml solution, and the mixture was stirred at room temperature for 2 hours and then potassium carbonate (3.80 g).
(27.5 mmol) and diethyl sulfate 2.70 ml (27.5 mmo)
l) was added. After stirring for 45 minutes, the mixture was extracted with chloroform and washed with a 10% aqueous citric acid solution and saturated saline. After drying over anhydrous magnesium sulfate and concentration, purification by silica gel column chromatography and recrystallization from acetone were performed to obtain 1.30 g of a reddish brown crystalline compound (yield 27%). It was confirmed to be compound 8 by ¹ H-NMR, ultraviolet-visible absorption spectrum and IR analysis. Chemical shift of ¹ H-NMR signal, IR
The wave number of the signal, the absorption wavelength of the UV-visible absorption spectrum and the mobility of silica gel thin layer chromatography are shown below, and the charts of ¹ H-NMR and the UV-visible absorption spectrum are shown in FIGS. 5 and 6. ¹ H-NMR (CDCl ₃ -DMSO-d ⁶ ) δ: 0.93
(3H, t) 3.97 (2H, q) 6.50-7.87 (9H, m) 8.07-8.27 (1H, m) IR (KBr) cm ⁻¹ : 1720, 1640, 1590 , 1
460, 1270, 1210, 1110. UV-visible absorption spectrum (methanol) λmax: 2
29, 498. Silica gel thin layer chromatography Rf: 0.37 (chloroform: methanol = 10:
1)

1.5 of compound 6 prepared as described above
6 g (3.21 mmol), 1.74 g of compound 8 (4.83 mmo)
l), triphenylphosphine 0.84 g (3.21 mmol) and tetra-n-butylammonium bromide 0.10 g (0.32 m)
2.22 g (16.1 mmol) of potassium carbonate was added to a suspension of (mol) N, N-dimethylformamide in 15 ml, and the mixture was stirred at 90 ° C. for 24 hours and then allowed to cool. A 10% aqueous sodium carbonate solution was added, extracted with chloroform, and washed with saturated saline. After drying over anhydrous magnesium sulfate and concentrating, the residue is dissolved in 20 ml of dichloromethane and imidazole 0.70 g (10.3 mm) is dissolved.
ol) and t-butyldimethylsilyl chloride 1.50 g (9.9
6 mmol) was added and the mixture was stirred at room temperature for 24 hours. Then, the extract was washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel column chromatography to obtain a brownish brown powdery compound 1.
11 g was obtained (yield 35%). ¹ H-NMR and IR
By analysis, it was confirmed to be the above compound 5. ¹ H-
The chemical shift of the NMR signal, the wave number of the IR signal and the mobility of silica gel thin layer chromatography are shown below, and the ¹ H-NMR chart is shown in FIG. 7. ¹ H-NMR (CDCl ₃ ) δ: 0.12 (12H, s) 0.83-2.15 (37H, m) 3.37-4.68 (11H, m) 5.77-6.18 ( 3H, m) 6.38-7.00 (6H, m) 7.22-7.78 (3H, m) 8.00-8.37 (3H, m) IR (KBr) cm ^-1 : 3330, 2930, 2860, 1
720, 1640, 1600, 1520, 1470, 1
250. Silica gel thin layer chromatography Rf: 0.51 (chloroform: methanol = 5: 1)

Example 5 A compound 9 represented by the following formula was synthesized as follows.

[0037]

[Chemical 12]

First, the compound 10 represented by the following formula was prepared as follows.

[0039]

[Chemical 13]

25.5 g (71.1 mmol) of 5'-O-t-butyldimethylsilyluridine in 100 ml of tetrahydrofuran
75.0 ml (150 mmol) of 2.0 M t-butylmagnesium chloride / tetrahydrofuran solution was added dropwise to the solution at 0 ° C.,
After stirring for 30 minutes, at the same temperature, 82.0 g of 1,5-dibromopentane
A solution of (357 mmol) in N, N-dimethylformamide (100 ml) was added, and the mixture was heated under reflux for 21 hours. After cooling down, methanol 2
After insoluble matter was dissolved by adding 0 ml, the mixture was extracted with ethyl acetate and washed with 10% aqueous citric acid solution and saturated saline. After drying over anhydrous magnesium sulfate and concentration, purification by silica gel column chromatography was performed to obtain a colorless liquid compound (yield 50%). ¹ H-NMR and I
It was confirmed to be compound 10 by R analysis. ¹ H-
The chemical shifts of NMR signals, the wave numbers of IR signals, and the mobilities of silica gel thin layer chromatography are shown below. ¹ H-NMR (CDCl ₃ ) δ: 0.08 (6H, s) 0.89 (9H, s) 1.33-2.17 (6H, m) 3.20-4.45 (10H, m) 5.60-5.90 (2H, m) 7.82 (1H, d) IR (KBr) cm ^-1 : 3430, 2930, 1710, ¹
660, 1460, 1100. Silica gel thin layer chromatography Rf: 0.53 (chloroform: methanol = 10:
1)

1. of compound 10 obtained as described above.
80 g (3.55 mmol), 1.35 g (3.75 g) of the compound 8
mmol), triphenylphosphine 0.93 g (3.55 mmol)
And tetra-n-butylammonium bromide 0.12 g (0.3
2.45 g of potassium carbonate (17.7 mmol) was added to a suspension of 6 mmol of N, N-dimethylformamide in 15 ml, and the mixture was added at 90 ° C. to 1
After stirring for 0 hour, the mixture was allowed to cool. Next, 1.0 M tetrafluoride
n-Butyl ammonium / tetrahydrofuran solution 5.0 m
l (5.0 mmol) was added, and the mixture was stirred at room temperature for 3.5 hours, extracted with chloroform, and washed with water and saturated brine. The extract was dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel column chromatography to obtain 1.15 g of a light brown powdery compound (yield 48%). It was confirmed to be the above compound 9 by ¹ H-NMR and IR analysis.
The chemical shift of the ¹ H-NMR signal, the wave number of the IR signal and the mobility of silica gel thin layer chromatography are shown below, and the IR chart is shown in FIG. 8. ¹ H-NMR (CDCl ₃ -DMSO-d ⁶ ) δ: 0.82
-2.05 (9H, m) 5.63 (1H, d) 5.88 (1H, d) 6.20-8.33 (11H, m) IR (KBr) cm ^-1 : 3390, 2940, 1710 , 1
660, 1600, 1500, 1460, 1250, 1
210, 1110. Silica gel thin layer chromatography Rf: 0.59 (chloroform: methanol = 5: 1)

Example 6 Compounds 11 and 12 represented by the following formulas were synthesized as follows.

[0043]

[Chemical 14]

[0044]

[Chemical 15]

673 mg of the compound 9 synthesized in Example 5
(1.00 mmol) was dissolved in 5 ml of trimethyl phosphate, and at -10 to -20 ° C, 0.10 ml of phosphorus oxychloride.
(1.07 mmol) was added dropwise, and after stirring at the same temperature for 30 minutes, 0.10 ml (1.07 mmol) of phosphorus oxychloride was further added.
After stirring at room temperature for 50 minutes, the reaction mixture was cooled to -10-
9.0 ml of a 1.0 M pyrophosphate-tris (tri-n-butylammonium) / N, N-dimethylformamide solution at 20 ° C
Was added dropwise. After stirring at room temperature for 2 hours, the reaction mixture was added to a solution of 1.40 ml of triethylamine in 20 ml of water at 0 ° C., stirred for 15 minutes and then left at 4 ° C. for 20 hours.
After extraction with chloroform, the organic layer was mixed with 25 ml of water and 0.1 ml.
5M sodium hydrogen carbonate / sodium carbonate aqueous solution (pH =
9.0) 10 ml and 25 ml of acetone were added and stirred,
The aqueous layer was separated. When the aqueous layer was analyzed by high performance liquid chromatography under the following conditions, the chart shown in FIG. 9 was obtained. Column: ODS-15 Elution solvent: Aqueous methanol solution 20-40% (20
Min) Detection wavelength: 254 nm Chart speed: 2.5 mm / min Peak I and peak II are separately collected and subjected to UV-visible absorption spectrum and ³¹ P-NMR analysis. Peak I is compound 11 and peak II is compound 12 Was confirmed. UV-visible absorption spectrum and ³¹ P-
The chemical shifts of NMR are shown below, and the charts of UV-visible absorption spectra are shown in FIGS. 10 and 11. Compound 11 UV-visible absorption spectrum (H ₂ O) λmax: 229,
254, 454, 477. ³¹ P-NMR (D ₂ O) δ: −22.7, −12.2
-7.4 Compound 12 UV-visible absorption spectrum (H ₂ O) λmax: 228,
253, 454, 477. ³¹ P-NMR (D ₂ O) δ: -11.9, -7.9

[0046]

INDUSTRIAL APPLICABILITY When the nucleoside or nucleotide derivative having a non-radioactive label at the 2'- or 3'-hydroxyl group provided by the present invention is used, the non-radioactive label can be introduced in any number at any position in the oligomer. What you can do, and
Compared with the conventional method of introducing into the nucleobase portion or the method of directly introducing a non-radioactive label into a specific portion of the furanose ring constituting the nucleoside, it has a feature that it is less likely to impair the original function of the nucleic acid. Therefore, its utility value is high. Further, it has the advantage that inexpensive fluorescein or its ester can be used without using expensive raw materials such as fluorescein isothiocyanate and can be manufactured at low cost.

[Brief description of drawings]

FIG. 1 shows IR of compound 1 obtained in Example 1.
chart.

FIG. 2 is an IR of compound 2 obtained in Example 1.
chart.

FIG. 3 shows ¹ H of compound 3 obtained in Example 2.
-NMR chart.

FIG. 4 shows ¹ H of compound 4 obtained in Example 3.
-NMR chart.

FIG. 5 shows ¹ H of compound 8 prepared in Example 4.
-NMR chart.

FIG. 6 is an ultraviolet-visible absorption spectrum chart of the compound 8 prepared in Example 4.

FIG. 7 shows ¹ H of compound 5 obtained in Example 4.
-NMR chart.

FIG. 8 shows IR of compound 9 obtained in Example 5.
chart.

FIG. 9 shows the HPL of the reaction product obtained in Example 6.
C chart.

FIG. 10 is an ultraviolet-visible absorption spectrum chart of the compound 11 obtained in Example 6.

FIG. 10 is an ultraviolet-visible absorption spectrum chart of the compound 12 obtained in Example 6.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // C12Q 1/68 A 7823-4B

Claims (1)

[Claims] 1. A nucleoside or nucleotide derivative represented by the following formula [I]: However, in the formula, B is a purine or pyrimidine base, R ¹ is a hydrogen atom, a protective group for a hydroxyl group, a phosphoric acid residue, a diphosphoric acid residue or a triphosphoric acid residue, and R ² and R ³ are either One is a hydrogen atom or a hydroxyl-protecting group, the other is (CH ₂ ) _n R ⁴ , n is an arbitrary integer of 3 or more, and R ⁴ is a phenolic hydroxyl group or carboxyl bonded through an oxygen atom. A non-radioactive label having a group is shown.