JP2010254616A - Method for producing ribonucleoside derivative and oligonucleic acid derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of selectively and efficiently removing the substituted carbamoyl group from the 2'-hydroxy group in a ribonucleoside derivative and/or an oligonucleic acid derivative including this ribonucleoside derivative as a constituting unit. <P>SOLUTION: The method for efficiently producing a high purity oligonucleic acid comprises reacting a ribonucleoside derivative having a carbamoyl protective group for the 2'-hydroxy group and/or an oligonucleic acid derivative including this ribonucleoside derivative as a constituting unit with R<SB>4</SB>NF and removing the carbamoyl protective group from the 2'-hydroxy group. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a ribonucleoside derivative and / or an oligonucleic acid derivative. More specifically, the present invention relates to a method for obtaining a ribonucleoside derivative and / or oligonucleic acid derivative by deprotecting a ribonucleoside derivative and / or oligonucleic acid having a protected 2'-hydroxyl group.

  Oligo RNA is a useful material mainly used for medical applications such as nucleic acid drugs and diagnostic drugs such as antisense drugs, ribozyme drugs, decoy oligo drugs, SiRNA drugs, miRNA drugs, and aptamer drugs. Oligo RNAs used for these applications are often produced using chemical synthesis methods.

  Among the chemical synthesis methods for oligo RNA, the chemical synthesis method that is currently mainstream is the phosphoramidite method. The feature of this phosphoramidite method is that the internucleotide formation reaction proceeds at a higher yield than other methods.

  The phosphoramidite monomer used in the phosphoramidite method has a protecting group that can be deprotected with an acid or a fluorine compound at the 5′-, 2′-hydroxyl group, and an alkoxy group and an amino group on the phosphorus atom at the 3′-hydroxyl group. have. An internucleotide bond is formed by reacting this phosphoramidite monomer with a desired nucleoside or nucleotide in the presence of an activating agent. Thereafter, the desired oligonucleotide can be obtained by oxidation.

  The oligonucleotide thus obtained must be deprotected because the base, phosphate, 5'-, 3'- and 2'-hydroxyl groups are all protected. At the time of this deprotection, it is necessary that the deprotection of other protective groups and the degradation of the oligonucleic acid do not occur under the predetermined deprotection conditions of the protective group. Particularly, since the deprotection of the 2'-hydroxyl group is carried out after deprotecting other protecting groups, particularly strict conditions are required. Specific conditions required for the protecting group of 2′-hydroxyl group are stable under the deprotection condition of 5′-hydroxyl group, do not inhibit the coupling reaction, and stable under the coupling reaction condition. In other words, it is stable under the deprotection conditions of the phosphoric acid and the base part, and is not decomposed by cleavage of the oligo chain under the deprotection conditions (Non-patent Document 1).

RNAi method and antisense method, Mitsuo Sekine, Kazumasa Tahira, 10

  The present inventors have recently found that when a carbamoyl group is introduced into the 2'-hydroxyl group of ribonucleoside, a protective group can be efficiently introduced into the 2'-hydroxyl group with high regioselectivity.

  The carbamoyl group is a protective group suitable for oligonucleic acid synthesis because it does not decompose under the detritylation reaction condition, the coupling reaction condition, or the deprotection condition of phosphoric acid and base moiety. However, no efficient deprotection method for the carbamoyl group introduced into the 2'-hydroxyl group of the oligonucleic acid has been reported so far. As a general deprotection condition for the decarbamoylation reaction, a method using a metal hydroxide or a metal alkoxide is known. For example, when sodium hydroxide is used, decomposition with an internucleotide via a phosphorus atom is performed. Or accompanied by rearrangement of the phosphate group from 3'- to 2'-hydroxyl group. In addition, as another decarbamoylation reaction, a method using hydride ions such as aluminum hydride has been reported. Under such conditions, there is a concern about degradation of oligonucleic acid.

As a result of intensive studies in order to solve such problems, the present inventors have obtained a ribonucleoside derivative having a carbamoyl protecting group at the 2′-hydroxyl group and / or an oligonucleic acid derivative containing this ribonucleoside derivative as a structural unit. It has been found that deprotection can be efficiently carried out by reacting with R ₄ NF, and the present invention has been completed.

  That is, the present invention relates to the general formula (1):

[Wherein R ¹ has an optionally substituted alkyl group having 1 to 18 carbon atoms, an optionally substituted cycloalkyl group having 1 to 18 carbon atoms, and a substituent. An aryl group having 6 to 18 carbon atoms, an aralkyl group having 7 to 18 carbon atoms which may have a substituent, a sulfonyl group which may have a substituent, or a substituent. Represents a good silyl group. R ² represents hydrogen, a hydroxyl protecting group, or an oligonucleic acid. R ³ represents an oligonucleic acid. X represents a hetero atom. Y represents hydrogen, a hydroxyl group, an alkoxy group, or a thiol group. B is the following formula:

R ⁴ represents adenine, guanine, cytosine, uracil represented by hydrogen or an amino-protecting group, or a group derived therefrom. m represents a natural number greater than 1, and a ribonucleoside derivative in which the 2′-hydroxyl group is protected and / or an oligonucleic acid derivative containing this ribonucleoside derivative as a constituent unit is represented by the general formula (2):
R ⁵ ₄ NF
[Wherein R ⁵ has an optionally substituted alkyl group having 1 to 18 carbon atoms, an optionally substituted cycloalkyl group having 1 to 18 carbon atoms, and a substituent. The 2′-hydroxyl group is deprotected by reacting with an aryl group having 6 to 18 carbon atoms, or an aralkyl group having 7 to 18 carbon atoms which may have a substituent. 3):

[Wherein R ² , R ³ , B, X, Y, m are the same as above. And / or a method for converting to an oligonucleic acid derivative containing the ribonucleoside as a constituent unit.

  It has not been reported so far to use a fluorine compound for the deprotection reaction of the substituted carbamoyl group introduced into the 2'-hydroxyl group of the oligonucleic acid.

  According to the method of the present invention, a ribonucleoside derivative in which a 2′-hydroxyl group is substituted and substituted with a carbamoyl and / or an oligonucleic acid derivative containing this ribonucleoside derivative as a constituent unit can be easily converted into a ribonucleoside derivative and / or this ribonucleoside derivative. An oligonucleic acid derivative containing as a structural unit can be produced.

  Hereinafter, the present invention will be described in detail.

  In the present invention, the general formula (1):

And / or an oligonucleic acid derivative containing the ribonucleoside derivative as a structural unit, and a general formula (2):
R ⁵ ₄ NF (2)
With general formula (3):

Or an oligonucleic acid derivative containing this ribonucleoside as a structural unit.

  First, the compound used for this reaction is demonstrated below.

  Oligonucleic acid derivatives refer to oligo DNA and oligo RNA, including those that have been chemically modified, natural and non-natural types, and peptides, sugars, polymers, etc. It may be included.

In the formula (1), R ¹ represents an optionally substituted alkyl group having 1 to 18 carbon atoms, an optionally substituted cycloalkyl group having 1 to 18 carbon atoms, and a substituent. An aryl group having 6 to 18 carbon atoms which may have, an aralkyl group having 7 to 18 carbon atoms which may have a substituent, a sulfonyl group which may have a substituent, or a substituent. It represents a silyl group that may be present.

Specific examples of R ¹ include alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, isoheptyl, octyl, isooctyl; cyclopropyl, cyclobutyl Cycloalkyl groups such as cyclopentyl and cyclohexyl; aralkyl groups such as benzyl, 1-phenylethyl, 2-phenylethyl, 1-methylnaphthyl, 2-methylnaphthyl, 1-ethylnaphthyl, 2-ethylnaphthyl; Aryl groups such as naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthryl, 9-phenanthryl, 10-phenanthryl; methanesulfonyl, ethanesulfonyl, benzenesulfonyl, toluenesulfur Sulfonyl groups such as nyl; silyl groups such as trimethylsilyl, triethylsilyl, triphenylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, dimethylphenylsilyl, dimethylbenzylsilyl, diphenylmethylsilyl, etc. Are an alkyl group and an aryl group, more preferably an aryl group, and particularly preferably a phenyl group.

  These may have a substituent. Examples of the substituent include halogen atoms such as fluorine, chlorine, bromine and iodine atoms; methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, Alkyl groups such as isopentyl, hexyl, isohexyl, heptyl, isoheptyl, octyl, isooctyl; cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; benzyl, 1-phenylethyl, 2-phenylethyl, 1-methylnaphthyl, 2 Aralkyl groups such as methyl naphthyl, 1-ethyl naphthyl and 2-ethyl naphthyl; aryl groups such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl and 1-phenanthryl; methyloxy , Ethyloxy, Alkoxy such as propyloxy, t-butyloxy, pentyloxy, isopentoxy, hexyloxy, heptyloxy, octyloxy, cyclopropyloxy, cyclobutyloxy, 2-methylcyclopropyloxy, cyclopropylmethyloxy, cyclopentyloxy, cyclohexyloxy Group; amino group; mono- or dialkylamino group such as N-methylamino, N-cyclohexylamino, N, N-dimethylamino, N, N-diethylamino, N, N-diisopropylamino; N-phenylamino, N- Mono- or diarylamino groups such as naphthylamino, N, N-diphenylamino, N-naphthyl-N-phenylamino groups; mono- or diaralkyl groups such as N-benzylamino, N, N-dibenzylamino groups; nitro groups To mention Kill. The substituent may be 1 or 2 or more, and the position of the substituent is not particularly limited.

R ² represents hydrogen, a hydroxyl protecting group, or an oligonucleic acid. Here, as a hydroxyl-protecting group, a general hydroxyl-protecting group, for example, a trityl group such as a 4,4′-dimethoxytrityl group or a 4-methoxytrityl group, a tert-butyldimethylsilyl group, or a tert-butyldiphenylsilyl group. And a silyl group such as a triisopropylsilyl group.

R ³ represents an oligonucleic acid.

  X represents a hetero atom, and specific examples include oxygen, sulfur, and boron.

  Y represents hydrogen, a hydroxyl group, an alkoxy group, or a thiol group. Examples of the alkoxy group include a methoxy group and a cyanoethyl group.

  B is the following formula:

(R ⁴ represents hydrogen or an amino-protecting group), and represents adenine, guanine, cytosine, uracil or a derivative thereof.

Among B, R ⁴ on the amino group contained in adenine, guanine, and cytosine represents a hydrogen atom or a protecting group for an amino group. As the amino-protecting group, commonly used amino-protecting groups are used. Specifically, for example, acyl groups such as acetyl, benzoyl and phenoxyacetyl; alkyl groups such as benzyl and dimethoxytrityl; -Carbamate groups such as butyl dicarbonate and benzyl carbamate; cyclic imide groups such as phthalimide; sulfo groups such as toluenesulfonyl; silyl groups such as tert-butyldimethylsilyl; N, N-dimethylaminomethyl and N, N-dibutylamino Examples thereof include imino groups such as methyl.

  m represents a natural number greater than 1.

In the formula (2), R ⁵ represents an optionally substituted alkyl group having 1 to 18 carbon atoms, an optionally substituted cycloalkyl group having 1 to 18 carbon atoms, and a substituent. An aryl group having 6 to 18 carbon atoms which may be present and an aralkyl group having 7 to 18 carbon atoms which may have a substituent are represented.

Specific examples of R ⁵ include alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, isoheptyl, octyl, isooctyl; cyclopropyl, cyclobutyl Cycloalkyl groups such as cyclopentyl and cyclohexyl; aralkyl groups such as benzyl, 1-phenylethyl, 2-phenylethyl, 1-methylnaphthyl, 2-methylnaphthyl, 1-ethylnaphthyl, 2-ethylnaphthyl; An aryl group such as naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthryl, 9-phenanthryl, 10-phenanthryl, and the like can be mentioned, preferably an alkyl group and an aryl group, Even better Properly is an alkyl group, particularly preferably a butyl group.

These may have a substituent, and examples of the substituent include the same as in R ¹ . The substituent may be 1 or 2 or more, and the position of the substituent is not particularly limited.

  In this reaction, a reaction solvent is usually used. The reaction solvent is not particularly limited as long as it does not inhibit the reaction. For example, nitrile solvents such as acetonitrile and propionitrile, tert-butyl methyl ether, diethyl ether, dimethyl ether, diisopropyl ether, tetrahydrofuran, dioxane and the like Ether solvents, amide solvents such as N, N-dimethylformamide and N, N-dimethylacetamide, sulfoxide solvents such as dimethyl sulfoxide, halogenation such as methylene chloride, 1,2-dichloroethylene, chloroform and carbon tetrachloride Examples thereof include hydrocarbon solvents, alcohol solvents such as methyl alcohol, ethyl alcohol, isopropanol, and phenol, and water, and tetrahydrofuran is preferred. Two or more of these may be mixed. When a mixed solvent is used, the mixing ratio is not particularly limited.

  Next, the reaction will be described in detail.

In this reaction, the oligoribonucleoside (oligoribonucleoside (1)) represented by the formula (1) is reacted with R ⁵ ₄ NF.

The amount of R ⁵ ₄ NF used in the present invention is not particularly limited. Usually, when m = 1, it is 1 to 3000 mol, preferably 1 to 1000 mol, with respect to the oligoribonucleoside (1). Particularly preferably, 5 to 500 moles may be used. When m = 1 or more, the amount of R ⁵ ₄ NF may be increased corresponding to the number of m.

The order of mixing each compound during the reaction is not particularly limited, but in general, R ⁵ ₄ NF may be added to the oligoribonucleoside (1) solution.

  The concentration of the compound in conducting the reaction varies depending on the reaction solvent to be used, but generally the reaction can be carried out at 0.01 to 100% by weight, preferably 1 to 50% by weight.

The reaction temperature during the reaction varies depending on the type of compound used and R ⁵ ₄ NF, but is usually in the range of the boiling point or lower from the freezing point of the reaction solvent used. In order to complete the reaction in a short time, it is better to carry out the reaction at a higher temperature. From the viewpoint of suppressing the progress of the decomposition of the oligoribonucleoside, it is better to carry out the reaction at a lower temperature. Preferably, it is -78 degreeC-100 degreeC, Most preferably, it is -10 degreeC-80 degreeC.

The reaction time during the reaction varies depending on the type of compound used and R ⁵ ₄ NF, but when the reaction temperature is carried out at −30 ° C. to 80 ° C., usually about 0.1 to 200 hours is preferable.

  The reaction can be carried out at any of normal pressure, reduced pressure, and increased pressure. Moreover, you may implement in air or you may implement in inert gas atmosphere, such as nitrogen, helium, and argon.

  The reaction may be performed in the presence of an additive for the purpose of accelerating the reaction rate. The additive is not particularly limited and may be either soluble or insoluble in the reaction solvent. Examples of the additive include tetrabutylammonium hydroxide, molecular sieves, inorganic fluorine compounds, and organic bases.

  As a post-treatment of this reaction, a general treatment for obtaining a product from the reaction solution may be performed. The obtained product may be subjected to general purification to further increase the purity.

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In addition, the apparatus used for the measurement of the physical property in each Example is as follows.
High performance liquid chromatography: Shimadzu Corporation SPD-10Avp, LC-10ADvp, SCL-10Avp, DGU-12A, CTO-10ASvp, C-R8A

Method for producing deprotected uridylic acid dimer 2′-O-phenylcarbamoyluridylic acid dimer (37.6 mg, 0.05 mmol) was added to 1M THF solution of TBAF (tetrabutylammonium fluoride) (1.5 mL). 1.5 mmol) and stirred at 66 ° C. for 16 hours. As confirmed by HPLC, a deprotected uridylic acid dimer was produced at a conversion rate of 99% or more.
[HPLC analysis conditions]
Column: Phenomenex, eluent: acetonitrile / 10 mM ammonium acetate aqueous solution = 0/100 → 90/10 (gradient), flow rate: 1.0 mL / min. , Detection wavelength: 254 nm, column temperature: 40 ° C., retention time: 2′-O-phenylcarbamoyluridylic acid dimer = 24 minutes, deprotected uridylic acid dimer = 12 minutes.

Method for producing deprotected uridylic acid dimer 2′-O-phenylcarbamoyluridylic acid dimer (37.6 mg, 0.05 mmol) was added to 1M THF solution of TBAF (tetrabutylammonium fluoride) (1.5 mL). 1.5 mmol) and stirred at 50 ° C. for 161 hours. As confirmed by HPLC, a deprotected uridylic acid dimer was produced at a conversion rate of 99%.

Method for producing deprotected uridylic acid dimer 2′-O-phenylcarbamoyluridylic acid dimer (37.6 mg, 0.05 mmol) was added to 1M THF solution of TBAF (tetrabutylammonium fluoride) (1.5 mL). ) Followed by a solution of tetrabutylammonium hydroxide in methanol (0.15 mL, 0.15 mmol). After stirring at 50 ° C. for 98 hours, when confirmed by HPLC, a deprotected uridylic acid dimer was produced at a conversion rate of 99% or more.

Method for producing deprotected uridylic acid dimer 2′-O-phenylcarbamoyluridylic acid dimer (30.1 mg, 0.04 mmol) was added to 1M THF solution of TBAF (tetrabutylammonium fluoride) (1.2 mL). , 1.2 mmol) followed by MS4A (5.0 mg). After stirring at 67 ° C. for 22 hours, it was confirmed by HPLC that a deprotected uridylic acid dimer was produced at a conversion rate of 96%.

Method for Producing 5′-O-dimethoxytrityluridylic Acid Dimer Deprotected with 2 ′ Hydroxyl 5′-O-Dimethoxytrityl-2′-O-phenylcarbamoyluridylic Acid Dimer (48.6 mg, 0.05 mmol) was added to a 1M THF solution (5.0 mL, 5 mmol) of TBAF (tetrabutylammonium fluoride), and the mixture was stirred at 66 ° C. for 15 hours. As confirmed by HPLC, 5'-O-dimethoxytrityl-protected uridylic acid dimer was produced at a conversion rate of 99% or more.
[HPLC analysis conditions]
Column: μ-BONDASPHERE, eluent: acetonitrile / 10 mM ammonium acetate aqueous solution = 10/90 → 90/10 (gradient), flow rate: 1.0 mL / min. Detection wavelength: 254 nm, column temperature: 40 ° C., retention time: 5′-O-dimethoxytrityl-2′-O-phenylcarbamoyluridylic acid dimer = 26 minutes, 2 ′ hydroxyl group deprotected 5 ′ Production method of -O-dimethoxytrityluridylic acid dimer = 24 minutes.

Method for Producing 5′-O-dimethoxytrityluridylic Acid Dimer Deprotected with 2 ′ Hydroxyl 5′-O-Dimethoxytrityl-2′-O-phenylcarbamoyluridylic Acid Dimer (48.6 mg, 0.05 mmol) was added to a 1M THF solution (5.0 mL, 5 mmol) of TBAF (tetrabutylammonium fluoride), and the mixture was stirred at 50 ° C. for 96 hours. As confirmed by HPLC, 5'-O-dimethoxytrityl-protected uridylic acid dimer was produced at a conversion rate of 99% or more.

Comparative example

  2′-O-phenylcarbamoyluridylic acid dimer (23.0 mg, 0.033 mmol) was added to a 1M aqueous solution of sodium hydroxide (1.0 mL, 1.0 mmol), and then 2.5 ° C. at 50 ° C. After stirring for a period of time, when confirmed by HPLC, the raw material was consumed to 0.1 area% or less, the deprotected uridylic acid dimer was 3 area%, and the decomposition product uridylic acid was 16 area%, 5 '-Phosphoric acid was produced in 24 area% and uridine in 39 area%.

Claims (3)

General formula (1):

[Wherein R ¹ has an optionally substituted alkyl group having 1 to 18 carbon atoms, an optionally substituted cycloalkyl group having 1 to 18 carbon atoms, and a substituent. An aryl group having 6 to 18 carbon atoms, an aralkyl group having 7 to 18 carbon atoms which may have a substituent, a sulfonyl group which may have a substituent, or a substituent. Represents a good silyl group, R ² represents a hydrogen or hydroxyl protecting group or oligonucleic acid, and R ³ represents an oligonucleic acid. X represents a hetero atom. Y represents hydrogen, a hydroxyl group, an alkoxy group, or a thiol group. B is the following formula:

R ⁴ represents adenine, guanine, cytosine, uracil represented by hydrogen or an amino protecting group, or a group derived therefrom. m represents a natural number greater than 1, and a ribonucleoside derivative in which the 2′-hydroxyl group is protected and / or an oligonucleic acid derivative containing this ribonucleoside derivative as a constituent unit is represented by the general formula (2):
R ⁵ ₄ NF (2)
[Wherein R ⁵ has an optionally substituted alkyl group having 1 to 18 carbon atoms, an optionally substituted cycloalkyl group having 1 to 18 carbon atoms, or a substituent. And an aryl group having 6 to 18 carbon atoms, which may have a substituent, and an aralkyl group having 7 to 18 carbon atoms which may have a substituent.], Represented by the general formula (3):

[Wherein R ² , R ³ , B, X, Y, m are the same as above. And / or an oligonucleic acid derivative containing the ribonucleoside as a constituent unit. The process according to claim 1, wherein R ⁵ is an alkyl group. The process according to claim 2, wherein the alkyl group of R ⁵ is a butyl group.