JP3032815B2 - 2'-O-silyl cyclic silylated nucleoside derivative, method for producing the same, and method for producing 2'-O-silyl nucleoside using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel 2'-O-
The present invention relates to a silyl cyclic siliconized nucleoside derivative, a method for producing the same, and a method for producing 2'-O-silyl nucleoside using the same. More specifically, the present invention relates to the synthesis of RNA-related compounds or nucleosides involved in genetic engineering. A 2′-O-silyl cyclic siliconized nucleoside derivative which is an important intermediate in the synthesis of related pharmaceuticals and the like, a production method for easily obtaining it from a nucleoside via a cyclic siliconized nucleoside in good yield, and a monomer for RNA synthesis The present invention relates to a method for producing a 2'-O-silylated nucleoside which is important as a starting material for preparation from the above 2'-O-silyl cyclic silylated nucleoside derivative. 2'-O- produced according to the present invention
Silyl cyclic siliconized nucleoside derivative and 2'-O-
In the silylated nucleoside, the hydroxyl group at the 2'-position is protected by a silyl group, and the deoxyribonucleic acid (DN
A) It is considered to be an important intermediate when applying technologies accumulated in related fields to the synthesis and modification of ribonucleic acids.

[0002]

2. Description of the Related Art Due to the widespread development of genetic engineering in recent years,
Various nucleic acid derivatives have attracted attention as functional substances or pharmaceuticals, and nucleoside derivatives constituting the basic skeleton of nucleic acids have been actively studied as starting materials or intermediates in their synthesis. Until now, the technology centered on deoxyribonucleic acid. While deoxyribonucleosides contain one primary and one secondary hydroxyl group in the molecule, their use is expanding. The ribonucleoside in the nucleic acid contains one primary and two secondary ribonucleotides in the molecule.
Has a total of three hydroxyl groups. For this reason, as a starting material for RNA synthesis, a compound in which the 2′-hydroxyl group has been protected in advance in order to imitate deoxyribonucleoside is preferable. Among the important 2'-O-silylated nucleosides, a compound in which a highly reactive primary hydroxyl group is protected in advance has been prepared, the remaining two hydroxyl groups are silylated, and the product is chromatographed. This was manufactured by fractionation by graphy and the like. In this method, it is difficult to increase the selectivity, and the desired compound could not be obtained without complicated treatment.

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to 2'-O-
It is an object of the present invention to provide a silyl cyclic silylated nucleoside and a method for producing the same, and to provide a method for producing 2'-O-silyl nucleoside using the 2'-O-silyl cyclic silylated nucleoside.

[0004]

Means for Solving the Problems The inventor of the present invention has proposed a simple 2 ′
To enable the production of -O-silylated nucleosides, intensive studies were conducted on protecting groups at the 3'-position and the 5'-position of each hydroxyl group. The product can be regioselectively introduced into each of the hydroxyl groups at the '-position and the 5'-position, the product is stable under various synthesis conditions, and the hydroxyl group at the 2'-position is further protected by a silyl group by a continuous silylation reaction. It has been found that a novel nucleoside derivative can be obtained. In addition, they have found that the cyclic siliconized group can be eliminated from this novel nucleoside derivative with high selectivity. The present invention has been completed based on these findings. That is, according to the present invention, the following general formula (1)

Embedded image (Wherein B is an unprotected or protected nucleobase, R ¹ and R ² are an aromatic group or a branched aliphatic group having 3 or more carbon atoms,
R ³ , R ⁴ and R ⁵ are an aromatic group or an aliphatic group). Further, according to the present invention, in order to produce the 2′-O-silyl cyclic silylated nucleoside derivative represented by the general formula (1), the following general formula (2)

Embedded image (Where B is an unprotected or protected nucleic acid base) represented by the following general formula (3)

In embedded image _{^{R 1 R 2 SiX 1 2 (}} 3) ( wherein, R ¹ and R ² are an aromatic group, or having 3 or more branched aliphatic group having a carbon, X ¹ is a residue of acid) After cyclic silylation using the represented bifunctional silicon compound, the remaining 2′-hydroxyl group is replaced with the following general formula (4)

R ³ R ⁴ R ⁵ SiX ² (4) (wherein R ³ , R ⁴ and R ⁵ are an aromatic group or an aliphatic group, X ²
Is a leaving group), and a silylation using a monofunctional silicon compound represented by the formula (I) is provided.
Further, according to the present invention, the 2′-O-silyl cyclic silylated nucleoside derivative represented by the general formula (1) is
The following general formula (5) characterized by being subjected to a decyclic siliconization treatment.

Embedded image (Wherein B, R ³ , R ⁴ and R ⁵ have the same meaning as described above).

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the above general formula (1) representing the 2′-O-silyl cyclic silylated nucleoside derivative of the present invention, R ¹ and R ² are an aromatic group or a branched fatty acid having 3 or more carbon atoms. Represents a group. As the aromatic group, phenyl,
And aryl groups such as tolyl and naphthyl, and arylalkyl groups such as benzyl and phenethyl. The branched aliphatic group having 3 or more carbon atoms includes an iso-form, a sec-form and a tert-form, and has 3 to 8, preferably 3 to 4 carbon atoms. Examples of such a branched aliphatic group include iso-propyl, iso-butyl, sec-butyl, tert-butyl, iso-pentyl, neopentyl, tert-pentyl, iso-hexyl, and tert.
-Hexyl, iso-octyl, tert-octyl,
2,3-dimethyl-2-butyl (also known as texil),
And cyclohexyl. A preferred branched aliphatic group is tert-butyl. R ³ , R ⁴ and R ⁵ are
It represents an aromatic group or an aliphatic group. Examples of the aromatic group include phenyl, tolyl, naphthyl, benzyl, phenethyl and the like. Aliphatic groups include linear and cyclic ones, and linear ones include linear and branched ones. The aliphatic group has 1 to 8, preferably 1 to 6, carbon atoms. Such aliphatic groups include methyl, ethyl, vinyl, propyl, isopropyl, propenyl, butyl, iso-butyl, tert-butyl, pentyl,
Isopentyl, neopentyl, hexyl, iso-hexyl, cyclohexyl, cyclohexenyl, octyl,
iso-octyl, cyclooctyl, 2,3-dimethyl-2-butyl and the like.

In the general formula (2) representing the nucleoside used as a reaction raw material in the present invention, the sugar may be arabinose, xylose or lyxose other than ribose. B represents an unprotected or protected nucleobase, including a pyrimidine base and a purine base. The glycosidic bond in which B binds to the sugar may be β-type or α-type. Further, the protecting group protects a functional group such as an amino group, an amide group, and a hydroxyl group contained in the nucleic acid base,
Those commonly used for protecting those functional groups can be used. For example, to protect amino or amide groups,
An acyl group such as a benzoyl group, an isobutyryl group, and a phenoxyacetyl group can be used.
An arylalkyl group such as a benzyl group and a phenethyl group can be used. Specific examples of the nucleic acid base B include, for example, adenine, guanine, cytosine, uracil, thymine, hypoxanthine, xanthine, pseudouracil and the like. Specific examples of the nucleoside of the general formula (2) include uridine, adenosine, cytidine, guanosine inosine, xanthosine, pseudouridine, and N-benzoylated, N-isobutyrylated, phenoxyacetylated compounds thereof.

In the above general formula (3), which represents a bifunctional silicon compound used as a reaction raw material in the present invention, R ¹ and R ² have the same meanings as described above. X ¹ represents a residue of an acid. In this case, the acid includes an inorganic acid and an organic acid, and nitric acid, perchloric acid, trifluoromethanesulfonic acid and the like can be preferably used. In the above general formula (4) showing the monofunctional silicon compound used as a reaction raw material in the present invention, R ³ , R ⁴ and R ⁵ have the same meaning as described above. X ² represents a leaving group such as an acid residue, and specific examples thereof include X ¹
And imidazole, N-methyltrifluoroacetamide, 3-penten-2-one and the like.

In order to produce the 2′-O-silyl cyclic silylated nucleoside represented by the general formula (1), the nucleoside represented by the general formula (2) is added to the nucleoside represented by the general formula (3). The resulting bifunctional silicon compound is reacted to form a cyclic silylation to obtain a silylated product represented by the following general formula (6).

Embedded image (In the above formula, R ¹ , R ² and B have the same meanings as described above.) Next, this silylated product is reacted with a monofunctional silicon compound represented by the aforementioned general formula (4) to obtain the silyl compound. the -OH remaining compound, is converted to -OSiR ³ R ⁴ R ^5.
Thus, a 2′-O-silyl cyclic silylated nucleoside derivative of the above general formula (1) is obtained. In the reaction,
The reaction temperature is -10 to 50C, preferably 10 to 30C.
° C, and the reaction pressure is 0.5 to 2 atm, preferably normal pressure. The reaction is preferably performed in an organic solvent. Any organic solvent may be used as long as it can dissolve or disperse the starting nucleoside, and any one can be used. However, a polar organic solvent such as dimethylformamide, dimethylsulfoxide, tetrahydrofuran, or dioxane is preferably used. The proportion of the bifunctional silicon compound represented by the general formula (3) is 0.9 to 2 mol, preferably 1.0 to 1. mol per mol of the nucleoside of the general formula (2).
1 mole ratio. When the method of the present invention is carried out, as the bifunctional silicon compound, a compound synthesized in advance outside the reaction system can be used, and a compound generated in the reaction system can also be used. In order to produce the acid in the reaction system, a silver salt of an acid may be reacted with dialkyldichlorosilane.

The 2'-O-silyl nucleoside represented by the general formula (4) is produced by subjecting the 2'-O-silyl cyclic silylated nucleoside derivative of the general formula (1) to decyclic siliconization. Is done. In this case, the decyclic siliconization treatment removes the bifunctional silicon compound bonded to the 3′-position and the 5′-position hydroxyl group of the nucleoside derivative of the general formula (1), and substitutes the substituted hydroxyl group. Is a process of converting to a free hydroxyl group. The reaction in this case is represented by the following equation.

Embedded image (In the above formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and B have the same meanings as described above.) The treating agent used in the decyclic siliconization treatment is a nucleoside derivative of the general formula (1) A reaction component having active hydrogen that decomposes the substituted hydroxyl groups at the 3′-position and the 5′-position to convert to a free hydroxyl group, or releasing hydrogen ions, and a silicon compound component generated by decomposing the substituted hydroxyl groups. And reacting components. Both of these reaction components may be contained in one compound or may be contained in separate compounds. like this,
Specific examples of the processing include, for example, the following. (1) A combination of a compound having active hydrogen and a tetraalkylammonium fluoride. In this case, as the compound having active hydrogen, an alcohol such as methanol or ethanol is used. (2) A combination of a compound having active hydrogen and a highly active fluorine compound. As the compound having active hydrogen in this case, an organic carboxylic acid such as acetic acid or propionic acid is preferably used. As the highly active fluorine compound, tetrabutylammonium fluoride or a salt of an inorganic fluoride dissolved in a polar organic solvent is preferably used. (3) A fluorine compound supported on a carrier. Examples of the carrier in this case include silica gel, alumina, zirconia, titania, activated carbon, ion exchange resins, porous substances such as polymers (for example, polyvinyl pyridine), and salts of inorganic fluorides such as sodium fluoride. Examples of the fluorine compound include hydrogen fluoride and a polymer thereof. (4) A combination of a base and hydrofluoric acid. As the base,
Examples include tributylamine and triethylamine. (5) Bifluoride compounds. Examples of the bifluoride compound include compounds represented by the following general formula (8).

Embedded image (Y represents an ionic basic substance) Examples of the ionic basic substance include tetrabutylammonium and the like.

In the present invention, a combination of a tertiary amine and hydrofluoric acid is preferably used. The proportion of hydrofluoric acid used is 0.5 to 2 mol, preferably 0.8 to 1.2 mol, as HF, per mol of the tertiary amine. Examples of the tertiary amine include a chain amine such as tributylamine and triethylamine, and a cyclic amine such as pyridine. The decyclic silation reaction of the nucleoside derivative of the general formula (1) is carried out at -10 to 50C, preferably 10 to 3O0C.
It is performed at a temperature of 0 ° C. The reaction pressure is not particularly limited,
Generally, normal pressure is employed.

[0011]

The 2'-O-silyl cyclic silylated nucleoside derivative of the present invention is a novel compound which has not been described in any literature, does not require a complicated operation for isolating an intermediate in the production thereof, and is free of nucleic acids. Since dimethylformamide (DMF), which is a good solvent, can be used in the reaction, it can be synthesized at once from the starting nucleoside, and can be used in the synthesis of RNA-related compounds related to genetic engineering or the synthesis of pharmaceuticals containing nucleosides. Can be used as a synthetic material, and can be used for simple and efficient production of 2′-O-silylated nucleosides such as 2′-O-tert-butyldimethylsilyl nucleoside which is an important intermediate in RNA synthesis. it can.

[0012]

Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention.

Example 1 Under anhydrous conditions, 0.4 mmol of uridine was dissolved in 2 ml of N, N-dimethylformamide, and the space in the reaction vessel was replaced with dry nitrogen. After adding 1.1 times mole of di-tert-butylsilyl bis (trifluoromethanesulfonate) and reacting at room temperature for 4 minutes, 1.1 times mole of tert
-Butyldimethylsilyl trifluoromethanesulfonate was added and treated for another 10 minutes. After adding triethylamine, the solvent and excess reagent were removed under reduced pressure, and 174 mg of a main product was obtained by silica gel column chromatography using hexane-ethyl acetate as an eluent. In the infrared absorption spectrum of this substance, the characteristic absorption of a hydroxyl group around 3400 cm ^-1 disappeared, and the molecular formula (C ₂₃ H ₄₂ N ₂ O ₆ Si ₂ , m
/ Z = 499.2628) was obtained. Also, ¹ H N
Each peak of the MR spectrum matched the expected structure for chemical shift and peak area. From the above analysis results, this substance was found to be 2'-O-tert-butyldimethylsilyl-3 ', 5'-O- (di-tert-
-Butylsilanediyl) uridine.

Example 2 Under anhydrous conditions, 0.4 mmol of adenosine was dissolved in 2 ml of N, N-dimethylformamide, and the space in the reaction vessel was replaced with dry nitrogen. After adding 1.1 times mole of di-tert-butylsilyl bis (trifluoromethanesulfonate) and reacting at room temperature for 4 minutes, 1.1 times mole of ter
t-Butyldimethylsilyl trifluoromethanesulfonate was added and the mixture was further treated for 10 minutes. After adding triethylamine, the solvent and excess reagent were removed under reduced pressure, and 166 mg of a main product was obtained by silica gel column chromatography using hexane-chloroform as an eluent. In the infrared absorption spectrum of this substance, the characteristic absorption of a hydroxyl group at around 3400 cm ^-1 disappeared, and the molecular formula (C ₂₄ H ₄₃ N ₅ O ₄ S) which coincides with the structure expected by double-focusing mass spectrometry
i ₂ , m / z = 522.2901). Also ¹
Each peak of the 1 H NMR spectrum was consistent with the assumed structure for chemical shift and peak area. From the above analysis results, this substance was found to be 2'-O-te
rt-butyldimethylsilyl-3 ′, 5-O- (di-t
tert-butylsilanediyl) adenosine.

Example 3 Under anhydrous conditions, 0.4 mmol of uridine was dissolved in 2 ml of N, N-dimethylformamide, and the space in the reaction vessel was replaced with dry nitrogen. After adding 1.1-fold molar amount of di-tert-butylsilyl bis (trifluoromethanesulfonate) and reacting at room temperature for 4 minutes, 1.1-fold molar amount of dimethylsexylsilyl trifluoromethanesulfonate was added and further 2 hours. Processed. After adding triethylamine, the solvent and excess reagent were removed under reduced pressure, and 188 mg of a main product was obtained by silica gel column chromatography using chloroform as an eluent. In the infrared absorption spectrum of this substance, the characteristic absorption of a hydroxyl group at around 3400 cm ^-1 disappeared, and each peak of the ¹ H NMR spectrum coincided with the expected structure in terms of chemical shift and peak area. From the above analysis results, this substance was found to be 2'-O
-Dimethylsexylsilyl-3 ', 5'-O- (di-t
tert-butylsilanediyl) uridine.

Example 4 Under anhydrous conditions, 0.4 mmol of uridine was dissolved in 2 ml of N, N-dimethylformamide, and the space in the reaction vessel was replaced with dry nitrogen. After adding 1.1 times mole of di-tert-butylsilyl bis (trifluoromethanesulfonate) and reacting at room temperature for 4 minutes, 1.1 times mole of triethylamine is added, and then 2.2 times mole of triisopropylamine. Silyl trifluoromethanesulfonate was added and the mixture was further treated for 48 hours. The solvent and excess reagent were removed under reduced pressure, and 194 mg of the main product was obtained by silica gel column chromatography using chloroform as an eluent. In the infrared absorption spectrum of this substance, the characteristic absorption of a hydroxyl group at around 3400 cm ^-1 disappeared, and each peak of the ¹ H NMR spectrum coincided with the expected structure in terms of chemical shift and peak area. From the above analysis results,
This material is 2'-O-triisopropylsilyl-
It was confirmed to be 3 ', 5'-O- (di-tert-butylsilanediyl) uridine.

Example 5 2'-O-tert-butyldimethylsilyl-3 ',
5'-O- (di-tert-butylsilanediyl) uridine was added with a twice molar solution of a mixture of tributylamine and hydrofluoric acid (molar ratio = 1: 1) in tetrahydrofuran (0.1 molar concentration). A decyclic siliconization treatment was performed for one hour. Calcium chloride was added, the mixture was allowed to stand, filtered, the solvent was removed under reduced pressure, and the main product was obtained by silica gel column chromatography using chloroform-methanol as an eluent. The behavior of this substance on various chromatographies was determined by 2'-O-te synthesized by a separately known method.
Consistent with rt-butyldimethylsilyl uridine. Further, the examination results of the instrumental analysis using ¹ H NMR, IR, double focusing mass spectrometry and the like show the assumed structure (C ₁₅ H ₂₆ N ₂ O ₆
Si, m / z = 359.1642). From the above analysis results, it was confirmed that this substance was 2'-O-tert-butyldimethylsilyl uridine.

Example 6 2'-O-tert-butyldimethylsilyl-3 ',
5'-O- (di-tert-butylsilanediyl) uridine is added with a 4-fold molar solution of a mixture of pyridine and hydrofluoric acid (molar ratio = 1: 1) in tetrahydrofuran (0.4 molar concentration) to give 30. A partial decyclic siliconization treatment was performed. Next, calcium chloride was added, the mixture was allowed to stand, and then filtered. The solvent was removed under reduced pressure, and the main product was obtained by silica gel column chromatography using chloroform-methanol as an eluent. The behavior of this substance on various chromatographies and the results of examination of the instrumental analysis were the same as in Example 5 with 2′-O-
Consistent with tert-butyldimethylsilyl uridine.

Claims (3)

(57) [Claims] [Claim 1] The following general formula (1) (Wherein B is an unprotected or protected nucleobase, R ¹ and R ² are an aromatic group or a branched aliphatic group having 3 or more carbon atoms,
R ³ , R ⁴ and R ⁵ are an aromatic group or an aliphatic group). 2. The following general formula (2): (Where B is an unprotected or protected nucleobase)
The in nucleoside represented by the following general formula (3) ## STR3 ## R ¹ R ² SiX ¹ ₂ (3) (wherein, R ¹ and R ² are an aromatic group, or having 3 or more branched aliphatic carbon Group, X ¹ is a residue of an acid), and the remaining 2′-hydroxyl group is converted into a cyclic group using a bifunctional silicon compound represented by the following general formula (4). R ³ R ⁴ R ⁵ SiX ² (4) (wherein R ³ , R ⁴ and R ⁵ are an aromatic group or an aliphatic group, X ²
Is a leaving group) and is silylated using a monofunctional silicon compound represented by the following general formula (1): (Wherein, B, R ¹ , R ² , R ³ , R ⁴ and R ⁵ have the same meanings as described above). 3. The following general formula (1): (Wherein B is an unprotected or protected nucleobase, R ¹ and R ² are an aromatic group or a branched aliphatic group having 3 or more carbon atoms,
R ³ , R ⁴ and R ⁵ are an aromatic group or an aliphatic group), wherein the 2′-O-silyl cyclic silylated nucleoside derivative represented by the following general formula: (5) (Wherein, B, R ³ , R ⁴ and R ⁵ have the same meanings as described above).