JP2010241836A - Phosphonate nucleoside derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new phosphonate and a 5'-H-phosphonate oligonucleotide derivative. <P>SOLUTION: The phosphate is expressed by formula (5) (wherein B is a base; R<SP>3</SP>is a hydrogen atom, hydroxy group or the like; and R<SP>4</SP>is a carrier). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a phosphitylated nucleoside monomer, a process for producing the same, a novel H-phosphonate oligonucleotide derivative and a phosphite oligonucleotide derivative obtained by using the phosphitylated nucleoside monomer, and a process for producing them.

  The first technique for DNA synthesis, the phosphodiester method, was developed by Khorana et al. In the 1960s, but it was a cumbersome method in which all reactions were performed in the liquid phase and the product had to be isolated at each stage. It was. Khorana, however, synthesized this yeast alanine tRNA gene consisting of 77 bases in 1972 by combining this method with the enzymatic method.

  By the early 1980s, these difficult and tedious liquid phase methods were replaced by much faster solid phase methods and oligonucleotide synthesis was automated. One of the uses of the synthetic oligonucleotide is a pharmaceutical, which uses not a natural DNA but a DNA analog obtained by chemically modifying it. Various DNA analogs have been developed, but only phosphorothioate DNA in which one of the non-bridging oxygen atoms of the phosphodiester bond is substituted with a sulfur atom has been put into practical use. However, phosphorothioate DNA has a strong interaction with proteins and has high cytotoxicity and side effects. Therefore, development of a new DNA analog to replace this is required.

  The most widely used method for the synthesis of DNA analogues today is a reaction devised by Letsinger and developed by Caruthers et al. And is called the phosphoramidite method (phosphorous acid triester method). The scheme is shown below.

（式中、「B^p」とは、保護基が付加された塩基を意味し、「DMTr」とは、ジメトキシトリチル基を指す。）

(In the formula, “B ^p ” means a base to which a protecting group is added, and “DMTr” means a dimethoxytrityl group.)

  In parallel with the development of the phosphoramidite method, Garegg and Matteucci independently developed the H-phosphonate method (Garegg, PJ et. Al, Chemica Scripta, 1985, 25, 280-282, Froehler, BC et. al, Tetrahedron Lett., 1986, 27, 469-472). The scheme is shown below.

（式中、「DBU」とは、1,8-ジアザビシクロ[5.4.0]-ウンデカ-1-エンを示す。）

(In the formula, “DBU” represents 1,8-diazabicyclo [5.4.0] -undec-1-ene.)

  When the phosphoramidite method is used to synthesize DNA analogs, modification on the phosphorus atom can only be done with group 16 elements oxygen, sulfur, selenium, and borane that forms coordinate bonds. On the other hand, using the H-phosphonate method, a wide range of modifications can be made, and DNA analogs that cannot be synthesized by the phosphoramidite method can be synthesized. This is because the phosphoramidite method modifies via phosphite, which is a phosphite triester, whereas the H-phosphonate method modifies oxidatively via a phosphite diester.

As described above, the H-phosphonate method is extremely useful as a DNA analog synthesis method. However, since various side reactions occur in the synthesis process, the synthesis yield of oligonucleotide is low compared to the phosphoramidite method. Disadvantages have been pointed out. As a side reaction,
1) Since pivaloyl chloride (PivCl) is used as a condensing agent, the amino group at the base site is acylated (Froehler, BC et. Al, Tetrahedron Lett., 1986, 27, 469-472).
2) Since a benzoyl group is used as a protecting group for adenosine, depletion, which is an adenine ring elimination reaction, occurs under acidic conditions (for example, Hayakawa, Y. et. Al, J. Am. Chem. Soc. 1998, 120 , 12395-12401).
3) Since I ₂ -H ₂ O is used in the oxidation reaction, the main chain is partially cleaved (Wada, T. et. Al, Tetrahedron Lett., 1999, 40, 915-918).
4) After deprotection, the nucleophilic attack of the terminal hydroxyl group on the adjacent phosphorus atom causes the cleavage reaction of the oligonucleotide chain (Wada, T. et. Al, Tetrahedron Lett., 1999, 40, 915- 918).

  Wada et al. Developed an unprotected H-phosphonate method that overcomes 1)-3) (Wada, T. et. Al, J. Am. Chem. Soc. 1997, 119, 12710-12721). In this method, first, the monomer unit is synthesized by functionally selectively phosphonylating the 3′-hydroxy group of a nucleoside in which the 5′-hydroxy group is protected with a dimethoxytrityl group (DMTr group) with diphenylphosphonate. Next, 2- (benzotriazol 1-yloxy) -1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphoridinium hexafluorophosphate (BOMP) (see formula below) as a condensing agent ) To selectively condense this monomer unit with a functional group. This is silylated with N, O-bistrimethylsilylacetamide (BSA) and then functional group-selectively with 2- (phenylsulfonyl) -3- (3-nitrophenyl) oxaziridine (PNO) (see formula below) in dichloromethane. Oxidize to obtain the oligonucleotide.

しかしながら、当該方法では、モノマー単位合成が多段階にわたり煩雑であり、また、上記問題4)が解決できないという問題点があった。

However, this method has a problem that the monomer unit synthesis is complicated in many steps and the problem 4) cannot be solved.

  An object of the present invention is to provide a method capable of easily performing monomer unit synthesis and chain extension utilizing the same while overcoming the problems 1) to 4) in the H-phosphonate method.

  As a result of diligent studies to solve the above problems, the present inventors can solve the above problems by using nucleoside 5′-phosphite as a monomer unit and forming a phosphonyl group at the end of the extending chain. The present invention was completed.

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R¹は炭化水素を含む保護基を表し、但し、互いに架橋して置換基を有していてもよい環を形成してもよく、R²は、水素原子、置換基を有していてもよいアシル基、置換基を有していてもよいアルキル基又は置換基を有していてもよいトリアルキルシリル基を表し、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表す。］ That is, in the first aspect of the present invention, a nucleotide monomer unit represented by the following general formula (1) is provided.

[Wherein, B represents a pyrimidine base, a purine base, or a derivative thereof, and R ¹ represents a protecting group containing a hydrocarbon, provided that they form a ring that may be substituted with each other to form a ring. at best, R ² represents a hydrogen atom, an optionally substituted acyl group, an optionally trialkylsilyl group optionally having an optionally substituted alkyl group or a substituted group , R ³ is a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group Represents an alkylthiocarbonyl group, an alkoxythiocarbonyl group, an arylthiocarbonyl group, an aralkylthiocarbonyl group, an aryloxythiocarbonyl group or an aralkyloxythiocarbonyl group, and Z ⁵ represents an electron-withdrawing group. ]

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R¹は炭化水素を含む保護基を表し、但し、互いに架橋して置換基を有していてもよい環を形成してもよく、R²は、水素原子、置換基を有していてもよいアシル基、置換基を有していてもよいアルキル基又は置換基を有していてもよいトリアルキルシリル基を表し、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表す。］活性化剤存在下、下記一般式（２）で示されるヌクレオシドと、

［式中、B、R²及びR³は、前記と同義を表す。］下記一般式（３）で示されるホスフィチル化剤とを

［式中、R¹は前記と同義を表す。A¹及びA²は、それぞれ、互いに独立し、同一または異なって、Ｃ₁〜Ｃ₆アルキル基を表す。］反応させることを特徴とする、ヌクレオチドモノマー単位の製造方法が提供される。 In the second aspect of the present invention, a method for producing a nucleotide monomer unit according to the first aspect of the present invention is provided. That is, in the second aspect of the present invention, there is provided a method for producing a nucleotide monomer unit represented by the following general formula (1),

[Wherein, B represents a pyrimidine base, a purine base, or a derivative thereof, and R ¹ represents a protecting group containing a hydrocarbon, provided that they form a ring that may be substituted with each other to form a ring. at best, R ² represents a hydrogen atom, an optionally substituted acyl group, an optionally trialkylsilyl group optionally having an optionally substituted alkyl group or a substituted group , R ³ is a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group Represents an alkylthiocarbonyl group, an alkoxythiocarbonyl group, an arylthiocarbonyl group, an aralkylthiocarbonyl group, an aryloxythiocarbonyl group or an aralkyloxythiocarbonyl group, and Z ⁵ represents an electron-withdrawing group. A nucleoside represented by the following general formula (2) in the presence of an activator;

[Wherein, B, R ² and R ³ are as defined above. A phosphitylating agent represented by the following general formula (3):

[Wherein, R ¹ has the same meaning as described above. A ¹ and A ² are each independently the same or different and each represents a C ₁ -C ₆ alkyl group. A method for producing a nucleotide monomer unit is provided, which is characterized by reacting.

In the first and second embodiments of the present invention, R ¹ is an alkyl group containing at least a secondary carbon or a tertiary carbon group, an arylalkyl group containing at least a secondary carbon or a tertiary carbon group, at least a secondary carbon or A diarylalkyl group containing a tertiary carbon group or a group containing a condensed hydrocarbon ring having two or more rings is preferred, and R ¹ is a t-butyl group, 2-phenyl-1,1-dimethyl Ethyl group, 2- (1-naphthyl) -1,1-dimethylethyl group, diphenylmethyl group, 9-fluorenylmethyl group, 2-cyano-1,1-dimethylethyl group, 9-fluorenyl-dimethylmethyl group 1-cyclohexyldimethylmethyl group, tribenzylmethyl group, tricyclohexylmethyl group, 1-adamantyl group, di (trifluoromethyl) phenylmethyl group, 2-trimethylsilyl-1,1-dimethylethyl group, Is 2- or triphenylsilyl-1,1-dimethylethyl group, or, it is more preferable to form a ring represented by the following formula together with the phosphorus atom and two oxygen atoms by crosslinking R ¹ each other.

［式中、Phはフェニル基を示す。］

[Wherein, Ph represents a phenyl group. ]

  In the second embodiment of the present invention, the activator is pyridine hydrochloride, pyridinesulfonate, pyridineborate, N- (cyanomethyl) aminesulfonate, N- (cyanomethyl) amineborate, N- ( Cyanomethyl) amine phosphate, imidazole sulfonate, triazole sulfonate, N-hydroxytriazole, benzimidazole sulfonate, imidazole, triazole or tetrazole are preferred.

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R¹は炭化水素 を含む保護基を表し、但し、互いに架橋して置換基を有していてもよい環を形成してもよく、R²は、水素原子、置換基を有していてもよいアシル基、置換基を有していてもよいアルキル基又は置換基を有していてもよいトリアルキルシリル基を表し、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表す。］下記一般式（４ａ）で示される化合物、

［式中、A³は、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基又は置換基を有していてもよいＣ₆〜Ｃ₁₀アリール基を表す。］
下記一般式（４ｂ）で示される化合物、

［式中、A⁴は、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基又は置換基を有していてもよいＣ₆〜Ｃ₁₀アリール基を表す。］
臭化亜鉛(ZnBr₂)、臭化マグネシウム(MgBr₂)、トリフルオロ酢酸、トリクロロ酢酸、ジクロロ酢酸、2-t-ブチルイミノ-2-ジエチルアミノ-1,3-ジメチル-1,3,2-ジアザホスホリナン、(t-ブチルイミノ)トリス(ジメチルアミノ)ホスホラン、トリエチルアミン、ジイソプロピルエチルアミン、及び1,8-ジアザビシクロ[5.4.0]-7-ウンデセンからなる群より選ばれる１種以上の脱保護試薬とを反応させて、R¹を脱離させることを特徴とする、保護基除去方法が提供される。 In the third aspect of the present invention, there is provided a method for removing R ¹ which is a protecting group from the nucleotide monomer unit according to the first aspect of the present invention. That is, in the third aspect of the present invention, a nucleotide monomer unit represented by the following general formula (1):

[In the formula, B represents a pyrimidine base, a purine base or a derivative thereof, and R ¹ represents a protecting group containing a hydrocarbon, provided that they form a ring which may have a substituent by bridging each other. at best, R ² represents a hydrogen atom, an optionally substituted acyl group, an optionally trialkylsilyl group optionally having an optionally substituted alkyl group or a substituted group , R ³ is a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group Represents an alkylthiocarbonyl group, an alkoxythiocarbonyl group, an arylthiocarbonyl group, an aralkylthiocarbonyl group, an aryloxythiocarbonyl group or an aralkyloxythiocarbonyl group, and Z ⁵ represents an electron-withdrawing group. A compound represented by the following general formula (4a):

Wherein, A ³ represents an optionally substituted C ₁ -C ₁₀ alkyl group or an optionally substituted C ₆ -C ₁₀ aryl group. ]
A compound represented by the following general formula (4b):

Wherein, A ⁴ represents an optionally substituted C ₁ -C ₁₀ alkyl group or an optionally substituted C ₆ -C ₁₀ aryl group. ]
Zinc bromide (ZnBr ₂ ), magnesium bromide (MgBr ₂ ), trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, 2-t-butylimino-2-diethylamino-1,3-dimethyl-1,3,2-diaza One or more deprotecting reagents selected from the group consisting of phosphorinan, (t-butylimino) tris (dimethylamino) phosphorane, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo [5.4.0] -7-undecene. Provided is a method for removing a protecting group, characterized by reacting to remove R ¹ .

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表し、R⁴は、担体を表す。］ Moreover, in the 4th aspect of this invention, the phosphonate shown by following General formula (5) is provided.

[Wherein, B represents a pyrimidine base, a purine base, or a derivative thereof, and R ³ represents a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or the following formula: Group

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group , alkylthiocarbonyl group, an alkoxythiocarbonyl group, arylthio group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, Z ⁵ represents a representative.) the electron-withdrawing group, R ⁴ Represents a carrier. ]

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R¹は炭化水素を含む保護基を表し、但し、互いに架橋して置換基を有していてもよい環を形成してもよく、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表し、R⁴は、担体を表す。ｎは１以上の整数を表す。］ In the fifth aspect of the present invention, a 5′-phosphite oligonucleotide derivative represented by the following general formula (7) is provided.

[Wherein, B represents a pyrimidine base, a purine base, or a derivative thereof, and R ¹ represents a protecting group containing a hydrocarbon, provided that they form a ring that may be substituted with each other to form a ring. R ³ may be a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula:

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group , alkylthiocarbonyl group, an alkoxythiocarbonyl group, arylthio group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, Z ⁵ represents a representative.) the electron-withdrawing group, R ⁴ Represents a carrier. n represents an integer of 1 or more. ]

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R¹は炭化水素 を含む保護基を表し、但し、互いに架橋して置換基を有していてもよい環を形成してもよく、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表す。］縮合剤存在下、下記一般式（５）で示されるホスホネートと

［式中、B及びR³は、前記と同義を表す。R⁴は、担体を表す。］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、B、R¹、R³及びR⁴は、前記と同義を表す。］ Further, in one aspect of the sixth aspect of the present invention, one aspect of the nucleotide monomer unit according to the first aspect of the present invention is condensed with the phosphonate provided in the fourth aspect of the present invention. A method for producing a dimer that is one aspect of the five aspects is provided. That is, in one aspect of the sixth aspect of the present invention, a nucleotide monomer unit represented by the following general formula (1a)

[In the formula, B represents a pyrimidine base, a purine base or a derivative thereof, and R ¹ represents a protecting group containing a hydrocarbon, provided that they form a ring which may have a substituent by bridging each other. R ³ may be a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula:

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group Represents an alkylthiocarbonyl group, an alkoxythiocarbonyl group, an arylthiocarbonyl group, an aralkylthiocarbonyl group, an aryloxythiocarbonyl group or an aralkyloxythiocarbonyl group, and Z ⁵ represents an electron-withdrawing group. In the presence of a condensing agent, a phosphonate represented by the following general formula (5)

[Wherein, B and R ³ have the same meaning as described above. R ⁴ represents a carrier. To produce a dimer represented by the following general formula (6).

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

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R¹は炭化水素を含む保護基を表し、但し、互いに架橋して置換基を有していてもよい環を形成してもよく、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表し、R⁴は、担体を表す。ｎは１以上の整数を表す。］
下記一般式（４ａ）で示される化合物、

［式中、A³は、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基又は置換基を有していてもよいＣ₆〜Ｃ₁₀アリール基を表す。］
下記一般式（４ｂ）で示される化合物、

［式中、A⁴は、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基又は置換基を有していてもよいＣ₆〜Ｃ₁₀アリール基を表す。］
臭化亜鉛(ZnBr₂)、臭化マグネシウム(MgBr₂)、トリフルオロ酢酸、トリクロロ酢酸、ジクロロ酢酸、2-t-ブチルイミノ-2-ジエチルアミノ-1,3-ジメチル-1,3,2-ジアザホスホリナン、(t-ブチルイミノ)トリス(ジメチルアミノ)ホスホラン、トリエチルアミン、ジイソプロピルエチルアミン、及び1,8-ジアザビシクロ[5.4.0]-7-ウンデセンからなる群より選ばれる１種以上の脱保護試薬とを反応させて、R¹を脱離させた後、縮合剤存在下、下記一般式（１ａ）で示されるヌクレオチドモノマー単位と

［式中、B、R¹及びR³は、前記と同義を表す。］
を反応させることを特徴とする、下記一般式（８）で示される5'-ホスファイト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、B、R¹、R³、R⁴及びｎは、前記と同義を表す。］ In another aspect of the sixth embodiment of the present invention, the protecting group is removed from the 5′-phosphite oligonucleotide derivative according to the fifth embodiment of the present invention according to the third embodiment of the present invention. There is provided a method for producing a 5′-phosphite oligonucleotide derivative which is condensed with one embodiment of the nucleotide monomer unit according to the first embodiment to extend the chain. That is, in another aspect of the sixth embodiment of the present invention, a 5′-phosphite oligonucleotide derivative represented by the following general formula (7):

[Wherein, B represents a pyrimidine base, a purine base, or a derivative thereof, and R ¹ represents a protecting group containing a hydrocarbon, provided that they form a ring that may be substituted with each other to form a ring. R ³ may be a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula:

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group , alkylthiocarbonyl group, an alkoxythiocarbonyl group, arylthio group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, Z ⁵ represents a representative.) the electron-withdrawing group, R ⁴ Represents a carrier. n represents an integer of 1 or more. ]
A compound represented by the following general formula (4a):

Wherein, A ³ represents an optionally substituted C ₁ -C ₁₀ alkyl group or an optionally substituted C ₆ -C ₁₀ aryl group. ]
A compound represented by the following general formula (4b):

Wherein, A ⁴ represents an optionally substituted C ₁ -C ₁₀ alkyl group or an optionally substituted C ₆ -C ₁₀ aryl group. ]
Zinc bromide (ZnBr ₂ ), magnesium bromide (MgBr ₂ ), trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, 2-t-butylimino-2-diethylamino-1,3-dimethyl-1,3,2-diaza One or more deprotecting reagents selected from the group consisting of phosphorinan, (t-butylimino) tris (dimethylamino) phosphorane, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo [5.4.0] -7-undecene. After reacting to remove R ¹ , in the presence of a condensing agent, a nucleotide monomer unit represented by the following general formula (1a)

[Wherein, B, R ¹ and R ³ have the same meaning as described above. ]
A process for producing a 5′-phosphite oligonucleotide derivative represented by the following general formula (8) is provided.

^{Wherein, B, R 1, R 3} , R 4 and n represent the same meaning. ]

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表し、R⁴は、担体を表す。ｎは１以上の整数を表す。］ In the seventh aspect of the present invention, a 5′-H-phosphonate oligonucleotide derivative represented by the following formula (9) is provided.

[Wherein, B represents a pyrimidine base, a purine base, or a derivative thereof, and R ³ represents a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or the following formula: Group

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group , alkylthiocarbonyl group, an alkoxythiocarbonyl group, arylthio group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, Z ⁵ represents a representative.) the electron-withdrawing group, R ⁴ Represents a carrier. n represents an integer of 1 or more. ]

［式中、Bは、ピリミジン塩基、プリン塩基またはそれらの誘導体を表し、R¹は炭化水素を含む保護基を表し、但し、互いに架橋して置換基を有していてもよい環を形成してもよく、R³は、水素原子、ヒドロキシ基、アルコキシ基、アルケニルオキシ基、アシルオキシ基、トリアルキルシリルオキシ基、または下記式で表される基

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表し、R⁴は、担体を表す。ｎは１以上の整数を表す。］
下記一般式（４ａ）で示される化合物、

［式中、A³は、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基又は置換基を有していてもよいＣ₆〜Ｃ₁₀アリール基を表す。］
下記一般式（４ｂ）で示される化合物、

［式中、A⁴は、置換基を有していてもよいＣ₁〜Ｃ₁₀アルキル基又は置換基を有していてもよいＣ₆〜Ｃ₁₀アリール基を表す。］
臭化亜鉛(ZnBr₂)、臭化マグネシウム(MgBr₂)、トリフルオロ酢酸、トリクロロ酢酸、ジクロロ酢酸、2-t-ブチルイミノ-2-ジエチルアミノ-1,3-ジメチル-1,3,2-ジアザホスホリナン、(t-ブチルイミノ)トリス(ジメチルアミノ)ホスホラン、トリエチルアミン、ジイソプロピルエチルアミン、及び1,8-ジアザビシクロ[5.4.0]-7-ウンデセンからなる群より選ばれる１種以上の脱保護試薬とを反応させて、R¹を脱離させる工程を含む、下記式（９）で示される5'-H-ホスホネートオリゴヌクレオチド誘導体を製造する方法が提供される。

［式中、B、R³、R⁴及びｎは、前記と同義を表す。］ The eighth aspect of the present invention also provides a method for producing the 5′-H-phosphonate oligonucleotide derivative according to the seventh aspect of the present invention. That is, in an eighth aspect of the present invention, a 5′-phosphite oligonucleotide derivative represented by the following general formula (7):

[Wherein, B represents a pyrimidine base, a purine base, or a derivative thereof, and R ¹ represents a protecting group containing a hydrocarbon, provided that they form a ring that may be substituted with each other to form a ring. R ³ may be a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula:

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group , alkylthiocarbonyl group, an alkoxythiocarbonyl group, arylthio group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, Z ⁵ represents a representative.) the electron-withdrawing group, R ⁴ Represents a carrier. n represents an integer of 1 or more. ]
A compound represented by the following general formula (4a):

Wherein, A ³ represents an optionally substituted C ₁ -C ₁₀ alkyl group or an optionally substituted C ₆ -C ₁₀ aryl group. ]
A compound represented by the following general formula (4b):

Wherein, A ⁴ represents an optionally substituted C ₁ -C ₁₀ alkyl group or an optionally substituted C ₆ -C ₁₀ aryl group. ]
Zinc bromide (ZnBr ₂ ), magnesium bromide (MgBr ₂ ), trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, 2-t-butylimino-2-diethylamino-1,3-dimethyl-1,3,2-diaza One or more deprotecting reagents selected from the group consisting of phosphorinan, (t-butylimino) tris (dimethylamino) phosphorane, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo [5.4.0] -7-undecene. by reacting, comprising the step of desorbing R ^1, a method for producing a 5'-H- phosphonate oligonucleotide derivative represented by the following formula (9) is provided.

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

  In the third to eighth embodiments of the present invention, B is preferably cytosine, thymine or a derivative thereof.

  According to the present invention, problems in the conventional H-phosphonate method can be overcome, and not only monomer unit synthesis but also oligonucleotide derivative synthesis can be carried out easily.

［式中、B、R¹、R²及びR³は、上記の意味を表す。］ In the first aspect of the present invention, a nucleotide monomer unit represented by the following general formula (1) is provided.

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

［式中、B、R¹、R²、R³、A¹及びA²は、上記の意味を表す。］ The nucleotide monomer unit represented by the above formula (1) provided in the first aspect of the present invention can be obtained by the method for producing the nucleotide monomer unit provided in the second aspect of the present invention represented by the following reaction formula. it can. That is, it is produced by reacting a nucleoside represented by the following general formula (2) with a phosphitylating agent represented by the following general formula (3) in the presence of an activator.

[Wherein, B, R ¹ , R ² , R ³ , A ¹ and A ² represent the above meanings. ]

  In the present invention, the nucleoside 5′-phosphite represented by the above formula (1) is used as the monomer unit, so that the synthesis of the monomer unit, which is a two-step process in the conventional method and the unprotected H-phosphonate method, is performed in accordance with Can be obtained in one step.

In the method for producing a nucleotide monomer unit according to the present invention, a phosphitylating agent represented by the following general formula (3) is used.

The phosphitylating agent represented by the above formula (3) is preferably one that regioselectively and functionally selectively phosphitylates the 5′-position of the nucleoside represented by the above formula (2). Moreover, when the obtained nucleotide monomer unit represented by the above formula (1) is used for oligonucleotide synthesis, it is preferable that R ^{1 as} a protecting group can be eliminated.

From the above viewpoint, in the above formula (3), R ¹ preferably contains a bulky hydrocarbon group.
For example, a hydrocarbon group containing at least a secondary carbon or tertiary carbon group (for example, an alkyl group containing at least a secondary carbon or a tertiary carbon group, an arylalkyl group, a diarylalkyl group, etc.), or A group containing a condensed hydrocarbon ring having two or more rings can be exemplified.
Or the hydrocarbon group shown by two R < ¹ > may mutually bridge | crosslink, and the ring (for example, 5-6 membered ring) which may have a substituent may be formed.

These hydrocarbon groups may have a substituent. As the substituent, for example, C ₁ -C ₁₀ alkyl group (e.g., methyl, ethyl, propyl, butyl, etc.), C ₆ -C ₁₀ aryl group (e.g., phenyl, etc.), C ₁ -C ₁₀ alkoxy group ( For example, methoxy, ethoxy, propoxy, butoxy, etc.), C ₆ -C ₁₀ aryloxy groups (eg, phenyloxy, naphthyloxy, biphenyloxy, etc.), amino groups, hydroxyl groups, cyano groups, halogen atoms (eg, fluorine, chlorine) , Bromine, iodine) or a silyl group. 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 present invention, R ¹ is t-butyl group, 2-phenyl-1,1-dimethylethyl group, 2- (1-naphthyl) -1,1-dimethylethyl group, diphenylmethyl group, 9-fluorene group. Nylmethyl, 2-cyano-1,1-dimethylethyl, 9-fluorenyl-dimethylmethyl, 1-cyclohexyldimethylmethyl, tribenzylmethyl, tricyclohexylmethyl, 1-adamantyl, di (trifluoro Preferred examples include methyl) phenylmethyl group, 2-trimethylsilyl-1,1-dimethylethyl group, or 2-triphenylsilyl-1,1-dimethylethyl group.

［式中、Phはフェニル基を示す。］ Alternatively, R ¹ may be bridged with each other to form a ring together with a phosphorus atom and two oxygen atoms, and preferred examples of the ring formed in this case include a ring represented by the following formula.

[Wherein, Ph represents a phenyl group. ]

In the above formula (3), A ¹ and A ² are the same or different and are C ₁ -C ₆ alkyl groups.
In the present specification, examples of the “C ₁ -C ₆ 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, A ¹ and A ² are the same or different and are preferably ethyl or isopropyl.

In the present invention, as the phosphitylating agent,
Di (t-butyl) N, N-diethyl phosphoramidite,
Di (t-butyl) N, N-diisopropyl phosphoramidite,
Di (2-phenyl-1,1-dimethylethyl) N, N-diethyl phosphoramidite,
Di (2-phenyl-1,1-dimethylethyl) N, N-diisopropyl phosphoramidite,
Di (2- (1'-naphthyl) -1,1-dimethylethyl) N, N-diethyl phosphoramidite,
Di (2- (1′-naphthyl) -1,1-dimethylethyl) N, N-diisopropyl phosphoramidite,
Di (1,2-diphenyl-1,2-dimethylpropyl) N, N-diethyl phosphoramidite,
Di (1,2-diphenyl-1,2-dimethylpropyl) N, N-diisopropyl phosphoramidite,
Di (diphenylmethyl) N, N-diethyl phosphoramidite,
Di (diphenylmethyl) N, N-diisopropyl phosphoramidite,
Di (9-fluorenylmethyl) N, N-diethyl phosphoramidite,
Di (9-fluorenylmethyl) N, N-diisopropyl phosphoramidite,
Di (2-cyano-1,1-dimethylethyl) N, N-diethyl phosphoramidite,
Di (2-cyano-1,1-dimethylethyl) N, N-diisopropyl phosphoramidite,
Di (9-fluorenyl-dimethylmethyl) N, N-diethyl phosphoramidite,
Di (9-fluorenyl-dimethylmethyl) N, N-diisopropyl phosphoramidite,
Di (1-cyclohexyldimethylmethyl) N, N-diethyl phosphoramidite,
Di (1-cyclohexyldimethylmethyl) N, N-diisopropyl phosphoramidite,
Di (tribenzylmethyl) N, N-diethyl phosphoramidite,
Di (tribenzylmethyl) N, N-diisopropyl phosphoramidite,
Di (tricyclohexylmethyl) N, N-diethyl phosphoramidite,
Di (tricyclohexylmethyl) N, N-diisopropyl phosphoramidite,
Di (1-adamantyl) N, N-diethyl phosphoramidite,
Di (1-adamantyl) N, N-diisopropyl phosphoramidite,
Di (di (trifluoromethyl) phenylmethyl) N, N-diethyl phosphoramidite,
Di (di (trifluoromethyl) phenylmethyl) N, N-diisopropyl phosphoramidite,
Di (2-trimethylsilyl-1,1-dimethylethyl) N, N-diethyl phosphoramidite,
Di (2-trimethylsilyl-1,1-dimethylethyl) N, N-diisopropyl phosphoramidite,
Di (2-triphenylsilyl-1,1-dimethylethyl) N, N-diethyl phosphoramidite, or
Di (2-triphenylsilyl-1,1-dimethylethyl) N, N-diisopropyl phosphoramidite can be preferably used.

上記式（２）中、R²は、水素原子、置換基を有していてもよいアシル基、置換基を有していてもよいアルキル基又は置換基を有していてもよいトリアルキルシリル基を表す。
本明細書において、「アシル基」としては、制限するわけではないが、Ｃ_1-6アルキル−カルボニル（たとえばメチルカルボニル、エチルカルボニル等）、Ｃ_6-10アリール−カルボニル（たとえばベンゾイル）などが挙げられる。
本明細書において、「トリアルキルシリル基」としては、制限するわけではないが、トリメチルシリル基、トリエチルシリル基、ｔ−ブチルジメチルシリル基などを挙げることができる。 In the present invention, the nucleoside represented by the following general formula (2) is phosphitylated with the phosphitylating agent represented by the above formula (3).

In the above formula (2), R ² is a hydrogen atom, an acyl group which may have a substituent, an alkyl group which may have a substituent, or a trialkylsilyl which may have a substituent. Represents a group.
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 “trialkylsilyl group” include, but are not limited to, a trimethylsilyl group, a triethylsilyl group, a t-butyldimethylsilyl group, and the like.

In the present invention, the “acyl group”, “alkyl group”, and “trialkylsilyl group” represented by R ² may have a substituent. Examples of the substituent include a C _{1 to} C ₁₀ alkoxy group (for example, methoxy, ethoxy, propoxy, butoxy and the like), a C _{6 to} C ₁₀ aryloxy group (for example, phenyloxy, naphthyloxy, biphenyloxy and the like), An amino group, a hydroxyl group, a cyano group, a halogen atom (for example, fluorine, chlorine, bromine, iodine) or a silyl group can be exemplified. 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 present invention, R ² is preferably a hydrogen atom, a benzoyl group, or a phenoxyacetyl group.

（式中、Ｚ¹はメチル基を示す。Ｚ²，Ｚ³及びＺ⁴は、それぞれ、互いに独立し、同一または異なって、水素原子、ハロゲン原子、又は、置換基を有していてもよいアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、アリール基、アラルキル基、アシル基、アルコキシ基、アリールオキシ基、アラルキルオキシ基、アルコキシカルボニル基、アリールオキシカルボニル基、アラルキルオキシカルボニル基、アルキルチオカルボニル基、アルコキシチオカルボニル基、アリールチオカルボニル基、アラルキルチオカルボニル基、アリールオキシチオカルボニル基若しくはアラルキルオキシチオカルボニル基を表し、Ｚ⁵は、電子吸引基を表す。）を表す。 In the above formula (2), R ³ is a hydrogen atom, a hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, or a group represented by the following formula

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group Represents an alkylthiocarbonyl group, an alkoxythiocarbonyl group, an arylthiocarbonyl group, an aralkylthiocarbonyl group, an aryloxythiocarbonyl group or an aralkyloxythiocarbonyl group, and Z ⁵ represents an electron-withdrawing 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-carbonyl (eg, benzoyloxy). ) 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 present specification, examples of the “alkenyl group” include, but are not limited to, vinyl, allyl, propenyl, isopropenyl, 2-methyl-1-propenyl, 2-methylallyl, 2-butenyl and the like.

In the present specification, examples of the “alkynyl group” include, but are not limited to, ethynyl, propynyl, butynyl and the like.
In the present specification, examples of the “cycloalkyl group” include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
In the present specification, examples of the “cycloalkenyl group” include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl and the like.

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 present specification, the “aralkyl group” is not limited, but includes benzyl, phenethyl, diphenylmethyl, triphenylmethyl, 1-naphthylmethyl, 2-naphthylmethyl, 2,2-diphenylethyl, 3-phenylpropylene. And 4-phenylbutyl, 5-phenylpentyl and the like.

In the present specification, examples of the “aryloxy group” include, but are not limited to, phenyloxy, naphthyloxy, biphenyloxy and the like.
In the present specification, the “aralkyloxy group” is not limited, but includes benzyloxy, phenethyloxy, diphenylmethyloxy, triphenylmethyloxy, 1-naphthylmethyloxy, 2-naphthylmethyloxy, 2,2 -Diphenylethyloxy, 3-phenylpropyloxy, 4-phenylbutyloxy, 5-phenylpentyloxy and the like can be mentioned.

In the present specification, examples of the “alkoxycarbonyl group” include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, 2-methoxyethoxycarbonyl, t-butoxycarbonyl and the like.
In the present specification, examples of the “aryloxycarbonyl group” include, but are not limited to, phenoxycarbonyl, naphthoxycarbonyl, phenylphenoxycarbonyl and the like.

  In the present specification, the “aralkyloxycarbonyl group” is not limited, but includes benzyloxycarbonyl, phenethyloxycarbonyl, diphenylmethyloxycarbonyl, triphenylmethyloxycarbonyl, 1-naphthylmethyloxycarbonyl, 2-naphthyl. Examples thereof include methyloxycarbonyl, 2,2-diphenylethyloxycarbonyl, 3-phenylpropyloxycarbonyl, 4-phenylbutyloxycarbonyl, 5-phenylpentyloxycarbonyl and the like.

In the present specification, the “alkylthiocarbonyl group” is not limited, but includes methylthiocarbonyl, ethylthiocarbonyl, propylthiocarbonyl, isopropylthiocarbonyl, n-butylthiocarbonyl, sec-butylthiocarbonyl, tert-butyl. Mention may be made of thiocarbonyl, pentylthiocarbonyl, hexylthiocarbonyl and the like.
In the present specification, examples of the “alkoxythiocarbonyl group” include, but are not limited to, methoxythiocarbonyl, ethoxythiocarbonyl, 2-methoxyethoxythiocarbonyl, t-butoxythiocarbonyl and the like.

In the present specification, examples of the “arylthiocarbonyl group” include, but are not limited to, phenylthiocarbonyl, naphthylthiocarbonyl, and the like.
In the present specification, the “aralkylthiocarbonyl group” is not limited, but includes benzylthiocarbonyl, phenethylthiocarbonyl, diphenylmethylthiocarbonyl, triphenylmethylthiocarbonyl, 1-naphthylmethylthiocarbonyl, 2-naphthylmethylthiocarbonyl, Examples include 2,2-diphenylethylthiocarbonyl, 3-phenylpropylthiocarbonyl, 4-phenylbutylthiocarbonyl, 5-phenylpentylthiocarbonyl, and the like.

In the present specification, examples of the “aryloxycarbonyl group” include, but are not limited to, phenoxythiocarbonyl, naphthoxythiocarbonyl and the like.
In the present specification, the “aralkyloxythiocarbonyl group” is not limited, but includes benzyloxythiocarbonyl, phenethyloxythiocarbonyl, diphenylmethyloxythiocarbonyl, triphenylmethyloxythiocarbonyl, 1-naphthylmethyloxy. Examples include thiocarbonyl, 2-naphthylmethyloxythiocarbonyl, 2,2-diphenylethyloxythiocarbonyl, 3-phenylpropyloxythiocarbonyl, 4-phenylbutyloxythiocarbonyl, 5-phenylpentyloxythiocarbonyl, and the like. .

  In this specification, examples of the electron withdrawing group include, but are not limited to, a cyano group, a nitro group, a trifluoromethyl group, a methanesulfonyl group, a benzenesulfonyl group, fluorine, and chlorine.

であって、式中、Ｚ¹がメチル基であり、Ｚ²，Ｚ³及びＺ⁴が、水素原子であり、Ｚ⁵が、シアノ基又はニトロ基であることが好ましい。 In the present invention, R ³ represents a hydrogen atom, a hydroxy group, a methoxy group, or a group represented by the following formula:

In the formula, it is preferable that Z ¹ is a methyl group, Z ² , Z ³ and Z ⁴ are hydrogen atoms, and Z ⁵ is a cyano group or a nitro group.

  In the above formula (2), B may be different from each other, pyrimidine bases such as thymine, cytosine and uracil; purine bases such as adenine and guanine; or 5-methylcytosine, 5-fluorouracil and 5-hydroxymethylcytosine And their derivatives.

  In order to improve the solubility in the solvent, the base site is dimethoxytrityl group (DMTr), benzoyl (Bz), isopropylcarbonyl (iBu), phenoxyacetyl (PAC), 4- (t-butyl) phenoxyacetyl (BPA) , Protecting groups such as allyloxycarbonyl (AOC), 2-[(t-butyldiphenylsilyloxy) methyl] benzoyl (SiOMB), 2- (acetylmethyl) benzoyl (AMB), 2-azidobenzoyl (AZMB) May be.

  In the present invention, B is preferably cytosine, thymine, adenine, guanine or a derivative thereof, or a group in which a protecting group is introduced.

  In the second embodiment of the present invention, the phosphitylating agent represented by the above formula (3) is used in an amount of 0.1 to 1 mol of the nucleoside represented by the above formula (2) in order to selectively react with the 5 ′ hydroxyl group. It is preferably used in an amount of from mol to 1.5 mol, more preferably from 0.3 mol to 1.2 mol, and still more preferably from 0.5 mol to 1.0 mol.

  In the second aspect of the present invention, an activator is used. Activating agents include pyridine hydrochloride; pyridine sulfonate such as pyridium triflate; pyridine borate such as pyridium tetrafluoroborate; imidazole such as 4,5-dicyanoimidazole and 4,5-dichloroimidazole; Tetrazole such as benzyltetrazole, 5-ethylthio-1H-tetrazole, 1H-tetrazole; imidazole sulfonate such as imidazolium triflate; benzimidazole sulfonate such as 5-nitrobenzimidazolium triflate; triazole such as triazolium triflate N-hydroxytriazole such as 1-hydroxybenztriazole; Triazole; N- (cyanomethyl) amine borate such as N- (cyanomethyl) pyrrolidinium tetrafluoroborate; N- (cyanomethyl) pyrrolidinium trifura N- (cyanomethyl) amine sulfonates such as N- (cyanomethyl) pyrrolidinium hexafluorophosphate, etc. N- (cyanomethyl) amine phosphates such as pyridine hydrochloride, pyridine sulfonate Pyridine borate, imidazole, or tetrazole can be preferably mentioned.

  In the second embodiment of the present invention, the activator is preferably used in an amount of 1 mol to 10 mol, based on 1 mol of the phosphitylating agent represented by the above formula (3), in order to complete the reaction. It is more preferable to use mol, and it is still more preferable to use 2 mol to 3 mol.

  In the second embodiment of the present invention, typically, the nucleoside solution represented by the above formula (2) is added with an activating agent and stirred, and then a phosphitylating agent is added and stirred, and then the above formula ( The nucleotide monomer unit represented by 1) is obtained. The nucleoside (2) need not be an isolated nucleoside, and a nucleoside prepared in a solution may be used as it is.

  In the second embodiment of the present invention, the regioselectivity / functional group selectivity in phosphitylating the nucleoside represented by the above formula (2) depends on the solubility of the nucleoside in the solvent. Accordingly, a solvent that can dissolve the nucleoside represented by the above formula (2) is preferable. Examples thereof include pyridine, a mixed solvent of pyridine and acetonitrile, and a mixed solvent of pyridine and N, N-dimethylformamide, and are preferably pyridine or a mixed solvent of pyridine and acetonitrile.

In the second embodiment of the present invention, when a mixed solvent of pyridine and acetonitrile is used, the volume ratio is preferably 2 to 0.5: 1, and more preferably about 1: 1.
The reaction temperature is preferably -78 ° C to 30 ° C, more preferably 0 ° C to 30 ° C, still more preferably 15 ° C to 25 ° C. If desired, the reaction may be allowed to proceed while blocking light.
The pressure is preferably atmospheric pressure.

In the third aspect of the present invention, there is provided a method for removing R ¹ which is a protecting group from the nucleotide monomer unit provided in the first aspect of the present invention. That is, a nucleotide monomer unit represented by the above general formula (1), a compound represented by the following general formula (4a), a compound represented by the following general formula (4b), zinc bromide (ZnBr ₂ ), magnesium bromide ( MgBr ₂ ), trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, 2-t-butylimino-2-diethylamino-1,3-dimethyl-1,3,2-diazaphosphorinane, (t-butylimino) tris (dimethylamino) ) R ¹ is eliminated by reacting with one or more deprotecting reagents selected from the group consisting of phosphorane, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo [5.4.0] -7-undecene. A method for protecting group removal is provided.

In the above formula (4a), A ³ represents optionally C ₁ may be -C ₁₀ alkyl group or optionally substituted C ₆ -C ₁₀ aryl group substituted. Further, in the above formula (4b), A ⁴ represents an optionally C ₁ may be -C ₁₀ alkyl group or optionally substituted C ₆ -C ₁₀ aryl group substituted.

In the present specification, examples of the “C ₆ -C ₁₀ aryl group” include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl, indenyl, biphenylyl, anthryl, phenanthryl and the like.

In the present invention, a substituent may be introduced into the “C ₁ -C ₁₀ alkyl group” and the “C ₆ -C ₁₀ aryl group” represented by A ³ and A ⁴ . Examples of the substituent include C _{1 to} C ₁₀ hydrocarbon groups (eg, methyl, ethyl, propyl, butyl, phenyl, tolyl, xylyl, cumenyl, mesityl, naphthyl, indenyl, tolyl, xylyl, benzyl, etc.) ₁ -C ₁₀ alkoxy group (e.g., methoxy, ethoxy, propoxy, butoxy, etc.), C ₆ -C ₁₀ aryloxy group (e.g., phenyloxy, naphthyloxy, biphenyloxy, etc.), an amino group, a hydroxyl group, a halogen atom (e.g. , Fluorine, chlorine, bromine, iodine) or a silyl group. 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 present invention, A ³ is preferably trifluoromethyl, phenyl, p-methylphenyl, methyl, ethyl, propyl, or isopropyl.
In the present invention, A ⁴ is preferably methyl, ethyl, or isopropyl.

In protecting group removal method of the present invention, the deprotecting reagent is appropriately selected depending on the protecting group R ¹ in formula (1) to release de. For example, R ¹ is a t-butyl group, 2-phenyl-1,1-dimethylethyl group, 2- (1-naphthyl) -1,1-dimethylethyl group, diphenylmethyl group, 1-cyclohexyldimethylmethyl group, Tribenzylmethyl group, tricyclohexylmethyl group, 1-adamantyl group, di (trifluoromethyl) phenylmethyl group, 2-trimethylsilyl-1,1-dimethylethyl group, 2-triphenylsilyl-1,1-dimethylethyl group Or when R ¹ is bridged with each other to form a ring with a phosphorus atom and two oxygen atoms, the deprotection reagent or zinc bromide (ZnBr) represented by the above formula (4a) or (4b) ₂ ), magnesium bromide (MgBr ₂ ), trifluoroacetic acid, trichloroacetic acid and dichloroacetic acid are preferably used. Preferred examples of the deprotection reagent represented by the above formulas (4a) and (4b) include trimethylsilyl trifluoromethanesulfonate (TMSOTf), trimethylsilylbenzenesulfonate, dimethylaluminum chloride, diethylaluminum chloride, and diisopropylaluminum chloride. it can.

Further, R ¹ is 9-fluorenylmethyl group, 9-fluorenyl - dimethyl methyl, when there like 2-cyano-1,1-dimethylethyl group, 2-t-butylimino-2-diethylamino -1 1,3-dimethyl-1,3,2-diazaphosphorinane, (t-butylimino) tris (dimethylamino) phosphorane, triethylamine, diisopropylethylamine, or 1,8-diazabicyclo [5.4.0] -7-undecene It is preferably used as a protective reagent.

  In the third embodiment of the present invention, the deprotection reagent is preferably used in an amount of 2 mol to 20 mol, based on 1 mol of the nucleotide monomer unit represented by the above formula (1), in order to complete the reaction. More preferably, it is used in an amount of 5 mol to 8 mol.

In the third embodiment of the present invention, R ² may have a substituent such as a benzoyl group or a phenoxyacetyl group from the viewpoint of protecting the 3 ′ position in the nucleotide monomer unit represented by the above formula (1). A good acyl group is preferred.

In the third embodiment of the present invention, typically, a deprotecting reagent is added to a solution of the nucleotide monomer unit represented by the above formula (1) and stirred to remove the protecting group R ¹ . The nucleotide monomer unit (1) need not be an isolated one, and a nucleotide monomer unit prepared in a solution may be used as it is.

  In the case where the deprotection reagent is a compound represented by the above formula (4a) such as TMSOTf, when the deprotection reagent is allowed to act on the nucleotide monomer unit represented by the above formula (1), the protecting group is eliminated and trimethylsilyl It becomes an ester and trifluoromethanesulfonic acid is produced. Therefore, optionally, the trimethylsilyl group can be removed by using methanol or the like, and further trifluoromethanesulfonic acid can be neutralized by using triethylamine (TEA) or the like to obtain a triethylamine salt of H-phosphonate monoester. In this case, methanol is preferably used in an amount of 6 mol to 20 mol, more preferably 10 mol to 16 mol, with respect to 1 mol of the nucleotide monomer unit represented by the above formula (1). The reaction mechanism is shown below.

［式中、B、R¹、R²及びR³は、上記の意味を表す。］

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

The above reaction mechanism is a hypothesis, and the present invention is not limited to this reaction mechanism.
As shown in the above reaction mechanism, 5′-H-phosphonate can be converted to a new nucleotide monomer unit (1a) as described later by removing the protecting group of the nucleotide monomer unit and then removing it. Therefore, chain extension is facilitated.
In the third aspect of the present invention, the solvent is preferably a solvent capable of dissolving the nucleotide monomer unit represented by the above formula (1). Examples thereof include collidine, pyridine, a mixed solvent of pyridine and acetonitrile, and a mixed solvent of pyridine and N, N-dimethylformamide, and are preferably pyridine or a mixed solvent of pyridine and acetonitrile.

In the third embodiment of the present invention, when a mixed solvent of pyridine and acetonitrile is used, the volume ratio is preferably 2 to 0.5: 1, and more preferably about 1: 1.
The reaction temperature is preferably −30 ° C. to 30 ° C., more preferably 0 ° C. to 30 ° C., and still more preferably 15 ° C. to 25 ° C. If desired, the reaction may be allowed to proceed while blocking light.
The pressure is preferably atmospheric pressure.

上記式中、B及びR³についての説明は、本発明の第１態様〜第２態様においてしたのと同様である。 In the fourth aspect of the present invention, a phosphonate represented by the following general formula (5) is provided.

In the above formula, the explanation about B and R ³ is the same as that in the first to second aspects of the present invention.

In the above formula, R ⁴ represents a carrier. As the carrier, for example, a known polymer support used in a solid phase method such as controlled pore glass (CPG) or highly cross-linkable polystyrene (HCP), which has as little swelling as possible and is used in excess. Examples thereof include those that can be easily removed by washing, and protecting groups such as a phenoxyacetyl group and a benzoyl group.
In the present invention, R ⁴ is preferably a phenoxyacetyl group.

The phosphonate represented by the above formula (5) can be prepared by, for example, introducing a carrier R ⁴ into the 3′-position of the nucleotide monomer unit synthesized according to the second embodiment, and then protecting the 5′-phosphite according to the third embodiment. Can be obtained by removing.

In the fifth aspect of the present invention, a 5′-phosphite oligonucleotide derivative represented by the following general formula (7) is provided.

In the above formula, the explanation for B, R ¹ and R ³ is the same as that in the first to second aspects of the present invention. Further, in the above formula, the explanation for R ⁴ is the same as that in the fourth embodiment of the present invention.
In the above formula, n represents an integer of 1 or more. n is preferably an integer of 1 to 100, and more preferably an integer of 10 to 30.

  In the sixth aspect of the present invention, as one aspect, the fifth aspect of the present invention is obtained by condensing one aspect of the nucleotide monomer unit according to the first aspect of the present invention and the phosphonate provided in the fourth aspect of the present invention. A method for producing a nucleotide dimer that is an aspect of an embodiment is provided. That is, in the presence of a condensing agent, a nucleotide monomer unit represented by the following general formula (1a) is reacted with a phosphonate represented by the following general formula (5) to produce a dimer represented by the following general formula (6). To do.

In the above formula, the explanation for B, R ¹ and R ³ is the same as that in the first to second aspects of the present invention. Further, in the above formula, the explanation for R ⁴ is the same as that in the fourth embodiment of the present invention.

  In the sixth aspect of the present invention, the phosphonate represented by the above formula (5) (H-phosphonate monoester) is used as the end of the extended chain. Therefore, intramolecular and intermolecular chain scission reactions, which are side reactions that have been confirmed by conventional methods, are suppressed.

  In the sixth aspect of the present invention, in order to produce the dimer (6), the nucleotide monomer unit represented by the above formula (1a) has the above formula (5) to allow the condensation reaction to proceed quantitatively. In the liquid phase method, it is preferably used in an amount of 1 mol to 10 mol, more preferably 1 mol to 5 mol, and still more preferably 1 mol to 1.5 mol. In the solid phase method, it is preferably used in an amount of 1 mol to 100 mol, more preferably 10 mol to 50 mol, more preferably 20 mol, relative to 1 mol of phosphonate bound to the solid phase carrier represented by the above formula (5). It is still more preferable to use ~ 30 mol.

  In the method for producing an oligonucleotide derivative according to the present invention, a condensing agent is used. Condensation agents include 2- (benzotriazol 1-yloxy) -1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphoridinium hexafluorophosphate (BOMP), N, N -Bis (2-oxo-3-oxazolidinyl) phosphonic chloride (BopCl) can be mentioned, and BOMP can be preferably used.

  In the sixth aspect of the present invention, the condensing agent is preferably used in an amount of 1 to 20 mol in the liquid phase method for 1 mol of the phosphonate represented by the above formula (5) in order to allow the reaction to proceed quantitatively. More preferably, it is used in an amount of 10 to 10 mol, and still more preferably 1 to 5 mol. In the solid phase method, it is preferably used in an amount of 1 mol to 200 mol, more preferably 20 mol to 100 mol, more preferably 40 mol, per mol of phosphonate bound to the solid phase carrier represented by the above formula (5). It is still more preferable to use ~ 60 mol.

  In the sixth aspect of the present invention, typically, the nucleotide monomer unit represented by the above formula (1a) is dissolved and added to the phosphonate (5) solution, the condensing agent is added, and the dimer is stirred. (6) is obtained. The phosphonate (5) need not be an isolated one, and a nucleotide monomer unit prepared in a solution may be used as it is.

  In the sixth aspect of the present invention, the solvent is preferably one that can dissolve the phosphonate represented by the above formula (5). Examples thereof include pyridine, a mixed solvent of pyridine and acetonitrile, and a mixed solvent of pyridine and N, N-dimethylformamide, and are preferably pyridine or a mixed solvent of pyridine and acetonitrile.

In the sixth embodiment of the present invention, when a mixed solvent of pyridine and acetonitrile is used, the volume ratio is preferably 2 to 0.5: 1, and more preferably about 1: 1.
The reaction temperature is preferably 0 ° C to 50 ° C, more preferably 10 ° C to 30 ° C, still more preferably 15 ° C to 25 ° C. If desired, the reaction may be allowed to proceed while blocking light.
The pressure is preferably atmospheric pressure.

  In another aspect of the sixth embodiment of the present invention, the protecting group is removed according to the third embodiment of the present invention from the 5′-phosphite oligonucleotide derivative according to the fifth embodiment of the present invention. A 5′-phosphite oligonucleotide derivative that is condensed with one embodiment of the nucleotide monomer unit according to the first embodiment of the present invention to extend the chain in the same manner as the condensation method according to one aspect of the sixth embodiment of the present invention A method of manufacturing is provided.

That is, in another aspect of the sixth embodiment of the present invention, a 5′-phosphite oligonucleotide derivative represented by the following general formula (7), a compound represented by the above general formula (4a) such as TMSOTf, Compound represented by formula (4b), zinc bromide (ZnBr ₂ ), magnesium bromide (MgBr ₂ ), trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, 2-t-butylimino-2-diethylamino-1,3-dimethyl -1,3,2-diazaphosphorinane, (t-butylimino) tris (dimethylamino) phosphorane, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo [5.4.0] -7-undecene by reacting with one or more deprotecting reagent, after the R ¹ desorbed, characterized in that the reaction in the presence a condensing agent, a nucleotide monomeric unit represented by the following general formula (1a), the following Method for producing 5'-phosphite oligonucleotide derivative represented by the general formula (8) is provided.

［式中、B、R¹、R³、R⁴及びｎ、上記の意味を表す。］

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

  For example, using the dimer obtained in the sixth embodiment as a starting material, a nucleotide trimer can be obtained by (a) a deprotection step and (b) a polycondensation step with a new monomer. Further, when the chain is extended, the chain can be easily extended by repeating the above steps (a) and (b).

In the sixth aspect of the present invention, the deprotection reagent and reaction conditions for removing R ¹ from the 5′-phosphite oligonucleotide derivative represented by the above formula (7) are described in the third aspect of the present invention. It is the same as that. After removing the protecting group, 5′-H-phosphonate oligonucleotide may be optionally used using methanol, TEA or the like.

  In the sixth aspect of the present invention, the condensing agent and the reaction conditions for the reaction with the nucleotide monomer unit represented by the above formula (1a) in the presence of the condensing agent after removing the protecting group are the same as those described above. This is the same as described in the aspect of the sixth aspect of the invention.

［式中、B、R³、R⁴及びｎ、上記の意味を表す。］ In a seventh aspect of the present invention, a 5′-H-phosphonate oligonucleotide derivative represented by the following formula (9) is provided.

[Wherein B, R ³ , R ⁴ and n represent the above meanings. ]

In the above formula, the explanation about B and R ³ is the same as that in the first to second aspects of the present invention. Further, in the above formula, the explanation for R ⁴ is the same as that in the fourth embodiment of the present invention.

According to an eighth aspect of the present invention, there is provided a method for producing a 5′-H-phosphonate oligonucleotide derivative according to the seventh aspect of the present invention. That is, in the eighth aspect of the present invention, a 5′-phosphite oligonucleotide derivative represented by the following general formula (7), a compound represented by the above general formula (4a) such as TMSOTf, and the above general formula (4b) Compound represented by the following: zinc bromide (ZnBr ₂ ), magnesium bromide (MgBr ₂ ), trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, 2-t-butylimino-2-diethylamino-1,3-dimethyl-1,3 , 2-diazaphosphorinane, (t-butylimino) tris (dimethylamino) phosphorane, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo [5.4.0] -7-undecene There is provided a method for producing a 5′-H-phosphonate oligonucleotide derivative represented by the following formula (9), which comprises a step of reacting with a deprotecting reagent to eliminate R ¹ .

［式中、B、R¹、R³、R⁴及びｎは、上記の意味を表す。］

[Wherein, B, R ¹ , R ³ , R ⁴ and n represent the above meanings. ]

In the eighth embodiment of the present invention, the deprotection reagent and reaction conditions for removing R ¹ from the 5′-phosphite oligonucleotide derivative represented by the above formula (7) are described in the third embodiment of the present invention. It is the same as that. After removing the protecting group, the 5′-H-phosphonate oligonucleotide derivative represented by the above formula (9) can be obtained by treatment with methanol, TEA or the like.

  The procedure for obtaining the 5′-H-phosphonate oligonucleotide derivative after removal of the protecting group is the same as that described in the third embodiment of the present invention. However, methanol is preferably used in an amount of 6 mol to 20 mol, more preferably 10 mol to 16 mol, relative to 1 mol of the nucleotide in the 5'-phosphite oligonucleotide derivative represented by the above formula (7). .

［式中、B、R³、R⁴及びｎは、上記の意味を表す。］ The oligonucleotide derivative (9) thus obtained can be widely modified and a useful DNA analog can be synthesized. For example, by reacting with R ⁵ OH in the presence of a condensing agent, an oligonucleotide derivative of the following formula (10) having the functional group R ⁵ introduced therein is obtained.

[Wherein, B, R ³ , R ⁴ and n represent the above meanings. ]

Examples of R ⁵ include functional residues such as hydrogen, nucleoside, alkyl, aryl, various fluorescent chromophores, and biotin.
Examples of the condensing agent include BOMP and BopCl, and BOMP can be preferably used.

  Subsequently, various DNA analogs can be obtained by oxidizing or chemically converting the obtained oligonucleotide derivative represented by the above formula (10). For example, various corresponding DNA analogs can be obtained by using the following reagents / solvents (1) to (7).

［式中、B、R³、R⁴、R⁵及びｎは、上記の意味を表す。Rとは、アルキル基を示す。］

[Wherein, B, R ³ , R ⁴ , R ⁵ and n represent the above meanings. R represents an alkyl group. ]

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. Tetrahydrofuran and diethyl ether were distilled in the presence of sodium benzophenone ketyl immediately before use.
Di-tert-butyl N, N-diethyl phosphoramidite, 3′-O-benzoylthymidine and 3′-O-tert-butyldimethylsilylthymidine were synthesized by known methods.

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 60 N from KANTO CHEMICAL was used as the silica gel packed in the column chromatography.

Example 1 (Synthesis of 3'-terminal)
3'-O-benzoyl-5'-O- (di-t-butyl) -phosphitylthymidine

Under an argon atmosphere, 3′-O-benzoylthymidine (693.3 mg, 2.0 mmol) was azeotroped three times with dry pyridine (5 ml). To this, 16 ml of a dry pyridine solution of pyridine hydrochloride prepared by azeotropic distillation with dry pyridine, adjusted to 0.25 M, and dried with Molecular Sieves 3A was added (A). To this, di-tert-butyl N, N-diethyl phosphoramidite was added dropwise at room temperature over 2 minutes. After completion of the dropwise addition, the mixture was stirred at room temperature for 1.5 hours, diluted with chloroform (50 ml), and washed with a saturated aqueous sodium hydrogen carbonate solution (40 ml). The aqueous layer was back-extracted with chloroform (50 ml), all the organic layers were collected and dried over anhydrous sodium sulfate, the solvent was distilled off, and column chromatography (hexane-ethyl acetate-triethylamine (75: 25: 2 After removing triethylamine under reduced pressure, the target compound was collected by azeotroping 3 times with dry toluene (2 ml) and 3 times with dry dichloromethane (2 ml). 91% of a white foamy solid.
¹ H NMR (CDCl ₃ ) δ1.4, 1.44 (2s, 18H), δ1.960, 1.964 (2s, 3H), δ2.31-2.41 (m, 1H), δ2.49-2.56 (m, 1H) , δ3.98-4.04 (m, 1H), δ4.27-4.36 (m, 2H), δ5.56-5.60 (m, 1H), δ6.53 (dd, 1H), δ7.44-7.50 (m , 2H), δ7.57-7.63 (m, 1H), δ7.800, 7.804 (2s, 1H), δ8.04-8.08 (m, 2H), δ8.54 (brs, 1H)
³¹ P NMR (CDCl ₃ ) δ134.4

Example 2 (Synthesis of 3'-end)
Triethylammonium 3'-O-benzoylthymidine-5'-yl phosphonate

Under an argon atmosphere, 3′-O-benzoyl-5′-O-bis (t-butyl) -phosphitylthymidine (156.8 mg, 0.3 mmol) obtained in Example 1 was added to dry pyridine (1 ml) with 3 Azeotropically dried three times with dry toluene (1 ml). This was made into a 3 ml dry acetonitrile solution, and trimethylsilyl triflate (110 μl, 0.6 mmol) was added dropwise over 30 seconds. After completion of the dropwise addition, the mixture was stirred at room temperature for 10 minutes, deprotected with methanol (49 μl, 1.2 mmol), and neutralized with triethylamine (170 μl, 1.2 mmol). This was separated and purified by column chromatography (dichloromethane-triethylamine (98: 2, v / v) -dichloromethane-methanol-triethylamine (96: 4: 2, v / v / v)) and purified with toluene (3 ml). Azeotropically with chloroform (3 ml) three times. Then, it was diluted with dichloromethane (2 ml) and washed with 1 M aqueous triethylammonium carbonate-pyridine (1: 1, v / v) (2 ml). This was dried over anhydrous sodium sulfate, the solvent was distilled off, and then azeotroped three times with toluene (1 ml) and three times with dichloromethane (1 ml) to obtain the target compound in a yield of 25%. White foamy solid.
¹ H-NMR (CDCl ₃ ) δ1.35 (t, 9H, -CH ₂ -of Et ₃ N, ³ J _HH = 7.2 Hz), δ1.97 (s, 3H, -CH ₃ of thymine), δ2. 47-2.50 (m, 2H, 2'-H), δ3.11 (q, 6H, -CH ₃ of Et ₃ N, ³ J _HH = 7.2 Hz), δ3.52-3.65 (ddd, 1H, 5 ' -H), δ4.12-4.30 (ddd, 1H, 5``-H), δ4.34 (m, 1H, 4'-H), δ5.64 (m, 1H, 3'-H), δ6 .53 (t, 1H, 1'-H), 6.91 (d, 1H, H of PH, ¹ J _PH = 583 Hz), δ7.43-7.49 (m, 2H, o-of Bz), δ7.57 -7.62 (m, 1H, p- of Bz), 7.99-8.05 (m, 2 H, m-of Bz)
³¹ P-NMR (CDCl ₃ ) δ4.53 ( ¹ J _PH = 618 Hz

Example 3 (Synthesis of monomer unit)
Thymidine 5'-O-bis (tert-butyl) phosphite

  Thymidine (74.5 mg, 0.3 mmol) was azeotropically dried three times with dry pyridine (1 ml). To this, dry pyridine (3.6 ml) and 10 molecular sieves 3A grains were added. Then, azeotropically with dry pyridine, adjusted to 0.25 M, 2.4 ml of a dry pyridine solution of pyridine hydrochloride dried with molecular sieves 3A was added. After stirring overnight, bis-tert-butyl N, N-diethyl phosphoramidite was added dropwise thereto over 3 minutes. After completion of the dropwise addition, the mixture was stirred at room temperature for 1.5 hours, then MS3A was removed by filtration, diluted with chloroform (10 ml), washed with saturated aqueous sodium hydrogen carbonate solution (10 ml), and then three times with chloroform (10 ml). Back extracted. This was dried over anhydrous sodium sulfate, the solvent was distilled off, and column chromatography (hexane-ethyl acetate-triethylamine (80: 20: 2, v / v / v) -hexane-ethyl acetate-triethylamine (50:50 : 2, v / v / v)). Triethylamine was removed under reduced pressure and then azeotroped three times with dry toluene (2 ml) and three times with chloroform (2 ml) to obtain the target compound in a yield of 58%. White foamy solid.

Example 4 (Synthesis of 3'-terminal)
3'-O- (t-butyldimethylsilyl) -5'-O-bis (t-butyl) -phosphitylthymidine

Under an argon atmosphere, 3′-O- (tert-butyldimethylsilyl) thymidine (107.1 mg, 0.3 mmol) was azeotroped three times with dry pyridine (2 ml). To this, dry pyridine (0.12 ml) and 15 MS3A grains were added. Thereafter, the mixture was azeotroped with dry pyridine, adjusted to 0.25 M, and 2.88 ml of a dry pyridine solution of pyridine hydrochloride dried with Molecular Sieves 3A was added. Bis-tert-butyl N, N-diethyl phosphoramidite was added dropwise thereto over 2 minutes. After completion of the dropwise addition, the mixture was stirred at room temperature for 15 minutes, and then MS3A was removed by filtration, diluted with chloroform (30 ml), and washed with a saturated aqueous sodium hydrogen carbonate solution (30 ml). This was dried over anhydrous sodium sulfate, the solvent was distilled off, and column chromatography (hexane-ethyl acetate-triethylamine (96: 16: 2, v / v / v) -hexane-ethyl acetate-triethylamine (80:20 : 2, v / v / v)). Triethylamine was removed under reduced pressure, and then azeotroped three times with dry toluene (2 ml) and three times with chloroform (2 ml) to obtain the target compound in 79% yield. White foamy solid.
¹ H-NMR (CDCl ₃ ) δ0.08 (s, 6H), δ0.87 (s, 9H), δ1.41 (s, 18H), δ1.93 (s, 3H), δ2.00-2.28 ( m, 2H), δ4.01-4.02 (m, 3H), δ4.40-4.41 (m, 1H), δ6.33-6.38 (dd, 1H), δ7.69 (s, 1H), δ8.26 (s, 1H)
³¹ P NMR (CDCl ₃ ) δ133.2

Example 5 (Synthesis of 3'-end)
Triethylammonium 3'-O- (t-butyldimethylsilyl) thymidine-5'-yl phosphonate

5 ml of 3′-O- (t-butyldimethylsilyl) -5′-O-bis (t-butyl) -phosphitylthymidine (4.26 mg, 8 mmol) obtained in Example 4 was added under an argon atmosphere. In a dry dichloromethane solution, trifluoroacetic acid (8.0 ml, 103 mmol) was added dropwise over 3 minutes. After completion of the dropwise addition, the mixture was stirred at room temperature for 1 hour and then neutralized with triethylamine (17.3 ml, 40 mmol). This was washed with 1 M aqueous triethylammonium carbonate (80 ml). This was dried over anhydrous sodium sulfate, the solvent was distilled off, and azeotroped with toluene (10 ml). Separation and purification by column chromatography (dichloromethane-triethylamine (100: 0.5, v / v) -dichloromethane-methanol-triethylamine (97: 3: 0.5, v / v / v)), 3 times with toluene (10 ml), Azeotropy with dichloromethane (10 ml) three times gave the target compound in 98% yield. White foamy solid.
¹ H-NMR (CDCl ₃ ) δ0.08 (s, 6H), δ0.89 (s, 9H), δ1.31-1.36 (t, 9H), δ1.96 (s, 3H), δ2.14- 2.19 (m, 2H), δ3.06-3.08 (m, 6H), δ4.01-4.01 (m, 3H), δ4.48 (m, 1H), δ6.35-6.40 (dd, 1H), δ6 .90 (s, 1H, J = 618.0 Hz), δ7.76 (s, 1H), δ8.76 (s, 1H)
³¹ P-NMR (CDCl ₃ ) δ 5.24 (s, J = 620.4 Hz)

Example 6 (Synthesis of dimer)
3'-O- (t-butyldimethylsilyl) thymidine-5'-yl- [5'-O- (bis-tert-butylphosphityl) thymidine-3'-yl]] phosphonate

Thymidine 5′-O- (ditert-butyl) phosphite (418.2 mg, 1 mmol) obtained in Example 3 was azeotropically dried three times with dry toluene (1 ml), and the resulting solution was added to 3 ml of dry pyridine solution. did. To this was added triethylammonium 3′-O-tert-butyldimethylsilylthymidine-5′-yl phosphonate (897.3 mg, 1.72 mmol) obtained in Example 5 dissolved in dry pyridine (7 ml), and BopCl ( 0.509 g, 2.00 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. Pyridine was distilled off under reduced pressure, diluted with chloroform (100 ml), washed 3 times with saturated aqueous sodium hydrogen carbonate solution (100 ml), and back-extracted once with chloroform (100 ml). After drying this over anhydrous sodium sulfate, the solvent was distilled off to obtain the target compound quantitatively. White foamy solid.
¹ H-NMR (CDCl ₃ ) δ0.09 (m, 6H), δ0.89 (s, 9H), δ1.40 (s, 3H), δ1.89-1.93 (m, 3H), δ2.14- 2.35 (m, 2H), δ2.43-2.61 (m, 2H), δ3.36-3.55 (m, 2H), δ3.80 (s, 6H), δ3.98-4.03 (m, 2H), δ4 .05-4.42 (m, 4H), δ5.25 (m, 2H), δ4.05-4.42 (dd, 1H), δ6.10-6.14 (t, 1H), δ6.19-6.23 (t, 1H ), δ6.43-6.46 (m, 2H), δ6.892 (d, 1H, 717 Hz), δ6.933 (d, 1H, 716 Hz), δ7.16-7.37 (m, 13H), δ8. 50-8.75 (m, 2H)
³¹ P-NMR (CDCl ₃ ) δ133.53, 133.46, δ9.16 (s, J = 704.8 Hz), 7.72 (s, J = 708.2 Hz)

Example 7 (Demer Deprotection)

3'-O- (t-butyldimethylsilyl) thymidine-5'-yl- [5'-O- (bis tert-butylphosphityl) thymidine-3'-yl]] phosphonate obtained in Example 6 (41.0 mg, 0.05 mmol) was azeotropically dried three times with dry toluene (1 ml) to give a 5 ml dry dichloromethane solution. To this was added trifluoroacetic acid (50 μl, 0.649 mmol), and the mixture was stirred at room temperature for 25 hours. The mixture was diluted with chloroform (30 ml), washed 3 times with 1 M aqueous triethylammonium carbonate solution (30 ml), and back-extracted 5 times with chloroform (30 ml). After drying this over anhydrous sodium sulfate, the solvent was distilled off to obtain the target compound. Yellow oily substance.
³¹ P-NMR (CDCl ₃ ) δ8.97 (m, ¹ J _PH = 708.3 Hz), 8.85 (m, ¹ J _PH = 708.3 Hz), δ4.90 (m, ¹ J _PH = 619.4 Hz), 4.781 ( _{^{m, 1 J PH = 619.4 Hz}} )

Example 8 (Synthesis of trimer)

The same procedure as in Example 6 was performed. However, the phosphonate obtained in Example 7 was used in place of the triethylammonium 3′-O-tert-butyldimethylsilylthymidin-5′-yl phosphonate obtained in Example 5. Yellow oily substance.
³¹ P-NMR (CDCl ₃ ) δ133.72 (t), δ133.69 (t), δ133.34 (t), δ133.31 (t), δ9.91 (m), δ9.73 (m), δ9.26 (m), δ9.07 (m), δ8.19 (m), δ8.07 (m), δ7.83 (m), δ7.77 (m)

アルゴン雰囲気下、3'-O-(フェノキシアセチル)-5'-O-ビスジメチルシアノエチルホスフィチルチミジン (36.2 mg, 0.06 mmol) をドライピリジン (1 ml) で3回、ドライトルエン (1 ml) で3回共沸した。これをドライ重アセトニトリル-アセトニトリル (1:4, v/v) (0.6 ml) 溶液にして、DBUを添加した。22時間後、クロロホルム (3 ml) で希釈し、DBU 重炭酸塩(3 ml) で洗浄した。これを無水硫酸ナトリウムで乾燥し、溶媒を留去して目的化合物を得た。
³¹P NMR (CDCl₃) δ0.15 (¹J_PH=584.1 Hz) Example 9 (deprotection under basic conditions)
1,8-diazabicyclo [5,4,0] 1-undecenium 3'-O- (t-phenoxyacetyl) thymidin-5'-yl phosphonate

3'-O- (phenoxyacetyl) -5'-O-bisdimethylcyanoethylphosphitylthymidine (36.2 mg, 0.06 mmol) in dry pyridine (1 ml) 3 times, dry toluene (1 ml) Azeotroped 3 times. This was made into a solution of dry heavy acetonitrile-acetonitrile (1: 4, v / v) (0.6 ml), and DBU was added. After 22 hours, it was diluted with chloroform (3 ml) and washed with DBU bicarbonate (3 ml). This was dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain the target compound.
³¹ P NMR (CDCl ₃ ) δ0.15 ( ¹ J _PH = 584.1 Hz)

Claims (2)

A phosphonate represented by the following general formula (5).

Wherein, B represents a pyrimidine base, purine base or a derivative thereof, R ³ is a hydrogen atom, hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyl group table or by the following formula, Group

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group , alkylthiocarbonyl group, an alkoxythiocarbonyl group, arylthio group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, Z ⁵ represents a representative.) the electron-withdrawing group, R ⁴ Represents a carrier. ] A 5′-H-phosphonate oligonucleotide derivative represented by the following formula (9):

Wherein, B represents a pyrimidine base, purine base or a derivative thereof, R ³ is a hydrogen atom, hydroxy group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyl group table or by the following formula, Group

(In the formula, Z ¹ represents a methyl group. Z ² , Z ³ and Z ⁴ are each independently the same or different and may have a hydrogen atom, a halogen atom or a substituent. Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group , alkylthiocarbonyl group, an alkoxythiocarbonyl group, arylthio group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, Z ⁵ represents a representative.) the electron-withdrawing group, R ⁴ Represents a carrier. n represents an integer of 1 or more. ]