JPWO2006043521A1 - Optically active oligonucleic acid compound having phosphorothioate bond - Google Patents

Abstract

An object of the present invention is to provide a novel nucleic acid dimer compound useful for producing an optically active oligonucleic acid compound having a phosphorothioate bond. An optically active nucleic acid dimer compound represented by the following general formula (2a) or (2b). [Wherein, Bx1 and Bx2 each independently represents a nucleobase optionally having a protecting group. R1a1 and R1a2 each independently represent hydrogen, alkoxy or silyloxy. R2 represents acyl or a substituent represented by the following general formula (3a) or (3b). [Wherein, R2a and R2b are the same or different and each represents alkyl, or represents a 5- to 6-membered saturated amino ring group formed by R2a and R2b together with an adjacent nitrogen atom. R2d represents aryl or a bicyclic heterocyclic group. R2c and R2e represent alkyl. R 3 represents alkyl, aryl, or a bicyclic heterocyclic group. R4 represents hydrogen or a group capable of leaving under acidic conditions. ]

Description

  The present invention relates to an optically active oligonucleic acid compound having a phosphorothioate bond, useful as a therapeutic agent, diagnostic agent or research reagent, and a method for producing the same.

  In recent years, an antisense method using oligo DNA is known as a method for suppressing the expression of a specific gene. Antisense molecules having a structure complementary to the target gene (mRNA) are theoretically capable of controlling the growth of cancer cells and the like at the gene level, and thus are expected to be used as new drugs. It has already been clarified that a natural oligo DNA having a structure complementary to a target gene (mRNA) forms a stable duplex with the target gene (mRNA) and functions as an antisense molecule. However, it has been clarified that natural oligo DNA loses its function as an antisense molecule due to metabolism of its phosphate cross-linking part and nucleobase part by various enzymes in the cell. Creation of nucleic acid molecules with features such as improved affinity for the target gene (mRNA), improved permeability to cells, and improved stability to nucleolytic enzymes to make the antisense method an effective gene therapy method Is effective.

In order to achieve the above object, various chemical modifications have been studied on oligo DNA. In particular, oligo DNA having a phosphorothioate bond is the most studied compound and has been confirmed to have an antisense effect on various target genes (mRNA).
However, oligo DNAs having phosphorothioate bonds synthesized by ordinary chemical synthesis are theoretically 2 ^N if each phosphorothioate binding site is a diastereomeric mixture of Rp and Sp, and N phosphorothioate bonds are present in the molecule. There are diastereomeric mixtures. This P-chiral problem is expected to greatly affect the affinity to the target gene (mRNA), the stability to nucleolytic enzymes, etc., and the synthesis of optically active nucleic acid compounds is desired.

Several research groups have reported that in oligo DNA, the Rp isomer enhances the binding ability to the target RNA, and the Sp isomer is sufficiently stable to exonuclease. .
Recently, an optically active oligo DNA having a phosphorothioate bond has been synthesized (see Patent Document 1 and Non-Patent Document 1). However, since the condensation step includes a step in which a phosphorothioate bond having an asymmetric center is formed, it is not always possible to obtain a highly pure oligo DNA. Moreover, an optically active oligo RNA having a phosphorothioate bond or an oligonucleic acid compound having RNA in the molecule has not been produced yet.

On the other hand, recently, it has been found that RNA itself has various functions and activities. For example, gene expression suppression as seen in RNA interference is known.
However, since an optically active oligo RNA having a phosphorothioate bond or an oligonucleic acid having RNA in the molecule has not been synthesized, the effect of the compound has not been sufficiently studied.

International Publication No. 01 / 40515A1 Pamphlet Wojciech J.H. Stec et al., Journal of American Chemical Society, 120 (29), 7156 (1998).

  An object of the present invention is to provide an optically active oligonucleic acid compound having a phosphorothioate bond that can be useful mainly as a therapeutic agent, a diagnostic agent or a research reagent, a method for producing the same, and a nucleic acid dimer as a raw material thereof.

As a result of intensive studies, the present inventors have found that the above object can be achieved by using an optically active nucleic acid dimer compound having a phosphorothioate bond, and have completed the present invention.

I. Process for producing optically active oligonucleic acid compound having phosphorothioate bond An optically active oligonucleic acid compound having a phosphorothioate bond represented by the following general formula (1) is used as a nucleic acid synthesis unit as the following general formula (2a) or (2b). It can be produced by using at least one optically active nucleic acid dimer compound represented by This will be described in detail below.
[In the formula, each B independently represents a nucleobase. n represents an integer within the range of 1 to 99. Each R ¹ independently represents hydrogen, a hydroxyl group or an alkoxy group, and each R ¹ represents at least one hydroxyl group. Each Y independently represents a substituent represented by the following general formula (1a), (1b) or (1c), and each Y represents at least one substituent (1a) or (1b). Represents. However, the case where Y of adjacent constituent monomers is any substituent (1a) or (1b) is excluded.
Z represents hydrogen or a phosphate group.
Bx ₁ and Bx ₂ are the same or different and represent a nucleobase which may have a protecting group. R ^1a1 and R ^1a2 are the same or different and each represents hydrogen, alkoxy or silyloxy. R ² represents acyl or a substituent represented by the following general formula (3a) or (3b).
[Wherein R ^2a and R ^2b are the same or different and each represents alkyl, or R ^2a and R ^2b are formed together with an adjacent nitrogen atom to form a 5- to 6-membered saturated amino ring group Represents. R ^2d represents aryl or a bicyclic heterocyclic group. R ^2c and R ^2e represent alkyl. ]
R ³ represents alkyl, aryl, or a bicyclic heterocyclic group. R ⁴ represents hydrogen or a group capable of leaving under acidic conditions. ]

The “nucleobase” according to B, B _X1 and B _X2 is not particularly limited as long as it is used for nucleic acid synthesis. For example, adenine, guanine, cytosine, uracil, thymine, or modifications thereof can be mentioned.
A “modified form” of a nucleobase refers to a compound in which the nucleobase is substituted with an arbitrary substituent.
Examples of the substituent according to the modified form of B, B _X1 and B _X2 include halogen, acyl, alkyl, arylalkyl, alkoxy, alkoxyalkyl, hydroxy, amino, monoalkylamino, dialkylamino, carboxy, cyano and nitro. One to three identical or different substituents can be selected from the group consisting of

Here, examples of “halogen” in the present invention include fluorine, chlorine, bromine and iodine.
In the present invention, examples of the “acyl” include linear or branched alkanoyl having 1 to 6 carbon atoms and aroyl having 7 to 13 carbon atoms. Specific examples include formyl, acetyl, n-propionyl, isopropionyl, n-butyryl, isobutyryl, tert-butyryl, valeryl, hexanoyl, benzoyl, naphthoyl, and levulinyl. The “acyl” may be substituted, and examples of the substituent include halogen, alkyl, alkoxy, cyano, nitro, and trimethylsilyl, and 1 to 3 of these are substituted. Among them, examples of the “acyl” related to R ² include levulinyl, acetyl, propionyl, butyryl, isobutyryl, benzoyl, 4-anisoyl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl. Can be mentioned.

In the present invention, examples of the “alkyl” include linear or branched ones having 1 to 5 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. The “alkyl” may be substituted, and examples of the substituent include halogen, alkyl, alkoxy, cyano, nitro, and trimethylsilyl, which are substituted by 1 to 3 of them. Among them, examples of the “alkyl” related to R ^2a and R ^2b include methyl, ethyl, n-propyl and isopropyl, and the “alkyl” related to R ^2c , R ^2d and R ³ includes, for example, , Methyl, 2-cyanoethyl, 2-nitroethyl, and 2-trimethylsilylethyl.
In the present invention, examples of the alkyl moiety of “arylalkyl”, “alkoxyalkyl”, “monoalkylamino” and “dialkylamino” are the same as the above “alkyl”.

In the present invention, examples of the “aryl” include those having 6 to 12 carbon atoms. Specific examples include phenyl, 1-naphthyl, 2-naphthyl, and biphenyl. The “aryl” may be substituted, and examples of the substituent include halogen, alkyl, alkoxy, cyano, and nitro, and 1 to 3 of these are substituted. Among them, examples of the “aryl” according to R ^2d and R ³ include 2-chlorophenyl and 2-chloro-4-tert-butylphenyl.
In the present invention, examples of the aryl moiety of “arylalkyl” include the same as the above “aryl”.

In the present invention, examples of the “alkoxy” include linear or branched ones having 1 to 4 carbon atoms. Specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. The “alkoxy” may be substituted, and examples of such substituent include alkyl and alkoxy, and 1 to 3 of these may be substituted. Among them, ^examples of “alkoxy” related to R ¹ , R ^1a1 , and R ^1a2 include methoxy, ethoxy, and methoxyethoxy.
In the present invention, the alkoxy moiety of “alkoxyalkyl” may be the same as the above “alkoxy”.

  In the present invention, “halogen”, “alkyl” and “alkoxy” which are substituents of “acyl”, “alkyl” and “aryl” may be the same as those described above.

The “nucleobase” according to B _X1 and B _X2 may be protected. Among them, a nucleic acid having an amino group, such as adenine, guanine and cytosine, preferably has an amino group protected. The “amino-protecting group” is not particularly limited as long as it is used as a protecting group for nucleic acids. Specifically, for example, benzoyl, 4-anisoyl, acetyl, propionyl, butyryl, isobutyryl, phenyl Examples include acetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, and 4-isopropylphenoxyacetyl.

^Examples of “silyloxy” ^according to R ^1a1 and R ^1a2 include trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, triisopropylsilyl, and tert-butyldiphenylsilyl.

Examples of the “1-2 bicyclic heterocyclic group” according to R ^2d and R ³ include 1 to 6 heteroatoms selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom. Examples thereof include a 5- to 12-membered monocyclic ring or condensed ring which may have one unsaturated bond and may be substituted. Examples of such substituents include 1 to 3 identical or different substituents selected from the group consisting of alkyl, alkoxy, halogen, and nitro. Of these, 1-benzotriazole and 1-morpholino are preferable.

Examples of the “group capable of leaving under acidic conditions” according to R ⁴ include trityl, 4,4′-dimethoxytrityl, 9-phenylxanthen-9-yl, 2-tetrahydropyranyl, 1-methoxy-4- Mention may be made of tetrahydropyranyl.
Examples of the “5- to 6-membered saturated amino ring group” related to R ^2a and R ^2b include pyrrolidin-1-yl, piperidin-1-yl, and morpholin-1-yl.

All the asymmetric centers on the phosphorous atom of the phosphorothioate binding site present in the oligonucleic acid compound produced in the present invention are introduced by the optically active nucleic acid dimer compound (2a) or (2b).
The nucleic acid dimer compound (2a) or (2b) used in the method for producing an oligonucleic acid compound according to the present invention differs depending on whether it is produced by a so-called solid phase method or a so-called liquid phase method.
Hereinafter, the production method by the solid phase method and the production method by the liquid phase method of the oligonucleic acid compound having a phosphorothioate bond will be described in detail.

FIG. 1 represents a chromatogram obtained by reverse phase HPLC analysis of (Rp) -AsA (1). In the figure, the vertical axis represents time (minutes) and the horizontal axis represents absorption intensity. FIG. 2 represents a chromatogram obtained by reverse phase HPLC analysis of (Sp) -AsA (2). In the figure, the vertical axis represents time (minutes) and the horizontal axis represents absorption intensity. FIG. 3 represents a chromatogram obtained by reverse phase HPLC analysis of a mixture of (Rp) -AsA (1) and (Sp) -AsA (2). In the figure, the vertical axis represents time (minutes) and the horizontal axis represents absorption intensity. FIG. 4 shows a chromatogram obtained by ion exchange HPLC analysis of the oligonucleic acid compound (3) synthesized in Example 8. In the figure, the vertical axis represents time (minutes) and the horizontal axis represents absorption intensity. FIG. 5 shows a chromatogram obtained by ion exchange HPLC analysis of the oligonucleic acid compound (4) synthesized in Example 9. In the figure, the vertical axis represents time (minutes) and the horizontal axis represents absorption intensity.

I (a). Method for producing optically active oligonucleic acid compound having phosphorothioate bond by solid phase method The method for producing oligonucleic acid compound having phosphorothioate bond by solid phase method is, for example, stepwise through the following steps 1 to 7. The nucleic acid synthesis unit is condensed in the 3 ′ to 5 ′ direction to produce an oligonucleic acid compound having a phosphorothioate bond having a nucleic acid polymerization degree of 2 to 100. The production method can be carried out by any method using a manual or an automatic synthesizer, but a method using an automatic synthesizer is desirable from the viewpoint of simplification of the operation method and the accuracy of synthesis. Further, among the compounds and reagents described in the reaction formula, those other than the nucleic acid synthesis unit are not particularly limited as long as they are generally used for oligo DNA synthesis.

(1) Step 1: A step of removing a protecting group for a hydroxyl group at the 5 ′ position by allowing an acid to act on a nucleic acid compound supported on a solid phase carrier.
This step is produced by carrying out the operations of the nucleic acid compounds represented by the following general formulas (6), (6a) and (6b) supported on a solid phase carrier, or steps A to D described later. It can be carried out by allowing an acid to act on oligo RNA or oligo DNA (hereinafter referred to as “nucleic acid compound supported on a solid phase carrier”) supported on a solid phase carrier.
[Wherein, Bx, B _x1 , B _x2 , R ^1a , R ^1a1 , R ^1a2 , R ³ , R ⁴ are as defined above. R ^2L is a substituent represented by the following general formula (7).
[Wherein L represents a single bond or a substituent represented by the following general formula (8).
[Wherein R ^2e has the same ^meaning as described above. ]]]

  Examples of the “solid support” include, for example, controlled pore glass (CPG), oxalylated-constant glass (see, for example, Alul et al., Nucleic Acids Research, 1991, 19, 1527), TentaGel support— Mention may be made of aminopolyethylene glycol derivatized supports (see eg Wright et al., Tetrahedron Letters, 1993, 34, 3373), Poros-polystyrene / divinylbenzene copolymer.

Examples of the “linker” include 3-aminopropyl, succinyl, 2,2′-diethanolsulfonyl, and long-chain alkylamino (LCAA).
Compounds (6), (6a), and (6b) are nucleic acid compounds that are produced according to known methods or are supported on a solid phase carrier that is commercially available. Preferable embodiments of R ^{2L in} the compounds (6), (6a), and (6b) include substituents represented by the following general formulas (9a) and (9b).
[Wherein, R ^2e has the same ^meaning as described above. ]
The compound (6a) or (6b) in which R ^2L is the substituent (9a) or (9b) can be obtained by, for example, a method known from the nucleic acid dimer compound (2a) or (2b) (for example, Steven P. Adams et al., Journal). of American Chemical Society, 105, 661 (1983), T. Horn et al., Tetrahedron Letters, 27, 4705 (1986), A. Guzaev et al., US Pat. No. 5,959,090).

Examples of the “acid” that can be used in this step include trifluoroacetic acid, trichloroacetic acid, acetic acid, and the like. The acid used in this step can also be diluted with an appropriate solvent. Examples of the reaction solvent include dichloromethane, acetonitrile, water, or a mixed solvent thereof. The reaction temperature in the above reaction is suitably 20 ° C to 50 ° C. The reaction time varies depending on the type of acid used and the reaction temperature, but usually 1 minute to 1 hour is appropriate. The amount of the reagent used is suitably 1 to 100 times the molar amount, preferably 1 to 10 times the molar amount of the nucleic acid compound supported on the solid phase carrier.

(2) Step 2: A step of condensing a nucleic acid synthesis unit using an activator to the nucleic acid compound carried in Step 1 and supported on a solid phase carrier.
This step can be performed by allowing a nucleic acid synthesis unit and an activator to act on a nucleic acid compound supported on a solid phase carrier.

  Examples of the “nucleic acid synthesis unit” used in the solid phase method include a nucleic acid monomer unit, a nucleic acid dimer unit, and a nucleic acid oligomer unit.

Examples of the “nucleic acid monomer unit” include a compound represented by the following general formula (10a), which can be produced or commercially obtained according to a known method.
[Wherein, Bx, R ^1a and R ⁴ are as defined above. R ^2pa represents a substituent represented by the general formula (3a).
[Wherein, R ^2a , R ^2b and R ^2c have the same ^meaning as described above. ]]

Examples of the “nucleic acid dimer unit” include an optically active nucleic acid dimer compound represented by the following general formula (2a ₁ ) or (2b ₁ ).
[Wherein, B _x1 , B _x2 , R ^1a1 , R ^1a2 , R ^2pa , R ³ , R ⁴ are as defined above. ]

Examples of the “oligonucleic acid unit” include an oligonucleic acid compound represented by the following general formula (11a).
[Wherein, B _x , R ^1a , R ^2pa , R ³ , R ⁴ are as defined above. q represents an integer in the range of 1 to 98. ]

Examples of the “activator” include 1H-tetrazole, 5- (4-nitrophenyl) -1H-tetrazole, or diisopropylaminotetrazolide. The reaction temperature in the above reaction is suitably 20 ° C to 50 ° C. The reaction time varies depending on the type of activator used and the reaction temperature, but usually 1 minute to 1 hour is appropriate. The amount of the reagent to be used is suitably 1 to 100-fold mol amount, more preferably 1 to 10-fold mol amount based on the nucleic acid compound supported on the solid phase carrier.

(3) Step 3: A step of capping the hydroxyl group at the 5′-position of the nucleic acid compound supported on the solid phase carrier that has not been reacted in Step 2.
This step is a reaction for protecting the 5′-position hydroxyl group of the nucleic acid compound supported on the solid phase carrier that has not been reacted in Step 2, and a capping agent is added to the nucleic acid compound supported on the solid phase carrier. It can be implemented by acting.

Examples of the “capping agent” include acetic anhydride or phenoxyacetic anhydride. The capping agent can be used by diluting with a suitable solvent so as to have a concentration of 0.05 to 1M. The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include pyridine, dichloromethane, acetonitrile, tetrahydrofuran, and mixed solvents thereof. Further, as necessary, for example, 4-dimethylaminopyridine and N-methylimidazole can be used as the “reaction accelerator”. The reaction temperature in the above reaction is suitably 20 ° C to 50 ° C. The reaction time varies depending on the type of capping agent used and the reaction temperature, but usually 1 minute to 30 minutes is appropriate. The amount of the reagent used is suitably 1 to 100 times the molar amount, preferably 1 to 10 times the molar amount of the nucleic acid compound supported on the solid phase carrier.

(4) Step 4: A step of converting a phosphite group into a phosphate group by allowing an oxidant to act on the nucleic acid compound produced in Step 2.
This step is a reaction for conversion from trivalent phosphorus to pentavalent phosphorus using an oxidizing agent, and can be performed by allowing an oxidizing agent to act on the nucleic acid compound supported on the solid phase carrier.

Examples of the “oxidant” include iodine and tert-butyl hydroperoxide. In addition, the oxidizing agent used in this step can be used by diluting with an appropriate solvent so as to have a concentration of 0.05 to 2M. The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include pyridine, tetrahydrofuran, water, or a mixed solvent thereof. For example, iodine / water / pyridine-tetrahydrofuran or iodine / pyridine-acetic acid, a peroxide (eg, tert-butyl hydroperoxide / dichloromethane) can be used. The reaction temperature is suitably 20 ° C to 50 ° C. The reaction time varies depending on the type of oxidant used and the reaction temperature, but usually 1 minute to 30 minutes is appropriate. The amount of the reagent to be used is suitably 1 to 100 times the molar amount, preferably 10 to 50 times the molar amount of the nucleic acid compound supported on the solid phase carrier.

(5) Step 5: An oligonucleic acid compound having a desired chain length phosphorothioate bond produced by repeating Steps 1 to 4 above is excised from the solid support, and each nucleobase moiety and each 2′-position hydroxyl group Removing the protecting group.
This step is a reaction in which an oligonucleic acid compound having a desired chain length phosphorothioate bond is removed from the solid phase carrier and linker by a cleaving agent. It can be carried out by adding a cleaving agent to the carrier. In this step, the protecting group of each nucleobase can be removed.

Examples of the “cutting agent” include concentrated aqueous ammonia and methylamine. The “cutting agent” used in this step can also be used after diluted with water, methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran, or a mixed solvent thereof. Of these, ethanol is preferred.
The reaction temperature is suitably 15 ° C to 75 ° C, preferably 15 ° C to 30 ° C, and more preferably 18 ° C to 25 ° C. The deprotection reaction time is suitably 1 to 30 hours, preferably 1 to 24 hours, and more preferably 12 to 24 hours. The concentration of ammonium hydroxide in the solution used for deprotection is suitably 20-30% by weight, preferably 25-30% by weight, more preferably 28-30% by weight. The amount of the reagent used is suitably 1 to 100 times the molar amount, preferably 10 to 50 times the molar amount of the nucleic acid compound supported on the solid phase carrier.

The step of removing the protecting group for the hydroxyl group at the 2′-position includes “reagent for removing the protecting group for the hydroxyl group at the 2′-position” such as tetra n-butylammonium fluoride, hydrogen trifluoride / triethylamine salt. This can be performed by acting on an oligonucleic acid compound having a phosphorothioate bond having a desired chain length cut out from the phase carrier. The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include tetrahydrofuran, N-methylpyrrolidone, pyridine, and a mixed solvent thereof. The reaction temperature is suitably 20 ° C to 80 ° C. The reaction time varies depending on the type of deprotecting agent to be used and the reaction temperature, but usually 1 hour to 100 hours is appropriate. The amount of the reagent to be used is suitably 50 to 500-fold mol amount, preferably 50 to 100-fold mol amount based on the protecting group to be removed.
Conventional separation means from the reaction mixture, for example, extraction, concentration, neutralization, filtration, centrifugation, recrystallization, silica gel column chromatography, ion exchange column chromatography, gel filtration column chromatography, dialysis, ultrafiltration, etc. Can be used to isolate an oligonucleic acid compound in which the 5′-position is protected.

(6) Step 6: A step of removing the protecting group for the 5′-hydroxyl group of the oligonucleic acid compound produced in Step 5.
This step is a reaction that finally removes the protecting group of the 5′-hydroxyl group of the oligonucleic acid compound, and is carried out by allowing an acid to act on the oligonucleic acid compound having a phosphorothioate bond cleaved from the solid phase carrier. Can do.

Examples of the “acid” include trichloroacetic acid, dichloroacetic acid, acetic acid and the like. The acid used in this step can also be diluted with an appropriate solvent. The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, water, a buffer solution having a pH of 2 to 5, and a mixed solvent thereof. Examples of the buffer solution include an acetate buffer solution. The reaction temperature is suitably 20 ° C to 50 ° C. The reaction time varies depending on the type of acid used and the reaction temperature, but usually 1 minute to 1 hour is appropriate. The amount of the reagent used is suitably 1 to 100 times the molar amount, preferably 1 to 10 times the molar amount of the nucleic acid compound supported on the solid phase carrier.

(7) Step 7: A step of separating and purifying the oligonucleic acid compound produced in Step 6.
The “separation and purification step” refers to the usual separation and purification means from the above reaction mixture, for example, extraction, concentration, neutralization, filtration, centrifugation, recrystallization, C ₈ to C ₁₈ reverse phase column chromatography, C ₈ Desired by using means such as _C18 reverse phase cartridge column, cation exchange column chromatography, anion exchange column chromatography, gel filtration column chromatography, high performance liquid chromatography, dialysis, ultrafiltration, alone or in combination. This is a step of isolating and purifying an oligonucleic acid compound having a phosphorothioate bond.

Examples of the “elution solvent” include acetonitrile, methanol, ethanol, isopropyl alcohol, water alone, or a mixed solvent of any ratio. In this case, for example, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, ammonium acetate, triethylammonium acetate, sodium acetate, potassium acetate, tris hydrochloric acid, ethylenediaminetetraacetic acid are added at a concentration of 1 mM to 2 M. The pH of the solution can be adjusted in the range of 1-9.

By repeating the steps 1 to 4, the oligonucleic acid compound (1) having a phosphorothioate bond having a desired chain length can be produced. In this production method, compounds (6), (6a), (6b) and the like can be used as starting materials for producing an oligonucleic acid compound having a phosphorothioate bond. However, when the compound (6) is used as a starting material, at least one of the nucleic acid dimer compounds (2a ₁ ) or (2b ₁ ) needs to be used as the nucleic acid synthesis unit.
In this production method, the oligo RNA is isolated and purified by performing the operation of the above step 6 before performing the operation of the above step 5, performing the operation of the above step 5 and then performing the operation of the above step 7. You can also.
Furthermore, in this production method, after the operation of the above step 5 is performed, the operation of the above step 7 is performed, and then the above step 6 and then the operation of the above step 7 are performed again to isolate and purify oligo RNA. it can.

I (b). Production of optically active oligonucleic acid compound having phosphorothioate bond by liquid phase method Production of oligonucleic acid compound having phosphorothioate bond by liquid phase method is carried out stepwise by, for example, the following steps a to d. An oligonucleic acid compound having a phosphorothioate bond having a nucleic acid polymerization degree of 2 to 100 can be produced by condensing nucleic acid synthesis units. In the synthesis method, among the compounds and reagents described in the reaction formula, those other than the nucleic acid synthesis unit are not particularly limited as long as they are generally used for oligo DNA synthesis.

  Examples of the “nucleic acid synthesis unit” used in the liquid phase method include a nucleic acid monomer unit, a nucleic acid dimer unit, and a nucleic acid oligomer unit.

Examples of the “nucleic acid monomer block” include compounds represented by the following general formula (10b) that can be produced or commercially obtained according to known methods.
[Wherein, Bx, R ^1a and R ⁴ are as defined above. R ^2pb represents acyl or a substituent represented by the following general formula (3b).
[Wherein, R ^2d and R ^2e are as defined above. ]]
^Examples of the “acyl” related to R ^2pb include the same as those described above. Among them may be the same as the R ^2.

Examples of the “nucleic acid dimer unit” include an optically active nucleic acid dimer compound represented by the following general formula (2a ₂ ) or (2b ₂ ).
[Wherein, B _x1 , B _x2 , R ^1a1 , R ^1a2 , R ^2pb , R ³ , R ⁴ are as defined above. ]

As the “oligonucleic acid unit”, for example, a compound represented by the following general formula (11b), or a nucleic acid monomer unit, a nucleic acid dimer unit or an oligonucleic acid unit (11b) is condensed through steps a and b. And an oligonucleic acid compound produced in the above manner.
[Wherein, Bx, R ^1a , R ^2pb , R ³ and R ⁴ have the same ^meanings as described above. q represents an integer in the range of 1 to 98. ]

(1) Step a: Selective deprotection step.
The selective deprotection step is a step of producing a selectively deprotected compound by allowing a reagent that removes the protecting group of the hydroxyl group at the 5′-position or 3′-position to act on the nucleic acid synthesis unit. Specifically, for example, in a nucleic acid monomer unit, a nucleic acid dimer unit or an oligonucleic acid unit which is a nucleic acid synthesis unit, for example, 4,4′-dimethoxytrityl which is a protecting group substituted on the 5′-position hydroxyl group Or a step of removing, for example, a cyanoethyl group, which is a protecting group on a phosphate group substituted with a hydroxyl group at the 3 ′ position.
Examples of the “reagent for removing cyanoethyl group” include a pyridine-triethylamine-water (3: 1: 1) solution.
Examples of the “reagent for removing 4,4′-dimethoxytrityl group” include trichloroacetic acid and acetic acid.
The amount of “reagent for removing 4,4′-dimethoxytrityl group” and “reagent for removing cyanoethyl group” is 1 to 20 times the amount of 4,4′-dimethoxytrityl group or cyanoethyl group to be removed. The molar amount, more preferably 1 to 10 times the molar amount. The reaction temperature in the above reaction is, for example, suitably from −20 ° C. to 100 ° C., preferably from 0 ° C. to 80 ° C., more preferably from 5 ° C. to 30 ° C. The reaction time varies depending on the type of raw material used and the reaction temperature, but usually 1 minute to 1 hour is appropriate.

(2) Step b: a condensation step.
This step is usually carried out in the presence of an excess organic amine in an appropriate solvent in the presence of two types of selectively deprotected nucleic acid synthesis units (for example, nucleic acid synthesis in which the 5'-position hydroxyl protecting group is eliminated). The reaction can be carried out by allowing a condensing reagent to act on the unit and the nucleic acid synthesis unit from which the protecting group for the hydroxyl group at the 3 ′ position has been eliminated.
The reaction solvent is not particularly limited as long as it does not participate in the reaction. For example, ethers such as tetrahydrofuran, diethyl ether and 1,4-dioxane, nitriles such as acetonitrile and propionitrile, hydrocarbons such as benzene and toluene , Organic amines such as pyridine, or a mixed solvent thereof. The amount of the condensing reagent to be used is suitably 1 to 20 times the molar amount and preferably 1 to 10 times the molar amount of the nucleic acid monomer unit, nucleic acid dimer unit or oligonucleic acid unit which is the nucleic acid synthesis unit. The reaction temperature is, for example, suitably from −20 ° C. to 100 ° C., preferably from 0 ° C. to 80 ° C., more preferably from 5 ° C. to 30 ° C. The reaction time varies depending on the type of raw material used and the reaction temperature, but usually 30 minutes to 100 hours is appropriate.
Examples of the “condensation reagent” include 1- (2-mesitylenesulfonyl) -3-nitro-1,2,4-triazole, 2,4,6-trimethylbenzenesulfonyltetrazole, and 1- (2,4,6- Mention may be made of triisopropylbenzenesulfonyl) -3-nitro-1,2,4-triazole.

(3) Step c: Step of removing all protecting groups.
This step is a step of removing all protecting groups of a protected oligonucleic acid compound having a desired chain length produced by repeating the steps a and b.
Examples of the “reagent for removing each protecting group” used in this step include ammonia, a reagent for removing a silyl group, and an acid.
Examples of the “ammonia” include an ammonium hydroxide aqueous solution and an ammonia / methanol solution. The reaction temperature is suitably 18 ° C to 75 ° C, preferably 18 ° C to 65 ° C, and more preferably 25 ° C to 55 ° C. The reaction time is suitably 1 to 30 hours, preferably 1 to 24 hours. The concentration of ammonium hydroxide in the solution used for deprotection is suitably 20-30% by weight, preferably 25-30% by weight. The amount of the reagent to be used is suitably 1 to 100-fold mol amount, preferably 10 to 50-fold mol amount based on the protecting group to be removed.
Examples of the “reagent for removing silyl group” include when the protecting group for the hydroxyl group at the 2 ′ position is a tert-butyldimethylsilyl group, for example, tetra n-butylammonium fluoride, hydrogen trifluoride / triethylamine salt. it can. The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include tetrahydrofuran, N-methylpyrrolidone, pyridine, triethylamine, or a mixed solvent thereof. The reaction temperature is suitably 20 ° C to 80 ° C, preferably 55 ° C to 70 ° C. The reaction time varies depending on the type of deprotecting agent to be used and the reaction temperature, but usually 1 hour to 100 hours is appropriate. The amount of the reagent to be used is suitably 50 to 500-fold mol amount, preferably 50 to 100-fold mol amount based on the protecting group to be removed.
Examples of the “acid” include trichloroacetic acid and acetic acid. The acid can be used by diluting with an appropriate solvent. Examples of the reaction solvent include dichloromethane, acetonitrile, water, or a mixed solvent thereof. The reaction temperature is suitably 20 ° C to 50 ° C. The reaction time varies depending on the type of acid used and the reaction temperature, but usually 1 minute to 1 hour is appropriate. The amount of the reagent to be used is suitably 1 to 100-fold mol amount, preferably 1 to 10-fold mol amount based on the compound supported on the solid phase carrier.

(4) Step d: Purification step.
“Purification step” refers to usual separation and purification means from the above reaction mixture, such as extraction, concentration, neutralization, filtration, centrifugation, recrystallization, C ₈ to C ₁₈ reverse phase column chromatography, C ₈ to C ₁₈ reverse phase cartridge columns, cation exchange column chromatography, anion exchange column chromatography, gel filtration column chromatography, high performance liquid chromatography, dialysis, ultrafiltration, etc. This is a step of isolating and purifying an oligonucleic acid compound having a phosphorothioate bond.
Examples of the “elution solvent” include acetonitrile, methanol, ethanol, isopropyl alcohol, water alone, or a mixed solvent of any ratio. In this case, for example, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, ammonium acetate, triethylammonium acetate, sodium acetate, potassium acetate, tris hydrochloric acid, ethylenediaminetetraacetic acid are added at a concentration of 1 mM to 2 M. The pH of the solution can be adjusted in the range of 1-9.

By repeating the above steps a and b, an oligonucleic acid compound having a desired chain length can be produced. In addition, in order to produce an oligonucleic acid compound having a phosphorothioate bond in this production method, at least one of the nucleic acid dimer compounds (2a ₂ ) or (2b ₂ ) is used as a nucleic acid synthesis unit, and as a constituent monomer of the oligonucleic acid compound It is necessary to use at least one ribonucleic acid compound.

I (c). Summary The present invention, at least one nucleic acid dimer compounds highly phosphorothioate binding site so as not to cause racemization protected (2a) or (2b) (case of the solid phase method, the nucleic acid dimer compound (2a ₁₎ Or (2b ₁ ), in the case of the liquid phase method, is a method for producing the oligonucleic acid compound (1) using the nucleic acid dimer compound (2a ₂ ) or (2b ₂ )). Further, the present invention is a method for forming only a phosphodiester bond, unlike the conventional method for producing an oligonucleic acid compound, unlike the conventional method for producing an oligonucleic acid compound having a phosphorothioate bond.
Therefore, this invention is a manufacturing method which can manufacture a highly purified oligonucleic acid compound easily by using a highly purified nucleic acid dimer compound (2a) or (2b) as one of the nucleic acid synthesis units. The purity of the oligonucleic acid compound produced according to the present invention is, for example, suitably from 50% to 100%, preferably from 95 to 99.99%, more preferably from 99 to 99.9%.

II. Optically Active Nucleic Acid Dimer Compound Having Phosphorothioate Bond The present invention includes an optically active nucleic acid dimer compound represented by the following general formula (2a) or (2b) (hereinafter referred to as “the nucleic acid dimer of the present invention”). Can do.
[Wherein, Bx ₁ , Bx ₂ , R ^1a1 , R ^1a2 , R ² , R ³ , R ⁴ are as defined above. ]

The nucleic acid dimer of the present invention has a feature that it does not cause racemization because it has a highly protected optically active phosphorothioate binding site. Therefore, the nucleic acid dimer of the present invention is useful as a raw material compound for producing, for example, an optically active oligonucleic acid compound (1) having a phosphorothioate bond.

Specific examples of the nucleic acid dimer of the present invention include the following 1. -14. Can be mentioned.
1. (Rp) -5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
2. (Rp) -5′-O- (4,4′-dimethoxytrityl) -2′-O-tert-butyldimethylsilyluridyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thiophosphoryl- 2'-O-tert-butyldimethylsilyluridine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
3. (Rp) -5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-2-N-isobutyryl-2'-O-tert-butyldimethylsilylguanosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
4). (Rp) -5'-O- (4,4'-dimethoxytrityl) -2-N-isobutyryl-2'-O-tert-butyldimethylsilylguanosyl- [3 '→ 5']-O- (2 -Cyanoethyl) thiophosphoryl-6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
5. (Rp) -5′-O- (4,4′-dimethoxytrityl) -2′-O-tert-butyldimethylsilyluridyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thiophosphoryl- 4-N- (4-Anisoyl) -2'-O-tert-butyldimethylsilylcytidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
6). (Rp) -5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-4-N- (4-anisoyl) -2'-O-tert-butyldimethylsilylcytidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
7). (Rp) -5'-O- (4,4'-dimethoxytrityl) -2-N-isobutyryl-2'-O-tert-butyldimethylsilylguanosyl- [3 '→ 5']-O- (2 -Cyanoethyl) thiophosphoryl-thymidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
8). (Sp) -5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
9. (Sp) -5′-O- (4,4′-dimethoxytrityl) -2′-O-tert-butyldimethylsilyluridyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thiophosphoryl- 2'-O-tert-butyldimethylsilyluridine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
10. (Sp) -5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-2-N-isobutyryl-2'-O-tert-butyldimethylsilylguanosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
11. (Sp) -5′-O- (4,4′-dimethoxytrityl) -2-N-isobutyryl-2′-O-tert-butyldimethylsilylguanosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
12 (Sp) -5′-O- (4,4′-dimethoxytrityl) -2′-O-tert-butyldimethylsilyluridyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thiophosphoryl- 4-N- (4-Anisoyl) -2'-O-tert-butyldimethylsilylcytidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
13. (Sp) -5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-4-N- (4-anisoyl) -2'-O-tert-butyldimethylsilylcytidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)
14 (Sp) -5′-O- (4,4′-dimethoxytrityl) -2-N-isobutyryl-2′-O-tert-butyldimethylsilylguanosyl- [3 ′ → 5 ′]-O— (2 -Cyanoethyl) thiophosphoryl-thymidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite)

III. Production Method of the Nucleic Acid Dimer of the Present Invention The nucleic acid dimer of the present invention can be produced as follows.
In the following production method, when there are substituents (for example, hydroxy, amino, carboxy) that are not desired to react with the raw material, the raw material is protected with a protecting group (for example, benzoyl or 4-anisoyl group) according to a known method in advance. It is common to use for reaction after protecting. After the reaction, the protecting group can be removed according to a known method such as catalytic reduction, alkali treatment, acid treatment or the like.
The nucleic acid dimer of the present invention can be produced from a known compound or an easily manufacturable intermediate through, for example, the following steps A to D.
Hereinafter, these steps will be described in detail.

(1) Step A: Step of producing a nucleic acid dimer compound (15).
[Wherein, B _x1 , B _x2 , R ^1a1 , R ^1a2 , R ³ , R ⁴ are as defined above. R ^2y represents acyl.
R ^2x represents a substituent represented by the following general formulas (13a) to (13e).
[Wherein, R ^3a and R ^3b are the same or different and each represents alkyl. R ^3c , R ^3d and R ^3e represent alkyl. R ^3f represents alkyl, aryl, or a bicyclic heterocyclic ring. q, r, s, and t each independently represent an integer in the range of 1 to 2. ]]
^Examples of the “alkyl” related to R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , and R ^3f include the same as described above. Especially, the "alkyl" concerning R ^{< 3a >} , R ^{< 3b>} , R ^<3e> can mention the same thing as the "alkyl" concerning said R ^<2a> , R ^<2b> . Moreover, the “alkyl” according to R ^3c , R ^3d , and R ^3f can be the same as the “alkyl” according to R ^2c .
Examples of the “aryl” and “1-2-bicyclic heterocycle” according to R ^3f include the same ones as described above. Among them, the “aryl” related to R ^3f can be the same as the “aryl” related to R ^2d .
^Examples of the “acyl” related to R ^2y include the same ones as described above. Among these, the same “acyl” according to R ² can be exemplified.

In this step, the nucleic acid compound (13) can be produced by dissolving the nucleic acid compound (12) in an appropriate solvent and reacting with an appropriate phosphorylating reagent. This reaction itself can be carried out according to a known method.
The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, toluene, and mixed solvents thereof. Examples of the “phosphorylation reagent” include 2-chlorophenyldichlorothiophosphate, 2,4-dichlorophenyldichlorothiophosphate, 2-cyanoethyl diisopropylchlorophosphoramidite, and 2-cyanoethyl tetraisopropylphosphorodiamidite. The amount of the phosphorylating reagent used is preferably 1 to 20 times the molar amount, more preferably 1 to 10 times the molar amount relative to the compound (12). The reaction temperature in the above reaction is preferably 0 ° C to 50 ° C. The reaction time varies depending on the type of raw material used and the reaction temperature, but usually 30 minutes to 100 hours is appropriate.

Next, the nucleic acid dimer compound (15) can be produced by dissolving the nucleic acid compound (13) in an appropriate solvent and reacting the nucleic acid compound (14) with an appropriate activator or condensing agent. This reaction itself can be performed by a known method.
Examples of the “activator” include 1H-tetrazole, 5-ethylthiotetrazole, 3,4-dichloroimidazole, 3,4-dicyanoimidazole, benzotriazole triflate, imidazole triflate, 5- (4-nitrophenyl)- Examples thereof include 1H-tetrazole and diisopropylaminotetrazolide. Examples of the “condensing agent” include 1- (2-methicylenesulfonyl) -3-nitro-1,2,4-triazole, 2,4,6-trimethylbenzenesulfonyltetrazole or 1- (2,4,6). -Triisopropylbenzenesulfonyl) -3-nitro-1,2,4-triazole. The amount of the activator or condensing agent used is preferably 1 to 20 times the molar amount, more preferably 1 to 10 times the molar amount with respect to the nucleic acid compound (13). The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, toluene, and mixed solvents thereof. The reaction temperature is suitably from 0 ° C to 50 ° C. The reaction time varies depending on the type of raw material used, reaction temperature, etc., but usually 30 minutes to 100 hours is appropriate.

At this time, when R ^2x is substituents (13a) to (13d), for example, the nucleic acid dimer compound (15) can be produced by oxidation with a sulfurizing reagent. Examples of the “sulfurizing reagent” include sulfur, 3H-1,2-benzodithiol-3-one 1,1-dioxide (view cage reagent), 3-amino-1,2,4-dithiazole-5-thione ( ADTT). The amount of the sulfurizing reagent used is preferably 1 to 20 times the molar amount, more preferably 1 to 10 times the molar amount with respect to the nucleic acid compound (13). The solvent used when oxidizing with the sulfurizing reagent is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, pyridine, and a mixed solvent thereof.

Usually, the nucleic acid dimer compound (15) produced in this step is a one-to-one diastereomer mixture because it has an asymmetric center at the phosphorothioate-linked phosphorus atom. When the nucleic acid compound (13) in which R ^2x is an optically active substituent (13d) is used, the ratio is shifted to one of the diastereomers, so that the separation step in the next step may be simplified.

(2) Step B: A step of producing an optically active nucleic acid dimer compound (15a) or (15b) by separation.
[Wherein, B _x1 , B _x2 , R ^1a1 , R ^1a2 , R ^2y , R ³ , R ⁴ are as defined above. ]
The diastereomer mixture nucleic acid dimer compound (15) can be separated into optically active nucleic acid dimer compounds (15a) and (15b) having a phosphorothioate bond by a separation operation such as silica gel chromatography column purification.
The nucleic acid dimer compounds (15a) and (15b) produced in this step are the nucleic acid dimers of the present invention in which R ² is acyl.

(3) Step C: a step of selectively removing R ^2y of the nucleic acid dimer compound (15a) or (15b).
[Wherein, B _x1 , B _x2 , R ^1a1 , R ^1a2 , R ^2y , R ³ , R ⁴ are as defined above. ]
The nucleic acid dimer compound (16a) or (16b) can be produced by dissolving the nucleic acid dimer compound (15a) or (15b) in an appropriate solvent and reacting with a reagent that selectively removes R ^2y .
The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include pyridine, acetonitrile, or a mixed solvent thereof. As the “reagent for selectively removing R ^2y ”, for example, R ^1a1 or R ^1a2 is tert-butyldimethylsilyl, R ⁴ is 4,4′-dimethoxytrityl, and R ³ is 2-cyanoethyl. When R ^2y is levulinyl, the reagent for selectively removing R ^2y may include a 0.5 M hydrazine monohydrate / acetic acid-pyridine (1: 4) solution. The amount of the “reagent for selectively removing R ^2y ” is 1 to 20 times by mole, more preferably 1 to 10 times by mole, relative to the nucleic acid dimer compound (15a) or (15b). . The reaction temperature is preferably 0 ° C to 50 ° C. The reaction time varies depending on the type of raw material used, reaction temperature, etc., but usually 30 minutes to 100 hours is appropriate.

(4) Process D: The process of manufacturing a nucleic acid dimer compound (17a) or (17b).
[Wherein, B _x1 , B _x2 , R ^1a1 , R ^1a2 , R ³ , R ⁴ are as defined above. R ^2p represents a substituent represented by the following general formula (3a) or (3b).
[Wherein, R ^2a , R ^2b , R ^2c , R ^2d , R ^2e have the same ^meaning as described above. ]]
In the present invention, the nucleic acid dimer compound (17a) or (17b) can be produced by dissolving the nucleic acid dimer compound (16a) or (16b) in an appropriate solvent and allowing a phosphorylating reagent to act.
The nucleic acid dimer compounds (17a) and (17b) produced in this step are the nucleic acid dimers of the present invention in which R ² is a substituent (3a) or (3b).
The reaction solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, toluene, and mixed solvents thereof. Examples of the “phosphorylation reagent” include 2-cyanoethyl diisopropylchlorophosphoramidite, 2-cyanoethyl tetraisopropylphosphorodiamidite, and 2-chlorophenylphosphoryl dichloride. A phosphorylation reagent is 1-20 times mole amount with respect to a nucleic acid dimer compound (16a) or (16b), More preferably, it is 1-10 times mole amount. The reaction temperature is, for example, suitably from −20 ° C. to 100 ° C., preferably from 0 ° C. to 80 ° C., more preferably from 5 ° C. to 30 ° C. The reaction time varies depending on the type of raw material used and the reaction temperature, but usually 30 minutes to 100 hours is appropriate.

IV. Oligonucleic acid of the present invention An optically active oligonucleic acid compound represented by the following general formula (1) (hereinafter referred to as “oligonucleic acid of the present invention”) will be described in detail.
[Wherein, B, n, R ¹ , Y, Z are as defined above. ]
n is suitably an integer in the range of 1 to 99, preferably an integer in the range of 10 to 50, and more preferably an integer in the range of 15 to 30.

The present invention also includes an optically active oligonucleic acid compound having a phosphorothioate bond represented by the following general formula (4) (also referred to as “the oligonucleic acid of the present invention” together with the oligonucleic acid compound (1)). Can do.
[Wherein, B and R ¹ are as defined above.
m represents an integer in the range of 1 to 49.
Y ¹ and Y ² are different from each other, one is a substituent represented by the following general formula (1a) or general formula (1b), and the other is represented by the following general formula (1c) Represents the substituent represented.
Represents.
T ¹ represents hydrogen or a substituent represented by the following general formula (5a).
[Wherein, B and R ¹ are as defined above. ]
T ² represents a hydroxyl group or a substituent represented by the following general formulas (5a) and (5b).
[Wherein, B and R ¹ are as defined above. ]]
m is suitably an integer in the range of 1 to 49, preferably an integer in the range of 5 to 25, and more preferably an integer in the range of 7 to 15.

The oligonucleic acid of the present invention can be used in the form of a free acid, but can also be used in the form of a salt according to a known method. Examples of such “salts” include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts, and tertiary organic amine salts such as triethylamine and pyridine.
In addition, examples of the present invention include the oligonucleic acid of the present invention or a salt thereof, in which all phosphorothioate bonds existing in the molecule have the Rp configuration or all the Sp configuration.
Furthermore, examples of the present invention include a highly pure oligonucleic acid of the present invention. The purity of the oligonucleic acid of the present invention is, for example, suitably from 50% to 100%, preferably from 95 to 99.99%, more preferably from 99 to 99.9%.

As a result of measuring the double-strand melting temperature of the oligonucleic acid of the present invention, the present inventors have the same or higher affinity for RNA that can form a complementary strand with that of the natural oligo RNA. It was revealed that a thermodynamically stable hybrid was formed (Test Example 1).
Therefore, it was revealed that the oligonucleic acid of the present invention is a useful compound as an antisense molecule. Among these, the oligonucleic acid of the present invention in which all phosphorothioate bonds are in the Rp configuration is particularly useful as an antisense molecule.
In addition, the present inventors have clarified that the oligonucleic acid of the present invention is more resistant to a nucleolytic enzyme (phosphodiesterase I, bovine serum) than a natural oligo RNA having the same sequence (test) Example 3, Test Example 4).

V. Double-stranded oligonucleic acid of the present invention As the present invention, a natural oligo RNA having a sequence complementary to the oligonucleic acid of the present invention or a double-stranded oligonucleic acid compound with the oligonucleic acid of the present invention can be mentioned.
In the present invention, the double-stranded oligonucleic acid compound is a pair of 12 to 52 base oligonucleic acid compounds having a duplex forming part of 10 to 50 base pairs, and this oligonucleic acid compound pair is introduced into the cell. It is defined as a pair of oligonucleic acids having an activity of cleaving mRNA complementary to the double-stranded oligonucleic acid compound by introduction to suppress the synthesis of the gene product encoded by the mRNA. Further, in the present specification, the double-stranded oligonucleic acid compound is not limited to siRNA reported by Tuschl et al. For example, in the double-stranded oligonucleic acid of the present invention, it is a pair of 12-52 base oligonucleic acid compounds, more preferably a pair of 15-30 base oligonucleic acid compounds.
The double-stranded oligonucleic acid of the present invention, when introduced into a cell as a complex with a liposome, exhibits RNA interference equivalent to a double-stranded oligoRNA of an oligoRNA consisting of all phosphodiester bonds having the same sequence, It was clarified that expression was suppressed (Test Example 2). Therefore, it was revealed that the double-stranded oligonucleic acid of the present invention is a useful compound as siRNA.
Furthermore, the present inventors examined the resistance to a nucleolytic enzyme (phosphodiesterase I) when a double-stranded oligonucleic acid compound was formed with a natural oligo RNA having a sequence complementary to the oligonucleic acid of the present invention. As a result, it was clarified that it has decomposition resistance (Test Example 5).

VI. Pharmaceutical composition The present invention includes a complex of the oligonucleic acid of the present invention and a carrier effective for transferring the oligonucleic acid into cells, or the double-stranded oligonucleic acid of the present invention and the Provided is a pharmaceutical composition comprising a complex with a carrier effective for transferring a double-stranded oligonucleic acid into a cell. The pharmaceutical composition of the present invention is used for the treatment and / or prevention of, for example, a disease caused by overexpression of Bcl-2 protein, a disease for which promotion of apoptosis is desired, or a hematological malignancy. Can be used. These diseases specifically include hematological malignancies including both lymphoma and leukemia, and solid tumors such as liver cancer, skin cancer, breast cancer, lung cancer, gastrointestinal cancer, prostate cancer, uterine cancer, bladder Such as cancer.
The carrier that forms a complex with the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention is a cationic liposome and cation that are effective for transferring the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention into cells. A cationic carrier such as a functional polymer or a carrier utilizing a viral envelope may be used. What is desirable as the cationic liposome is a liposome containing 2-O- (2-diethylaminoethyl) carbamoyl 1,3-O-dioleoylglycerol (hereinafter referred to as liposome A), oligofectamine (Invitrogen), lipofectin (Invitrogen), Lipofectamine (Invitrogen), Lipofectamine 2000 (Invitrogen), DMRIE-C (Invitrogen), GeneSilencer (Gene Therapeutics Systems), TransMessenger (QIAGEN), TransMist. Desirable examples of the cationic polymer include JetSI (Qbiogene), Jet-PEI (polyethyleneimine; Qbiogene), and the like. A desirable carrier using a viral envelope is GenomeONE (HVJ-E liposome; Ishihara Sangyo).
The complex of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention and a carrier contained in the pharmaceutical composition of the present invention can be prepared by methods known to those skilled in the art. Briefly, it is prepared by mixing a carrier dispersion having an appropriate concentration and a solution of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention. When a cationic carrier is used, the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention is negatively charged in an aqueous solution. A strand oligonucleic acid and a cationic carrier easily form a complex. As the aqueous solvent used for forming the complex, electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution may be used. Moreover, those skilled in the art can appropriately select conditions such as pH and temperature when forming the complex. For example, in the case of liposome A, a solution of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention in a 10% maltose aqueous solution is added to a 16 mg / ml liposome dispersion in a 10% maltose aqueous solution at pH 7.4, 25 ° C. It can be prepared by gradually adding while stirring.
The composite can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersion device or a high-pressure emulsification device, if necessary. A person skilled in the art uses the optimal method and conditions for preparing the complex of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention and a carrier, depending on the carrier to be used. The optimum method for the carrier can be selected.
The compounding ratio of the oligonucleic acid of the present invention or the complex of the present invention double stranded oligonucleic acid and the carrier contained in the pharmaceutical composition of the present invention is 1 part by weight of the oligonucleic acid of the present invention or double stranded oligonucleic acid of the present invention 1 to 200 parts by weight of carrier is suitable. The amount is preferably 2.5 to 100 parts by weight, more preferably 10 to 20 parts by weight, based on 1 part by weight of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention.
The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent in addition to the complex of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention and a carrier. Pharmaceutically acceptable carriers or diluents and the like are essentially chemically inert and harmless compositions that do not affect the biological activity of the pharmaceutical composition of the present invention at all. Examples of such carriers or diluents include but are not limited to salt solutions, sugar solutions, glycerol solutions, ethanol and the like.
The pharmaceutical composition of the present invention is provided in a form that contains a therapeutically and / or prophylactically effective amount of the complex and can be appropriately administered to a patient. The preparation form of the pharmaceutical composition of the present invention may be, for example, liquid preparations such as injections and drops, external preparations such as ointments and lotions, or freeze-dried preparations.
In the case of a liquid agent, it is appropriate that the present complex is present in a concentration range of 0.001 to 25% (w / v), preferably a concentration of 0.01 to 5% (w / v). It is suitable that it exists within the range, more preferably within the concentration range of 0.1 to 2% (w / v). The pharmaceutical composition of the present invention may contain an appropriate amount of any pharmaceutically acceptable additive such as an emulsification aid, a stabilizer, an isotonic agent, and a pH adjuster. Specifically, fatty acids having 6 to 22 carbon atoms (for example, cabrylic acid, cabric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, docosahexaenoic acid) and pharmaceutically Acceptable salts (for example, sodium salts, potassium salts, calcium salts); emulsification aids such as albumin and dextran; stabilizers such as cholesterol and phosphatidic acid; sodium chloride, glucose, maltose, lactose, sucrose, trehalose, etc. Isotonizing agents; pH adjusting agents such as hydrochloric acid, nitric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine; and the like. Any pharmaceutically acceptable additive can be added in a suitable step either before or after dispersion of the complex.
The lyophilized preparation can be prepared by dispersing a complex of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention and a carrier, followed by lyophilization. The lyophilization treatment can be performed by a conventional method. For example, a predetermined amount of the complex solution after the dispersion treatment described above is dispensed into a vial in an aseptic state, preliminarily dried for about 2 hours under conditions of about −40 to −20 ° C., and about 0 to 10 ° C. Primary drying under reduced pressure, followed by secondary drying under reduced pressure at about 15-25 ° C. and lyophilization. In general, the inside of the vial is replaced with nitrogen gas and stoppered to obtain a lyophilized preparation of the pharmaceutical composition of the present invention.
In general, the lyophilized preparation of the present invention can be re-dissolved and used by adding any appropriate solution (re-dissolved solution). Examples of such redissolved solution include electrolyte solutions such as water for injection and physiological saline, glucose solution, and other general infusion solutions. The amount of the redissolved solution varies depending on the application and is not particularly limited, but is suitably 0.5 to 2 times the amount of the solution before lyophilization or 500 ml or less.
The pharmaceutical composition of the present invention is preferably administered in a dosage unit form, and is administered intravenously, intraarterially, orally, intratissue, transdermally, transmucosally or rectally to animals including humans. It can be administered and may be administered by an appropriate method according to the patient's symptoms. Particularly preferred are intravenous administration, transdermal administration, and transmucosal administration. Moreover, local administration, such as local administration in cancer, can also be performed. Of course, it is administered in dosage forms suitable for these administration methods, for example, various injections, oral preparations, drops, absorbents, eye drops, ointments, lotions, suppositories and the like.
For example, the dose of the pharmaceutical composition of the present invention is preferably determined in consideration of the drug, dosage form, patient condition such as age and weight, administration route, nature and degree of disease, etc. The amount of the oligonucleic acid of the present invention or the oligonucleic acid of the present invention for adults is generally in the range of 0.1 mg to 10 g / day / human, preferably in the range of 1 mg to 500 mg / day / human. In some cases, this may be sufficient, or vice versa. In addition, it can be administered once to several times a day, or at intervals of 1 to several days.
In another embodiment of the present invention, the DNA used to produce the double-stranded oligonucleic acid of the present invention can be made into a pharmaceutical composition together with pharmaceutically acceptable additives. Here, the DNA used to generate the double-stranded oligonucleic acid of the present invention has the base sequence of the duplex forming part of the double-stranded oligonucleic acid of the present invention as a deoxyribonucleotide (where uracil in the nucleotide sequence is It means a plasmid or the like for producing the double-stranded oligonucleic acid of the present invention containing DNA (converted to thymine). When such a pharmaceutical composition is administered to a patient, the double-stranded oligonucleic acid of the present invention is produced in the patient's living body. Therefore, the pharmaceutical composition containing the double-stranded oligonucleic acid of the present invention and an appropriate carrier described above is used. That is, it is effective for the treatment and / or prevention of diseases caused by overexpression of Bcl-2. The dosage form and administration route may be administered by an appropriate method according to the patient's symptoms, as in the case of the pharmaceutical composition containing the complex of the oligonucleic acid of the present invention or the double-stranded oligonucleic acid of the present invention and a carrier. The dose may also be determined in consideration of the drug, dosage form, patient condition such as age and weight, route of administration, nature and extent of illness, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
Here, DMTr is 4,4-dimethoxytrityl, ps is O- (2-cyanoethyl) thiophosphoryl, s is thiophosphoryl, Lev is levuniryl, and CEP is 2-cyanoethyl-N, N-diisopropyl phosphoramidite, TBDMS, tert-butyldimethylsilyl, A, adenosine, C, cytidine, G, guanosine, U, uridine, dT, thymidine, ^p A , ^P C, ^p G, and ^p U each represent a protected nucleic acid. Rp and Sp represent a stereo on the phosphorus atom of the phosphorothioate bond.
The number after the compound name represents an optically active compound. When the diastereomer is first divided, 1 is assigned to a compound having a large Rf value, and 2 is assigned to a compound having a small Rf value. It was attached. The name of the compound produced using these raw material compounds is followed by the number of the raw material compound and the number.

Example 1
Process 1
3'-O- [5'-O- (4,4'-dimethoxytrityl) -6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosyl] -5'-O- [6-N - benzoyl -2'-O-tert-butyldimethylsilyl-3'-O-levulinate sulfonyl adenosyl] phosphothioate O-(2-cyanoethyl) ester ^{(DMTrO- p Aps p a-OLev} ) diastereomer separation step

6-N-benzoyl-2'-O-tert-butyldimethylsilyl-3'-O-levulinyl adenosine 2g and 5'-O- (4,4'-dimethoxytrityl) -6-N-benzoyl-2 '-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropylphosphoramidite) 4.06 g, 1H-tetrazole 577 mg was dissolved in 30 ml of acetonitrile under an argon atmosphere, and 40 minutes Stir at room temperature. To this was added 932 mg of 3-amino-1,2,4-dithiazole-5-thione (ADTT) dissolved in 270 ml of acetonitrile and 30 ml of pyridine under an argon atmosphere, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was added to saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, further evaporated to dryness under reduced pressure to obtain a crude product as a yellow solid as a diastereo mixture. This was purified by silica gel chromatography, Rf value is greater diastereomers ^{TLC (DMTrO- p Aps p A-} OLev-1; Example 1-1) (1.7g, 66% yield) as small Jia diastereomer ^{(DMTrO- p Aps p a-OLev} -2; example 1-2) (2.18 g, 85% yield) was separated into, the target compound was obtained as a white foam.

Step 2-1
5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosine-3′-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -1 (DMTrO- ^p Aps ^p A-OCEP -1; Example 1-3)

^{DMTrO- p Aps p A-OLev-} 1 0.7g of pyridine: acetic acid = 4: 0.5M hydrazine solution 9ml prepared at 1, and the mixture was stirred for 20 minutes at room temperature. Under ice-cooling, 2 ml of acetone was added to the reaction solution, stirred as it was for 5 minutes, poured into a saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was washed with saturated brine, then dried with anhydrous magnesium sulfate, filtered, evaporated to remove the solvent, and dried under reduced pressure. The resulting crude product was purified by silica gel chromatography, 0.53 g (81% yield, ESI-Mass (m / z) = 1404.4 (M ⁺ )) of the delevulinyl compound was obtained as a foam.
Next, 0.5 g of the delevulinyl compound was dissolved in 4 ml of acetonitrile under an argon atmosphere, and 0.73 ml of diisopropylethylamine was added under ice cooling. To this solution, 0.68 g of 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite was added dropwise and stirred for 2 hours at room temperature. The reaction mixture was poured into a saturated aqueous sodium hydrogen carbonate solution and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, evaporated to dryness under reduced pressure, and the resulting crude product was purified by silica gel chromatography to give a white foam. As a substance, 0.93 g (82% yield) of the target compound was obtained.
ESI-Mass (m / z) = 1604.6 (M ⁺ )

Step 2-2
5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosine-3′-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -2 (DMTrO- ^p Aps ^p A-OCEP -2; Example 1-4)

Using ^{DMTrO- p Aps p A-OLev-} 2, to give the desired compound by the same method as in Example 1, Step 2-1.
ESI-Mass (m / z) = 1604.7 (M ⁺ )

Step 3-1
Step of removing all protecting groups from DMTrO- ^p Aps ^p A-OLev-1 to obtain AsA-1

DMTrO- ^p Aps ^p dissolving 7mg the A-OLev-1 in pyridine 2 ml, was stirred for 16 hours at 55 ° C. by the addition of concentrated aqueous ammonia 10 ml. The reaction solution was concentrated to dryness, 1 ml of 1M tetra n-butylammonium fluoride in THF was added to the residue, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated, dissolved in 50% pyridine water, added with DOWEX 50W (pyridine type), and filtered after 10 minutes. The filtrate was concentrated and then purified using reverse phase chromatography. Fractions containing 4,4′-dimethoxytrityl group were collected and concentrated to dryness. To this was added 2 ml of 80% acetic acid, and the mixture was stirred at room temperature for 1 hour and concentrated. After separation with ethyl acetate-water, the aqueous layer was lyophilized. This was further purified using reverse phase chromatography and lyophilized to give 11 mg of the desired compound.
ESI-Mass (m / z) = 613.2 (M ⁺ )

Step 3-2
DMTrO- ^p Aps ^p a A-OLev-2 of any protecting groups eliminated, obtaining AsA-2 step

The target compound was prepared using DMTrO-ApsA-OLev-2 in the same manner as in Example 1, Step 3-1.
ESI-Mass (m / z) = 613.1 (M ⁺ )

Process 4
Structure determination <Identification of Rp and Sp>

Example 1 AsA-1 and AsA-2 obtained in Step 3-1 and Example 1 Step 3-2 are described in literature (B. Karwowski et al. Bioorg. Med. Chem. Lett. 11 (2001) 1001. In accordance with the method of -1003), snake venom phosphodiesterase (svPDE) and nuclease P1 (NP1) were treated, and AsA-1 hydrolyzed by svPDE was identified as Rp, and AsA-2 hydrolyzed by NP1 was identified as Sp.
That is, the compound produced in Example 1 has the chemical structure shown in Table 1.
Moreover, from the result of the reverse phase HPLC analysis shown in FIGS. 1 to 3, AsA-1 and AsA-2 obtained in Example 1 Step 3-1 and Example 1 Step 3-2 are each a single diastereomer. It was confirmed that the product was high purity. The measurement conditions are as follows.

Measurement condition:
HPLC instrument
Liquid feeding unit: LC-6A (manufactured by Shimadzu Corporation)
Detector: SPD-6A (manufactured by Shimadzu Corporation)
Reversed phase HPLC column Mightysil RP-18GP <4.6 mmφ × 15 cm> (manufactured by Kanto Chemical Co., Inc.)
Column temperature: 35 ° C
Mobile phase Gradient: Linear gradient 20 minutes (Liquid B: 0% -70%)
Solution A: 50 mM triethylamine-acetate buffer containing 5% acetonitrile Solution B: 50 mM triethylamine-acetate buffer containing 90% acetonitrile Flow rate of mobile phase: 1 ml / min

Example 2
Process 1
3'-O- [5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyluridyl] -5'-O- [2'-O-tert-butyldimethyl Diastereomeric separation step of silyl-3′-O -levulinyluridyl ] phosphothioate O- (2-cyanoethyl) ester (DMTrO- ^p Ups ^p U-OLev)

2'-O-tert-butyldimethylsilyl-2-N-isobutyryl-3'-O-levulinylguanosine 3g and 5'-O- (4,4'-dimethoxytrityl) -6-N-benzoyl-2 Using 1.85 g of '-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite), the target compound was prepared in the same manner as in Example 1, Step 1. Manufactured.
Diastereomer having a large Rf value (DMTrO- ^p Ups ^p U-OLev-1; Example 2-1): 1.05 g, diastereomer having a small 77% yield (DMTrO- ^p Ups ^p U-OLev-2; Example 2-2): 1.12 g, 82% yield

Step 2-1
5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyluridyl- [3 '→ 5']-O- (2-cyanoethyl) thiophosphoryl-2'-O -tert- butyldimethylsilyl-uridine-3'-O-(2-cyanoethyl -N, N-diisopropyl ^{phosphoramidite) -1 (DMTrO- p Ups p U} -OCEP-1; example 2-3)

Using DMTrO- ^p Ups ^p U-OLev-1 and using the same method as in Example 1 step 2-1, 1.37 g of the target compound was obtained as a white foam.
ESI-Mass (m / z) = 1350.7 (M ⁺ )

Step 2-2
5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyluridyl- [3 '→ 5']-O- (2-cyanoethyl) thiophosphoryl-2'-O -3'-O--tert-butyldimethylsilyl-uridine (2-cyanoethyl -N, N-diisopropyl ^{phosphoramidite) -2 (DMTrO- p Ups p U} -OCEP-2; example 2-4)

The target compound was produced using DMTrO- ^p Ups ^p U-OLev-2 in the same manner as in Example 1, step 2-1.
ESI-Mass (m / z) = 1350.7 (M ^<+> ), 1332.6 ([M + Na] ^< +>)

Process 3
Example 1 DMTrO- ^p Ups ^p U-OCEP-1 to UsU-1 and DMTrO- ^p Ups ^p U-OCEP-2 to UsU-2 were produced in the same manner as in Step 3-1, respectively. In the same manner as in Step 4, the structures of UsU-1 and UsU-2 were determined. The results are shown in Table 2.
Moreover, from the result of reverse phase HPLC analysis, it was confirmed that the compounds UsU-1 and UsU-2 obtained in Step 3 of Example 2 were each a single diastereomer and high purity.

Example 3
Process 1
3'-O- [5'-O- (4,4'-dimethoxytrityl) -6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosyl] -5'-O- [2-N - isobutyryl -2'-O-tert-butyldimethylsilyl-3'-O-levulinate sulfonyl guano sill] phosphothioate O-(2-cyanoethyl) ester ^{(DMTrO- p Aps p G-OLev} ) diastereomer separation step

2'-O-tert-butyldimethylsilyl-2-N-isobutyryl-3'-O-levulinylguanosine 3g and 5'-O- (4,4'-dimethoxytrityl) -6-N-benzoyl-2 Using 1.85 g of '-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite), the target compound was prepared in the same manner as in Example 1, Step 1. Manufactured.
Rf value is greater diastereomer ^{(DMTrO- p Aps p G-OLev} -1; EXAMPLE 3-1): 3.14g, ESI-Mass (m / z) = 1484.7 (M +)
Rf value is less diastereomer ^{(DMTrO- p Aps p G-OLev} -2; Example 3-2): 3.51g, ESI-Mass (m / z) = 1484.5 (M +)

Step 2-1
5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio phosphoryl -2-N-isobutyryl -2'-O-tert-butyldimethylsilyl-guanosine -3'-O- (2- cyanoethyl -N, N-diisopropyl ^{phosphoramidite) -1 (DMTrO- p Aps p G} -OCEP -1; Example 3-3)

Using ^{DMTrO- p Aps p G-OLev-} 1, to obtain the desired compound in the same manner as in Example 1, Step 2-1.
ESI-Mass (m / z) = 1586.8 (M ⁺ )

Step 2-2
5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio phosphoryl -2-N-isobutyryl -2'-O-tert-butyldimethylsilyl-guanosine -3'-O- (2- cyanoethyl -N, N-diisopropyl ^{phosphoramidite) -2 (DMTrO- p Aps p G} -OCEP -2; Example 3-4)

Using ^{DMTrO- p Aps p G-OLev-} 2, was prepared the desired compound using the same method as in Example 1, Step 2-1.
ESI-Mass (m / z) = 1586.8 (M ⁺ )

Process 3
In the same manner as in Example 1, Step ^3-1, DMTrO- the ^p Aps p AsG-1 from ^{G-OCEP-1, DMTrO- p} Aps p G-OCEP-2 from AsG-2 were prepared, respectively, in Example 1 In the same manner as in step 4, the structures of AsG-1 and AsG-2 were determined. The results are shown in Table 3.
From the results of reverse phase HPLC analysis, it was confirmed that AsG-1 and AsG-2 obtained in Step 3 of Example 3 were each a single diastereomer and high purity.

Example 4
Process 1
3'-O- [5'-O- (4,4'-dimethoxytrityl) -2-N-isobutyryl-2'-O-tert-butyldimethylsilylguanosyl] -5'-O- [6-N -Benzoyl-2'-O-tert-butyldimethylsilyl-3'-O -levulinyl adenosyl] phosphothioate O- (2-cyanoethyl) ester (DMTrO- ^p Gps ^p A-OLev)

6-N-benzoyl-2'-O-tert-butyldimethylsilyl-3'-O-levulinyl adenosine 2.5g and 5'-O- (4,4'-dimethoxytrityl) -2'-O- Example 1 Using 7.38 g of tert-butyldimethylsilyl-2-N-isobutyrylguanosine-3′-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) Example 1 The target compound was prepared using this.
Diastereomer having a large Rf value (DMTrO- ^p Gps ^p A-OLev-1; Example 4-1): 1.40 g, ESI-Mass (m / z) = 1484.6 (M ⁺ )
Diastereomer having a small Rf value (DMTrO- ^p Gps ^p A-OLev-2; Example 4-2): 1.89 g, ESI-Mass (m / z) = 1484.7 (M ⁺ )

Step 2-1
5′-O- (4,4′-dimethoxytrityl) -2-N-isobutyryl-2′-O-tert-butyldimethylsilylguanosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -1 (DMTrO- ^p Gps ^p A-OCEP -1; Example 4-3)

The target compound was produced in the same manner as in Example 1, step 2-1, using DMTrO- ^p Gps ^p A-OLev-1.
ESI-Mass (m / z) = 1587.8 (M ⁺ )

Step 2-2
5′-O- (4,4′-dimethoxytrityl) -2-N-isobutyryl-2′-O-tert-butyldimethylsilylguanosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -2 (DMTrO- ^p Gps ^p A-OCEP -2; Example 4-4)

The target compound was produced in the same manner as in Example 1, Step 2-1, using DMTrO- ^p Gps ^p A-OLev-2.
ESI-Mass (m / z) = 1587.7 (M ⁺ )

Process 3
Example 1 In the same manner as in Step 3-1, DMTrO- ^p Gps ^p A-OCEP-1 to GsA-1 and DMTrO- ^p Gps ^p A-OCEP-2 to GsA-2 were produced, respectively. In the same manner as in Step 4, the structures of GsA-1 and GsA-2 were determined. The results are shown in Table 4.
Moreover, from the result of reverse phase HPLC analysis, it was confirmed that GsA-1 and GsA-2 obtained in Step 3 of Example 4 were each a single diastereomer and high purity.

Example 5
Process 1
3'-O- [5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyluridyl] -5'-O- [4-N- (4-anisoyl) -2'-O-tert-butyldimethylsilyl-3'-O-levulinate sulfonyl cytidine Jill] phosphothioate O-(2-cyanoethyl) ester ^{(DMTrO- p Ups p C-OLev} )

4-N- (4-Anisoyl) -2′-O-tert-butyldimethylsilyl-3′-O-levulinyl cytidine 3.5 g and 5′-O- (4,4′-dimethoxytrityl) -2 The target compound was prepared in the same manner as in Example 1, Step 1, using 7.7 g of '-O-tert-butyldimethylsilyluridine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite). Manufactured.
Diastereomer with a large Rf value (DMTrO- ^p Ups ^p C-OLev-1; Example 5-1): 3.12 g
Diastereomer with a large Rf value (DMTrO- ^p Ups ^p C-OLev-2; Example 5-2): 3.70 g

Step 2-1
5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyluridyl- [3 '→ 5']-O- (2-cyanoethyl) thiophosphoryl-4-N- (4-anisoyl) -2'-O-tert- butyldimethylsilyl cytidine-3'-O-(2-cyanoethyl -N, N-diisopropyl ^{phosphoramidite) -1 (DMTrO- p Ups p C} -OCEP-1 Example 5-3)

The target compound was produced in the same manner as in Example 1, Step 2-1, using DMTrO- ^p Ups ^p C-OLev-1.
ESI-Mass (m / z) = 1483.5 (M ⁺ )

Step 2-2
5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyluridyl- [3 '→ 5']-O- (2-cyanoethyl) thiophosphoryl-4-N- (4-Anisoyl) -2'-O-tert-butyldimethylsilylcytidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -2 (DMTrO- ^p Ups ^p C-OCEP-2 Example 5-4)

The target compound was produced in the same manner as in Example 1, step 2-1, using DMTrO- ^p Ups ^p C-OLev-2.
ESI-Mass (m / z) = 1483.8 (M ⁺ )

Process 3
Example 1 In the same manner as in Step 3-1, DMTrO- ^p Ups ^p C-OCEP-1 to UsC-1 and DMTrO- ^p Ups ^p C-OCEP-2 to UsC-2 were produced, respectively. In the same manner as in Step 4, the structures of UsC-1 and UsC-2 were determined. The results are shown in Table 5.
Moreover, from the result of the reverse phase HPLC analysis, it was confirmed that UsC-1 and UsC-2 obtained in Step 3 of Example 5 were each a single diastereomer and high purity.

Example 6
Process 1
3'-O- [5'-O- (4,4'-dimethoxytrityl) -6-N-benzoyl-2'-O-tert-butyldimethylsilyladenosyl] -5'-O- [4-N - (4-anisoyl) -2'-O-tert-butyldimethylsilyl-3'-O-levulinate sulfonyl cytidine Jill] phosphothioate O-(2-cyanoethyl) ester ^{(DMTrO- p Aps p C-OLev} )

4-N- (4-Anisoyl) -2'-O-tert-butyldimethylsilyl-3'-O-levulinyl cytidine 3g and 5'-O- (4,4'-dimethoxytrityl) -6-N Example 1 Using 6.03 g of benzoyl-2′-O-tert-butyldimethylsilyladenosine-3′-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) The target compound was prepared using this.
Diastereomer having a large Rf value (DMTrO- ^p Aps ^p C-OLev-1; Example 6-1): 3.57 g
Diastereomer with a small Rf value (DMTrO- ^p Aps ^p C-OLev-2; Example 6-2): 3.64 g

Step 2-1
5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-4-N- (4-anisoyl) -2'-O-tert-butyldimethylsilylcytidine-3'-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -1 (DMTrO- ^p Aps ^p C-OCEP-1; Example 6-3)

Using ^{DMTrO- p Aps p C-OLev-} 1, was prepared the desired compound using the same method as in Example 1, Step 2-1.
ESI-Mass (m / z) = 1610.6 (M ⁺ )

Step 2-2
5′-O- (4,4′-dimethoxytrityl) -6-N-benzoyl-2′-O-tert-butyldimethylsilyladenosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-4-N- (4-anisoyl) -2′-O-tert-butyldimethylsilylcytidine-3′-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -2 (DMTrO- ^p Aps ^p C-OCEP-2; Example 6-4)

Using ^{DMTrO- p Aps p C-OLev-} 2, was prepared the desired compound using the same method as in Example 1, Step 2-1.
ESI-Mass (m / z) = 1610.5 (M ⁺ )

Process 3
In the same manner as in Example 1, Step ^3-1, DMTrO- the ^p Aps p ASC-1 from ^{C-OCEP-1, DMTrO- p} Aps p C-OCEP-2 from ASC-2 were prepared, respectively, in Example 1 In the same manner as in Step 4, the structures of AsC-1 and AsC-2 were determined. The results are shown in Table 6.
From the results of reverse phase HPLC analysis, it was confirmed that AsC-1 and AsC-2 obtained in Step 6 of Example 6 were each a single diastereomer and high purity.

Example 7
Process 1
3'-O- [5'-O- (4,4'-dimethoxytrityl) -2-N-isobutyryl-2'-O-tert-butyldimethylsilylguanosyl] -5'-O- [3'- O -levulinylthymidyl ] phosphothioate O- (2-cyanoethyl) ester (DMTrO- ^p GpsdT-OLev)

2 g of 3'-O-levulinylthymidine and 5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyl-2-N-isobutyrylguanosine-3'-O Using 6.84 g of-(2-cyanoethyl-N, N-diisopropyl phosphoramidite), the target compound was produced in the same manner as in Example 1, Step 1.
Diastereomer with a large Rf value (DMTrO- ^p GpsdT-OLev-1; Example 7-1): 3.18 g
Diastereomer with a large Rf value (DMTrO- ^p GpsdT-OLev-2; Example 7-2): 2.86 g

Step 2-1
5′-O- (4,4′-dimethoxytrityl) -2-N-isobutyryl-2′-O-tert-butyldimethylsilylguanosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-thymidine-3′-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -1 (DMTrO- ^p GpsdT-OCEP-1; Example 7-3)

The target compound was produced using DMTrO- ^p GpsdT-OLev-1 in the same manner as in Example 1, step 2-1.
ESI-Mass (m / z) = 1343.6 (M ^<+> ), 1365.6 ([M + Na] ^< +>)

Step 2-2
5′-O- (4,4′-dimethoxytrityl) -2-N-isobutyryl-2′-O-tert-butyldimethylsilylguanosyl- [3 ′ → 5 ′]-O- (2-cyanoethyl) thio Phosphoryl-thymidine-3′-O- (2-cyanoethyl-N, N-diisopropyl phosphoramidite) -2 (DMTrO- ^p GpsdT-OCEP-2; Example 7-4)

The target compound was produced in the same manner as in Example 1, step 2-1, using DMTrO- ^p GpsdT-OLev-2.
ESI-Mass (m / z) = 1343.7 (M ^<+> ), 1366.6 ([M + Na] ^< +>)

Process 3
Example 1 In the same manner as in Step 3-1, DMTrO- ^p GpsdT-OCEP-1 to GsdT-1 and DMTrO- ^p GpsdT-OCEP-2 to GsdT-2 were produced, respectively. Thus, the structures of GsdT-1 and GsdT-2 were determined. The results are shown in Table 7.
Moreover, from the result of the reverse phase HPLC analysis, it was confirmed that GsdT-1 and GsdT-2 obtained in Step 3 of Example 7 were each a single diastereomer and high purity.

<Manufacture of oligonucleic acid compounds>

Example 8
(Rp) -5′-AsAAsAAsAAsAAsAAsAAsAAsAAsAAsAA-3 ′ (SEQ ID NO: 1)

The ^{DMTrO- p Aps p A-OCEP-} 1 is a Rp body was dissolved in acetonitrile, was synthesized oligo nucleic acid compound by a nucleic acid automatic synthesizer (Expedite ^TM). A commercially available adenine CPG column was used as the solid phase carrier, tetrazole was used as the condensation catalyst, an iodine solution was used as the oxidizing agent, and acetic anhydride and an N-methylimidazole solution were used as the capping solution. After completion of the synthesis, the CPG column (DMTr-ON) was treated with concentrated aqueous ammonia / ethanol (3/1) and cut out from the solid support, and then incubated at 55 ° C. for 17 hours to deprotect the base protecting group. This was concentrated to dryness, and stirred at 55 ° C. for 2 hours in a mixed solution of 3HF / TEA, TEA, N-methylpyrrolidine and N-methylpyrrolidone to deprotect the hydroxyl-protecting group at the 2 ′ position. The reaction solution was cooled to room temperature, poured into 2-propanol, and the resulting precipitate was collected by centrifugation. This was purified using reverse phase chromatography, and fractions containing 4,4′-dimethoxytrityl group were collected and concentrated to dryness. Subsequently, the 4,4′-dimethoxytrityl group was deprotected with 80% acetic acid, concentrated to dryness, then separated with ethyl acetate-water, and the aqueous layer was lyophilized. The obtained crude product was purified by ion exchange chromatography. Fractions containing the target compound were collected and lyophilized, then desalted and lyophilized again to obtain the target compound.
MALDI TOF MS: Calculated 7012.05
Found 7011.3

From the results of the ion exchange HPLC analysis in FIG. 4, it is clear that the target compound is almost single. The measurement conditions are as follows.
Measurement condition:
HPLC instrument
Liquid feeding unit: LC-6A (manufactured by Shimadzu Corporation)
Detector: SPD-6A (manufactured by Shimadzu Corporation)
Ion exchange HPLC column DEAE-NPR <4.5 mmφ × 3.5 cm> (manufactured by Tosoh Corporation)
Column temperature: 35 ° C
Mobile phase Gradient: Linear gradient 15 minutes (B solution: 25% -90%), B solution 90% 5 minutes A solution: 10 mM phosphate buffer (pH 7)
Solution B: 10 mM phosphate buffer (pH 7) containing 1 M NaCl
Mobile phase flow rate: 1 ml / min UV-visible spectrometer Detection wavelength: 260 nm

Example 9
(Sp) -5′-AsAAsAAsAAsAAsAAsAAsAAsAAsAAsAA-3 ′ (SEQ ID NO: 2)

The target compound was synthesized in the same manner as in Example 8 using DMTrO-ApsA-OCEP-2 which is an Sp form.
MALDI TOF MS: Calculated 7012.05
Found 7013.72
From the results of the ion exchange HPLC analysis in FIG. 5, it is clear that the target compound is almost single. The same measurement conditions as in Example 8 were used.

Example 10
(Rp) -5′-UsUUsUUsUUsUUsUUsUUsUUsUUsUUsUU-3 ′ (SEQ ID NO: 3)

The target compound was synthesized in the same manner as in Example 8 using DMTrO-UpsU-OCEP-1 which is an Rp form. Ion exchange HPLC analysis was used to confirm that the target compound was almost single. The same measurement conditions as in Example 8 were used.
MALDI TOF MS: Calculated 6528.21
Found 6530.3

Example 11
(Sp) -5′-UsUUsUUsUUsUUsUUsUUsUUsUUsUUsUU-3 ′ (SEQ ID NO: 4)

The target compound was synthesized in the same manner as in Example 8, using DMTrO-UpsU-OCEP-2 which is an Sp form. Ion exchange HPLC analysis was used to confirm that the target compound was almost single. The same measurement conditions as in Example 8 were used.
MALDI TOF MS: Calculated 6528.21
Found 6529.6

(Synthesis of real sequence)
5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′

Example 12
(Rp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 5)

It is Rp body ^{DMTrO- p Aps p G-OCEP-} 1, is Rp body ^{DMTrO- p Gps p A-OCEP-} 1, is Rp body ^{DMTrO- p Aps p A-OCEP-} 1, is Rp body DMTrO - dissolved ^{p Ups p C-OCEP-1} , is Rp body ^{DMTrO- p Aps p C-OCEP-} 1, is Rp body DMTrO- ^p GpsdT-OCEP-2 in acetonitrile so that each becomes 0.1M Using a commercially available thymidine CPG column as the amidite solution and solid phase carrier, tetrazole as the condensation catalyst, dichloromethane solution of 2.0M tert-butyl hydroperoxide as the oxidizing agent, acetic anhydride and N-methylimidazole solution as the capping solution In the same manner as in Example 8, using an automated nucleic acid synthesizer (Expeditite ^™ ) The target compound was obtained by synthesizing a rigo nucleic acid compound. Reverse phase HPLC analysis was used to confirm that the target compound was almost single. The measurement conditions were the same as in Example 1.
MALDI TOF MS: Calculated 6986.93
Found 6990.946

Example 13
(Sp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 6)

A Sp body ^{DMTrO- p Aps p G-OCEP-} 2, a Sp body ^{DMTrO- p Gps p A-OCEP-} 2, a Sp body ^{DMTrO- p Aps p A-OCEP-} 2, a Sp body DMTrO - with ^{p Ups p C-OCEP-2} , a Sp body ^{DMTrO- p Aps p C-OCEP-} 2, a Sp body DMTrO- ^p GpsdT-OCEP-1, using the same method as in example 8 Thus, an oligonucleic acid compound was synthesized to obtain the target compound. Reverse phase HPLC analysis was used to confirm that the target compound was almost single. The measurement conditions were the same as in Example 1.
MALDI TOF MS: Calculated 6986.93
Found 6989.943

Test example 1
Nucleic acid duplex stability evaluation <Tm value>
The same amount as the sample so that the final concentration of the _21- mer (A ₂₁ ) of the oligonucleic acid compound synthesized in Example 8, the oligonucleic acid compound synthesized in Example 9 or natural adenosine is 1.6 μM. A phosphate buffer solution (0.1 M sodium chloride, 10 mM sodium phosphate, pH 7.5) containing polyuridylic acid (Poly (U)) was prepared. Each sample solution thus prepared was heated to 70 ° C. and then slowly cooled to room temperature, and then heated from 20 ° C. to 80 ° C. over 120 minutes using a Hitachi U-3210 self-recording spectrophotometer. Absorbance at 260 nm was measured. Each double chain melting temperature (Tm value) was determined from the obtained absorbance-temperature curve.
Similarly, the Tm value of the oligonucleic acid compound produced in Example 10, the oligonucleic acid compound produced in Example 11, or the ₂₁ mer (U ₂₁ ) of natural uridine and polyadenylic acid (Poly (A)) was determined. It was. Table 8 shows the results.
From the results of Table 8, it was revealed that the oligonucleic acid of the present invention has an affinity for RNA similar to or higher than that of natural oligo RNA. Especially, it became clear that the oligonucleic acid compound in which all phosphorothioate bonds in the oligonucleic acid compound are Rp significantly improves the affinity.

Test example 2
Evaluation of protein expression inhibitory activity A double-stranded oligonucleic acid compound was introduced into A431 cells together with a carrier, and Bcl-2 protein expression inhibitory activity was quantitatively evaluated by Western blotting.

Preparation of complex of double-stranded oligonucleic acid compound and carrier Using liposome A containing 2-O- (2-diethylaminoethyl) carbamoyl 1,3-O-dioleoylglycerol and purified egg yolk lecithin as the carrier, double-stranded A complex with an oligonucleic acid compound was prepared.
A complex was prepared by mixing 16 parts by weight of liposome A with 1 part by weight of a double-stranded oligonucleic acid compound. Hereinafter, preparation of 2 ml of a complex solution of a double-stranded oligonucleic acid compound having a final concentration of 300 nM will be described. However, the concentration of the double-stranded oligonucleic acid compound complex is the molarity of the double-stranded oligonucleic acid compound contained in the complex when it is assumed that the double-stranded oligonucleic acid compound is completely double-stranded. Shown in concentration.
The sense strand and the antisense strand dissolved in water for injection were diluted with 10% maltose to prepare a double-stranded oligonucleic acid compound solution containing both strands at a concentration of 600 nM. A 16 mg / ml liposome A dispersion was diluted with 10% maltose to 0.130 mg / ml. The same volume of the double-stranded oligonucleic acid compound solution was gradually added to the liposome dispersion while stirring. By the above operation, a double-stranded oligonucleic acid compound-liposome complex solution having a final concentration of double-stranded oligonucleic acid compound of 300 nM was prepared. This composite was dispersed for 15 seconds using a 600 W bath-type ultrasonic device to make the composite particles uniform.

Western blotting By Western blotting, whether the expression of Bcl-2 protein is suppressed by transfection of a double-stranded oligonucleic acid compound into cells using the above-described double-stranded oligonucleic acid compound-liposome complex. Evaluation was made by changing the amount of Bcl-2 protein.
A431 cells (epithelial cancer cells) were seeded at 2 × 10 ⁵ cells / 6 cm dish in a petri dish having a diameter of 6 cm, and 10% FBS (fetal calf serum) in DMEM medium (Sigma, D6046) at 37 ° C., 5%. Cultivated overnight under CO ₂ conditions. On the next day, the medium was aspirated from the culture dish, and 2.7 ml of 10% FBS-containing DMEM medium (Sigma, D6046) was added to change the medium. Thereto was added 0.3 ml of a double-stranded oligonucleic acid compound / liposome A complex (weight ratio: double-stranded oligonucleic acid compound: liposome A = 1: 16) mixed in a 10% maltose aqueous solution. Was 3 ml. At this time, the final concentration of the double-stranded oligonucleic acid compound is 10 nM. The cells were cultured for 72 hours in a 37 ° C. 5% CO ₂ incubator. The cells were washed twice with PBS (phosphate buffered saline) and transferred to a 1.5 ml tube using a cell scraper. After centrifuging at 1000 × g for 2 minutes and removing the supernatant, 60 μl of Lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40) was added to lyse the cells. After standing for 30 minutes on ice and centrifuging at 100,000 × g for 20 minutes, the supernatant was transferred to a new tube to prepare a sample for electrophoresis.
For electrophoresis, polyacrylamide gel (ATTO E-T520L) was used, and 15 μg of sample was applied per lane. After completion of electrophoresis, the protein in the gel was transferred to a polyvinylidene fluoride (PVDF) membrane, and PBST containing 5% skim milk (PBST-MLK; where PBST was composed of PBS buffer containing 0.1% Tween 20) Blocked for 1 hour at room temperature. First, Bcl-2 protein was detected. After blocking, the PVDF membrane was shaken overnight at 4 ° C. in a mouse anti-human Bcl-2 monoclonal antibody (DAKO M0887) diluted 500 times with PBST-MLK to bind to the primary antibody. The PVDF membrane was washed with PBST and then shaken in HRP (horseradish peroxidase) -labeled anti-mouse Ig antibody (DAKO PO260) diluted 2000 times with PBST-MLK at room temperature for 90 minutes to bind to the secondary antibody. After washing with PBST, light was emitted using WesternLighting Chemiluminescence Reagent Plus (Perkin Elmer), and Bcl-2 protein was quantified by ChemiDoc (BioRad).
After the detection of Bcl-2 protein, the same PVDF membrane was washed with MilliQ water, and the Actin protein was detected in the same manner as Bcl-2. A goat anti-human Actin antibody (Santa Cruz sc-1616) was used as the primary antibody, and an HRP-labeled anti-goat Ig antibody (DAKO P0449) was used as the secondary antibody. Each was diluted 500-fold and 1500-fold with PBST-MLK and used.
The amount of Bcl-2 protein when transfected with a double-stranded oligonucleic acid compound (GL3) that suppresses luciferase expression was used as a negative control.
The double-stranded oligonucleic acid compound used is shown below.
B622:
Sense strand 5′-CCUCUGUUUGAUUUCUCCUdTdT-3 ′ (SEQ ID NO: 7)
Antisense strand 5′-AGGAGAAAUCAACAGAGGdTdT-3 ′ (SEQ ID NO: 8)
B622-Rp: double-stranded oligonucleic acid compound in which the antisense strand is the oligonucleic acid compound of Example 12 sense strand 5′-CCUCUGUUUGAUUCUCCUdTdT-3 ′ (SEQ ID NO: 7)
Antisense strand (Rp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 8; Example 12)
B622-Sp: Double-stranded oligonucleic acid compound in which the antisense strand is the oligonucleic acid compound of Example 13 Sense strand 5′-CCUCUGUUUGAUUCUCCUdTdT-3 ′ (SEQ ID NO: 7)
Antisense strand (Sp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 6; Example 13)

Table 9 shows the results of evaluating the activity of these double-stranded oligonucleic acid compounds by Western blotting.
It was clarified that the double-stranded oligonucleic acid compound in which the thioate bonds constituting the antisense strand are all Rp suppresses the expression of Bcl-2 protein almost as much as the natural type.

Test Example 3
Evaluation of degradation resistance of single-stranded oligonucleic acid compound to exonuclease The 5 ′ end of single-stranded oligonucleic acid compound was labeled with ³² P by T4 polynucleotide kinase. Unincorporated ³² P-ATP was removed with Microspin G-25 Columns (Amersham Bioscience). In addition to 3.3 pmol of ³² P-labeled RNA in 180 μl of 25 mM Tris-HCl (pH 8.9) containing 1 mM MgCl ₂ , add 0.2 mU of Phosphoesterase I (Exonclease from Crotus Adamanteus Venom) at 37 ° C. Incubated for minutes. The reaction was stopped by extracting 40 μl with phenol / chloroform. Analysis was by polyacrylamide gel electrophoresis (PAGE; 16% polyacrylamide / 8M urea; acrylamide: bis-acrylamide 19: 1; 89 mM Tris / 89 mM boric acid / 2 mM EDTA). After drying the gel, 21-base RNA was quantified using BAS-2500 (FUJI FILM). The single-stranded oligonucleic acid compound collected immediately after the enzyme addition was defined as a residual rate of 100%.
The single-stranded oligonucleic acid compound used is shown below.

5'-AGGAGAAAUCAACAGAGGGGdTdT-3 '(SEQ ID NO: 8)
(Rp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 5; Example 12)
(Sp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 6; Example 13)

Table 10 shows the results of the degradation resistance of these single-stranded oligonucleic acid compounds to exonuclease.
In the oligonucleic acid of the present invention having a phosphorothioate bond, it was found that an extremely large number of compounds remained and the degradation resistance to exonuclease was high as compared with the natural type. Among these, the oligonucleic acid of the present invention in which all thioate bonds are Sp was found to have a remarkable effect.

Test example 4
³² P-labeled single-stranded oligonucleic acid compound was obtained in the same manner as in Test Example 3 for evaluation of degradation resistance of bovine serum to single-stranded oligonucleic acid compound. 3.3 pmol of ³² P-labeled RNA was incubated for 30 minutes at 37 ° C. in 40 μl of 10% bovine serum. The reaction was stopped by collecting 9 μl and extracting with phenol / chloroform. PAGE was performed by the method of Test Example 3 to quantify 21-base RNA. The single-stranded oligonucleic acid compound collected immediately after addition of bovine serum was defined as a residual rate of 100%.
The single-stranded oligonucleic acid compound used is shown below.

5'-AGGAGAAAUCAACAGAGGGGdTdT-3 '(SEQ ID NO: 8)
(Rp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 5; Example 12)
(Sp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 6; Example 13)

Table 11 shows the results of degradation resistance of these single-stranded oligonucleic acid compounds against bovine serum.
In the oligonucleic acid of the present invention having a phosphorothioate bond, it was found that an extremely large number of compounds remained and the degradation resistance to exonuclease was high as compared with the natural type. Among these, the oligonucleic acid of the present invention in which all thioate bonds are Sp was found to have a remarkable effect.

Test Example 5
Evaluation of degradation resistance of double-stranded RNA to exonuclease 20 μM each of sense and antisense strands in Annealing Buffer (100 mM potassium acetate, 30 mM HEPES-KOH, 2 mM magnesium acetate, pH 7.4) at 90 ° C. for 1 minute at 70 ° C. Double-stranded RNA was formed by lowering the temperature from 70 ° C. to 37 ° C. over 1 hour every 1 minute. Desalted with MicroSpin G-25 Columns (Amersham Bioscience).
Into 33 mM Tris-HCl (pH 8.9), 0.67 mM MgCl ₂ , 400 pmol of double-stranded RNA was added with 258 mU of Phosphoesterase I (Exonclease from Crotrus Adamanteus Venom; Worthington Biochemical 37 minutes at 37 ° C.). The reaction was stopped by extracting 10 μl with phenol / chloroform after incubation. The aqueous layer was confirmed by PAGE.
Double-stranded RNA was analyzed by polyacrylamide gel electrophoresis (PAGE; 12% polyacrylamide; acrylamide: bis-acrylamide 19: 1; 89 mM Tris / 89 mM boric acid / 2 mM EDTA). After staining with ethidium bromide, 19 base pair RNA was quantified using ChemiDoc (BioRad). The residual rate of 19 base pair RNA recovered immediately after enzyme addition was taken as 100%.
The double-stranded oligonucleic acid compound used is shown below.

B622:
Sense strand 5′-CCUCUGUUUGAUUUCUCCUdTdT-3 ′ (SEQ ID NO: 7)
Antisense strand 5′-AGGAGAAAUCAACAGAGGdTdT-3 ′ (SEQ ID NO: 8)
B622-Rp: double-stranded oligonucleic acid compound in which the antisense strand is the oligonucleic acid compound of Example 12 sense strand 5′-CCUCUGUUUGAUUCUCCUdTdT-3 ′ (SEQ ID NO: 7)
Antisense strand (Rp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 5; Example 12)
B622-Sp: double-stranded oligonucleic acid compound in which the antisense strand is the oligonucleic acid compound of Example 12 sense strand 5′-CCUCUGUUUGAUUCUCCUdTdT-3 ′ (SEQ ID NO: 7)
Antisense strand (Sp) -5′-AsGGsAGsAAsAUsCAsAAsCAsGAsGGsdTdT-3 ′ (SEQ ID NO: 6; Example 13)

Table 12 shows the results of degradation resistance of these double-stranded oligonucleic acid compounds to exonuclease.
The natural double-stranded oligonucleic acid compound has no degradation resistance to exonuclease, but the present oligonucleic acid having a phosphorothioate bond has a very large number of compounds remaining and is resistant to degradation against exonuclease. Was found to be very high. Among these, the oligonucleic acid of the present invention in which all thioate bonds are Sp was found to have a remarkable effect.

The method for producing an optically active oligonucleic acid compound having a phosphorothioate bond according to the present invention is different from a conventionally known method for producing an oligonucleic acid compound having a phosphorothioate bond, and is similar to a method for producing a normal oligonucleic acid compound. It is a method of forming only bonds.
Therefore, by using an optically active nucleic acid dimer compound having a high purity phosphorothioate bond as a nucleic acid synthesis unit, an optically active oligonucleic acid compound having a high purity phosphorothioate bond can be easily produced. .
Moreover, since the nucleic acid dimer of the present invention is a nucleic acid dimer compound having a phosphorothioate bond, it is useful as a raw material compound capable of producing an optically active oligonucleic acid compound having a phosphorothioate bond.
The optically active oligonucleic acid compound having a phosphorothioate bond, which can be produced using at least one nucleic acid dimer of the present invention, is similar to or more than a natural oligo RNA with respect to an RNA capable of forming a complementary strand thereto. It became clear that it has the above affinity.
Further, an optically active oligonucleic acid compound having a phosphorothioate bond, which can be produced using at least one of the nucleic acid dimers of the present invention, is an exonuclease or bovine as compared with a natural oligonucleic acid compound having the same sequence. It showed very high degradation resistance against serum.
Furthermore, the double-stranded oligonucleic acid compound containing the oligonucleic acid compound also showed a very high degradation resistance against exonuclease compared to the natural double-stranded oligonucleic acid compound having the same sequence.
That is, an optically active oligonucleic acid compound having a phosphorothioate bond and having a sequence similar to that of a natural oligo RNA having a cleavage activity for a target gene (mRNA) is almost the same as or higher than that of a natural oligo RNA. The oligonucleic acid of the present invention having various enzyme resistances is useful as a therapeutic agent, diagnostic agent or research reagent because it has an affinity and a cleavage activity for almost the same target gene (mRNA).

Claims (20)

The optical system represented by the following general formula (1), wherein at least one optically active nucleic acid dimer compound represented by the following general formula (2a) or (2b) is used as the nucleic acid synthesis unit. Production of active oligonucleic acid compounds.
[In the formula, each B independently represents a nucleobase. n represents an integer within the range of 1 to 99. Each R ¹ independently represents hydrogen, a hydroxyl group or alkoxy, and at least one of each R ¹ represents a hydroxyl group. Each Y independently represents a substituent represented by the following general formula (1a), general formula (1b) or general formula (1c), at least one of which is a substituent (1a) or a substituent ( 1b). However, the case where Y of adjacent constituent monomers is all a substituent (1a) or a substituent (1b) is excluded.
Z represents hydrogen or a phosphate group.
Bx ₁ and Bx ₂ are the same or different and represent a nucleobase which may have a protecting group. R ^1a1 and R ^1a2 are the same or different and each represents hydrogen, alkoxy or silyloxy. R ² represents acyl or a substituent represented by the following general formula (3a) or (3b).
[Wherein R ^2a and R ^2b are the same or different and each represents alkyl, or R ^2a and R ^2b are formed together with an adjacent nitrogen atom to form a 5- to 6-membered saturated amino ring group Represents. R ^2d represents aryl or a bicyclic heterocyclic group. R ^2c and R ^2e represent alkyl. ]
R ³ represents alkyl, aryl, or a bicyclic heterocyclic group. R ⁴ represents hydrogen or a group capable of leaving under acidic conditions. ] An optically active nucleic acid dimer compound represented by the following general formula (2a) or (2b).
[Wherein, Bx ₁ and Bx ₂ are the same or different and each represents a nucleobase which may have a protecting group. R ^1a1 and R ^1a2 are the same or different and each represents hydrogen, alkoxy or silyloxy. R ² represents acyl or a substituent represented by the following general formula (3a) or (3b).
[Wherein R ^2a and R ^2b are the same or different and each represents alkyl, or R ^2a and R ^2b are formed together with an adjacent nitrogen atom to form a 5- to 6-membered saturated amino ring group Represents. R ^2d represents aryl or a bicyclic heterocyclic group. R ^2c and R ^2e represent alkyl. ]
R ³ represents alkyl, aryl, or a bicyclic heterocyclic group. R ⁴ represents hydrogen or a group capable of leaving under acidic conditions. ] An optically active oligonucleic acid compound represented by the following general formula (1):
[In the formula, each B independently represents a nucleobase. n represents an integer within the range of 1 to 99. Each R ¹ independently represents hydrogen, a hydroxyl group or alkoxy, and at least one of each R ¹ represents a hydroxyl group. Each Y independently represents a substituent represented by the following general formula (1a), general formula (1b) or general formula (1c), at least one of which is a substituent (1a) or a substituent ( 1b). However, the case where Y of adjacent constituent monomers is all a substituent (1a) or a substituent (1b) is excluded.
Z represents hydrogen or a phosphate group. ] The oligonucleic acid compound of Claim 3 represented by following General formula (4).
[In the formula, each B independently represents a nucleobase. m represents an integer in the range of 1 to 49. Each R ¹ independently represents hydrogen, a hydroxyl group or alkoxy, and at least one of each R ¹ represents a hydroxyl group.
Y ¹ and Y ² are different from each other, one of them represents a substituent represented by the following general formula (1a) or general formula (1b), and the other is represented by the following general formula (1c). Represents a substituent.
T ¹ represents hydrogen or a substituent represented by the following general formula (5a).
[Wherein, B and R ¹ are as defined above. ]
T ² represents a hydroxyl group or a substituent represented by the following general formula (5b) or (5c).
[Wherein, B and R ¹ are as defined above. ]] The oligonucleic acid compound according to claim 3, wherein n is an integer in the range of 10-50. The oligonucleic acid compound according to claim 3, wherein n is an integer in the range of 15-30. The oligonucleic acid compound according to claim 4, wherein m is an integer within a range of 5 to 25. The oligonucleic acid compound according to claim 4, wherein m is an integer in the range of 7 to 15. The oligonucleic acid compound having a phosphorothioate bond according to any one of claims 5 to 8, wherein phosphorothioate bonds existing in the oligonucleic acid compound are all in the Rp configuration or all in the Sp configuration. A pharmaceutical composition comprising a complex of a carrier effective for transferring an oligonucleic acid compound into cells and the oligonucleic acid compound according to any one of claims 5 to 9. The pharmaceutical composition according to claim 10, wherein the carrier effective for transferring the oligonucleic acid compound into cells is a cationic carrier. The pharmaceutical composition according to claim 10 or 11, comprising 1 to 200 parts by weight of a carrier effective for transferring the oligonucleic acid compound into cells with respect to 1 part by weight of the oligonucleic acid compound. The pharmaceutical composition according to claim 10 or 11, comprising 2.5 to 100 parts by weight of a carrier effective for transferring the oligonucleic acid compound into cells with respect to 1 part by weight of the oligonucleic acid compound. The pharmaceutical composition according to claim 10 or 11, comprising 10 to 20 parts by weight of a carrier effective for transferring the oligonucleic acid compound into cells with respect to 1 part by weight of the oligonucleic acid compound. A double-stranded oligonucleic acid compound which forms a double strand in part or in whole using the oligonucleic acid compound according to any one of claims 5 to 9. A pharmaceutical composition comprising a complex of a carrier effective for transferring a double-stranded oligonucleic acid compound into a cell and the double-stranded oligonucleic acid compound according to claim 15. The pharmaceutical composition according to claim 16, wherein the carrier effective for transferring the double-stranded oligonucleic acid compound into cells is a cationic carrier. The pharmaceutical composition according to claim 16 or 17, comprising 1 to 200 parts by weight of a carrier effective for transferring the double-stranded oligonucleic acid compound into cells with respect to 1 part by weight of the double-stranded oligonucleic acid compound. The pharmaceutical composition according to claim 16 or 17, comprising 2.5 to 100 parts by weight of a carrier effective for transferring the double-stranded oligonucleic acid compound into cells with respect to 1 part by weight of the double-stranded oligonucleic acid compound. object. The pharmaceutical composition according to claim 16 or 17, comprising 10 to 20 parts by weight of a carrier effective for transferring the double-stranded oligonucleic acid compound into cells with respect to 1 part by weight of the double-stranded oligonucleic acid compound.