JPH06256381A - Nucleoside derivative - Google Patents

Abstract

PURPOSE:To obtain derivative, having a low possibility for impairing the function essential to nucleic acid and important for synthesizing a modified DNA in which an optional number of thiol linkers are transduced into optional positions of an oligomer. CONSTITUTION:This nucleoside derivative is expressed by formula I [B is purine or pyrimidine; R1 is H, etc.; either of R2 and R3 is H and the other is (CH2)nR4; (n) is an integer of >=3; R4 is thiol which may have a protecting group or a nonradioactive label]. The derivative expressed by formula I is obtained by reacting a derivative, prepared from a ribonucleoside and expressed by formula II (R5 is protecting group of hydroxyl group; B is purine or pyrimidine, preferably adenine, guanine, cytosine or uracil) with a base such as a metallic hydride in an organic solvent such as tetrahydrofuran, adding a compound expressed by the formula X(CH2)nX (X is halogen, preferably Br) and adding N,N- dimethylformamide to the resultant alkoxide, thermally refluxing the obtained mixture for several hr to several days, isolating and purifying the prepared product of formula III and reacting the isolated and purified product with a thiol, a thiocarboxylic acid, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention provides a modified D in which an arbitrary number of thiol linkers are introduced at arbitrary positions in an oligomer, which are required for development of a DNA diagnostic agent.
It provides a nucleoside derivative which is important in the synthesis of NA, and is widely used in the pharmaceutical industry.

[0002]

2. Description of the Related Art In recent years, along with the development of molecular biology, active research has been conducted on diagnostic agents using the so-called DNA probe method. In these fields, regardless of organic chemical synthesis or biochemical synthesis, not only natural type DNA but also DNA with a specific modification, that is, a thiol or amino linker, or a DNA with a non-isotopic label introduced is a research material. It is being used extensively as, and the demand for it is increasing rapidly. In order to meet this demand, in the field of synthetic organic chemistry, synthetic studies of various nucleoside derivatives which can be used as a synthetic raw material for modified DNA have been actively conducted. 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 (1
988) A. Roget, et al., Nucleic Acids Res. , 17 , 7643 (198
9) Co-Ori Ori Interstitial Society Anonym, Published Patent Publication No. 3-5052
09 These achievements have made a great contribution to the field of molecular biology by providing new modified DNA. However, the nucleoside derivatives described in the above-mentioned documents and the like require expensive halogenated or thiolated nucleosides or nucleobases in the synthetic route thereof, and thus the modified DNA to be synthesized also becomes expensive, and Introducing 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, Patent Publication No. Hei 4-60600 Hoechst Actigenzelzyaft, Unexamined Patent Publication No. Hei 4-290896 However, these derivatives are obtained by converting the hydroxyl group of the furanose ring into an amino group or a thiol group. It has its structural features and does not include a linker (spacer) moiety. Therefore, these derivatives are satisfactory for 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.

[0004]

[Problems to be Solved by the Invention] A nucleoside required for providing a modified DNA, which is rapidly in demand in the field of molecular biology, has a furanose ring which constitutes a nucleoside, not a thiol linker. It is an important subject to provide a nucleoside derivative bound to a specific moiety and to provide a method for synthesizing the nucleoside derivative, which is easily available, as a starting material.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a thiol linker or a linker is used not at a nucleobase portion but at a specific portion of a furanose ring constituting a nucleoside. Thus, we succeeded in developing a nucleoside derivative having a non-radioactive label attached thereto, and completed the present invention. That is, the present invention relates to a nucleoside derivative represented by the following formula [I].

[0006]

[Chemical 2]

However, in the formula, B is a purine or pyrimidine base, R ¹ is a hydrogen atom or a hydroxyl-protecting group, R ² and R
³ is a hydrogen atom on one side and (CH ₂ ) _n R ⁴ on the other side, n is an arbitrary integer of 3 or more, and R ⁴ is a thiol group which may have a protecting group or a non-radioactive label. Indicates.

Synthesis Method of Nucleoside Derivative Represented by Formula [I] The nucleoside derivative represented by formula [I] can be synthesized, for example, as follows. Commercially available ribonucleosides can be prepared by known methods (for example, HG Khorana, et.al.
JAChem.Soc., 84 , 430 (1962) and 86 , 4188 (196
4), KKOgilvie, et.al. Can.J.Chem., 56, 2768 (197
8) and 57, 2230 (1979)) prepared by the following formula [II] to the nucleoside derivative having a protected 5'-hydroxyl group,
The base is allowed to act in a suitable organic solvent to form an alkoxide.

[0009]

[Chemical 3]

In the formula, R ⁵ is a hydroxyl-protecting group, B is a purine or pyrimidine residue, preferably an adenine residue, a guanine residue, a cytosine residue and a uracil residue.

The alkoxide-formed nucleoside derivative contains X (CH ₂ ) _n X (X is a halogen atom, preferably a bromine atom, and n is an integer of 3 or more) and N, N-
When dimethylformamide was added and heated under reflux for several hours to several days, two types represented by the following formula [III] or [IV] in which a (CH ₂ ) _n X group was introduced into the hydroxyl group of the nucleoside derivative represented by the above formula The derivative of is produced as a mixture. This mixture can be purified by silica gel column chromatography to isolate each compound.

[0012]

[Chemical 4]

[0013]

[Chemical 5]

The organic solvent used in this reaction may be any one that does not interfere with the progress of this reaction.
Tetrahydrofuran, 1,4-dioxane, pyridine, dimethoxyethane, diethyl ether and mixtures thereof are preferred. As the base, a metal hydride, an alkyl metal, a Grignard reagent and the like can be used, and preferably tert-butylmagnesium chloride, sec-butylmagnesium chloride, tert-butyllithium, sec-butyllithium. Also, as catalysts, tetra-n-butylammonium bromide, tris [2- (2-methoxyethoxy) ethyl]
An amine or the like can be added. Examples of the hydroxyl protecting group include a trityl protecting group, a benzyl and allyl protecting group, an acyl protecting group, a silyl protecting group, and the like.
Preferred for the present invention are 4,4′-dimethoxytrityl group, 4-methoxytrityl group, and tert-butyldimethylsilyl group. Using the above formula [III] or [IV], a reaction with a thiol or thiocarboxylic acid or the like is carried out by a conventional method, and the purified product is isolated to give the 2'-represented by the above formula [I] via an alkylene group. Thus, various nucleoside derivatives to which a thiol group, a thiol group having a protective group, or a thiol group having a non-radioactive label are added can be obtained. The protecting group of the thiol group is used in a usual organic synthesis method, and examples thereof include a trityl type protecting group, a benzyl and allyl type protecting group, an acyl type protecting group, a malonate type protecting group, and a carbonic acid ester type protecting group. Preferred for the present invention is an acetyl group, a benzoyl group or a trityl group. Examples of non-radioactive labels include those that can be used for detection of probes, and examples thereof include biotin derivatives, dinitrophenyl derivatives, dansyl derivatives, fluoroscein derivatives, rhodamine derivatives, aminofluorene derivatives, and the like. , Biotinyl, dansyl, fluorocein isothiocyanyl, sulforhodaminyl (Texas Red), or 2,4-dinitrophenyl group. 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 alkyl group, represents an arbitrary integer of 3 or more, but an integer of 20 or less is preferable for the present invention in consideration of difficulty in obtaining raw materials.

Utilization method The nucleoside derivative having a 2′- or 3′-hydroxyl group represented by the formula [I] of the present invention having a thiol linker or a non-radioactive label through the linker is an oligomer such as DNA or RNA. A thiol linker or a non-radioactive label is introduced at an arbitrary position in it to be used for the preparation of a covalent bond with another substance or the detection of a probe. A known method can be used to introduce the compound into the oligomer of phosphonate, an organic chemical method such as a phosphoric acid triester method, a phosphoramidite method, an H-phosphonate method or a thiophosphite method, and a 5'-triphosphate derivative. A biochemical method utilizing the action of an enzyme after conversion into a can be exemplified.

[0016]

In the nucleoside derivative having a linker or a non-radioactive label on the 2'- or 3'-hydroxyl group of the present invention, the linker or the non-radioactive label is introduced in any number at any position in the oligomer. And the possibility of impairing the original function of nucleic acid is lower than that of the conventional method of introducing a nucleic acid base moiety or the method of directly introducing a non-radioactive label to a specific portion of the furanose ring constituting a nucleoside. Shows the action.

[0017]

[Examples] 2'provided in the present invention by the following examples
A synthesis example of a nucleoside derivative having a -or a 3'-hydroxyl group 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, DMTr = 4,4'-dimethoxytrityl group.

Example 5 First, the compound 1 represented by the following formula is synthesized.

[0019]

[Chemical 6]

5'-O-4,4'-dimethoxytrityl-adenosine
2.37 g (4.16 mmol) of tetrahydrofuran dissolved in 30 ml of tetrahydrofuran
2.0 M sec-butylmagnesium chloride /
Add 2.10 ml of diethyl ether solution and stir for 15 minutes.
After that, 6.24 g (20.8 mmol) of 1,10-dibromodecane,
And tetra-n-butylammonium bromide 1.34 g (4.16 mm)
ol) in N, N-dimethylformamide (15 ml) and added
The mixture was heated under reflux for 0 hours. After allowing to cool, extract with ethyl acetate and
Washed with Japanese salt water. Dried over anhydrous magnesium sulfate, concentrated
After shrinking, purification by silica gel column chromatography
To give 0.25 g of a light brown powdery compound (yield 8%).
Obtained.¹H-NMR,¹³By C-NMR and IR analysis
It was confirmed to be the above compound 1.¹H-NMR
And¹³Chemical shift of C-NMR signal, IR sig
Null wavenumber and silica gel thin layer chromatography
The mobility is shown below,¹The H-NMR chart is shown in FIG.¹³
The C-NMR chart is shown in FIG. 2 and the IR chart is shown in FIG.
You ¹ H-NMR (CDCl₃) Δ: 0.83 -2.00 (16
H, m) 3.00-3.80 (13H, m) 4.00-4.57 (3H, m) 5.83-6.34 (3H, m) 6.50-6.80 (4H, m) 6.93-7.80 (9H, m) 7.88 (1H, s), 8.11 (1H, s)¹³ C-NMR (CDCl₃) Δ: 25.90, 28.15,
28.71, 29.28, 29.33, 29.37, 29.57,
32.81, 34.02, 55.23, 63.08, 69.90,
71.45, 81.64, 83.96, 86.61, 86.96,
 113.20, 120.13, 126.94, 127.89,
128.22, 130.14, 135.77, 139.14, 1
44.57, 149.63, 153.09, 155.36, 15
8.58 IR (KBr) cm^-1: 3340, 2930, 161
0,1510,1250 Silica gel thin layer chromatography: Rf: 0.42
Loroform: methanol = 10: 1) A compound represented by the following formula of the present invention using the obtained compound 1
Compound 2 is synthesized.

[0021]

[Chemical 7]

Trityl thiol 100 mg (0.362 mmol)
In 2 ml of N, N-dimethylformamide solution at room temperature at 60
% Oily sodium hydride (15.0 mg, 0.375 mmol) was added.
After stirring for 15 minutes, 140 mg (0.17 mg) of Compound 1 was obtained.
7 mmol) and stirred at room temperature for 16 hours, then
It was extracted with formul and washed with water and saturated saline. Nothing
After drying over magnesium sulfate and concentration, silica gel column
Purified by chromatography to give a pale yellow powder.
0.12 g of the compound was obtained (yield 69%).¹H-NMR
And the above compounds by IR analysisTwoMake sure that
It was¹H-NMR signal chemical shift, IR sig
Null wavenumber and silica gel thin layer chromatography
The mobility is shown below and the IR chart is shown in FIG. ¹ H-NMR (CDCl₃) δ: 0.87 -1.83 (16
H, m) 1.97 -2.33 (2H, m) 2.77 -4.40 (14H, m) 5.73 -6.20 (3H, m) 6.53 -6.93 (4H, m) m) 7.03-7.60 (28H, m) 8.00 (1H, s), 8.23 (1H, s) IR (KBr) cm^-1: 3340, 2930, 161
0,1510,1250 Silica gel thin layer chromatography: Rf: 0.50
Loloform: methanol = 10: 1)

[0023]

INDUSTRIAL APPLICABILITY In the nucleoside derivative having a linker or a non-radioactive label at the 2'- or 3'-hydroxyl group provided by the present invention, the linker or the non-radioactive label can be added at any position in the oligomer. Of the nucleic acid base, and compared with the conventional method of introducing into the nucleic acid base portion or the method of directly introducing a non-radioactive label into a specific portion of the furanose ring constituting the nucleoside, Its utility value is high because it has the characteristic of being unlikely to damage it.

[Brief description of drawings]

FIG. 1 is the ¹ H—N of the compound 1 obtained in Example 1.
MR chart.

FIG. 2 shows ¹³ C—N of compound 1 obtained in Example 1.
MR chart.

FIG. 3 is an IR chart of compound 1 obtained in Example 1.

FIG. 4 is an IR chart of compound 2 obtained in Example 1.

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

Claims (1)

[Claims] 1. A nucleoside derivative represented by the following formula [I]: [Chemical 1] However, B in the formula is purine or pyrimidine base, R ¹
Is a hydrogen atom or a hydroxyl-protecting group, and one of R ² and R ³ is a hydrogen atom and the other is (CH ₂ ) _n R ⁴ , and n is 3
Any of the above integers is shown, and R ⁴ is a thiol group which may have a protecting group or a non-radioactive label.