JP5341956B2 - Boranophosphate monomer - Google Patents

Abstract

A boranophosphate oligomer which is useful as an antisense molecule, etc. and can withstand practical use; and a boranophosphate monomer represented by the following formula (1). The oligomer is obtained by condensing the monomer in the presence of a given strong base and a given condensation agent. (1) (In the formula, B<1> represents a pyrimidine base, purine base, or derivative of either; R<1> represents dimethoxytrityl or monomethoxytrityl; and R<2> represents hydrogen, alkoxy, alkenyloxy, acyloxy, or trialkylsilyloxy.)

Description

  The present invention relates to a boranophosphate monomer, a method for producing the same, and a method for producing an oligonucleotide derivative using the boranophosphate monomer.

  One of the methods currently attracting attention in the field of gene therapy is the antisense method. The antisense method is a method of controlling only target protein synthesis by selectively forming a duplex with mRNA transcribed from DNA using an antisense molecule having a base sequence complementary to mRNA. .

  Here, as necessary conditions for the effective functioning of antisense molecules, firstly, it must have high cell membrane permeability, secondly, it is difficult to be hydrolyzed by nucleases in cells, and thirdly, It is possible to selectively form a stable duplex only with the mRNA.

  However, when natural antisense molecules are introduced into a living body, they are rapidly hydrolyzed by nucleases and have a problem of low cell membrane permeability. Therefore, there has been a demand for the design of more useful antisense molecules and the development of efficient production methods thereof.

  Many antisense molecules have been designed and synthesized so far, but phosphorothioate DNA, which is currently being studied for practical use, has problems such as cytotoxicity and low selectivity. It was.

In recent years, boranophosphate oligomers, which are new backbone antisense molecules, have been developed. This molecule is, for example,
(1) Since the phosphorus atom is coordinated to borane, it is highly lipophilic compared to the natural type, and high cell membrane permeability can be expected.
(2) High resistance to nucleases,
(3) High selectivity for target mRNA,
(4) The double strand formed by boranophosphate DNA and RNA becomes a substrate for RNase H, and target mRNA is effectively degraded.
(5) Application to boron neutron capture therapy (BNCT) can be expected,
It has been attracting attention as a practical antisense molecule.

［式中、ＤＭＴｒは、ジメトキシトリチル基を示す。］ The inventors have found the problems found in previously developed boranophosphate oligomers, namely:
(I) boranoization must be repeated each time the chain is extended,
(Ii) A method that eliminates the problem of side reaction to the nucleobase site by a boranoating reagent, that is, a method for synthesizing oligonucleotides using a boronated monomer represented by the following formula has been reported (Japan) Chemical Society 78th Annual Proceedings 746 pages (2000), Chemical Society of Japan 79th Annual Proceedings 942 (2001), Chemical Society of Japan 81st Annual Proceedings 937 pages (2002), Antisense DNA / RNA Study Group Proceedings, page 48 (2002), Chemical Society of Japan 83rd Annual Proceedings, page 944 (2003), Tetrahedron Lett. 43 (2002) 4237).

[Wherein, DMTr represents a dimethoxytrityl group. ]

  However, according to this technique, although the above problems (i) and (ii) are solved, when removing the protecting group on the phosphorus atom, there is a fatal problem that the oligonucleotide is decomposed. I understood it.

  Therefore, there has been a demand for a method for providing a boranophosphate oligomer that can solve the above problems (i) and (ii) and can withstand practical use.

As a result of intensive studies to solve the above problems, the present inventors have found that by using a cyanoethyl group as a protecting group on a phosphorus atom, the protecting group can be removed without decomposing the oligonucleotide. Furthermore, it has been found that the condensation of the oligomer is inhibited by using such a bulky protective group, and this point can be solved by performing a condensation reaction in the presence of a base and a special condensing agent. The present invention was completed.
That is, in the first aspect of the present invention, a boranophosphate monomer represented by the following formula (1) is provided.

［式中、Ｂ¹は、ピリミジン塩基、プリン塩基、又は、それらの誘導体を表し、Ｒ¹は、ジメトキシトリチル基、又は、モノメトキシトリチル基であり、Ｒ²は、水素原子、アルコキシ基、アルケニルオキシ基、アシルオキシ基、又は、トリアルキルシリルオキシ基を表す。］

[Wherein, B ¹ represents a pyrimidine base, a purine base, or a derivative thereof, R ¹ represents a dimethoxytrityl group or a monomethoxytrityl group, and R ² represents a hydrogen atom, an alkoxy group, or an alkenyl. An oxy group, an acyloxy group, or a trialkylsilyloxy group is represented. ]

［式中、Ｂ¹は、ピリミジン塩基、プリン塩基、又は、それらの誘導体を表し、Ｒ¹は、ジメトキシトリチル基、又は、モノメトキシトリチル基であり、Ｒ²は、水素原子、アルコキシ基、アルケニルオキシ基、アシルオキシ基、又は、トリアルキルシリルオキシ基を表す。］
シアノエチル基をβ脱離させない強塩基、及び、下記式（４ａ）又は、下記式（４ｂ）で示される縮合剤存在下、

［式中、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基であり、ただし、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよく、Ａ^7-は、求核性のないアニオン種であり、Ｑ環基は、置換基を有していてもよい炭化水素芳香環若しくは置換基を有していてもよい窒素含有芳香環と縮合した１，２，４−トリアゾール−１−イルオキシ基、又は、置換基を有していてもよい、窒素原子を２以上含む５員環基である。］

［式中、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基であり、ただし、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよく、Ａ^15-は、求核性のないアニオン種であり、Ｓ環基は、置換基を有していてもよい炭化水素芳香環若しくは置換基を有していてもよい窒素含有芳香環と縮合した１，２，４−トリアゾール−１−イルオキシ基、又は、置換基を有していてもよい、窒素原子を２以上含む５員環基である。］
下記式（２）で示されるボラノホスホリル化剤と、

下記式（３）で示されるヌクレオシド誘導体と

［式中、Ｂ¹、Ｒ¹及びＲ²は、上記の意味を有する。］
を反応させ反応生成物を得る工程と、前記反応生成物とトリエチルアミンとを反応させる工程とを含むことを特徴とする、ボラノホスフェートモノマーの製造方法が提供される。 According to a second aspect of the present invention, there is provided one aspect of a method for producing a boranophosphate monomer according to the first aspect of the present invention. That is, in the second aspect of the present invention, there is provided a method for producing a boranophosphate monomer represented by the following formula (1):

[Wherein, B ¹ represents a pyrimidine base, a purine base, or a derivative thereof, R ¹ represents a dimethoxytrityl group or a monomethoxytrityl group, and R ² represents a hydrogen atom, an alkoxy group, or an alkenyl. An oxy group, an acyloxy group, or a trialkylsilyloxy group is represented. ]
In the presence of a strong base that does not cause β-elimination of the cyanoethyl group and a condensing agent represented by the following formula (4a) or the following formula (4b):

[Wherein, A ¹ , A ² , A ³ , A ⁴ , A ⁵ and A ⁶ are each independently the same or different and may have a C ₁ -C ₁₀ alkyl group which may have a substituent. Provided that any combination of A ¹ , A ² , A ³ , A ⁴ , A ⁵ and A ⁶ may be bridged with each other to form a saturated ring or an unsaturated ring, and A ^7- is an anionic species having no nucleophilicity, and the Q ring group may have a hydrocarbon aromatic ring or a substituent which may have a substituent. It is a 1,2,4-triazol-1-yloxy group condensed with a nitrogen-containing aromatic ring, or a 5-membered ring group containing two or more nitrogen atoms which may have a substituent. ]

[Wherein, A ¹¹ , A ¹² , A ¹³ and A ¹⁴ are each independently the same or different, and are optionally substituted C ₁ -C ₁₀ alkyl groups, provided that A ^11, any combination of a ^12, a ¹³ and a ¹⁴ are, may form a saturated or unsaturated ring crosslinked to each other, and may have a substituent, a ^15-is An anionic species having no nucleophilicity, and the S ring group is a 1,2,4 condensed with a hydrocarbon aromatic ring which may have a substituent or a nitrogen-containing aromatic ring which may have a substituent. -A triazol-1-yloxy group or a 5-membered ring group containing two or more nitrogen atoms which may have a substituent. ]
A boranophosphorylating agent represented by the following formula (2);

A nucleoside derivative represented by the following formula (3):

[Wherein B ¹ , R ¹ and R ² have the above-mentioned meanings. ]
There is provided a method for producing a boranophosphate monomer, comprising the steps of: reacting to obtain a reaction product; and reacting the reaction product with triethylamine.

得られたトリス−２−シアノエチルボラノホスフェートとトリエチルアミンを反応させることにより得られることが好ましい。 In the second aspect of the present invention, the boranophosphorylating agent boranoates tris-2-cyanoethyl phosphite to obtain tris-2-cyanoethyl boranophosphate represented by the following formula:

It is preferably obtained by reacting the obtained tris-2-cyanoethylboranophosphate with triethylamine.

［式中、Ｂ¹は、ピリミジン塩基、プリン塩基、又は、それらの誘導体を表し、Ｒ¹は、ジメトキシトリチル基、又は、モノメトキシトリチル基であり、Ｒ²は、水素原子、アルコキシ基、アルケニルオキシ基、アシルオキシ基、又は、トリアルキルシリルオキシ基を表す。］
上記式（１）中のシアノエチル基をβ脱離させない強塩基、及び、下記式（４ａ）又は、下記式（４ｂ）で示される縮合剤存在下、

［式中、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基であり、ただし、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよく、Ａ^7-は、求核性のないアニオン種であり、Ｑ環基は、置換基を有していてもよい炭化水素芳香環若しくは置換基を有していてもよい窒素含有芳香環と縮合した１，２，４−トリアゾール−１−イルオキシ基、又は、置換基を有していてもよい、窒素原子を２以上含む５員環基である。］

［式中、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基であり、ただし、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよく、Ａ^15-は、求核性のないアニオン種であり、Ｓ環基は、置換基を有していてもよい炭化水素芳香環若しくは置換基を有していてもよい窒素含有芳香環と縮合した１，２，４−トリアゾール−１−イルオキシ基、又は、置換基を有していてもよい、窒素原子を２以上含む５員環基である。］
下記一般式（５）で示されるヌクレオシド誘導体と

［式中、Ｂ²は、ピリミジン塩基、プリン塩基、又は、それらの誘導体を表し、Ｒ³は、保護基を表し、Ｒ⁴は、水素原子、アルコキシ基、アルケニルオキシ基、アシルオキシ基、又は、トリアルキルシリルオキシ基を表す。］
を反応させ、下記一般式（６）で示される二量体を製造する方法が提供される。

［式中、Ｂ¹、Ｂ²、Ｒ¹、Ｒ²、Ｒ³及びＲ⁴は、前記と同義を表す。］ In the third aspect of the present invention, an aspect of a method for producing a dimer using the boranophosphate monomer according to the first aspect of the present invention is provided. That is, in the third aspect of the present invention, a boranophosphate monomer represented by the following general formula (1) and

[Wherein, B ¹ represents a pyrimidine base, a purine base, or a derivative thereof, R ¹ represents a dimethoxytrityl group or a monomethoxytrityl group, and R ² represents a hydrogen atom, an alkoxy group, or an alkenyl. An oxy group, an acyloxy group, or a trialkylsilyloxy group is represented. ]
In the presence of a strong base that does not β-eliminate the cyanoethyl group in the above formula (1), and a condensing agent represented by the following formula (4a) or the following formula (4b):

[Wherein, A ¹ , A ² , A ³ , A ⁴ , A ⁵ and A ⁶ are each independently the same or different and may have a C ₁ -C ₁₀ alkyl group which may have a substituent. Provided that any combination of A ¹ , A ² , A ³ , A ⁴ , A ⁵ and A ⁶ may be bridged with each other to form a saturated ring or an unsaturated ring, and A ^7- is an anionic species having no nucleophilicity, and the Q ring group may have a hydrocarbon aromatic ring or a substituent which may have a substituent. It is a 1,2,4-triazol-1-yloxy group condensed with a nitrogen-containing aromatic ring, or a 5-membered ring group containing two or more nitrogen atoms which may have a substituent. ]

[Wherein, A ¹¹ , A ¹² , A ¹³ and A ¹⁴ are each independently the same or different, and are optionally substituted C ₁ -C ₁₀ alkyl groups, provided that A ^11, any combination of a ^12, a ¹³ and a ¹⁴ are, may form a saturated or unsaturated ring crosslinked to each other, and may have a substituent, a ^15-is An anionic species having no nucleophilicity, and the S ring group is a 1,2,4 condensed with a hydrocarbon aromatic ring which may have a substituent or a nitrogen-containing aromatic ring which may have a substituent. -A triazol-1-yloxy group or a 5-membered ring group containing two or more nitrogen atoms which may have a substituent. ]
A nucleoside derivative represented by the following general formula (5):

[Wherein, B ² represents a pyrimidine base, a purine base, or a derivative thereof, R ³ represents a protecting group, and R ⁴ represents a hydrogen atom, an alkoxy group, an alkenyloxy group, an acyloxy group, or Represents a trialkylsilyloxy group. ]
Is reacted to produce a dimer represented by the following general formula (6).

[Wherein, B ¹ , B ² , R ¹ , R ² , R ³ and R ⁴ have the same meaning as described above. ]

［式中、Ｘ¹、Ｘ²、Ｘ³及びＸ⁴は、それぞれ、互いに独立し、同一または異なって、Ｃ₁〜Ｃ₁₀アルキル基であり、Ｘ⁵、Ｘ⁶、Ｘ⁷、Ｘ⁸、Ｘ⁹及びＸ¹⁰は、それぞれ、互いに独立し、同一または異なって、電子供与基である。］ In the third aspect of the present invention, the strong base may be a naphthalene derivative represented by the following formula (7a).

[Wherein, X ¹ , X ² , X ³ and X ⁴ are each independently the same or different and are C ₁ -C ₁₀ alkyl groups, and X ⁵ , X ⁶ , X ⁷ , X ⁸ , X ⁹ and X ¹⁰ are each independently the same or different and are electron donating groups. ]

In this case, in the formula (7a), X ¹ , X ² , X ³ and X ⁴ are each independently the same or different and are a methyl group or an ethyl group, and X ⁵ , X ⁶ , X ⁷ , X ⁸ , X ⁹ and X ¹⁰ are preferably a hydrogen atom, a C ₁ -C ₁₀ alkyl group, or a C ₁ -C ₁₀ alkoxy group.

In the third aspect of the present invention, the condensing agent may be a compound represented by the formula (4a). In this case, the Q ring group in the formula (4a) has a substituent. An imidazolyl group which may have a substituent, a triazolyl group which may have a substituent, a tetrazolyl group which may have a substituent, or a benzene ring or a substituent which may have a substituent. A triazolyloxy group condensed with a good pyridine ring, and A ⁷⁻ is PF ₆ ⁻ , BF ₄ ⁻ , ClO ₄ ⁻ , CF ₃ SO ₂ O ⁻ , or (CF ₃ SO ₂ ) ₂ N ^−. It is preferable that

［式中、Ｙ¹は、水素原子、Ｃ₁〜Ｃ₁₀アルキル基、又は、Ｃ₁〜Ｃ₁₀アルコキシ基であり、Ｙ²及びＹ³は、それぞれ、互いに独立し、同一または異なって、Ｃ₁〜Ｃ₁₀アルキル基である。］ In the third aspect of the present invention, the condensing agent may be a compound represented by the formula (4b), and the strong base may be a pyridine derivative represented by the following formula (7b).

[Wherein Y ¹ is a hydrogen atom, a C ₁ -C ₁₀ alkyl group, or a C ₁ -C ₁₀ alkoxy group, and Y ² and Y ³ are each independently the same or different, and a ₁ -C ₁₀ alkyl group. ]

In this case, the S ring group in the formula (4b) may have an imidazolyl group which may have a substituent, a triazolyl group which may have a substituent, or a tetrazolyl group which may have a substituent. Or a triazolyloxy group condensed with an optionally substituted benzene ring or an optionally substituted pyridine ring, and A ^15- represents PF ₆ ⁻ , BF ₄ ⁻ , ClO _{4. ^{-, CF 3 SO 2 O -}} , _{or, (CF 3 SO 2) 2} N - is preferably, also, the formula (7b), Y ¹ is a hydrogen atom or a methyl group, Y ² And Y ³ is preferably a methyl group.

［式中、Ｂ¹及びＢ²は、それぞれ、互いに独立し、同一または異なって、ピリミジン塩基、プリン塩基、又は、それらの誘導体を表し、Ｒ¹は、ジメトキシトリチル基、又は、モノメトキシトリチル基であり、Ｒ²及びＲ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、アルコキシ基、アルケニルオキシ基、アシルオキシ基、又は、トリアルキルシリルオキシ基であり、Ｒ³は、保護基であり、ｎは１以上の整数を示す。］
脱保護試薬とを反応させて、Ｒ¹を脱離させた後、上記式（８）中のシアノエチル基をβ脱離させない強塩基、及び、下記式（４ａ）、又は、下記式（４ｂ）で示される縮合剤存在下、

［式中、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基であり、ただし、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよく、Ａ^7-は、求核性のないアニオン種であり、Ｑ環基は、置換基を有していてもよい炭化水素芳香環若しくは置換基を有していてもよい窒素含有芳香環と縮合した１，２，４−トリアゾール−１−イルオキシ基、又は、置換基を有していてもよい、窒素原子を２以上含む５員環基である。］

［式中、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基であり、ただし、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよく、Ａ^15-は、求核性のないアニオン種であり、Ｓ環基は、置換基を有していてもよい炭化水素芳香環若しくは置換基を有していてもよい窒素含有芳香環と縮合した１，２，４−トリアゾール−１−イルオキシ基、又は、置換基を有していてもよい、窒素原子を２以上含む５員環基である。］
下記式（１）で示されるボラノホスフェートモノマーと

［式中、Ｂ¹、Ｒ¹及びＲ²は、上記と同義である。］
を反応させることを特徴とする、下記式（９）で示されるオリゴヌクレオチド誘導体の製造方法が提供される。

［式中、Ｂ¹、Ｂ²、Ｒ¹、Ｒ²、Ｒ³、Ｒ⁴及びｎは、前記と同義を表す。］ According to a fourth aspect of the present invention, there is provided one aspect of a method for producing an oligonucleotide derivative using a boranophosphate monomer according to the first aspect of the present invention. That is, an oligonucleotide derivative represented by the following formula (8):

[Wherein, B ¹ and B ² are each independently the same or different and represent a pyrimidine base, a purine base, or a derivative thereof, and R ¹ represents a dimethoxytrityl group or a monomethoxytrityl group. R ² and R ⁴ are each independently the same or different and are a hydrogen atom, an alkoxy group, an alkenyloxy group, an acyloxy group, or a trialkylsilyloxy group, and R ³ is a protecting group And n represents an integer of 1 or more. ]
After reacting with a deprotecting reagent to remove R ¹ , a strong base that does not cause β elimination of the cyanoethyl group in the above formula (8) and the following formula (4a) or the following formula (4b) In the presence of a condensing agent

[Wherein, A ¹ , A ² , A ³ , A ⁴ , A ⁵ and A ⁶ are each independently the same or different and may have a C ₁ -C ₁₀ alkyl group which may have a substituent. Provided that any combination of A ¹ , A ² , A ³ , A ⁴ , A ⁵ and A ⁶ may be bridged with each other to form a saturated ring or an unsaturated ring, and A ^7- is an anionic species having no nucleophilicity, and the Q ring group may have a hydrocarbon aromatic ring or a substituent which may have a substituent. It is a 1,2,4-triazol-1-yloxy group condensed with a nitrogen-containing aromatic ring, or a 5-membered ring group containing two or more nitrogen atoms which may have a substituent. ]

[Wherein, A ¹¹ , A ¹² , A ¹³ and A ¹⁴ are each independently the same or different, and are optionally substituted C ₁ -C ₁₀ alkyl groups, provided that A ^11, any combination of a ^12, a ¹³ and a ¹⁴ are, may form a saturated or unsaturated ring crosslinked to each other, and may have a substituent, a ^15-is An anionic species having no nucleophilicity, and the S ring group is a 1,2,4 condensed with a hydrocarbon aromatic ring which may have a substituent or a nitrogen-containing aromatic ring which may have a substituent. -A triazol-1-yloxy group or a 5-membered ring group containing two or more nitrogen atoms which may have a substituent. ]
A boranophosphate monomer represented by the following formula (1):

[Wherein, B ¹ , R ¹ and R ² have the same meanings as described above. ]
There is provided a method for producing an oligonucleotide derivative represented by the following formula (9), which comprises reacting

[Wherein, B ¹ , B ² , R ¹ , R ² , R ³ , R ⁴ and n are as defined above] ]

  In the fourth aspect of the present invention, the reaction is preferably performed in a solid phase.

  According to the present invention, it is possible to provide a boranophosphate oligomer that can withstand practical use while solving the problems found in conventionally developed boranophosphate oligomers. The boranophosphate oligomer obtained by the present invention is useful as an antisense molecule.

［式中、Ｂ¹、Ｒ¹及びＲ²は、上記の意味を有する。］ In the first aspect of the present invention, a boranophosphate monomer represented by the following formula (1) is provided.

[Wherein B ¹ , R ¹ and R ² have the above-mentioned meanings. ]

  In the boranophosphate monomer represented by the above formula (1), since the protecting group on the phosphorus atom is a cyanoethyl group, when the protecting group is removed using ammonia or the like, the cyanoethyl group is removed by β elimination. For this reason, even if this protective group is removed after obtaining the oligomer using the boranophosphate monomer, the oligomer is not decomposed. Therefore, according to the present invention, it is possible to provide a boranophosphate oligomer that can withstand practical use.

In the above formula, B ¹ represents a pyrimidine base such as thymine, cytosine or uracil; a purine base such as adenine or guanine; or a derivative thereof such as 5-methylcytosine, 5-fluorouracil or 5-hydroxymethylcytosine.

  In order to improve the solubility in a solvent, a dimethoxytrityl group (DMTr), acetyl (Ac), benzoyl (Bz), isobutyryl (iBu), phenoxyacetyl (PAC), 4- (t-butyl) phenoxy is added to the base site. Acetyl (BPA), allyloxycarbonyl (AOC), 2-[(t-butyldiphenylsilyloxy) methyl] benzoyl (SiOMB), 2- (acetylmethyl) benzoyl (AMB), 2-azidobenzoyl (AZMB), diphenyl A protecting group such as carbamoyl (DPC) or phenylacetyl (PA) may be introduced.

In the first embodiment of the present invention, B ¹ is preferably cytosine, thymine, adenine, guanine or a derivative thereof, or a group in which a protecting group is introduced.
In the above formula, R ¹ is a dimethoxytrityl group (DMTr) or a monomethoxytrityl group.

In the first embodiment of the present invention, R ¹ is preferably a dimethoxytrityl group.

In the above formula, R ² represents a hydrogen atom, an alkoxy group, an alkenyloxy group, an acyloxy group, or a trialkylsilyloxy group.

  In the present specification, examples of the “alkoxy group” include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy and the like.

  In the present specification, the “alkenyloxy group” is not limited, but includes vinyloxy, allyloxy, 1-propenyloxy, isopropenyloxy, 2-methyl-1-propenyloxy, 2-methylallyloxy, 2- Examples include butenyloxy.

In the present specification, the “acyloxy group” is not limited, but includes C _1-6 alkyl-carbonyloxy (eg, methylcarbonyloxy, ethylcarbonyloxy, etc.), C _6-10 aryl-carbonyloxy (eg, benzoyl). Oxy) and the like.

  In the present specification, examples of the “trialkylsilyloxy group” include, but are not limited to, a trimethylsilyloxy group and a triethylsilyloxy group.

In the first embodiment of the present invention, R ² is preferably a hydrogen atom, an alkoxy group, or a trialkylsilyloxy group.

When the boranophosphate monomer according to the first aspect of the present invention is used for RNA derivative synthesis, R ² is preferably a trialkylsilyloxy group, more preferably a t-butyldimethylsilyl group. preferable.

  According to a second aspect of the present invention, there is provided one aspect of a method for producing a boranophosphate monomer according to the first aspect of the present invention. That is, in the second aspect of the present invention, in the presence of a strong base that does not eliminate β of the cyanoethyl group and a predetermined condensing agent, a boranophosphorylating agent represented by the following formula (2) and the following formula (3): Production of a boranophosphate monomer represented by the following formula (1), which comprises a step of reacting the nucleoside derivative shown to obtain a reaction product and a step of reacting the reaction product with triethylamine. A method is provided.

［式中、Ｂ¹、Ｒ¹及びＲ²は、上記の意味を有する。］

[Wherein B ¹ , R ¹ and R ² have the above-mentioned meanings. ]

In the second aspect of the present invention, the explanation of B ¹ , R ¹ and R ² in the above formula is the same as that in the first aspect to the second aspect of the present invention.

In the second embodiment of the present invention, a boranophosphorylating agent represented by the following formula (2) is used.

［式中、Ｂ¹、Ｒ¹及びＲ²は、上記の意味を有する。］ In the second embodiment of the present invention, a nucleoside derivative represented by the following general formula (3) is used.

[Wherein B ¹ , R ¹ and R ² have the above-mentioned meanings. ]

  In the second embodiment of the present invention, the amount of the boranophosphorylation agent represented by the above formula (2) used is represented by the above formula (3) in order to allow the boranophosphorylation reaction to proceed quantitatively. It is preferable to use 1 mol to 3 mol, more preferably 1.2 mol to 1.5 mol, per 1 mol of the nucleoside derivative.

  Further, in the second aspect of the present invention, when the boranophosphorylating agent represented by the above formula (2) and the nucleoside derivative represented by the above formula (3) are reacted, the cyanoethyl group is not strongly β-eliminated. A base is used.

  In the present specification, examples of the “strong base that does not cause β-elimination of the cyanoethyl group” include a base that captures only dissociated protons and has an equilibrium on the capturing side.

上記式（７ａ）中、Ｘ¹、Ｘ²、Ｘ³及びＸ⁴は、それぞれ、互いに独立し、同一または異なって、Ｃ₁〜Ｃ₁₀アルキル基である。 As one embodiment of such a strong base, for example, a naphthalene derivative represented by the following formula (7a) can be given.

In the above formula ^{(7a), X 1, X} 2, X 3 and X ⁴ are each, independently of one another, identical or different, are C ₁ -C ₁₀ alkyl group.

In the present specification, examples of the “C ₁ -C ₁₀ alkyl group” include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like. be able to.

In the present invention, X ¹ , X ² , X ³ and X ⁴ are each independently the same or different and are preferably a methyl group or an ethyl group.

In the above formula (7a), X ⁵ , X ⁶ , X ⁷ , X ⁸ , X ⁹ and X ¹⁰ are each independently the same or different and are electron donating groups.

  In the present specification, examples of the “electron donating group” include, but are not limited to, a hydrogen atom, a hydroxy group, an alkoxy group, an amino group, a dialkylamino group, an alkyl group, and an aryl group.

  In the present specification, examples of the “aryl group” include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl, indenyl, biphenylyl, anthryl, phenanthryl and the like.

In the second embodiment of the present invention, X ⁵ , X ⁶ , X ⁷ , X ⁸ , X ⁹ and X ¹⁰ are each independently of each other, the same or different, a C ₁ -C ₁₀ alkyl group, or C _1- A C ₁₀ alkoxy group is preferable, and a hydrogen atom, a methyl group, an ethyl group, a methoxy group, and a dimethylamino group are more preferable.

上記式（７ｂ）中、Ｙ¹は、水素原子、Ｃ₁〜Ｃ₁₀アルキル基、又は、Ｃ₁〜Ｃ₁₀アルコキシ基である。また、Ｙ²及びＹ³は、それぞれ、互いに独立し、同一または異なって、Ｃ₁〜Ｃ₁₀アルキル基である。 In the present specification, as another embodiment of “a strong base that does not allow β-elimination of a cyanoethyl group”, for example, a pyridine derivative represented by the following formula (7b) can be given.

In the above formula (7b), Y ¹ is a hydrogen atom, C ₁ -C ₁₀ alkyl group, or a C ₁ -C ₁₀ alkoxy group. Y ² and Y ³ are each independently the same or different and each represent a C _{1 to} C ₁₀ alkyl group.

  Pyridine eliminates borane because of its nitrogen atom nucleophilicity, but it can be made into a compound having no nucleophilicity by introducing an alkyl group at the 2,6-position.

In the second embodiment of the present invention, Y ¹ is preferably a hydrogen atom or a C ₁ -C ₁₀ alkyl group, more preferably a hydrogen atom or a methyl group.
In the second embodiment of the present invention, Y ² and Y ³ are preferably methyl groups.

  In the second aspect of the present invention, in order to complete the reaction, it is preferable to use an excessive amount of the strong base that does not cause β-elimination of the cyanoethyl group relative to the boranophosphorylating agent represented by the above formula (2). For example, it is preferably used in an amount of 5 mol or more, more preferably 7 to 20 mol, per 1 mol of the boranophosphorylating agent represented by the above formula (2).

  In the second aspect of the present invention, a condensing agent is used when the boranophosphorylating agent represented by the above formula (2) is reacted with the nucleoside derivative represented by the above formula (3).

  The condensing agent used in the present invention needs to allow the condensation reaction to proceed even if the protecting group on the phosphorus atom is a bulky group such as a cyanoethyl group. An example of such a condensing agent is a condensing agent represented by the following formula (4a).

上記式（４ａ）中、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基（例えば、メチル基、エチル基であることが好ましい）であり、Ａ¹、Ａ²、Ａ³、Ａ⁴、Ａ⁵及びＡ⁶のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよい。

In the above formula ^{(4a), A 1, A} 2, A 3, A 4, A 5 and A ⁶ are each, independently of one another, identical or different, which may have a substituent C ₁ -C ₁₀ alkyl group (for example, preferably a methyl group or an ethyl group), and any combination of A ¹ , A ² , A ³ , A ⁴ , A ⁵ and A ⁶ is bridged with each other to form a saturated ring or An unsaturated ring may be formed and may have a substituent.

  In the present specification, the “saturated ring or unsaturated ring” is preferably a 4-membered to 10-membered saturated ring or unsaturated ring, and a 4-membered to 7-membered saturated ring or unsaturated ring. It is more preferable that it is a 5-membered saturated or unsaturated ring.

For example, as represented by the following formula, when A ¹ and A ² , A ³ and A ⁴ , and A ⁵ and A ⁶ are bridged with each other to form a 5-membered saturated ring, A ² and A 4 ³ and A ⁵ and A ⁶ are bridged to form a 5-membered saturated ring, and A ¹ and A ⁴ are methyl groups.

［式中、Ａ^7-及びＱ環基は、上記の意味を有する。］

[Wherein the A ^7- and Q ring groups have the above-mentioned meanings. ]

In the above formula (4a), A ⁷⁻ is an anionic species having no nucleophilicity.
In the present specification, examples of the “non-nucleophilic anionic species” include PF ₆ ⁻ , BF ₄ ⁻ , ClO ₄ ⁻ , CF ₃ SO ₂ O ⁻ , or (CF ₃ SO ₂ ) ₂ N ⁻ . Can be mentioned.
In a second aspect the present invention, A ^7- is, PF ₆ ^- it is preferably ^- or BF _4.

  In the above formula (4a), the Q ring group is a 1,2,4-triazole-fused with an optionally substituted hydrocarbon aromatic ring or an optionally substituted nitrogen-containing aromatic ring. It is a 1-yloxy group or a 5-membered cyclic group containing two or more nitrogen atoms which may have a substituent.

  In the present specification, examples of the “hydrocarbon aromatic ring” include a monocyclic hydrocarbon aromatic ring such as benzene; a polycyclic hydrocarbon aromatic ring such as biphenyl, triphenyl, naphthalene, indene, anthracene, and phenanthrene. Can be mentioned.

  In the present specification, examples of the “nitrogen-containing aromatic ring” include monocyclic nitrogen-containing aromatic rings such as pyrrole, imidazole, triazole, pyrazole, pyridine, pyridazine, pyrimidine, and pyrazine; indole, quinoline, isoquinoline, purine, Mention may be made of polycyclic nitrogen-containing aromatic rings such as cinnoline, quinoxaline, quinazolidine, phthalazine, carbazole, acridine, phenazine, phenanthridine.

  In the present specification, examples of the 5-membered cyclic group containing two or more nitrogen atoms include a pyrrolyl group, an imidazolyl group, a triazolyl group, and a tetrazolyl group.

In the second embodiment of the present invention, a substituent is introduced into the “hydrocarbon aromatic ring”, “nitrogen-containing aromatic ring”, and “5-membered ring group containing two or more nitrogen atoms” represented by the Q ring group. Also good. Examples of the substituent include a C _{1 to} C ₁₀ hydrocarbon group (for example, methyl, ethyl, propyl, butyl, phenyl, naphthyl, indenyl, tolyl, xylyl, benzyl, etc.), C _{1 to} C ₁₀ alkoxy group (for example, , Methoxy, ethoxy, propoxy, butoxy, etc.), C ₆ -C ₁₀ aryloxy groups (eg, phenyloxy, naphthyloxy, biphenyloxy, etc.), nitro groups, cyano groups, trifluoromethyl groups, hydroxyl groups, or halogen atoms ( For example, fluorine, chlorine, bromine, iodine) can be used. In this case, one or more substituents may be introduced at substitutable positions, and preferably 1 to 4 substituents may be introduced. When the number of substituents is 2 or more, each substituent may be the same or different.

  In the second embodiment of the present invention, the Q ring group is an imidazolyl group which may have a substituent, a triazolyl group which may have a substituent, a tetrazolyl group which may have a substituent, or A triazolyloxy group condensed with a benzene ring which may have a substituent or a pyridine ring which may have a substituent, such as a nitro group, a halogen atom, a cyano group or a trifluoromethyl group. It is more preferably an imidazolyl group, triazolyl group or tetrazolyl group having an electron-withdrawing type substituent, or a triazolyloxy group condensed with a pyridine ring.

上記式（４ｂ）中、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴は、それぞれ、互いに独立し、同一または異なって、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基（例えば、メチル基、エチル基であることが好ましい）であり、Ａ¹¹、Ａ¹²、Ａ¹³及びＡ¹⁴のいずれか組み合わせが、互いに架橋して飽和環又は不飽和環を形成してもよく、かつ、置換基を有していてもよい。 Moreover, the condensing agent shown by following formula (4b) can be mentioned as another aspect of the condensing agent used by the 2nd aspect of this invention.

In the above formula (4b), A ¹¹ , A ¹² , A ¹³ and A ¹⁴ are each independently the same or different, and may be a C ₁ -C ₁₀ alkyl group (for example, Any combination of A ¹¹ , A ¹² , A ¹³ and A ¹⁴ may be bridged with each other to form a saturated or unsaturated ring, and It may have a substituent.

［式中、Ａ^15-及びＳ環基は、上記の意味を有する。］ For example, as represented by the following formula, a case where A ¹² and A ¹³ are cross-linked with each other to form a 5-membered saturated ring, and A ¹¹ and A ¹⁴ are methyl groups is preferred.

[Wherein the A ^15- and S ring groups have the above-mentioned meanings. ]

In the above formula (4b), A ¹⁵⁻ is an anionic species having no nucleophilicity.
In a second aspect of the present invention, A ^15-is, PF ₆ ^- is preferably ^- or BF _4.

  In the above formula (4b), the S ring group is a 1,2,4-triazole-fused with an optionally substituted hydrocarbon aromatic ring or an optionally substituted nitrogen-containing aromatic ring. It is a 1-yloxy group or a 5-membered cyclic group containing two or more nitrogen atoms which may have a substituent.

In the second embodiment of the present invention, the “hydrocarbon aromatic ring”, “nitrogen-containing aromatic ring”, and “5-membered ring group containing two or more nitrogen atoms” represented by the S ring group have a substituent introduced. Also good. Examples of the substituent include a C _{1 to} C ₁₀ hydrocarbon group (for example, methyl, ethyl, propyl, butyl, phenyl, naphthyl, indenyl, tolyl, xylyl, benzyl, etc.), C _{1 to} C ₁₀ alkoxy group (for example, , Methoxy, ethoxy, propoxy, butoxy, etc.), C ₆ -C ₁₀ aryloxy groups (eg, phenyloxy, naphthyloxy, biphenyloxy, etc.), nitro groups, cyano groups, trifluoromethyl groups, hydroxyl groups, or halogen atoms ( For example, fluorine, chlorine, bromine, iodine) can be used. In this case, one or more substituents may be introduced at substitutable positions, and preferably 1 to 4 substituents may be introduced. When the number of substituents is 2 or more, each substituent may be the same or different.

  In the second embodiment of the present invention, the S ring group is an imidazolyl group which may have a substituent, a triazolyl group which may have a substituent, a tetrazolyl group which may have a substituent, or A triazolyloxy group condensed with a benzene ring which may have a substituent or a pyridine ring which may have a substituent, such as a nitro group, a halogen atom, a cyano group or a trifluoromethyl group. It is more preferably an imidazolyl group, triazolyl group or tetrazolyl group having an electron-withdrawing type substituent, or a triazolyloxy group condensed with a pyridine ring.

  In the second aspect of the present invention, the amount of the condensing agent used is from 1 mol to 1 mol of the boranophosphorylating agent represented by the above formula (2) in order to allow the condensation reaction to proceed quantitatively. It is preferable to use 20 mol, more preferably 1.5 mol to 10 mol, still more preferably 2 mol to 3 mol.

  In the second aspect of the present invention, when the condensing agent is a compound represented by the above formula (4a), a naphthalene derivative represented by the above formula (7a) having a stronger basicity may be used as the strong base. preferable.

  In the second embodiment of the present invention, when the condensing agent is a compound represented by the formula (4b), a naphthalene derivative represented by the above formula (7a) having a stronger basicity is used as the strong base. Alternatively, a pyridine derivative represented by the above formula (7b) which is slightly weak in basicity may be used. From the viewpoint of avoiding the reaction between the anionized nucleoside derivative and the condensing agent, it is preferable to use a pyridine derivative represented by the above formula (7b) having a slightly weak basicity.

  In the second aspect of the present invention, the reaction product obtained by reacting the boranophosphorylating agent with the nucleoside derivative is isolated and purified, and then reacted with triethylamine. Thereby, one cyanoethyl group on the phosphorus atom is removed, and a boranophosphate monomer represented by the above formula (1) can be obtained.

  In the second aspect of the present invention, in order to complete the reaction, it is preferable to use an excess amount of triethylamine with respect to the boranophosphorylating agent represented by the above formula (2). For example, it is preferably used in an amount of 5 mol or more, more preferably 7 to 20 mol, per 1 mol of the boranophosphorylating agent represented by the above formula (2).

  In the second aspect of the present invention, typically, a condensing agent and a strong base are added to a solution of the boranophosphorylating agent represented by the above formula (2) and the nucleoside derivative represented by the above formula (3) and stirred. The product obtained is isolated and purified, and then reacted with triethylamine to obtain a boranophosphate monomer represented by the above formula (1). Specifically, it is considered to proceed according to the following scheme.

［式中、Ｂ¹、Ｒ¹及びＲ²は、上記の意味を有する。］

[Wherein B ¹ , R ¹ and R ² have the above-mentioned meanings. ]

  In the second aspect of the present invention, the solvent is preferably a solvent capable of dissolving the boranophosphorylating agent represented by the above formula (2), the nucleoside derivative represented by the above formula (3) and the condensing agent. For example, acetonitrile, tetrahydrofuran, dichloromethane, N, N-dimethylformamide and the like can be mentioned, and acetonitrile or N, N-dimethylformamide is preferable.

  In the second embodiment of the present invention, the reaction temperature is preferably −30 ° C. to 50 ° C., more preferably 0 ° C. to 40 ° C., and still more preferably 15 ° C. to 30 ° C. If desired, the reaction may be allowed to proceed while blocking light.

  In the second aspect of the present invention, the pressure is preferably atmospheric pressure. The atmosphere is preferably an inert gas atmosphere such as argon.

The boranophosphorylating agent represented by the above formula (2) used in the second embodiment of the present invention is, for example, boranoylated tris-2-cyanoethyl phosphite with a BH ₃ .THF complex, a BH ₃ .SMe ₂ complex or the like. Thus, tris-2-cyanoethylboranophosphate can be obtained, and the obtained tris-2-cyanoethylboranophosphate can be reacted with triethylamine.

In the method for producing a boranophosphorylating agent, the amount of the boranoating agent such as BH ₃ · THF complex, BH ₃ · SMe ₂ complex, etc. used is tris-2- It is preferably 1 mol to 2 mol, more preferably 1.2 mol to 1.5 mol, per 1 mol of cyanoethyl phosphite.

  In the method for producing a boranophosphorylating agent, in order to complete the reaction, it is preferable to use an excess amount of triethylamine with respect to the obtained tris-2-cyanoethylboranophosphate. For example, it is preferably used in an amount of 5 mol or more, more preferably 7 mol to 20 mol, relative to 1 mol of tris-2-cyanoethylboranophosphate.

In the method for producing a boranophosphorylating agent, typically, a BH ₃ · THF complex is added to a solution of tris-2-cyanoethyl phosphite and stirred, and the resulting reaction product is isolated and purified, and then triethylamine is obtained. Is reacted with boranophosphorylating agent represented by the above formula (2).

  In the method for producing a boranophosphorylating agent, a solvent capable of dissolving tris-2-cyanoethyl phosphite is preferable. For example, acetonitrile, tetrahydrofuran, dichloromethane, N, N-dimethylformamide can be mentioned, and acetonitrile or N, N-dimethylformamide is preferable.

  In the method for producing a boranophosphorylating agent, the reaction temperature is preferably -30 ° C to 50 ° C, more preferably 0 ° C to 40 ° C, and still more preferably 15 ° C to 30 ° C. If desired, the reaction may be allowed to proceed while blocking light.

  In the method for producing a boranophosphorylating agent, the pressure is preferably atmospheric pressure. The atmosphere is preferably an inert gas atmosphere such as argon.

  In the third aspect of the present invention, an aspect of a method for producing a dimer using the boranophosphate monomer according to the first aspect of the present invention is provided. That is, in the third aspect of the present invention, a boranophosphate monomer represented by the following general formula (1), a strong base that does not cause β-elimination of the cyanoethyl group in the above formula (1), and a predetermined condensing agent are present. There is provided a method for reacting a nucleoside derivative represented by the following general formula (5) to produce a dimer represented by the following general formula (6).

［式中、Ｂ¹、Ｒ¹、Ｒ²は、上記の意味を有する。］

[Wherein B ¹ , R ¹ and R ² have the above-mentioned meanings. ]

In the above formula, the explanation of B ¹ , R ¹ and R ² is the same as that in the first aspect to the second aspect of the present invention.

上記式（５）中、Ｂ²は、チミン、シトシン、ウラシル等のピリミジン塩基；アデニン、グアニン等のプリン塩基；または５−メチルシトシン、５−フルオロウラシル、５−ヒドロキシメチルシトシン等のそれらの誘導体を表す。 In the third aspect of the present invention, a nucleoside derivative represented by the following formula (5) is used.

In the above formula (5), B ² represents a pyrimidine base such as thymine, cytosine or uracil; a purine base such as adenine or guanine; or a derivative thereof such as 5-methylcytosine, 5-fluorouracil or 5-hydroxymethylcytosine. Represent.

  In order to improve the solubility in a solvent, a dimethoxytrityl group (DMTr), benzoyl (Bz), isobutyryl (iBu), phenoxyacetyl (PAC), 4- (t-butyl) phenoxyacetyl (BPA), Allyloxycarbonyl (AOC), 2-[(t-butyldiphenylsilyloxy) methyl] benzoyl (SiOMB), 2- (acetylmethyl) benzoyl (AMB), 2-azidobenzoyl (AZMB), diphenylcarbamoyl, phenylacetyl, etc. A protective group of may be introduced.

In the third aspect of the present invention, B ² is preferably cytosine, thymine, adenine, guanine or a derivative thereof, or a group in which a protective group is introduced.

In the above formula (5), R ³ represents a protecting group.
In this specification, the protective group is a protective group (support) for solid phase reaction when the dimer production method is solid phase reaction, and various protections for liquid phase reaction when liquid phase reaction is used. The group can be mentioned.

  In the present specification, examples of the protective group for liquid phase reaction include an alkyl group, an alkenyl group, an acyl group, an optionally substituted silyl group, an alkoxyacyl group, and an aryloxyacyl group. .

  In the present specification, examples of the “alkenyl group” include, but are not limited to, vinyl, allyl, 1-propenyl, isopropenyl, 2-methyl-1-propenyl, 2-methylallyl, 2-butenyl and the like. it can.

In the present specification, examples of the “acyl group” include, but are not limited to, C _1-6 alkyl-carbonyl (eg, methylcarbonyl, ethylcarbonyl, etc.), C _6-10 aryl-carbonyl (eg, benzoyl), and the like. It is done.

  In the present specification, examples of the “silyl group optionally having a substituent” include, but are not limited to, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, and the like.

In the present specification, the “alkoxyacyl group” is not limited, but includes C _1-6 alkoxy-C _1-6 alkyl-carbonyl (eg, methoxymethylcarbonyl, ethoxyethylcarbonyl, etc.), C _1-6 alkoxy. _-C6-10 aryl-carbonyl (for example, methoxybenzoyl) and the like.

In the present specification, the “aryloxyacyl group” is not limited, but includes C _6-10 aryloxy-C _1-6 alkyl-carbonyl (eg, phenoxyacetyl, phenoxyethylcarbonyl, etc.), C _6-10. Aryloxy-C _6-10 aryl-carbonyl (for example, phenoxybenzoyl) and the like can be mentioned.

  In the present specification, examples of the carrier for solid-phase reaction include solid glass such as aminoalkylated porous glass (CPG) with controlled pore size and aminoalkylated highly cross-linkable polystyrene (HCP). Examples of known polymer carriers used in the phase method that are not swellable as much as possible and that can easily remove excess reagents by washing can be mentioned without particular limitation.

  The hydroxyl group of the carrier and the nucleoside may be bonded via a linker such as succinate, oxalate, or phthalate.

In the third aspect of the present invention, R ³ is preferably CPG or HCP or a carrier having a linker when the method for producing a dimer is a solid-phase reaction. In the case of a liquid phase reaction, a benzoyl group or a phenoxyacetyl group is preferable.

In the above formula (5), R ⁴ is a hydrogen atom, an alkoxy group, an alkenyloxy group, an acyloxy group, or a trialkylsilyl group.

In the third aspect of the present invention, R ⁴ is preferably a hydrogen atom, a methoxy group, or an acyloxy group.

  In the third aspect of the present invention, when the reaction is a solid phase reaction, the boranophosphate monomer represented by the above formula (1) is used in an excess amount relative to the nucleoside derivative represented by the above formula (5). preferable. For example, the boranophosphate monomer represented by the above formula (1) is preferably used in an amount of 1 mol to 50 mol, more preferably 10 mol to 40 mol based on 1 mol of the nucleoside derivative represented by the above formula (5). Preferably, it is still more preferable to use 20 mol-30 mol.

  In the third aspect of the present invention, when the reaction is a liquid phase reaction, the boranophosphate monomer represented by the above formula (1) is represented by the above formula (5) in order to allow the condensation reaction to proceed quantitatively. It is preferably used in an amount of 1 mol to 3 mol, more preferably 1.2 mol to 1.5 mol, per 1 mol of the nucleoside derivative.

  In the third aspect of the present invention, when the boranophosphate monomer represented by the above formula (1) is reacted with the nucleoside derivative represented by the above formula (5), a strong base that does not cause β-elimination of the cyanoethyl group is obtained. Used.

  In the third aspect of the present invention, the explanation of the strong base is the same as in the second aspect of the present invention.

  In the third aspect of the present invention, a strong base that does not cause β-elimination of the cyanoethyl group is represented by the above formula (1) in order to complete the reaction regardless of whether the reaction is in a liquid phase or a solid phase. It is preferable to use an excess amount with respect to the boranophosphate monomer. For example, it is preferably used in an amount of 5 mol or more, more preferably 7 to 20 mol, per 1 mol of the boranophosphate monomer represented by the above formula (1).

  In the third aspect of the present invention, when the boranophosphate monomer represented by the above formula (1) is reacted with the nucleoside derivative represented by the above formula (5), a predetermined condensing agent is used.

  In the third aspect of the present invention, the description of the predetermined condensing agent is the same as that described in the second aspect of the present invention.

In the third aspect of the present invention, the condensing agent used is represented by the above formula (1) in order to allow the condensation reaction to proceed quantitatively regardless of whether the reaction is a liquid phase or a solid phase. It is preferable to use 1 mol to 20 mol, more preferably 1.5 mol to 10 mol, still more preferably 2 mol to 3 mol, relative to 1 mol of boranophosphate monomer.
Also in the third aspect of the present invention, as in the second aspect of the present invention, when the condensing agent is a compound represented by the above formula (4a), the strong base is the above-mentioned formula ( It is preferable to use a naphthalene derivative represented by 7a).

  In the third aspect of the present invention, when the condensing agent is a compound represented by the above formula (4b), a naphthalene derivative represented by the above formula (7a) having a stronger basicity is used as the strong base. Alternatively, a pyridine derivative represented by the above formula (7b) which is slightly weak in basicity may be used. From the viewpoint of avoiding the reaction between the anionized nucleoside derivative and the condensing agent, it is preferable to use a pyridine derivative represented by the above formula (7b) having a slightly weak basicity.

  In the third embodiment of the present invention, typically, a condensing agent and a strong base are added to a solution of the boranophosphate monomer represented by the above formula (1) and the nucleoside derivative represented by the above formula (5) and stirred. Thus, the dimer represented by the above formula (6) is obtained.

  In the third aspect of the present invention, the solvent is preferably a solvent in which the boranophosphate monomer represented by the above formula (1), the nucleoside derivative represented by the above formula (5) and the condensing agent can be dissolved. For example, acetonitrile, tetrahydrofuran, dichloromethane, N, N-dimethylformamide can be mentioned, and acetonitrile or N, N-dimethylformamide is preferable.

  In the third aspect of the present invention, the reaction temperature is preferably −30 ° C. to 50 ° C., more preferably 0 ° C. to 40 ° C., and still more preferably 15 ° C. to 30 ° C. If desired, the reaction may be allowed to proceed while blocking light.

  In the third aspect of the present invention, the pressure is preferably atmospheric pressure. The atmosphere is preferably an inert gas atmosphere such as argon.

According to a fourth aspect of the present invention, there is provided one aspect of a method for producing an oligonucleotide derivative using a boranophosphate monomer according to the first aspect of the present invention. That is, after reacting an oligonucleotide derivative represented by the following formula (8) and a deprotection reagent to remove R ¹ , a strong base that does not cause β elimination of the cyanoethyl group in the above formula (8), And the manufacturing method of the oligonucleotide derivative shown by following formula (9) characterized by making the boranophosphate monomer shown by following formula (5) react with presence of a predetermined condensing agent is provided.

［式中、Ｂ¹、Ｂ²、Ｒ¹、Ｒ²、Ｒ³及びＲ⁴は、上記の意味を有する。］

[Wherein B ¹ , B ² , R ¹ , R ² , R ³ and R ⁴ have the above-mentioned meanings. ]

  By repeating the deprotection reaction and the condensation reaction according to the fourth aspect of the present invention, the oligonucleotide chain can be extended to an arbitrary chain length. Moreover, the oligomer which has arbitrary base sequences is compoundable by using the boranophosphate monomer (1) which has arbitrary nucleobases.

In the fourth aspect of the present invention, in the above formulas, B ¹ , B ² , R ¹ , R ² , R ³ and R ⁴ are the same as those described in the first to third aspects of the present invention. is there.

In the fourth embodiment of the present invention, first, the oligonucleotide derivative represented by the above formula (8) is reacted with a deprotecting reagent to desorb R ¹ .

Here, in said formula (8), n shows an integer greater than or equal to 1, It is preferable that it is 1-100, It is more preferable that it is 10-70, It is still more preferable that it is 10-30.
In the fourth aspect of the present invention, preferred examples of the deprotection reagent include trifluoroacetic acid, trichloroacetic acid, and dichloroacetic acid.

  In the fourth aspect of the present invention, the deprotection reagent is preferably used in an amount of 2 mol to 100 mol with respect to 1 mol of the oligonucleotide derivative represented by the above formula (8) in order to complete the reaction. It is more preferable to use 50 mol, and even more preferable to use 10 mol to 20 mol.

In the fourth aspect of the present invention, since the trityl cation generated by the deprotection reaction may act on the boranophosphate to cause a decomposition reaction, the deprotection reaction is present with a reagent (trityl cation scavenger) that reduces the trityl cation. It is preferable to carry out below. Examples of such a reagent include triethylsilane and BH ₃ · pyridine complex.

  In the fourth aspect of the present invention, the reagent for reducing the trityl cation is preferably used in a large excess relative to the oligonucleotide derivative represented by the above formula (8). For example, it is preferable to use 2 mol to 100 mol, more preferably 5 mol to 50 mol, still more preferably 10 mol to 20 mol, with respect to 1 mol of the oligonucleotide derivative represented by the above formula (8). preferable.

In the fourth embodiment of the present invention, after reacting the oligonucleotide derivative represented by the above formula (8) with a deprotecting reagent to remove R ¹ , the same reaction as in the condensation reaction according to the third embodiment of the present invention is performed. A condensation reaction is performed to produce an oligonucleotide derivative represented by the above formula (9).

  In the fourth aspect of the present invention, when the method for producing an oligonucleotide derivative is a solid phase reaction, the amount of the boranophosphate monomer represented by the above formula (1) used is quantitatively advanced through the condensation reaction. Therefore, it is preferable to use 1 mol to 50 mol, more preferably 10 mol to 40 mol, and further preferably 20 mol to 30 mol with respect to 1 mol of the oligonucleotide derivative represented by the above formula (8). It is preferable.

  In the fourth aspect of the present invention, when the method for producing an oligonucleotide derivative is a liquid phase reaction, the amount of the boranophosphate monomer represented by the above formula (1) used is quantitatively advanced through the condensation reaction. Therefore, it is preferably used in an amount of 1 mol to 3 mol, more preferably 1.2 mol to 1.5 mol, based on 1 mol of the oligonucleotide derivative represented by the above formula (8).

The strong base used in the fourth aspect of the present invention is the same as described in the second aspect of the present invention.
In the fourth aspect of the present invention, the strong base is a boranophosphate monomer represented by the above formula (1) in order to allow the reaction to proceed quantitatively regardless of whether the reaction is a liquid phase or a solid phase. It is preferable to use an excessive amount with respect to. For example, it is preferably used in an amount of 5 mol or more, more preferably 7 to 20 mol, per 1 mol of the boranophosphate monomer represented by the above formula (1).

The condensing agent used in the fourth aspect of the present invention is the same as described in the third aspect of the present invention.
In the fourth aspect of the present invention, the condensing agent is a boranophosphate monomer represented by the above formula (1) in order to allow the reaction to proceed quantitatively regardless of whether the reaction is a liquid phase or a solid phase. It is preferable to use 1 mol to 20 mol, more preferably 1.5 mol to 10 mol, still more preferably 2 mol to 3 mol, relative to 1 mol.

  In the fourth aspect of the present invention, typically, a deprotection reagent is added to a solution of the oligonucleotide derivative represented by the above formula (8) to obtain a reaction product. Next, the nucleoside derivative represented by the above formula (5) is added to the reaction product solution, a strong base and a condensing agent are added, and the mixture is stirred to obtain the oligonucleotide derivative (9).

  In the fourth aspect of the present invention, the solvent is preferably a solvent capable of dissolving the oligonucleotide derivative represented by the above formula (8) and the condensing agent. For example, acetonitrile, tetrahydrofuran, dichloromethane, N, N-dimethylformamide can be mentioned, and acetonitrile or N, N-dimethylformamide is preferable.

  In the fourth embodiment of the present invention, the reaction temperature is preferably −30 ° C. to 50 ° C., more preferably 0 ° C. to 40 ° C., and still more preferably 15 ° C. to 30 ° C. If desired, the reaction may be allowed to proceed while blocking light.

  In the fourth aspect of the present invention, the pressure is preferably atmospheric pressure. The atmosphere is preferably an inert gas atmosphere such as argon.

When an oligonucleotide derivative having an arbitrary chain length is obtained according to the fourth aspect of the present invention, first, R ¹ at the 5′-terminal is removed by the deprotection reaction described in the fourth aspect of the present invention.

Next, from the obtained oligomer having a free hydroxyl group at the 5′-position, a 2-cyanoethyl group, which is a protecting group on the phosphorus atom, was converted into DBU (1,8-diazabicyclo [5.4.0] -undec-7-7. E). Next, by treatment with ammonia, the protecting group of the nucleobase and the carrier represented by R ⁴ are removed, and a boranophosphate oligomer is obtained.

Hereinafter, the present invention will be described based on examples. However, the present invention is not limited to the following examples.
As the solvent used in the reaction, a commercially available product was distilled and then dried with sodium or molecular sieve 4A. Other reagents used were commercially available.

The following types of analyzers were used.
¹ H-nuclear magnetic resonance spectrum ( ¹ H-NMR): Varian Mercury 300 (300 MHz)
³¹ P-nuclear magnetic resonance spectrum ( ³¹ P-NMR): Varian Mercury 300 (121.5 MHz)
Silica Gel 60N from KANTO CHEMICAL was used as the silica gel packed in the column chromatography.

Reference example 1
Preparation of tris-2-cyanoethyl boranophosphate

To a solution of tris-2-cyanoethyl phosphite (24.7 g, 102.4 mmol) in anhydrous THF (103 mL) was added a 0.93 M solution of BH ₃ .THF / THF (121.5 mL, 113 mmol) under an argon atmosphere. Added dropwise at 0 ° C. The reaction mixture was stirred for 1 hour at room temperature and the reaction mixture was dried under reduced pressure. The residue was purified on a silica gel column (150 g). Column chromatography was performed using ethyl acetate. The portion containing tris-2-cyanoethylboranophosphate was collected and dried under reduced pressure to give the title compound as a colorless oil (22.9 g, 88%).
¹ H NMR (CDCl ₃ ) δ 4.31 (6H, m), 2.79 (6H, t, J = 5.7 Hz), 1-0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) [delta] 119.8-116.9 (m).

Reference example 2
Preparation of triethylammonium bis-2-cyanoethylboranophosphate

To an anhydrous CH ₂ Cl ₂ solution (89.4 mL) of tris-2-cyanoethylboranophosphate (22.8 g, 89.4 mmol) obtained in Reference Example 1 was added Et ₃ N (124 mL, 894 mmol) under an argon atmosphere. Was added dropwise at room temperature, heated to reflux. After stirring for 1 hour, the reaction mixture was cooled to room temperature. The residue was dried under reduced pressure and azeotropy repeated with anhydrous toluene and CHCl ₃ to give the title compound as a colorless oil (27.1 g, quantitative).

¹ H NMR (CDCl ₃ ) δ 12.58 (1H, bs), 4.14-4.04 (4H, m), 3.07 (6H, q, J = 7.3 Hz), 2.74-2. 66 (4H, m) 1.34 (9H, t, J = 7.2 Hz), 1-0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 96.64 (q, J _PB = 127 .37Hz)

［式中、「ＣＥ」はシアノエチル基を表す。］ The reaction scheme of Reference Example 1 to Reference Example 2 is shown below.

[Wherein “CE” represents a cyanoethyl group. ]

５'−Ｏ−ジメトキシトリチル−Ｎ⁶ベンゾイル−２'−デオキシアデノシン（１．３１ｇ，２．００ｍｍｏｌ）及び参考例２で得られたトリエチルアンモニウム ビス−２−シアノエチルボラノホスフェート（０．７３０ｇ，２．４０ｍｍｏｌ）を、無水ピリジンによる共沸、次いで無水トルエンによる共沸を繰り返すことで乾燥させた後、無水アセトニトリル（２０．０ｍＬ）に溶解させた。溶液に、引き続いて、２，６−ルチジン（２．３０ｍＬ，２０．０ｍｍｏｌ）、ＰｙＮＴＰ（トリス（ピロリジン−１−イル）３−ニトロ−１，２，４−トリアゾール−１−イル ホスホニウム ヘキサフルオロホスフェート）（２．４０ｇ，４．８０ｍｍｏｌ）を加えた。室温で１時間攪拌した後、反応混合物をＣＨＣｌ₃（５０ｍＬ）で希釈した。反応混合物を飽和ＮａＨＣＯ₃（３ｘ５０ｍＬ）で洗浄し、水層をＣＨＣｌ₃（２ｘ５０ｍＬ）で逆抽出した。有機層及び洗浄液を集めて、Ｎａ₂ＳＯ₄上で乾燥させ、濾過し、減圧下で乾燥させた。残渣を無水トルエンで共沸を繰り返し、シリカゲルカラムで精製した（１００ｇ）。カラムクロマトグラフィーは、酢酸エチルを用いて行った。ビス−２−シアノエチル ５'−Ｏ−ジメトキシトリチル−Ｎ⁶−ベンゾイル−２'−デオキシアデノシン−３'−イル ボラノホスフェートを含む部分を集めて、減圧下で乾燥させ、表題化合物を無色泡状物として得た（１．２９ｇ，７７％）。 Reference example 3
Bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate

5′-O-dimethoxytrityl-N ⁶ benzoyl-2′-deoxyadenosine (1.31 g, 2.00 mmol) and triethylammonium bis-2-cyanoethylboranophosphate (0.730 g, 2) obtained in Reference Example 2 .40 mmol) was dried by repeating azeotropy with anhydrous pyridine and then azeotropy with anhydrous toluene, and then dissolved in anhydrous acetonitrile (20.0 mL). To the solution, subsequently, 2,6-lutidine (2.30 mL, 20.0 mmol), PyNTP (tris (pyrrolidin-1-yl) 3-nitro-1,2,4-triazol-1-yl phosphonium hexafluorophosphate ) (2.40 g, 4.80 mmol) was added. After stirring at room temperature for 1 hour, the reaction mixture was diluted with CHCl ₃ (50 mL). The reaction mixture was washed with saturated NaHCO ₃ ( ₃ × 50 mL) and the aqueous layer was back extracted with CHCl ₃ (2 × 50 mL). The organic layer and washings were collected, dried over Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was repeatedly azeotroped with anhydrous toluene and purified on a silica gel column (100 g). Column chromatography was performed using ethyl acetate. The portion containing bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate was collected and dried under reduced pressure to give the title compound as a colorless foam Obtained as a product (1.29 g, 77%).

¹ H NMR (CDCl ₃ ) δ 9.03 (1H, bs), 8.72 (1H, s), 8.17 (1H, s), 8.07-7.18 (14H, m), 6.81 (4H, d, J = 8.1 Hz), 6.52 (1H, t, J = 7.1 Hz), 5.25 (1H, m), 4.38 (1H, m), 4.29-4 .17 (4H, m), 3.78 (6H, s), 3.44 (2H, m), 2.82-2.57 (6H, m), 1-0 (3H, bq, BH ₃ ) ³¹ P NMR (CDCl ₃ ) δ 120.0-116.8 (m).

Reference example 4
Bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ⁴ -benzoyl-2′-deoxycytidin-3′-yl boranophosphate

The same procedure as in Reference Example 3 was performed. However, 5′-O-dimethoxytrityl-N ⁴ -benzoyl-2′-deoxycytidine was used in place of 5′-O-dimethoxytrityl-N ⁶ benzoyl-2 ”-deoxyadenosine. Obtained as a crude product (crude product).

¹ H NMR (CDCl ₃ ) δ 8.66 (1H, s), 8.19 (1H, d, J = 7.5 Hz), 7.94-7.18 (15H, m), 6.86 (4H, d, J = 4.2 Hz), 6.34 (1H, t, J = 6.5 Hz), 5.16-5.06 (1H, m), 4.35-4.29 (1H, m), 4.25-4.13 (2H, m), 3.78 (6H, s), 3.51-3.47 (2H, m), 2.94-2.37 (4H, m), 1- 0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 119.8-116.4 (m).
Reference Example 5
Bis-2-cyanoethyl 5′-O-dimethoxytrityl-O ⁶ -diphenylcarbamoyl-N ² -phenylacetyl-2′-deoxyguanosine-3′-yl boranophosphate

The same procedure as in Reference Example 3 was performed. However, 5′-O-dimethoxytrityl-O ⁶ diphenylcarbamoyl-N ² phenylacetyl-2′-deoxyguanosine was used in place of 5′-O-dimethoxytrityl-N ⁶ benzoyl-2′-deoxyadenosine. The title compound was obtained as a pale yellow foam (yield 90%).

¹ H NMR (CDCl ₃ ) δ 8.10 (1H, s), 7.91 (1H, s), 7.46-7.14 (24H, m), 6.78 (4H, d, J = 9. 0 Hz), 6.41 (1 H, t, J = 7.5 Hz), 5.21 (1 H, m), 4.32 (1 H, m), 4.22 to 4.08 (4 H, m), 3 .98 (2H, s, CH ₂ of Pa), 3.74 (6H, s), 3.46-3.31 (2H, m), 2.99-2.50 (6H, m), 1- 0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 119.02-115.61 (m).

Reference Example 6
Bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ³ -benzoylthymidine-3′-ylboranophosphate

The same procedure as in Reference Example 3 was performed. However, 5′-O-dimethoxytrityl-N ³ benzoylthymidine was used in place of 5′-O-dimethoxytrityl-N ⁶ benzoyl-2′-deoxyadenosine. The title compound was obtained as a colorless foam (yield 98%).

¹ H NMR (CDCl ₃ ) δ 7.95, 7.93 (2H, m), 7.72 (1H, s), 7.68-7.21 (12H, m), 6.87 (4H, d, J = 9.0 Hz), 6.45, 6.42 (1H, dd, J _{1 ′, 2 ′} / J _{1 ′, 2 ″} = 5.7 Hz and 8.7 Hz), 5.21 (1H, t, J = 7.5 Hz), 4.29-4.11 (5H, m), 3.80 (6H, s), 3.48-3.44 (2H, m, 5'-H and 5 "-H ), 2.82-2.46 (6H, m) , 1.46 (3H, s, 5-CH 3), 1-0 (3H, bq, BH 3); 31 P NMR (CDCl 3) δ118. 79-115.48 (m).

Example 1
Triethylammonium 2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate

Bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate (1.29 g, 1.53 mmol) obtained in Reference Example 3 in anhydrous CH ₂ To a Cl ₂ solution (2 mL), Et ₃ N (2.8 mL, 20 mmol) was added dropwise at room temperature under an argon atmosphere. The reaction mixture was stirred for 1 hour at room temperature and the reaction mixture was dried under reduced pressure. The residue was purified with a silica gel column (40). Column chromatography elution was performed with CH ₂ Cl ₂ containing 0.5% Et ₃ N and a gradient was provided with methanol (0-4%). The portion containing triethylammonium 2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate was collected and dried. Excess Et ₃ N was removed by azeotroping several times with anhydrous toluene to give the title compound as a colorless foam (698.6 mg, 51%).

¹ H NMR (CDCl ₃ ) δ 9.18 (1H, bs), 8.71 (1H, s, 8-H), 8.20 (1H, s), 8.02, 8.05 (2H, 2m) 7.65-7.15 (12H, m), 6.79 (4H, d, J = 9.0 Hz), 6.58 (1H, m), 5.14 (1H, m), 4.43 (1H, m), 4.08-3.91 (2H, m), 3.77 (6H, s), 3.41 (2H, m), 3.04 (6H, q, J = 7.2 Hz) ), 2.96-2.46 (4H, m), 1.31 (9H, t, J = 7.35 Hz), 1-0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 98 .55-93.80 (m).

Example 2
Triethylammonium 2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxycytidin-3′-yl boranophosphate

The same procedure as in Example 1 was performed. However, in place of the bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate obtained in Reference Example 3, the bis obtained in Reference Example 4 was used. 2-Cyanoethyl 5′-O-dimethoxytrityl-N ⁴ -benzoyl-2′-deoxycytidin-3′-yl boranophosphate was used. The title compound was obtained as a colorless foam (yield 56%, 2 steps).

¹ H NMR (CDCl ₃ ) δ 12.50 (1H, bs), 8.70 (1H, bs), 8.24 (1H, q, J = 7.0 Hz), 7.90, 7.87 (2H, 2m), 7.65-7.11 (13H, m), 6.94-6.79 (4H, m), 6.36-6.26 (1H, m), 5.04 (1H, m) , 4.41-4.31 (1H, m), 4.00-3.88 (2H, m), 3.79 (6H, s), 3.52-3.44 (2H, m), 3 .04 (6H, q, J = 7.0 Hz), 2.90-2.78 (1H, m), 2.72-2.47 (2H, m), 2.39-2.24 (1H, m), 1.31 (9 H, t, J = 7.5 Hz, CH ₃ of Et ₃ NH ⁺ ), 1-0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 98.93-93 .67 (m) .

Example 3
Triethylammonium 2-cyanoethyl 5′-O-dimethoxytrityl-O ⁶ -diphenylcarbamoyl-N ² -phenylacetyl-2′-deoxyguanosine-3′-yl boranophosphate

The same procedure as in Example 1 was performed. However, in place of the bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate obtained in Reference Example 3, the bis obtained in Reference Example 5 was used. 2-Cyanoethyl 5′-O-dimethoxytrityl-O ⁶ -diphenylcarbamoyl-N ² -phenylacetyl-2′-deoxyguanosine-3′-yl boranophosphate was used. The title compound was obtained as a colorless foam (yield 90%).

¹ H NMR (CDCl ₃ ) δ 12.52 (1 H, bs), 8.08 (1 H, bs), 8.02 (1 H, d, J = 9.0 Hz), 7.51 to 7.09 (24 H, m), 6.75 (4H, d, J = 9.0 Hz), 6.45, 6.41 (1H, dd, J _{1 ′, 2 ′} / J _{1 ′, 2 ″} = 6.3 Hz and 12. 9 Hz), 5.10 (1 H, m), 4.37 (1 H, d, J = 15.0 Hz), 4.07-3.86 (4 H, m), 3.69 (6 H, s), 3 .38-3.28 (2H, m), 2.98 (6H, q, J = 7.3 Hz), 2.87-2.44 (4H, m), 1.26 (9H, t, J = 7.4 Hz), 1-0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 99.09-93.23 (m).

Example 4
Triethylammonium 2-cyanoethyl 5′-O-dimethoxytrityl-N ³ -benzoylthymidin-3′-yl boranophosphate

The same procedure as in Example 1 was performed. However, in place of the bis-2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxyadenosine-3′-yl boranophosphate obtained in Reference Example 3, the bis obtained in Reference Example 6 was used. 2-Cyanoethyl 5′-O-dimethoxytrityl-N ³ -benzoylthymidin-3′-yl boranophosphate was used. The title compound was obtained as a colorless foam (74% yield).

¹ H NMR (CDCl ₃ ) δ 12.42 (1H, bs), 7.97-7.20 (16H, m), 6.84 (4H, d, J = 9.0 Hz), 6.46, 6. 43 (1H, dd, J _{1 ′, 2 ′} / J _{1 ′, 2 ″} = 5.7 Hz and 8.1 Hz), 5.21-5.10 (1H, m, 3′-H), 4.29 (1H, d, J = 12.0 Hz, 4′-H), 4.03-3.86 (2H, m), 3.79 (6H, s), 3.45-3.42 (2H, m ), 2.96 (6H, q, J = 7.2 Hz), 2.68-2.39 (4H, m), 1.39, 1.35 (3H, 2s), 1.25 (9H, t , J = 7.2 Hz), 1-0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 98.31-93.59 (m).

Example 5
2-cyanoethyl 5′-O-dimethoxytrityl-N ⁴ -benzoyl-2′-deoxycytidin-3′-yl 3′-benzoyl-N ³ -benzoylthymidin-5′-yl boranophosphate

3′-O-benzoyl-N ³ -benzoylthymidine (45.0 mg, 0.100 mmol) and triethylammonium 2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′- obtained in Example 2 Deoxycytidin-3′-yl boranophosphate (103.9 mg, 0.120 mol) was dried by repeating azeotropy with anhydrous pyridine and then azeotropy with anhydrous toluene, and then anhydrous CH ₃ CN (1.00 mL). ). Subsequently, 2,6-lutidine (116 μL, 1.00 mmol) and PyNTP (120 mg, 0.240 mmol) were added to the solution. After stirring at room temperature for 1 hour, the reaction mixture was diluted with CHCl ₃ (10 mL). The reaction mixture was washed with saturated NaHCO ₃ ( ₃ × 10 mL) and the aqueous layer was back extracted with CHCl ₃ (2 × 10 mL). The organic layer and washings were collected, dried over Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified on a silica gel column (10 g). Elution of the column chromatography was carried out with CH ₂ Cl _2, it gave a gradient methanol (0-1.5%).

A moiety containing 2-cyanoethyl 5′-O-dimethoxytrityl-N ⁴ -benzoyl-2′-deoxycytidin-3′-yl 3′-O-benzoyl-N ³ -benzoylthymidin-5′-yl boranophosphate Collected and dried under reduced pressure to give the title compound as a colorless foam (113.4 mg, 95%).

¹ H NMR (CDCl ₃ ) δ 8.20-8.13 (1H, m), 8.06-7.16 (26H, m), 6.91-6.82 (4H, m), 6.54- 6.46 (1H, m), 6.34 (1 H, t, J = 6.0 Hz), 5.55-5.40 (1 H, m), 5.23-5.14 (1 H, m), 4.54-4.09 (6H, m), 3.93-3.64 (6H, m), 3.56-3.46 (2H, m), 3.06-2.35 (6H, m) ), 2.01, 1.99 (3H, 2s), 1-0 (3H, bq, BH ₃ ); ³¹ P NMR (CDCl ₃ ) δ 121.21-1117.55 (m).

［式中、「ＣＥ」はシアノエチル基を表す。］ The reaction scheme of Reference Example 3 to Example 5 is shown below.

[Wherein “CE” represents a cyanoethyl group. ]

Example 6
N ⁴ -benzoyl-2′-deoxycytidin-3′-yl 3′-benzoyl-N ³ -benzoylthymidin-5′-yl boranophosphate

A solid support (29.2 mg, 0.5 μmol) supported on CPG via a succinyl linker on the 3′-hydroxyl group of 5′-O-dimethoxytrityl-N ³ -benzoylthymidine was CH ₂ Cl ₂ -Et ₃ SiH. (3: 1, v / v) with 3% DCA (dichloroacetic acid) solution for 5 seconds and 3 times each, and then each 0.5 ml × 3 times with anhydrous CH ₂ Cl ₂ solution and anhydrous CH ₃ CN solution, It was dried under reduced pressure. After 10 minutes, MNTP (1,3-dimethyl-2-pyrrolidin-1-yl-2- (3-nitro-1,2,4-triazol-1-yl) -1,3,2-diazaphosphoridine (Hexafluorophosphate) (8.7 mg, 10 μmol), triethylammonium 2-cyanoethyl 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-deoxycytidin-3′-yl borano obtained in Example 2 Phosphate (8.9 mg, 20 μmol) was added and dried again under reduced pressure. 100 μl of 0.5 M 2,6-lutidine / CH ₃ CN solution was added and stirred for 3 minutes (synthesis of dimer).

Thereafter, it was washed with an anhydrous CH ₃ CN solution and an anhydrous CH ₂ Cl ₂ solution, and then washed with a 3% DCA solution of CH ₂ Cl ₂ -Et ₃ SiH (1: 1, v / v) for 5 seconds and 3 times each (dimethoxy). Removal of trityl group).
Next, it was washed with an anhydrous CH ₂ Cl ₂ solution and an anhydrous THF solution, an N-methylimidazole / acetic anhydride / THF solution (2: 1: 7, v / v / v) solution was added, and the mixture was stirred for 30 seconds. Thereafter, the mixture was washed with anhydrous THF solution and anhydrous CH ₃ CN solution, 0.2M DBU / CH ₃ CN solution was added, and the mixture was stirred for 5 minutes (removal of 2-cyanoethyl group).

After washing with anhydrous CH ₃ CN solution and drying under reduced pressure, 0.5 ml of ammonia / methanol solution is added and sealed. After stirring at room temperature for 12 hours, suction filtration was performed to separate from CPG, dried under reduced pressure, and diluted with water (3 mL) (removal of carrier).
The reaction mixture was washed with diethyl ether (5 × 3 mL) and the ether layer was back extracted with water (1 × 3 mL). The aqueous layer and washings were collected and dried under reduced pressure (product purification).
The residue was analyzed by reverse phase HPLC. (Linear gradient with 0-15% acetonitrile in 0.1 M ammonium acetate buffer (pH 7.0, 45 minutes, 50 ° C., flow rate 1 ml / min) The yield calculated from the integrated value of the peak of HPLC is 95%.

Example 7
Boranophosphate tetramer and 12 Synthesis of mer (1) 4 Synthesis 5'-O- dimethoxytrityl -N amount body ³ - a solid was carried on CPG through a succinyl linker to the 3'-hydroxyl group of benzoyl thymidine Boranophosphate tetramer d (C _PB A _PB G _PB T) was synthesized on the phase support (0.5 μmol) by the synthesis cycle shown in Table 1 below.

Table 1 The crude purified tetramer deprotected in Step 10 was subjected to suction filtration, separated from CPG, dried under reduced pressure, and diluted with water (3 mL). The reaction mixture was washed with diethyl ether (5 × 3 mL) and the ether layer was back extracted with water (1 × 3 mL). The aqueous layer and washings were collected and dried under reduced pressure. After separation and purification by anion exchange HPLC, desalting was carried out with a _C18 reverse phase column cartridge. As a result, the target boranophosphate tetramer; d (C _PB A _PB G _PB T )

(2) Synthesis of 12-mer Table 1 shows a solid phase carrier (0.5 μmol) supported on CPG via a succinyl linker on the 3′-hydroxyl group of 5′-O-dimethoxytrityl-N3-benzoylthymidine. Boranophosphate 12-mer d (C _PB A _PB G _PB T) ₃ was synthesized by the indicated synthesis cycle.
The crudely purified 12-mer deprotected in Table 1-Step 10 was subjected to suction filtration, separated from CPG, dried under reduced pressure, and diluted with water (3 mL). The reaction mixture was washed with diethyl ether (5 × 3 mL) and the ether layer was back extracted with water (1 × 3 mL). The aqueous layer and washings were collected and dried under reduced pressure. When purification was performed using 15% PAGE / 7M Urea (1 mm thickness, 40 × 20 cm), the desired boranophosphate 12-mer; d (C _PB A _PB G _PB T) ₃ was obtained with an isolation yield of 16%. Could get.

The synthesis scheme in Example 7 is shown below.

In Table 1 and the synthesis scheme, the structural formulas of “monomer”, “MNTP”, and “DMAN” are as follows.

  According to the present invention, boranophosphate oligomers are provided. The boranophosphate oligomer obtained by the present invention is useful as an antisense molecule and can be used as a reagent or a medicine.

Claims (1)

A boranophosphate monomer represented by the following formula (1).

[ Wherein B ¹ represents a base selected from thymine, cytosine, uracil, adenine, guanine, 5-methylcytosine, 5-fluorouracil, or 5-hydroxymethylcytosine ;
R ¹ is a dimethoxytrityl group or a monomethoxytrityl group,
R ² represents a hydrogen atom, an alkoxy group, an alkenyloxy group, an acyloxy group, or a trialkylsilyloxy group. ]