JP5228002B2 - Method for producing ribonucleotide or ribonucleotide derivative - Google Patents

Description

  The present invention relates to a method for producing ribonucleotides or ribonucleotide derivatives.

  Oligoribonucleic acid (oligo RNA) used in various applications such as gene cloning probes, gene expression analysis probes, antisense oligonucleotides, and RNAi can be chemically synthesized by the phosphoramidite method.

  In the phosphoramidite method, the hydroxyl group at the 2 ′ position of the phosphoramidite unit (monomer unit) is used to prevent rearrangement of 3′-5 ′ internucleotide bonds to 2′-5 ′ bonds and cleavage of the nucleotide chain. It is important to protect (2 ′ hydroxyl group) with an appropriate protecting group, and the 2 ′ hydroxyl protecting group includes removal of the 5 ′ hydroxyl group (5 ′ hydroxyl group) protecting group, phosphoric acid diester Characteristic of stable removal under conditions such as removal of protecting groups, removal of protecting groups of nucleobases, excision of nucleotide chains from solid phase carriers, etc., but removal in a short time under conditions different from these conditions Is required.

Conventionally, as the protecting group for the 2 ′ hydroxyl group, for example, an acetal protecting group, a silyl protecting group, and the like are known.
However, the conventional acetal-based protecting groups can be used under conditions such as removal of the 5 'hydroxyl protecting group, removal of the phosphate diester protecting group, removal of the nucleobase protecting group, and cleavage of the nucleotide chain from the solid phase carrier. Thus, the 3′-5 ′ internucleotide bond could not be transferred to the 2′-5 ′ bond or the nucleotide chain could not be prevented. For example, a 4,4′-dimethoxytrityl group (DMTr group) generally used as a protecting group for a 5 ′ hydroxyl group can be removed by acid treatment, but conventional acetal protecting groups can also be removed by acid treatment. Therefore, when an acetal protecting group and a DMTr group are used in combination, elimination of the DMTr group results in elimination of the acetal protecting group, and the 3′-5 ′ internucleotide bond is converted to the 2′-5 ′ bond. Rearrangement and nucleotide chain breakage could not be prevented. On the other hand, 2′-O-1- (2-cyanoethoxy) ethyl group (CEE group) has been known as an acetal protecting group that is stable against acid (Wolfgang Pfleiderer et al., HELVETICA CHIMICA ACTA, 81st). Vol. 1545-1566, 1998; Japanese Patent Application Laid-Open No. 7-196683). This acetal protecting group is too stable to acid, and therefore cannot be removed under acidic conditions that do not degrade RNA. It was considered unsuitable as a protecting group for the 2 ′ hydroxyl group.

For this reason, silyl-based protecting groups such as TBDMS are generally used as 2′-hydroxyl protecting groups. Further, as an acetal-based protecting group that is stable under the DMTr group deprotection conditions and can be removed under acidic conditions different from the DMTr group deprotection conditions, 1- (2-fluorophenyl) -4-methoxypiperidine-4- An yl (Fpmp) group and a 1- (4-chlorophenyl) -4-ethoxypiperidin-4-yl (Cpep) group have been developed.
However, silyl protecting groups such as TBDMS have problems such as low introduction efficiency to the 2 ′ hydroxyl group and steric hindrance due to bulky molecules, resulting in low condensation reaction efficiency.

  The present invention can be efficiently introduced into the 2 ′ hydroxyl group and does not cause steric hindrance in the condensation reaction, and further removes the 5 ′ hydroxyl group protecting group, the phosphoric acid diester protecting group, and the nucleobase protecting group. Ribonucleotides in which the 2 'hydroxyl group is protected by a protecting group that can be removed in a short time under conditions other than these conditions, such as removal of nucleotides and excision of nucleotide chains from solid phase carriers. It aims at providing the method of manufacturing a ribonucleotide or a ribonucleotide derivative using a derivative.

  In order to solve the above problems, the present invention provides the following methods for producing ribonucleotides or ribonucleotide derivatives.

［式中、Ｂは核酸塩基又はその誘導体を表し、Ｘはオキソアニオン又はチオアニオンを表し、Ｙ及びＺは互いに独立して水素原子、固相担体又は水酸基の保護基を表し、Ｒ¹は水素原子又は置換基を有していてもよいトリチル基若しくは９−フェニルキサンテニル基を表し、Ｒ²、Ｒ³及びＲ⁴は互いに独立して水素原子、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、Ｒ⁵は電子吸引基を表し、ｎは１以上の整数を表す。］
で表されるリボヌクレオチド誘導体（II）をフッ化物処理することにより、
次式（Ｉ）：

［式中、Ｂ、Ｘ、Ｙ、Ｚ、Ｒ¹及びｎは前記と同義である。］
で表されるリボヌクレオチド又はリボヌクレオチド誘導体（Ｉ）を製造する工程を含む、リボヌクレオチド又はリボヌクレオチド誘導体（Ｉ）の製造方法。 (1) The following formula (II):

[In the formula, B represents a nucleobase or a derivative thereof, X represents an oxoanion or a thioanion, Y and Z each independently represent a hydrogen atom, a solid phase carrier or a hydroxyl-protecting group, and R ¹ represents a hydrogen atom. Alternatively, it represents a trityl group or 9-phenylxanthenyl group which may have a substituent, and R ² , R ³ and R ⁴ may independently 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, alkoxythiocarbonyl group, arylthiocarbonyl group, aralkyl O carbonyl group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, R ⁵ represents an electron withdrawing group, n represents an integer of 1 or more. ]
By treating the ribonucleotide derivative (II) represented by
Formula (I):

[Wherein, B, X, Y, Z, R ¹ and n are as defined above. ]
The manufacturing method of ribonucleotide or ribonucleotide derivative (I) including the process of manufacturing the ribonucleotide or ribonucleotide derivative (I) represented by these.

(2) The production method according to (1), wherein tetrabutylammonium fluoride is used as the fluoride.

［式中、Ｂは核酸塩基又はその誘導体を表し、Ｘはオキソアニオン又はチオアニオンを表し、Ｙ及びＺは互いに独立して水素原子、固相担体又は水酸基の保護基を表し、Ｒ¹は水素原子又は置換基を有していてもよいトリチル基若しくは９−フェニルキサンテニル基を表し、Ｒ²、Ｒ³及びＲ⁴は互いに独立して水素原子、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、Ｒ⁵は電子吸引基を表し、ｎは１以上の整数を表す。］
で表されるリボヌクレオチド誘導体（II）を、無水溶媒中、シリル化剤存在下で第３級アミン処理又はホスファゼン塩基処理した後、中性条件又は酸性条件下で加水分解することにより、
次式（Ｉ）：

［式中、Ｂ、Ｘ、Ｙ、Ｚ、Ｒ¹及びｎは前記と同義である。］
で表されるリボヌクレオチド又はリボヌクレオチド誘導体（Ｉ）を製造する工程を含む、リボヌクレオチド又はリボヌクレオチド誘導体（Ｉ）の製造方法。 (3) The following formula (II):

[In the formula, B represents a nucleobase or a derivative thereof, X represents an oxoanion or a thioanion, Y and Z each independently represent a hydrogen atom, a solid phase carrier or a hydroxyl-protecting group, and R ¹ represents a hydrogen atom. Alternatively, it represents a trityl group or 9-phenylxanthenyl group which may have a substituent, and R ² , R ³ and R ⁴ may independently 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, alkoxythiocarbonyl group, arylthiocarbonyl group, aralkyl O carbonyl group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, R ⁵ represents an electron withdrawing group, n represents an integer of 1 or more. ]
By subjecting the ribonucleotide derivative (II) represented by the following formula to a tertiary amine treatment or phosphazene base treatment in the presence of a silylating agent in an anhydrous solvent, and then hydrolyzing under neutral or acidic conditions,
Formula (I):

[Wherein, B, X, Y, Z, R ¹ and n are as defined above. ]
The manufacturing method of ribonucleotide or ribonucleotide derivative (I) including the process of manufacturing the ribonucleotide or ribonucleotide derivative (I) represented by these.

(4) The method according to (3) above, wherein 1,8-diazabicyclo [5.4.0] -7-undecene is used as the tertiary amine.

［式中、Ｂは核酸塩基又はその誘導体を表し、Ｗは酸素原子又は硫黄原子を表し、Ｙ及びＺは互いに独立して水素原子、固相担体又は水酸基の保護基を表し、Ｒ¹は水素原子又は置換基を有していてもよいトリチル基若しくは９−フェニルキサンテニル基を表し、Ｒ²、Ｒ³及びＲ⁴は互いに独立して水素原子、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、Ｒ⁵は電子吸引基を表し、Ｒ⁶はリン酸保護基を表し、ｎは１以上の整数を表す。］
で表されるリボヌクレオチド誘導体（III）をアンモニア処理又は第１級アミン処理することにより、前記リボヌクレオチド誘導体（II）を製造する工程を含む、前記（１）〜（４）のいずれかに記載の製造方法。 (5) The following formula (III):

[Wherein, B represents a nucleobase or a derivative thereof, W represents an oxygen atom or a sulfur atom, Y and Z each independently represent a hydrogen atom, a solid phase carrier or a hydroxyl-protecting group, and R ¹ represents hydrogen. Represents a trityl group or 9-phenylxanthenyl group which may have an atom or a substituent, and R ² , R ³ and R ⁴ may independently have a hydrogen atom, a halogen atom or a substituent. Good 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, alkoxythiocarbonyl group, arylthiocarbonyl group, aralkylthiocarboro group Represents a group, aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, R ⁵ represents an electron withdrawing group, R ⁶ represents a phosphate protecting group, n represents an integer of 1 or more. ]
The method according to any one of (1) to (4) above, comprising a step of producing the ribonucleotide derivative (II) by treating the ribonucleotide derivative (III) represented by formula (II) with ammonia treatment or primary amine treatment. Manufacturing method.

［式中、Ｒ⁷はフェノキシアセチル基、フェニルアセチル基、アセチル基又はベンゾイル基を表し、Ｒ⁸はイソブチリル基又はベンゾイル基を表し、Ｒ⁹はフェノキシアセチル基、フェニルアセチル基、アセチル基又はイソブチリル基を表し、Ｒ¹⁰は２−シアノエチル基を表し、Ｒ¹¹はベンゾイル基、４−メトキシベンゾイル基又は４−メチルベンゾイル基を表し、Ｒ¹²はジメチルアミノメチレン基を表す。］
で表される保護基を有する核酸塩基である前記（５）記載の製造方法。 (6) The nucleobase represented by B in the formula (III) is represented by the following formula:

[Wherein R ⁷ represents a phenoxyacetyl group, a phenylacetyl group, an acetyl group or a benzoyl group, R ⁸ represents an isobutyryl group or a benzoyl group, and R ⁹ represents a phenoxyacetyl group, a phenylacetyl group, an acetyl group or an isobutyryl group. R ¹⁰ represents a 2-cyanoethyl group, R ¹¹ represents a benzoyl group, a 4-methoxybenzoyl group or a 4-methylbenzoyl group, and R ¹² represents a dimethylaminomethylene group. ]
The production method according to the above (5), which is a nucleobase having a protecting group represented by the formula:

(7) The manufacturing method in any one of said (1)-(6) including the process of removing the protecting group of 5 'hydroxyl group represented by R < ¹ > by acid treatment.

［式中、Ｂは核酸塩基又はその誘導体を表し、Ｗは酸素原子又は硫黄原子を表し、Ｙ及びＺは互いに独立して水素原子、固相担体又は水酸基の保護基を表し、Ｒ¹は水素原子又は置換基を有していてもよいトリチル基若しくは９−フェニルキサンテニル基を表し、Ｒ²、Ｒ³及びＲ⁴は互いに独立して水素原子、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、Ｒ⁵は電子吸引基を表し、Ｒ⁶はリン酸保護基を表し、ｎは１以上の整数を表す。］
で表されるリボヌクレオチド誘導体（III）を、無水溶媒中、シリル化剤存在下で第３級アミン処理又はホスファゼン塩基処理した後、中性条件又は酸性条件下で加水分解することにより、
次式（Ｉ）：

［式中、Ｂ、Ｘ、Ｙ、Ｚ、Ｒ¹及びｎは前記と同義である。］
で表されるリボヌクレオチド又はリボヌクレオチド誘導体（Ｉ）を製造する工程を含む、リボヌクレオチド又はリボヌクレオチド誘導体（Ｉ）の製造方法。 (8) The following formula (III):

[Wherein, B represents a nucleobase or a derivative thereof, W represents an oxygen atom or a sulfur atom, Y and Z each independently represent a hydrogen atom, a solid phase carrier or a hydroxyl-protecting group, and R ¹ represents hydrogen. Represents a trityl group or 9-phenylxanthenyl group which may have an atom or a substituent, and R ² , R ³ and R ⁴ may independently have a hydrogen atom, a halogen atom or a substituent. Good 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, alkoxythiocarbonyl group, arylthiocarbonyl group, aralkylthiocarboro group Represents a group, aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, R ⁵ represents an electron withdrawing group, R ⁶ represents a phosphate protecting group, n represents an integer of 1 or more. ]
The ribonucleotide derivative (III) represented by the formula (1) is treated with a tertiary amine or phosphazene base in an anhydrous solvent in the presence of a silylating agent, and then hydrolyzed under neutral or acidic conditions.
Formula (I):

[Wherein, B, X, Y, Z, R ¹ and n are as defined above. ]
The manufacturing method of ribonucleotide or ribonucleotide derivative (I) including the process of manufacturing the ribonucleotide or ribonucleotide derivative (I) represented by these.

(9) The method according to (8) above, wherein 1,8-diazabicyclo [5.4.0] -7-undecene is used as the tertiary amine.

(10) The production method according to the above (8) or (9), comprising a step of removing the protecting group for the 5 ′ hydroxyl group represented by R ¹ by acid treatment.

FIG. 1 shows 2′-O- [1- (2-cyanoethoxy) ethyl]]-3 ′, 5′-O- (1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) Synthesis steps of uridine (2) and 2′-O- [1- (2-cyanoethoxy) ethyl]]-5′-O- (4,4′-dimethoxytrityl) -uridine (4) FIG. FIG. 2 shows 2′-O- [1- (2-cyanoethoxy) ethyl]]-5′-O- (4,4′-dimethoxytrityl) -uridine-3 ′-(2-cyanoethyl-N, N FIG. 3 is a view showing a synthesis step of (-diisopropyl phosphoramidite) (5). FIG. 3 is a diagram showing a synthesis process of a protected uridylic acid dimer by a liquid phase method. FIG. 4 is a diagram showing a deprotection step of a uridylic acid dimer.

  In the above formula (I), (II) or (III), the nucleobase represented by B or a derivative thereof is not particularly limited. Examples of the nucleobase include pyrimidine bases such as cytosine and uracil, adenine And purine bases such as guanine. Examples of nucleobase derivatives include 5-methylcytosine, 5-hydroxymethylcytosine, 5-fluorouracil, thiouracil, 6-azauracil, 5-hydroxyuracil, and 2,6-diamino. Examples include base analogs such as purine, azaadenine, azaguanine, and isoguanine. Nucleobase or derivatives thereof are, for example, halogen atoms, alkyl groups, haloalkyl groups, alkenyl groups, haloalkenyl groups, alkynyl groups, amino groups, alkylamino groups, hydroxyl groups, hydroxyamino groups, aminooxy groups, alkoxy groups, mercapto groups , May have a substituent such as an alkyl mercapto group, an aryl group, an aryloxy group, or a cyano group. The nucleobase or a derivative thereof may have a protecting group. Examples of the protecting group for the amino group include aliphatic acyl groups such as an acetyl group, a propionyl group, a butyryl group, and an isobutyryl group; a benzoyl group, 4-methyl Aromatic acyl groups such as benzoyl group, 4-methoxybenzoyl group, phenylacetyl group, phenoxyacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropylphenoxyacetyl group; halogen atoms, alkyl groups, alkyloxy groups, etc. An aliphatic acyl group or aromatic acyl group having a substituent; an electron that can be removed by β-elimination such as 2-cyanoethyl group, 2- (p-nitrophenyl) ethyl group, 2- (benzenesulfonyl) ethyl group, etc. Ethyl group having a suction group; dialkylamino such as dimethylaminomethylene group and dibutylaminomethylene group An methylene group; an alkoxycarbonyl group having an electron-withdrawing group that can be removed by β-elimination such as 2-cyanoethoxycarbonyl group, 2- (p-nitrophenyl) ethoxycarbonyl group, 2- (benzenesulfonyl) ethoxycarbonyl group, etc. Etc.

The protecting group for the 2 ′ hydroxyl group of the ribonucleotide derivative (III) is the removal of the protecting group of the nucleobase or its derivative, the removal of the phosphate protecting group represented by R ⁶ , and the excision of the nucleotide chain from the solid phase carrier. Although it is stable with respect to ammonia or primary amine generally used in the case of, etc., there is a possibility that it may be slightly detached when the treatment time with ammonia or primary amine is long. Therefore, when the nucleobase represented by B in the above formula (III) or a derivative thereof has a protecting group, the protecting group is represented by R ⁶ by ammonia treatment or primary amine treatment of the nucleotide derivative (III). It can be removed in a short time together with the phosphate protecting group. Examples of such protecting groups include aliphatic acyl groups, aromatic acyl groups, aliphatic acyl groups having substituents or aromatic acyl groups as described above, and electron withdrawing groups that can be removed by β elimination. And an ethyl group having a dialkylaminomethylene group. The nucleobase having such a protecting group or a derivative thereof is exemplified below.

［式中、Ｒ⁷はフェノキシアセチル基、フェニルアセチル基、アセチル基又はベンゾイル基を表し、Ｒ⁸はイソブチリル基又はベンゾイル基を表し、Ｒ⁹はフェノキシアセチル基、フェニルアセチル基、アセチル基又はイソブチリル基を表し、Ｒ¹⁰は２−シアノエチル基を表し、Ｒ¹¹はベンゾイル基、４−メトキシベンゾイル基又は４−メチルベンゾイル基を表し、Ｒ¹²はジメチルアミノメチレン基を表す。］

[Wherein R ⁷ represents a phenoxyacetyl group, a phenylacetyl group, an acetyl group or a benzoyl group, R ⁸ represents an isobutyryl group or a benzoyl group, and R ⁹ represents a phenoxyacetyl group, a phenylacetyl group, an acetyl group or an isobutyryl group. R ¹⁰ represents a 2-cyanoethyl group, R ¹¹ represents a benzoyl group, a 4-methoxybenzoyl group or a 4-methylbenzoyl group, and R ¹² represents a dimethylaminomethylene group. ]

  Uracil or its derivatives usually do not require a protecting group, but by introducing a benzoyl group, a 4-methoxybenzoyl group or a 4-methylbenzoyl group, the lipophilicity and crystallinity of the synthetic intermediate are improved, and a silica gel column Purification and crystallization by chromatography are facilitated.

The nucleobase represented by B or a derivative thereof can be removed by the elimination of an aliphatic acyl group, aromatic acyl group, substituted aliphatic acyl group or aromatic acyl group as described above as a protecting group, or β In the case of having an ethyl group having an electron withdrawing group, a dialkylaminomethylene group or the like, the protective group can be removed by ammonia treatment or primary amine treatment. In addition, when the nucleobase represented by B or a derivative thereof has a trityl group such as a dimethoxytrityl group or a monomethoxytrityl group as a protective group, the protective group can be removed by acid treatment. In addition, when the nucleobase represented by B or a derivative thereof has an alkoxycarbonyl group having an electron withdrawing group that can be removed by β elimination as described above, the protecting group is treated with a tertiary amine. Alternatively, it can be removed by phosphazene base treatment.

  The above formula (I), (II) or (III) includes a plurality of nucleobases represented by B or derivatives thereof, but these may be the same type or different types. There may be.

In the above formula (I), (II) or (III), the trityl group or 9-phenylxanthenyl group represented by R ¹ may have a substituent or may not have a substituent. Also good. The trityl group or 9-phenylxanthenyl group represented by R ¹ is a protecting group for the 5 ′ hydroxyl group, and the protecting group can be removed by acid treatment.

  The trityl group can have a substituent on one or more of the three phenyl groups. The trityl group or 9-phenylxanthenyl group can have a substituent at any one or more of the 2-position, 3-position or 4-position of the phenyl group. When the trityl group or 9-phenylxanthenyl group has a plurality of substituents, the types of the substituents may be the same or different.

  The substituent that the trityl group or 9-phenylxanthenyl group has is not particularly limited, and specific examples thereof include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, alkyl groups such as s-butyl group, t-butyl group, n-pentyl group, isopentyl group, t-pentyl group, neopentyl group; methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, Examples thereof include alkoxy groups such as isobutoxy group, s-butoxy group, and t-butoxy group.

  Examples of the trityl group having a substituent include a 4-methoxytrityl group, 4,4′-dimethoxytrityl group, 4,4 ′, 4 ″ -trimethoxytrityl group, 4-methyltrityl group, 4,4 ′. -A dimethyltrityl group etc. are mentioned, Among these, a 4,4'- dimethoxytrityl group is preferable.

  Examples of the 9-phenylxanthenyl group having a substituent include a 9- (4′-methoxyphenyl) xanthenyl group and a 9- (4′-methylphenyl) xanthenyl group. A (4′-methoxyphenyl) xanthenyl group is preferred.

In the above formula (II) or (III), the halogen atom represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include fluorine atom, chlorine atom, bromine atom and the like. Can be mentioned.

In the above formula (II) or (III), the alkyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include a methyl group, an ethyl group, and an n-propyl group. Linear or branched having 1 to 5 carbon atoms such as isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, isopentyl group, t-pentyl group, neopentyl group, etc. A chain alkyl group is exemplified.

In the above formula (II) or (III), the alkenyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include vinyl group, allyl group, crotyl (2- Butenyl) groups and alkenyl groups having 2 to 5 carbon atoms such as isopropenyl (1-methylvinyl) group.

In the above formula (II) or (III), the alkynyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include an ethynyl group, a 1-propynyl group, and a propargyl group. C2-C5 alkynyl groups, such as these, are mentioned.

In the above formula (II) or (III), the cycloalkyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. And a cycloalkyl group having 3 to 6 carbon atoms.

In the above formula (II) or (III), the cycloalkenyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include the number of carbon atoms such as cyclopentenyl and cyclohexenyl. Examples include 5-6 cycloalkenyl groups.

In the above formula (II) or (III), the aryl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include a phenyl group, a p-methoxyphenyl group, 3 , 5-dimethoxyphenyl group, p-chlorophenyl group, p-fluorophenyl group, 3,5-dimethylphenyl group, 2,4,6-trimethylphenyl group, naphthyl group and other substituted or unsubstituted aromatic hydrocarbon groups Substituted or non-substituted such as furyl group, thienyl group, pyridyl group, pyrrolyl group, oxazolyl group, isoxazolyl group, thiazolyl group, isothiazolyl group, imidazolyl group, pyrazolyl group, pyrimidinyl group, pyridazinyl group, pyralidinyl group, quinolyl group, isoquinolyl group Examples thereof include substituted aromatic heterocyclic groups.

In the above formula (II) or (III), the aralkyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include benzyl group, α-methylbenzyl group, phenethyl group. And an aralkyl group such as a group.

In the above formula (II) or (III), the acyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include an acetyl group, a trifluoroacetyl group, and a propionyl group. An aliphatic acyl group having 1 to 5 carbon atoms such as butyryl group, isobutyryl group, pivaloyl group; benzoyl group, 3,5-dimethylbenzoyl group, 2,4,6-trimethylbenzoyl group, 2,6-dimethoxybenzoyl group 2,4,6-trimethoxybenzoyl group, 2,6-diisopropoxybenzoyl group, naphthylcarbonyl group, anthrylcarbonyl group and the like.

In the above formula (II) or (III), the alkoxy group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include a methoxy group, an ethoxy group, and an n-propoxy group. C1-C5 linear or branched alkoxy groups such as isopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group and t-butoxy group.

In the above formula (II) or (III), the aryloxy group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include aryl such as phenoxy group and naphthyloxy group. An oxy group is mentioned.

In the above formula (II) or (III), the aralkyloxy group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include benzyloxy group, phenethyloxy group and the like. An aralkyloxy group is mentioned.

In the above formula (II) or (III), the alkoxycarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, n Examples include alkoxycarbonyl groups such as -butoxycarbonyl group and n-octyloxycarbonyl group.

In the above formula (II) or (III), the aryloxycarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include phenoxycarbonyl group, naphthyloxycarbonyl And aryloxycarbonyl groups such as a group.

In the above formula (II) or (III), the aralkyloxycarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include benzyloxycarbonyl group, phenethyloxycarbonyl And an aralkyloxycarbonyl group such as a group.

In the above formula (II) or (III), the alkylthiocarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include a methylthiocarbonyl group, an ethylthiocarbonyl group, Examples thereof include alkylthiocarbonyl groups such as n-butylthiocarbonyl group and n-octylthiocarbonyl group.

In the above formula (II) or (III), the alkoxythiocarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include methoxythiocarbonyl group, ethoxythiocarbonyl group And alkoxythiocarbonyl groups such as n-butoxythiocarbonyl group and n-octyloxythiocarbonyl group.

In the above formula (II) or (III), the arylthiocarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include phenylthiocarbonyl group, naphthylthiocarbonyl group And arylthiocarbonyl group such as a group.

In the above formula (II) or (III), the aralkylthiocarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include benzylthiocarbonyl group, phenethylthiocarbonyl group And an aralkylthiocarbonyl group such as a group.

In the above formula (II) or (III), the aryloxythiocarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include phenoxythiocarbonyl group, naphthyloxy An aryloxythiocarbonyl group such as a thiocarbonyl group can be mentioned.

In the above formula (II) or (III), the aralkyloxythiocarbonyl group represented by R ² , R ³ or R ⁴ is not particularly limited, and specific examples thereof include benzyloxythiocarbonyl group, phenethyl An aralkyloxythiocarbonyl group such as an oxythiocarbonyl group may be mentioned.

In the above formula (II) or (III), an alkyl group, alkenyl group, alkynyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl group, acyl group, alkoxy represented by R ² , R ³ or R ⁴ Group, aryloxy group, aralkyloxy group, alkoxycarbonyl group, aryloxycarbonyl group, aralkyloxycarbonyl group, alkylthiocarbonyl group, alkoxythiocarbonyl group, arylthiocarbonyl group, aralkylthiocarbonyl group, aryloxythiocarbonyl group or aralkyl The oxythiocarbonyl group may have a substituent or may not have a substituent.

In the formula (II) or (III), and R ² (or R ³⁾ and R ^4, linked to one another, R ² (or R ³⁾ and jointly with the carbon atom to which R ⁴ is attached, respectively To form a ring.

In the above formula (II) or (III), the electron withdrawing group (electron withdrawing group) represented by R ⁵ is not particularly limited, and specific examples thereof include a cyano group, a nitro group, and p-nitro. Phenyl group, alkylsulfonyl group (eg, methylsulfonyl group, trifluoromethylsulfonyl group, ethylsulfonyl group, etc.), arylsulfonyl group (eg, phenylsulfonyl etc.), trifluoromethyl group, halogen atom, alkoxycarbonyl (eg, methoxy) Carbonyl, ethoxycarbonyl, etc.), hydroxycarbonyl, aryloxycarbonyl (eg, phenoxycarbonyl, etc.) and the like, and a cyano group is preferred.

In the above formula (III), the phosphate protecting group represented by R ⁶ is not particularly limited, and specific examples thereof include a methyl group, 2-cyanoethyl group, 2- (trimethylsilyl) ethyl group, 2- (P-nitrophenyl) ethyl group and the like can be mentioned, and among these, a methyl group or a 2-cyanoethyl group is preferable.

The above formula (I) or (II) includes a plurality of oxoanions (O ⁻ ) or thioanions (S ⁻ ) represented by X, and these may be the same kind of anions. Different types of anions may be used.

  Although the formula (III) includes a plurality of oxygen atoms or sulfur atoms represented by W, these may be the same type of atom or different types of atoms.

In the above formula (I), (II) or (III), the hydroxyl protecting group represented by Y and Z is not removed under the condition that the 5 ′ hydroxyl protecting group represented by R ¹ is removed. Although not particularly limited, specific examples thereof include silyl groups such as t-butyldimethylsilyl group and t-butyldiphenylsilyl group; levulinyl group, acetyl group, phenylacetyl group, phenoxyacetyl group, butyryl group, Examples include acyl groups such as propionyl group and benzoyl group; cyclic protecting groups such as tetrahydropyranyl and tetrahydrofuranyl; 2′3′-cyclic acetal type protecting groups such as isopropylidene group, methoxymethylidene group and benzylidene group. Of these, a phenoxyacetyl group or a t-butyldiphenylsilyl group is preferred. When both Y and Z represent a hydroxyl-protecting group, the hydroxyl-protecting groups represented by Y and Z may be of the same type or different types.

  When the protecting group for the hydroxyl group represented by Y and Z is an acyl group as described above, the protecting group can be removed by ammonia treatment or primary amine treatment. Further, when the protecting group for the hydroxyl group represented by Y and Z is a cyclic protecting group as described above, a 2'3'-cyclic acetal type protecting group or the like, it can be removed by acid treatment with formic acid or the like. Moreover, when the hydroxyl-protecting group represented by Y and Z is a silyl group as described above, the protecting group can be removed by fluoride treatment such as tetrabutylammonium fluoride (TBAF).

  In the above formula (I), (II) or (III), the solid phase carrier represented by Y and Z is not particularly limited. Specific examples thereof include aminoalkylated polymer carriers ( For example, polystyrene), porous spherical glass beads (controlled pore glass: CPG), polyethylene glycol, and the like. The hydroxyl group of the nucleotide and the solid phase carrier represented by Y and Z may be directly bonded, succinate, oxalate, phthalate, 4-carboxyphenyl (diisopropyl) silyl group (A. Kobori, et al. Chem. Lett., 16-17 (2002)) and the like may be linked via a spacer (linker). When Y and Z both represent a solid phase carrier, the solid phase carriers represented by Y and Z may be of the same type or different types.

  When the solid phase carrier represented by Y and Z is bonded to a hydroxyl group through a spacer such as succinate, oxalate, or phthalate, the solid phase carrier is treated with ammonia or primary amine. Can be removed.

  When the solid phase carrier represented by Y and Z is bonded to a hydroxyl group via a 4-carboxyphenyl (diisopropyl) silyl group, the solid phase carrier is treated with fluoride such as tetrabutylammonium fluoride (TBAF). Can be removed.

  In the above formula (I), (II) or (III), n is not particularly limited as long as it is an integer of 1 or more, but is usually an integer of 1 to 100, preferably an integer of 1 to 30.

Hereinafter, the ribonucleotide derivative (I), (II) or (III) in which R ¹ is an optionally substituted trityl group or 9-phenylxanthenyl group is referred to as “ribonucleotide derivative (I-1)”, respectively. ”,“ Ribonucleotide derivative (II-1) ”or“ ribonucleotide derivative (III-1) ”, and ribonucleotide derivative (I), (II) or (III) wherein R ¹ is a hydrogen atom, It is referred to as “ribonucleotide derivative (I-2)”, “ribonucleotide derivative (II-2)” or “ribonucleotide derivative (III-2)”.

［式中、Ｂは前記と同義である。］
で表されるリボヌクレオシド又はリボヌクレオシド誘導体（ａ）をピリジン、Ｎ，Ｎ−ジメチルホルムアミド、Ｎ−メチルピロリドン、テトラヒドロフラン、アセトニトリル等の有機溶媒に溶解し、これに１，３−ジクロロ−１，１，３，３−テトライソプロピルジシロキサン、ジ−tert−ブチルジクロロシラン又はジ−tert−ブチルシリルビス（トリフルオロメタンスルホン酸）を添加するとともに、発生する塩化水素やトリフルオロメタンスルホン酸の捕捉剤としてピリジン、トリエチルアミン等を添加して反応させることにより、次式（ｂ）：

［式中、Ｂは前記と同義であり、Ｒ¹³は１，１，３，３−テトライソプロピルジシロキサン−１，３−ジイル基又はジ−tert−ブチルシランジイル基を表す。］
で表されるリボヌクレオシド誘導体（ｂ）を製造することができる。この際の反応温度は通常０〜４０℃、好ましくは１５〜２５℃であり、反応時間は通常１〜１２時間、好ましくは４〜８時間である。また、１，３−ジクロロ−１，１，３，３−テトライソプロピルジシロキサン、ジ−tert−ブチルジクロロシラン又はジ−tert−ブチルシリルビス（トリフルオロメタンスルホン酸）の添加量は、リボヌクレオシド又はリボヌクレオシド誘導体（ａ）に対して通常１〜２モル当量、好ましくは１〜１.２モル当量であり、捕捉剤の添加量は、リボヌクレオシド又はリボヌクレオシド誘導体（ａ）に対して通常１〜２モル当量、好ましくは１〜１.２モル当量である。 [Production of ribonucleic acid synthesis monomer]
Formula (a):

[Wherein, B has the same meaning as described above. ]
Is dissolved in an organic solvent such as pyridine, N, N-dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, acetonitrile or the like, and 1,3-dichloro-1,1 is dissolved in the ribonucleoside or ribonucleoside derivative (a). , 3,3-tetraisopropyldisiloxane, di-tert-butyldichlorosilane or di-tert-butylsilylbis (trifluoromethanesulfonic acid) and pyridine as a scavenger for the generated hydrogen chloride and trifluoromethanesulfonic acid By adding triethylamine or the like and reacting, the following formula (b):

[Wherein, B is as defined above, and R ¹³ represents a 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl group or a di-tert-butylsilanediyl group. ]
The ribonucleoside derivative (b) represented by these can be manufactured. The reaction temperature at this time is usually 0 to 40 ° C., preferably 15 to 25 ° C., and the reaction time is usually 1 to 12 hours, preferably 4 to 8 hours. In addition, the amount of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, di-tert-butyldichlorosilane or di-tert-butylsilylbis (trifluoromethanesulfonic acid) added may be ribonucleoside or The amount is usually 1 to 2 molar equivalents, preferably 1 to 1.2 molar equivalents, relative to the ribonucleoside derivative (a), and the addition amount of the scavenger is usually 1 to 2 molar equivalents, preferably 1 to 1.2 molar equivalents.

［式中、Ｒ²、Ｒ³、Ｒ⁴及びＲ⁵は前記と同義である。］
で表される化合物（ｃ）を添加するとともに、触媒としてｐ−トルエンスルホン酸水和物、ｐ−トルエンスルホン酸ピリジニウム塩、トリフルオロ酢酸、トリクロロ酢酸、ジクロロ酢酸、塩化水素、硫酸等を添加して反応させることにより、次式（ｄ）：

［式中、Ｂ、Ｒ²、Ｒ³、Ｒ⁴、Ｒ⁵及びＲ¹³は前記と同義である。］
で表されるリボヌクレオシド誘導体（ｄ）を製造することができる。この際の反応温度は通常０〜４０℃、好ましくは１５〜２５℃であり、反応時間は通常１０分間〜１時間、好ましくは１０〜２０である。また、化合物（ｃ）の添加量は、リボヌクレオシド誘導体（ｂ）に対して通常１.１〜１０モル当量、好ましくは１.１〜５モル当量であり、触媒の添加量は、リボヌクレオシド誘導体（ｂ）に対して通常０.１〜１０モル当量、好ましくは１.１〜５モル当量である。 Next, the ribonucleoside derivative (b) is dissolved in an organic solvent such as 1,4-dioxane, tetrahydrofuran, toluene, acetonitrile, and the following formula (c):

[Wherein, R ² , R ³ , R ⁴ and R ⁵ are as defined above. ]
As a catalyst, p-toluenesulfonic acid hydrate, p-toluenesulfonic acid pyridinium salt, trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, hydrogen chloride, sulfuric acid and the like are added as a catalyst. To react the following formula (d):

[Wherein, B, R ² , R ³ , R ⁴ , R ⁵ and R ¹³ are as defined above. ]
The ribonucleoside derivative (d) represented by these can be manufactured. The reaction temperature at this time is usually 0 to 40 ° C., preferably 15 to 25 ° C., and the reaction time is usually 10 minutes to 1 hour, preferably 10 to 20. Moreover, the addition amount of a compound (c) is 1.1 to 10 molar equivalent normally with respect to a ribonucleoside derivative (b), Preferably it is 1.1 to 5 molar equivalent, The addition amount of a catalyst is a ribonucleoside derivative. The amount is usually 0.1 to 10 molar equivalents, preferably 1.1 to 5 molar equivalents, relative to (b).

［式中、Ｂ、Ｒ²、Ｒ³、Ｒ⁴及びＲ⁵は前記と同義である。］
で表されるリボヌクレオシド誘導体（ｅ）を製造することができる。この際の反応温度は通常０〜４０℃、好ましくは１５〜２５℃であり、反応時間は通常１５〜２０時間、好ましくは１６〜１８時間である。また、混合試薬におけるフッ素化剤と酸との混合比は、通常１：１〜１：２、好ましくは１：１〜１：１.５であり、混合試薬の添加量は、リボヌクレオシド誘導体（ｄ）に対して通常２〜１０モル当量、好ましくは３〜５モル当量である。 Next, the ribonucleoside derivative (d) is dissolved in an organic solvent such as tetrahydrofuran, 1,4-dioxane, dichloromethane, acetonitrile, and a fluorinating agent (for example, tetrabutylammonium fluoride, triethylamine trihydrofluoride, hydrogen fluoride pyridine). Etc.) and an acid (for example, acetic acid, hydrochloric acid, sulfuric acid) as a mixed reagent having an arbitrary mixing ratio, the following formula (e):

[Wherein, B, R ² , R ³ , R ⁴ and R ⁵ have the same meanings as described above. ]
The ribonucleoside derivative (e) represented by these can be manufactured. The reaction temperature at this time is usually 0 to 40 ° C., preferably 15 to 25 ° C., and the reaction time is usually 15 to 20 hours, preferably 16 to 18 hours. In addition, the mixing ratio of the fluorinating agent and the acid in the mixed reagent is usually 1: 1 to 1: 2, preferably 1: 1 to 1: 1.5, and the addition amount of the mixed reagent is the ribonucleoside derivative ( It is usually 2 to 10 molar equivalents, preferably 3 to 5 molar equivalents with respect to d).

［式中、Ｒ^1a、Ｒ²、Ｒ³、Ｒ⁴及びＲ⁵は前記と同義である。］
で表されるリボヌクレオシド誘導体（ｆ）を製造することができる。この際の反応温度は通常０〜４０℃、好ましくは１５〜２５℃であり、反応時間は通常４〜１２時間、好ましくは６〜８時間である。また、Ｒ^1a−Ｃｌの添加量は、リボヌクレオシド誘導体（ｅ）に対して通常１〜２モル当量、好ましくは１〜１.１モル当量である。
こうして製造されたリボヌクレオシド誘導体（ｆ）は、リボ核酸合成モノマーであるリボヌクレオシド誘導体（ｈ）を製造するにあたっての中間体である。 Then, the ribonucleoside derivative (e), dissolved in an organic solvent such as pyridine, to which R ^1a -Cl [wherein, R ^1a is an optionally substituted trityl group or 9 phenylxylyl Sante sulfonyl group Represents. ] Is added and reacted, the following formula (f):

[Wherein, R ^1a , R ² , R ³ , R ⁴ and R ⁵ have the same meanings as described above. ]
The ribonucleoside derivative (f) represented by these can be manufactured. The reaction temperature at this time is usually 0 to 40 ° C., preferably 15 to 25 ° C., and the reaction time is usually 4 to 12 hours, preferably 6 to 8 hours. The amount of R ^1a -Cl added is usually 1 to 2 molar equivalents, preferably 1 to 1.1 molar equivalents, relative to the ribonucleoside derivative (e).
The ribonucleoside derivative (f) thus produced is an intermediate for producing the ribonucleoside derivative (h) that is a ribonucleic acid synthesis monomer.

［式中、Ｒ⁶は前記と同義であり、Ｒ¹⁴及びＲ¹⁵は同一の又は異なるアルキル基を表す。］
で表される化合物（ｇ）及び亜リン酸化剤（例えば、２−シアノエチル−Ｎ，Ｎ−ジイソプロピルホスホロクロリダイト、メチル−Ｎ，Ｎ−ジイソプロピルホスホロクロリダイト等）を添加して反応させることにより、次式（ｈ）：

［式中、Ｒ^1a、Ｒ²、Ｒ³、Ｒ⁴、Ｒ⁵、Ｒ⁶、Ｒ¹⁴及びＲ¹⁵は前記と同義である。］
で表されるリボヌクレオシド誘導体（ｈ）を製造することができる。この際の反応温度は通常０〜２５℃、好ましくは１５〜２５℃であり、反応時間は通常１〜３時間、好ましくは２〜３時間である。また、化合物（ｇ）の添加量は、リボヌクレオシド誘導体（ｆ）に対して通常１〜２モル当量、好ましくは１〜１.１モル当量であり、亜リン酸化剤の添加量は、リボヌクレオシド誘導体（ｆ）に対して通常１〜２モル当量、好ましくは１〜１.１モル当量である。 Next, the ribonucleoside derivative (f) is dissolved in an organic solvent such as dichloromethane, tetrahydrofuran, 1,4-dioxane, dichloromethane, acetonitrile, and the following formula (g):

[Wherein, R ⁶ has the same meaning as described above, and R ¹⁴ and R ¹⁵ represent the same or different alkyl groups. ]
(G) and a phosphorylating agent (for example, 2-cyanoethyl-N, N-diisopropyl phosphorochloridite, methyl-N, N-diisopropyl phosphorochloridite, etc.) According to the following formula (h):

[Wherein, R ^1a , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ¹⁴ and R ¹⁵ are as defined above]. ]
The ribonucleoside derivative (h) represented by these can be manufactured. The reaction temperature at this time is usually 0 to 25 ° C., preferably 15 to 25 ° C., and the reaction time is usually 1 to 3 hours, preferably 2 to 3 hours. Moreover, the addition amount of a compound (g) is 1-2 mol equivalent normally with respect to a ribonucleoside derivative (f), Preferably it is 1-1.1 mol equivalent, The addition amount of a phosphorylating agent is ribonucleoside. The amount is usually 1 to 2 molar equivalents, preferably 1 to 1.1 molar equivalents, relative to the derivative (f).

In the above formulas (g) and (h), the alkyl group represented by R ¹⁴ and R ¹⁵ is not particularly limited, and specific examples thereof include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. Linear or branched chain having 1 to 5 carbon atoms such as n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, isopentyl group, t-pentyl group, neopentyl group, etc. Although an alkyl group is mentioned, Of these, an ethyl group or an isopropyl group is preferable. The alkyl groups represented by R ¹⁴ and R ¹⁵ may be the same or different. In addition, the alkyl groups represented by R ¹⁴ and R ¹⁵ may be combined to form a ring.
The ribonucleoside derivative (h) thus produced becomes a monomer for ribonucleic acid synthesis.

［式中、Ｂ、Ｒ^1a、Ｙ及びＺは前記と同義である。］
で表されるリボヌクレオシド誘導体（ｉ）を酸処理することにより、Ｒ^1aで表される５’水酸基の保護基を除去し、５'水酸基の脱保護を行う。この際、酸処理は、例えば、トリフルオロ酢酸、ジクロロ酢酸、トリクロロ酢酸等を１〜５％の濃度になるようにジクロロメタン、クロロホルム、１，２−ジクロロエタン等の有機溶媒に溶解した溶液を用いて行うことができる。当該溶液を用いた処理温度は通常０〜４０℃、好ましくは１５〜２５℃であり、処理時間は通常０.５〜１０分、好ましくは０.５〜１分である。 [Production of Ribonucleotide Derivative (III-1)]
(Step 1) Deprotection of 5 ′ hydroxyl group The following formula (i) obtained according to a conventional method:

[Wherein, B, R ^1a , Y and Z are as defined above. ]
The ribonucleoside derivative (i) represented by the formula (1) is acid-treated to remove the 5′-hydroxyl protecting group represented by R ^1a and deprotect the 5′-hydroxyl. In this case, the acid treatment is performed using, for example, a solution obtained by dissolving trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid or the like in an organic solvent such as dichloromethane, chloroform, 1,2-dichloroethane so as to have a concentration of 1 to 5%. It can be carried out. The processing temperature using the said solution is 0-40 degreeC normally, Preferably it is 15-25 degreeC, and processing time is 0.5-10 minutes normally, Preferably it is 0.5-1 minute.

(Step 2) Activation of phosphoramidite and chain elongation by condensation reaction A proton is given to the ribonucleoside derivative (h) to activate it, and a condensation reaction is performed with the ribonucleoside derivative (i) in which the 5 ′ hydroxyl group is deprotected. At this time, as the reaction solvent, for example, acetonitrile, tetrahydrofuran, dichloromethane or the like can be used, and as the activator, for example, 1H-tetrazole, 5-ethylthiotetrazole, 5-benzylthiotetrazole, 5-nitrophenyl. Tetrazole, 3,4-dicyanoimidazole, 3,4-dichloroimidazole, 3,4-dicyanoimidazole, benzotriazole triflate, imidazole triflate, N-cyanomethylammonium salt and the like can be used. Moreover, reaction temperature is 0-40 degreeC normally, Preferably it is 15-25 degreeC, and reaction time is 5-15 minutes normally, Preferably it is 3-5 minutes.

  As an activator in the condensation reaction, 1H-tetrazole is generally used. However, in the condensation reaction, the reduction of the condensation reaction rate and the condensation reaction yield due to the steric hindrance of the protecting group of the 2 ′ hydroxyl group becomes a problem, so that the condensation reaction rate and the condensation reaction yield are improved as compared with 1H-tetrazole. Expected activators (eg, 5-ethylthiotetrazole, 5-benzylthiotetrazole, 5-nitrophenyltetrazole, 3,4-dicyanoimidazole, 3,4-dichloroimidazole, 3,4-dicyanoimidazole, benzotriazole triflate Imidazole triflate, N-cyanomethylammonium salt, etc.) are preferably used. Examples of the N-cyanomethylammonium salt include N-cyanomethyldimethylammonium tetrafluoroboric acid, N-cyanomethyldimethylammonium hexafluorophosphoric acid, N-cyanomethyldimethylammonium trifluoromethanesulfonic acid, N-cyanomethyldimethylammonium. Bistrifluoromethanesulfonimide, N-cyanomethyldimethylperchloric acid, N-cyanomethylpyrrolidine tetrafluoroboric acid, N-hexafluorophosphoric acid, N-cyanomethylpyrrolidine trifluoromethanesulfonic acid, N-cyanomethylpyrrolidine trifluoromethanesulfone Imido, N-cyanomethylpyrrolidine perchloric acid, N-cyanomethylpiperidinetetrafluoroboric acid, N-cyanomethylpiperidinehexafluorophosphoric acid, N-silane Nomethylpiperidine trifluoromethanesulfonic acid, N-cyanomethylpiperidine trifluoromethanesulfonimide, N-cyanomethylpiperidine perchloric acid, N-cyanomethyldiisopropylammonium tetrafluoroboric acid, N-cyanomethyldiisopropylammonium hexafluorophosphoric acid, N -Cyanomethyldiisopropylammonium trifluoromethanesulfonic acid, N-cyanomethyldiisopropylammonium trifluoromethanesulfonimide, N-cyanomethyldiisopropylammonium perchloric acid, etc. can be used.

  Then, if necessary, the unreacted hydroxyl group is capped. At this time, as a capping reagent, a solution prepared by dissolving 4-dimethylaminopyridine or N-methylimidazole in an arbitrary mixed solvent such as pyridine, acetonitrile, tetrahydrofuran or the like to a concentration of 0.05 to 1M, acetic anhydride , Methoxyacetic anhydride and the like mixed at an appropriate mixing ratio (for example, 9: 1 to 19: 1) can be used. The reaction time using the solution is usually 0 to 40 ° C., preferably 15 to 25 ° C., and the reaction time is usually 0.5 to 5 minutes, preferably 0.5 to 1 minute.

(Step 3) Oxidation of phosphite bond to phosphate triester bond Oxidation of phosphite bond to phosphate triester bond includes, for example, iodine as an inorganic solvent such as pyridine, water, acetonitrile, tetrahydrofuran, organic solvent, etc. Or it can carry out using the solution which melt | dissolved in those arbitrary mixed solvents so that it might become a density | concentration of 0.05-2M, or the solution which melt | dissolved the t-butyl alcohol peroxide in the methylene chloride, toluene. The reaction time using the solution is usually 0 to 40 ° C., preferably 15 to 25 ° C., and the reaction time is usually 0.5 to 5 minutes, preferably 0.5 to 1 minute.

Oxidation of a phosphite bond to a phosphate triester bond should be performed in an anhydrous solvent to prevent hydrolysis of intermediates generated during the oxidation reaction and reduction in condensation reaction efficiency due to H ₂ O in the solvent. Is preferred. Examples of the reagent capable of oxidizing a phosphite bond to a phosphate triester bond in an anhydrous solvent include, for example, t-butyl alcohol peroxide, 2- (phenylsulfonyl) -3- (3-nitrophenyl) oxaziridine, 2- ( Phenylsulfonyl) -3-phenyloxaziridine, (1S)-(+)-camphorsulfonyloxyzaziridine, (1S)-(+)-8,8-dichlorocamphorsulfonyloxyzaziridine, and the like can be used.

［式中、Ｂ、Ｒ^1a、Ｒ²、Ｒ³、Ｒ⁴、Ｒ⁵、Ｒ⁶、Ｙ及びＺは前記と同義である。］
で表されるリボヌクレオチド誘導体（ｊ−１）（ダイマー）を製造することができる。 By the above steps 1 to 3, the following formula (j-1):

[Wherein, B, R ^1a , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , Y and Z are as defined above] ]
The ribonucleotide derivative (j-1) (dimer) represented by these can be manufactured.

［式中、Ｂ、Ｒ^1a、Ｒ²、Ｒ³、Ｒ⁴、Ｒ⁵、Ｒ⁶、Ｙ及びＺは前記と同義である。］
で表されるリボヌクレオチド誘導体（ｊ−２）（ダイマー）を製造することができる。硫化反応は、例えば、硫黄、Beaucage試薬（３Ｈ−１,２−ベンゾジチオール−３−オン−１，１−ジオキシド）等を用いて行うことができる。 Moreover, in the said process 3, by performing a sulfurization reaction instead of an oxidation reaction, following Formula (j-2):

[Wherein, B, R ^1a , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , Y and Z are as defined above] ]
The ribonucleotide derivative (j-2) (dimer) represented by these can be manufactured. The sulfurization reaction can be performed using, for example, sulfur, Beaucage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide) or the like.

［式中、Ｂ、Ｒ^1a、Ｒ²、Ｒ³、Ｒ⁴、Ｒ⁵、Ｒ⁶、Ｗ、Ｙ及びＺは前記と同義である。]
で表されるリボヌクレオチド誘導体（ｋ）（トリマー）を製造することができる。
同様にして上記工程１〜３を繰り返すことにより、リボヌクレオチド誘導体（III−１）（オリゴマー）を製造することができる。
上記工程１〜３は、市販の核酸合成機を用いて行ってもよい。 Next, by reacting the ribonucleotide derivative (j-1) or (j-2) (dimer) with the ribonucleoside derivative (h) (monomer) according to the above steps 1 to 3, the following formula (k):

[Wherein, B, R ^1a , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , W, Y, and Z are as defined above]. ]
The ribonucleotide derivative (k) (trimer) represented by these can be manufactured.
Similarly, by repeating the above steps 1 to 3, the ribonucleotide derivative (III-1) (oligomer) can be produced.
The above steps 1 to 3 may be performed using a commercially available nucleic acid synthesizer.

[Production of ribonucleotide derivative (III-2)]
The ribonucleotide derivative (III-2) can be produced by acid-treating the ribonucleotide derivative (III-1) to remove the 5 ′ hydroxyl protecting group represented by R ^1a . As the acid, for example, trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid and the like can be used, but dichloroacetic acid is preferably used. Specifically, the acid treatment can be performed using a solution in which the above acid is dissolved in dichloromethane, chloroform, 1,2-dichloroethane or the like so as to have a concentration of 1 to 5%. The processing temperature using the said solution is 0-40 degreeC normally, Preferably it is 15-25 degreeC, and processing time is 0.5 to 5 minutes normally, Preferably it is 0.5 to 1 minute.

In the ribonucleotide derivative (III-1), when the nucleobase represented by B or a derivative thereof has a trityl group such as a dimethoxytrityl group or a monomethoxytrityl group as a protective group, the protective group is a nucleotide derivative (III- It is removed together with the protecting group for the 5 ′ hydroxyl group represented by R ^1a by the acid treatment of 1).

[Production of Ribonucleotide Derivative (II-1)]
The ribonucleotide derivative (II-1) can be produced by treating the ribonucleotide derivative (III-1) with a base and removing the phosphate protecting group represented by R ⁶ . As the base, a base that removes the phosphate protecting group represented by R ⁶ but does not remove the protecting group of the 2 ′ hydroxyl group can be used. As such a base, for example, ammonia, a primary amine or the like can be used, and as the primary amine, for example, a primary alkylamine such as methylamine, ethylamine or propylamine can be used. . Specifically, the base treatment can be performed using a solution in which the above base is dissolved in water and ethanol so as to have a concentration of 15 to 30%. The processing temperature using the said solution is 0-50 degreeC normally, Preferably it is 10-25 degreeC, and processing time is 1 to 8 hours normally, Preferably it is 1-2 hours.

In the ribonucleotide derivative (III-1), the nucleobase represented by B or a derivative thereof is an aliphatic acyl group such as an acetyl group, a propionyl group, a butyryl group, and an isobutyryl group as a protecting group; a benzoyl group, a 4-methylbenzoyl group Aromatic acyl groups such as 4-methoxybenzoyl group, phenylacetyl group, phenoxyacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropylphenoxyacetyl group; substituents such as halogen atom, alkyl group, alkyloxy group An electron-withdrawing group that can be removed by β elimination such as 2-cyanoethyl group, 2- (p-nitrophenyl) ethyl group, 2- (benzenesulfonyl) ethyl group, etc. An ethyl group having a dimethylaminomethylene group, a dibutylaminomethylene group or the like When having aminomethylene groups, the protecting groups, the ammonia treatment or a primary amine treatment nucleotide derivative (III-1), it is removed with phosphoric acid protecting group represented by R ^6.

In the ribonucleotide derivative (III-1), the hydroxyl protecting group represented by Y and Z is an acyl group such as a levulinyl group, an acetyl group, a phenylacetyl group, a phenoxyacetyl group, a butyryl group, a propionyl group, or a benzoyl group. In this case, the protecting group is removed together with the phosphate protecting group represented by R ⁶ by ammonia treatment or primary amine treatment of the nucleotide derivative (III-1).

In the ribonucleotide derivative (III-1), when the solid phase carrier represented by Y and Z is bonded to a hydroxyl group via a spacer such as succinate, oxalate or phthalate, the solid phase carrier Is removed together with the phosphate protecting group represented by R ⁶ by ammonia treatment or primary amine treatment of the ribonucleotide derivative (III-1).

[Production of Ribonucleotide Derivative (II-2)]
(1) First production method The ribonucleotide derivative (II-2) can be produced by treating the ribonucleotide derivative (III-2) with a base and removing the phosphate protecting group represented by R ^6. it can. The base treatment can be carried out in the same manner as in [Production of ribonucleotide derivative (II-1)].

In the ribonucleotide derivative (III-2), the nucleobase represented by B or a derivative thereof is an aliphatic acyl group such as acetyl group, propionyl group, butyryl group, isobutyryl group; benzoyl group, 4-methylbenzoyl group, 4- Aromatic acyl groups such as methoxybenzoyl group, phenylacetyl group, phenoxyacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropylphenoxyacetyl group; fats having substituents such as halogen atom, alkyl group, alkyloxy group Ethyl having an electron-withdrawing group that can be removed by β elimination such as 2-cyanoethyl group, 2- (p-nitrophenyl) ethyl group, 2- (benzenesulfonyl) ethyl group, etc. Group: Dialkylaminomethyl such as dimethylaminomethylene group and dibutylaminomethylene group If having a down group, the protecting group is by ammonia treatment or a primary amine treatment nucleotide derivative (III-2), is removed with phosphoric acid protecting group represented by R ^6.

In the ribonucleotide derivative (III-2), the hydroxyl protecting group represented by Y and Z is an acyl group such as a levulinyl group, an acetyl group, a phenylacetyl group, a phenoxyacetyl group, a butyryl group, a propionyl group, or a benzoyl group. In this case, the protecting group is removed together with the phosphate protecting group represented by R ⁶ by ammonia treatment or primary amine treatment of the nucleotide derivative (III-2).

In the ribonucleotide derivative (III-2), when the solid phase carrier represented by Y and Z is bonded to a hydroxyl group via a spacer such as succinate, oxalate, or phthalate, the solid phase carrier Is removed together with the phosphate protecting group represented by R ⁶ by ammonia treatment or primary amine treatment of the ribonucleotide derivative (III-2).

(2) Second Production Method Ribonucleotide derivative (II-2) is produced by acid-treating ribonucleotide derivative (II-1) and removing the protecting group for the 5 ′ hydroxyl group represented by R ^1a. be able to. The acid treatment can be carried out in the same manner as in [Production of ribonucleotide derivative (III-2)].
In the ribonucleotide derivative (II-1), when the nucleobase represented by B or a derivative thereof has a trityl group such as a dimethoxytrityl group or a monomethoxytrityl group as a protective group, the protective group is a ribonucleotide derivative (II It is removed together with the protecting group for the 5 ′ hydroxyl group represented by R ^1a by the acid treatment of -1).

[Production of Ribonucleotide Derivative (I-1)]
(1) First production method The ribonucleotide derivative (I-1) can be produced by treating the ribonucleotide derivative (II-1) with a fluoride to remove the protecting group of the 2 'hydroxyl group. As the fluoride, for example, tetrabutylammonium fluoride (TBAF), a mixed reagent of potassium fluoride and 18-crown-6, or the like can be used, but TBAF is preferably used. This is because when TBAF is used, the protecting group for the 2 ′ hydroxyl group can be removed in a short time. When a mixed reagent of potassium fluoride and 18-crown-6 is used, potassium ions are captured by the crown ether and fluoride ions are liberated, and the liberated fluoride ions are converted into 2 ′ hydroxyl groups as in TBAF. Protecting groups can be removed. Specifically, the fluoride treatment can be performed using a solution or the like in which TBAF is dissolved in tetrahydrofuran or the like so as to have a concentration of 0.5 to 1M. The treatment temperature using the solution is usually from 0 to 60 ° C., preferably from 15 to 25 ° C., and the treatment time is usually from 0.1 to 30 hours, preferably from 0.2 to 0.5 hours.

  In the ribonucleotide derivative (II-1), when the protecting group for the hydroxyl group represented by Y and Z is a silyl group such as t-butyldimethylsilyl group or t-butyldiphenylsilyl group, the protecting group is a ribonucleotide. The derivative (II-1) is removed together with a protecting group for the 2 'hydroxyl group by fluoride treatment.

  In the ribonucleotide derivative (II-1), when the solid phase carrier represented by Y and Z is bonded to a hydroxyl group via a 4-carboxyphenyl (diisopropyl) silyl group, the solid phase carrier is a ribonucleotide derivative. By the fluoride treatment of (II-1), it is removed together with a protecting group for the 2 ′ hydroxyl group.

(2) Second production method The ribonucleotide derivative (I-1) is hydrolyzed under neutral conditions after the ribonucleotide derivative (II-1) is treated with a base in the presence of a silylating agent in an anhydrous solvent. And can be produced by removing the protecting group for the 2 ′ hydroxyl group. As the base, for example, an organic base such as a tertiary amine, a phosphazene base (Schwesinger base), or the like can be used. In addition, when a primary amine or secondary amine and a silylating agent are used in combination, it is converted into the corresponding silylamine and is not effective because the basicity of the primary amine or secondary amine is extremely reduced. Conceivable. As the tertiary amine, for example, 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), diisopropylethylamine, triethylamine, tributylamine, N-methylpiperidine and the like can be used. Is preferably used. This is because when DBU is used, the 2′-hydroxyl protecting group can be removed in a short time. Examples of the phosphazene base include 2-tert-butylimino-2-diethylamino-1,3-dimethyl-1,3,2-diazaphosphorinane, (tert-butylimino) tris (dimethylamino) phosphorane, tert-butyltris [ Tris (dimethylamino) phosphoranylidene] phosphorimidic triamide and the like can be used. As the silylating agent, for example, a trimethylsilylating agent such as N, O-bistrimethylsilylacetamide, N, O-bistrimethylsilyltrifluoroacetamide, N, O-bistrimethylsilylbenzamide or the like can be used. As the anhydrous solvent, for example, acetonitrile, nitromethane, tetrahydrofuran, dimethylformamide and the like can be used. Specifically, the base treatment is performed using a solution in which 1,8-diazabicyclo [5.4.0] -7-undecene (DBU) is dissolved in acetonitrile or the like so as to have a concentration of 0.5 to 1M. It can be carried out. The treatment temperature using the solution is usually from 0 to 60 ° C., preferably from 15 to 25 ° C., and the treatment time is usually from 0.1 to 30 hours, preferably from 0.2 to 0.5 hours. Hydrolysis is performed under neutral conditions after base treatment. Hydrolysis can be carried out, for example, after neutralizing the reaction solvent after the base treatment by adding an acid and removing the base remaining in the reaction solvent. Although the acid is not particularly limited, for example, an acetonitrile solution of acetic acid, an acetonitrile solution of hydrogen chloride, or the like can be used. It is necessary to neutralize the remaining base with an acid under anhydrous conditions.

  When the base treatment is carried out in the absence of a silylating agent, the deprotected 2 'hydroxyl group nucleophilically attacks the phosphorus atom of the phosphate ester, resulting in the degradation of the nucleotide chain. Therefore, base treatment is performed in the presence of a silylating agent. The silylating agent suppresses an attack on the adjacent phosphorus atom of the hydroxyl group by silylating the 2′-O-2-hydroxyethyl group generated as an intermediate of the 2 ′ hydroxyl group or the deprotection reaction. Is prevented from being decomposed.

By subjecting the ribonucleotide derivative (II-1) to base treatment in the presence of a silylating agent in an anhydrous solvent, the phosphate ester is converted to a phosphate silyl ester, and the 2 ′ hydroxyl group or intermediate of the deprotection reaction The 2′-O-2-hydroxyethyl group produced as a body is silylated. When water is added, silyl phosphate is immediately hydrolyzed and converted to free phosphate. When water is added, the silylated 2 ′ hydroxyl group is rapidly hydrolyzed by the intramolecular acid catalysis of the adjacent free phosphoric acid and converted to the free 2 ′ hydroxyl group. When the 2′-O-2-hydroxyethyl group produced as an intermediate of the deprotection reaction is silylated and converted to a 2-trimethylsiloxyethyl group, the silyl ether is extremely unstable, so water is added. Then, it is immediately hydrolyzed and converted into an unstable 2-hydroxyethyl group, and then immediately decomposed with elimination of acetaldehyde and converted into a free 2 ′ hydroxyl group. The hydrolysis treatment can also be carried out under acidic conditions. However, when the hydrolysis treatment is carried out under acidic conditions, the 5′-hydroxyl protecting group represented by R ^1a is removed, so that a ribonucleotide derivative (I- In the production method 1), hydrolysis is performed under neutral conditions. Neutral conditions are usually pH 7.0 and acidic conditions are usually pH 2.0-6.9.

  In the ribonucleotide derivative (II-1), the protecting group of the nucleic acid represented by B or a derivative thereof is 2-cyanoethoxycarbonyl group, 2- (p-nitrophenyl) ethoxycarbonyl group, 2- (benzenesulfonyl) ethoxy In the case of an alkoxycarbonyl group having an electron withdrawing group that can be removed by β-elimination such as a carbonyl group, the protecting group is removed by tertiary amine treatment or phosphazene base treatment of the ribonucleotide derivative (II-1). Is done.

(3) Third production method The ribonucleotide derivative (I-1) is hydrolyzed under neutral conditions after the ribonucleotide derivative (III-1) is treated with a base in the presence of a silylating agent in an anhydrous solvent. The phosphoric acid protecting group represented by R ⁶ and the protecting group for the 2 ′ hydroxyl group can be removed. The base treatment in the presence of a silylating agent in an anhydrous solvent and the hydrolysis treatment under neutral conditions can be carried out in the same manner as in the second production method.

  In the ribonucleotide derivative (III-1), the protecting group of the nucleic acid represented by B or its derivative is 2-cyanoethoxycarbonyl group, 2- (p-nitrophenyl) ethoxycarbonyl group, 2- (benzenesulfonyl) ethoxy In the case of an alkoxycarbonyl group having an electron-withdrawing group that can be removed by β-elimination such as a carbonyl group, the protecting group is removed by tertiary amine treatment or phosphazene base treatment of the ribonucleotide derivative (III-1). Is done.

[Production of Ribonucleotide Derivative (I-2)]
(1) First Production Method The ribonucleotide derivative (I-2) can be produced by treating the ribonucleotide derivative (II-2) with a fluoride and removing the protecting group for the 2 ′ hydroxyl group. The fluoride treatment can be carried out in the same manner as in [Production of ribonucleotide derivative (I-1)].

  In the ribonucleotide derivative (II-2), when the protecting group for the hydroxyl group represented by Y and Z is a silyl group such as t-butyldimethylsilyl group, t-butyldiphenylsilyl group, the protecting group is ribonucleotide The derivative (II-2) is removed together with the protecting group for the 2 ′ hydroxyl group by fluoride treatment.

  In the ribonucleotide derivative (II-2), when the solid phase carrier represented by Y and Z is bonded to a hydroxyl group via a 4-carboxyphenyl (diisopropyl) silyl group, the solid phase carrier is a ribonucleotide derivative It is removed together with the protecting group for the 2 ′ hydroxyl group by the fluoride treatment of (II-2).

(2) Second Production Method The ribonucleotide derivative (I-2) is produced by acid-treating the ribonucleotide derivative (I-1) and removing the protecting group for the 5 ′ hydroxyl group represented by R ^1a. be able to. At this time, it is preferable to perform an acid treatment so as not to decompose the nucleotide chain. Such an acid treatment is, for example, an aqueous solution in which acetic acid or the like is dissolved in water to a concentration of 80%, pH 1.5-2. 5 (preferably pH 2.0) aqueous solution (for example, hydrochloric acid aqueous solution-dioxane mixed solution) or the like. The processing temperature using the said solution is 0-50 degreeC normally, Preferably it is 20-30 degreeC, and processing time is 10-120 minutes normally, Preferably it is 20-40 minutes.

In the ribonucleotide derivative (I-1), when the nucleobase represented by B or a derivative thereof has a trityl group such as a dimethoxytrityl group or a monomethoxytrityl group as a protective group, the protective group is a ribonucleotide derivative (I It is removed together with the protecting group for the 5 ′ hydroxyl group represented by R ^1a by the acid treatment of -1).

(3) Third production method The ribonucleotide derivative (I-2) is prepared by subjecting the ribonucleotide derivative (II-2) to base treatment in the presence of a silylating agent in an anhydrous solvent, under neutral conditions or acidic conditions. And can be produced by removing the protecting group of the 2 ′ hydroxyl group. The base treatment in the presence of a silylating agent in an anhydrous solvent can be carried out in the same manner as in [Production of ribonucleotide derivative (I-1)]. The hydrolysis treatment can be performed in the same manner as in [Production of ribonucleotide derivative (I-1)] except that the hydrolysis treatment is performed under neutral conditions or acidic conditions.

  When the 2 'hydroxyl group or 3' hydroxyl group at the 3 'end of the nucleotide chain is silylated, the hydrolysis reaction under neutral conditions is considered to be slow because there is no involvement of the adjacent group of phosphoric acid. Therefore, in this case, it is preferable to hydrolyze under acidic conditions.

  In the ribonucleotide derivative (II-2), the protecting group possessed by the nucleic acid represented by B or a derivative thereof is 2-cyanoethoxycarbonyl group, 2- (p-nitrophenyl) ethoxycarbonyl group, 2- (benzenesulfonyl) ethoxy In the case of an alkoxycarbonyl group having an electron-withdrawing group that can be removed by β-elimination such as a carbonyl group, the protective group is removed by tertiary amine treatment or phosphazene base treatment of the ribonucleotide derivative (II-2). Is done.

(4) Fourth Production Method Ribonucleotide derivative (I-2) is obtained by subjecting ribonucleotide derivative (II-1) to base treatment in the presence of a silylating agent in an anhydrous solvent and then hydrolyzing under acidic conditions. It can be produced by removing the protecting group for 2 ′ hydroxyl group and the protecting group for 5 ′ hydroxyl group represented by R ^1a . The base treatment in the presence of a silylating agent in an anhydrous solvent can be carried out in the same manner as in [Production of ribonucleotide derivative (I-1)]. The hydrolysis treatment can be performed in the same manner as in [Production of ribonucleotide derivative (I-1)] except that the hydrolysis treatment is performed under acidic conditions.

  In the ribonucleotide derivative (II-1), the protecting group of the nucleic acid represented by B or a derivative thereof is 2-cyanoethoxycarbonyl group, 2- (p-nitrophenyl) ethoxycarbonyl group, 2- (benzenesulfonyl) ethoxy In the case of an alkoxycarbonyl group having an electron-withdrawing group that can be removed by β-elimination such as a carbonyl group, the protecting group is removed by tertiary amine treatment or phosphazene base treatment of the nucleotide derivative (II-1). The

(5) Fifth Production Method The ribonucleotide derivative (I-2) is prepared by subjecting the ribonucleotide derivative (III-2) to base treatment in the presence of a silylating agent in an anhydrous solvent, and then under neutral or acidic conditions. And the phosphate protecting group represented by R ⁶ and the protecting group of the 2 ′ hydroxyl group are removed. The base treatment in the presence of a silylating agent in an anhydrous solvent can be carried out in the same manner as in [Production of ribonucleotide derivative (I-1)]. The hydrolysis treatment can be performed in the same manner as in [Production of ribonucleotide derivative (I-1)] except that the hydrolysis treatment is performed under neutral conditions or acidic conditions.

  In the ribonucleotide derivative (III-2), the protecting group possessed by the nucleic acid represented by B or a derivative thereof is 2-cyanoethoxycarbonyl group, 2- (p-nitrophenyl) ethoxycarbonyl group, 2- (benzenesulfonyl) ethoxy In the case of an alkoxycarbonyl group having an electron-withdrawing group that can be removed by β-elimination such as a carbonyl group, the protecting group is removed by tertiary amine treatment of the ribonucleotide derivative (III-2) or a phosphazene base. The

(6) Sixth Production Method The ribonucleotide derivative (I-2) is obtained by subjecting the ribonucleotide derivative (III-1) to base hydrolysis in the presence of a silylating agent in an anhydrous solvent and then hydrolyzing under acidic conditions. And a protecting group for the 5 ′ hydroxyl group represented by R ^1a , a phosphate protecting group represented by R ⁶ and a protecting group for the 2 ′ hydroxyl group. The base treatment in the presence of a silylating agent in an anhydrous solvent can be carried out in the same manner as in [Production of ribonucleotide derivative (I-1)]. The hydrolysis treatment can be performed in the same manner as in [Production of ribonucleotide derivative (I-1)] except that the hydrolysis treatment is performed under acidic conditions.

  In the ribonucleotide derivative (III-1), the protecting group of the nucleic acid represented by B or its derivative is 2-cyanoethoxycarbonyl group, 2- (p-nitrophenyl) ethoxycarbonyl group, 2- (benzenesulfonyl) ethoxy In the case of an alkoxycarbonyl group having an electron-withdrawing group that can be removed by β-elimination such as a carbonyl group, the protecting group is removed by tertiary amine treatment or phosphazene base treatment of the ribonucleotide derivative (III-1). Is done.

In producing ribonucleotide derivatives (I-1) and (I-2), a nucleotide chain from which a protecting group of a nucleobase or a derivative thereof, a phosphate protecting group represented by R ⁶ and a protecting group of a 2 ′ hydroxyl group are removed Is placed under basic conditions, the nucleotide chain will be degraded. When removal of the protecting group of the nucleobase or its derivative, the 2 'hydroxyl group at the 3' end of the nucleotide chain or the protecting group of the 3 'hydroxyl group and the phosphate protecting group represented by R ⁶ is carried out by base treatment, When the protecting group of the derivative, the 2 ′ hydroxyl group at the 3 ′ end of the nucleotide chain or the protecting group of the 3 ′ hydroxyl group and the phosphate protecting group represented by R ⁶ are removed after the removal of the protecting group of the 2 ′ hydroxyl group The nucleotide chain from which the protecting group of the nucleobase or its derivative, the phosphate protecting group represented by R ⁶ and the protecting group of the 2 ′ hydroxyl group have been removed is placed under basic conditions, and the nucleotide chain is decomposed. End up. Therefore, when removal of the protecting group of the nucleobase or its derivative, the 2 ′ hydroxyl group at the 3 ′ end of the nucleotide chain or the protecting group of the 3 ′ hydroxyl group and the phosphate protecting group represented by R ⁶ is carried out by base treatment, The base treatment is preferably performed before the removal of the 2′-hydroxyl protecting group. According to the above production method, the protecting group of the nucleobase or its derivative, the 2 ′ hydroxyl group at the 3 ′ end of the nucleotide chain or the protecting group at the 3 ′ hydroxyl group and the phosphate protecting group represented by R ⁶ are removed by 2 'Because it can be carried out before the removal of the protecting group of the hydroxyl group, the nucleotide chain is not decomposed, and the ribonucleotide derivatives (I-1) and (I-2) can be produced efficiently.

The phosphate protecting group represented by R ⁶ and the protecting group for the 2 ′ hydroxyl group can exist stably under the removal conditions (acidic conditions) for the protecting group for the 5 ′ hydroxyl group represented by R ^1a . Therefore, in the production of the ribonucleotide derivatives (I-1) and (I-2), the time point at which the 5′-hydroxyl protecting group represented by R ^1a is removed is not particularly limited. It may be before the removal of the protecting group of the derivative, the 2 ′ hydroxyl group or the 3 ′ hydroxyl protecting group at the 3 ′ end of the nucleotide chain, the phosphate protecting group represented by R ⁶ and the protecting group of the 2 ′ hydroxyl group. However, it may be after these removals or during these removals, but the protecting group of the nucleobase or its derivative, the 2 ′ hydroxyl group at the 3 ′ end of the nucleotide chain or 3 It is preferably before removal of the hydroxyl protecting group and the phosphate protecting group represented by R ⁶ . This is for the purpose of preventing the 5 ′ hydroxyl group deprotected by the removal of the 5 ′ hydroxyl protecting group represented by R ^1a from attacking the phosphate ester bond and breaking the nucleotide chain.

  Purification of ribonucleotides or ribonucleotide derivatives can be accomplished by using methods such as C8-C18 reverse phase column chromatography, C8-C18 reverse phase cartridge column, cation exchange column chromatography, anion exchange column chromatography alone. Or by using in combination with a high performance liquid chromatography apparatus.

Hereinafter, the present invention will be described in more detail based on examples.
[Example 1] 2'-O- [1- (2-cyanoethoxy) ethyl]]-3 ', 5'-O- (1,1,3,3-tetraisopropyldisiloxane-1,3-diyl ) Synthesis of Uridine (2) The synthesis process of this example is shown in FIG.
To 3 ', 5'-O- (1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) uridine (1) (1.41 g, 3 mmol) dissolved in 45 ml of 1,4-dioxane On the other hand, vinyloxypropanenitrile (9.92 g, 30 mmol) and p-toluenesulfonic acid-hydrate (630 mg, 3.3 mmol) were added and stirred. The reaction was completed in 10 minutes, and an excess amount of pyridine was added to terminate the reaction. The reaction system was concentrated and diluted with dichloromethane, and the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution. The organic layer was separated, dried over anhydrous sodium sulfate, and purified with a silica gel column (methylene chloride, methanol). The target product (2) (1.60 g, 2.8 mmol) was a mixture of two diastereomers and was obtained in a yield of 94%. The yields when acetylated adenine, acetylated cytosine, and acetylated guanine were used as nucleobases instead of uridine were 96%, 97%, and 65%, respectively.
¹ H NMR (CDCl ₃ ) δ 8.84 (1H, br.s, HN (3)); 7.92 (1H, d, HC (6)); 5.72 (1H, d, HC (5)); 5.67 (1H , d, HC (1 ')); 5.13 (1H, m, MeCH (O) ₂ ); 4.30-3.71 (7H, m, CH ₂ CH ₂ O, HC (2'), HC (3 '), HC (4 '), HC (5'); 2.65 (2H, m, CH ₂ CH ₂ O); 1.44 (3H, d, MeCH (O) ₂ ); 1.10-0.86 (28H, m, 4 Me ₂ CH) .

[Example 2] Synthesis of 2'-O- [1- (2-cyanoethoxy) ethyl]]-5'-O- (4,4'-dimethoxytrityl) -uridine (4) Synthesis process of this example Is shown in FIG.
2'-O- [1- (2-Cyanoethoxy) ethyl]]-3 ', 5'-O- (1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) uridine (2) (568 mg, 1 mmol) was dissolved in 5 ml of tetrahydrofuran, and 5 ml of 1M tetrabutylammonium fluoride and 1.2 M acetic acid in tetrahydrofuran were added thereto, followed by reaction at room temperature for 17 hours. The oily residue obtained by concentrating the reaction solution under reduced pressure was dissolved in a small amount of methylene chloride, and purified by silica gel column chromatography (methylene chloride, methanol) to remove by-produced tetrabutylammonium acetate. The eluted 2′-O- [1- (2-cyanoethoxy) ethyl] uridine (3) was concentrated and then azeotropically dried with pyridine, and further 15 ml of pyridine was added. Thereto was added 4,4′-O-dimethoxytrityl chloride (367 mg, 1.1 mmol) to initiate the reaction. Water was added to the solution reacted at room temperature for 8 hours to complete the reaction. The solution after the reaction was concentrated under reduced pressure and dissolved in dichloromethane. The organic layer was washed twice with a saturated aqueous sodium bicarbonate solution and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. Further purification was performed by silica gel column chromatography (1% pyridine, methylene chloride, methanol), and the desired 2'-O- [1- (2-cyanoethoxy) ethyl] -5'-O- (4,4'- Dimethoxytrityl) -uridine (4) (543 mg, 0.85 mmol) was obtained as a mixture of two diastereomers in 85% yield. The yields when acetylated adenine, acetylated cytosine, and acetylated guanine were used as nucleobases instead of uridine were 96%, 90%, and 93%, respectively.
¹ H NMR (CDCl ₃ ) δ8.63 (1H, br.s, HN (3)); 8.00 (1H, d, HC (6)); 7.40-6.83 (13H, m, DMTr) 5.96 (1H, d , HC (1 ')); 5.32-5.27 (1H, d, HC (5)); 5.20-5.05 (1H, m, MeCH (O) ₂ ); 4.49-4.43 (1H, m, HC (2') ): 4.39-4.23 (1H, m, HC (3 ') 4.07-4.05 (1H, m, HC (4')); 3.96-3.71 (8H, m, HC (5 '), MeO); 2.66-2.57 _{(2H, m, CH 2 CH} 2 O); 1.43 (3H, d, MeCH (O) 2).

Example 3 2'-O- [1- (2-cyanoethoxy) ethyl]]-5'-O- (4,4'-dimethoxytrityl) -uridine-3 '-(2-cyanoethyl-N, Synthesis of N-diisopropyl phosphoramidite) (5) The synthesis process of this example is shown in FIG.
2′-O- [1- (2-Cyanoethoxy) ethyl]]-5′-O- (4,4′-dimethoxytrityl) -uridine (4) (644 mg, 1 mmol) with pyridine and toluene three times each Azeotropically and dried. Dissolved in 5 ml of dichloromethane and cooled to 0 ° C. A dichloromethane solution (447 μl, 2 mmol / 5 ml) of diisopropylethylamine and phosphite 2-cyanoethyl-N, N-diisopropyl phosphorochloridite was added dropwise thereto. The reaction solution after dropping was returned to room temperature and reacted for 1 hour. The reaction was quenched with dry ethanol and the reaction mixture was diluted with dichloromethane. The organic layer was washed 5 times with a phosphate buffer solution (pH 7.0), and the organic layer was dried over anhydrous sodium sulfate and further concentrated under reduced pressure. Purification is performed by silica gel column chromatography. The desired product (5) (692 mg, 0.82 mmol) was obtained as a mixture of four diastereomers in a yield of 82%.
¹ H NMR (CDCl ₃ ) δ 8.65 (1H, br.s, HN (3)); 8.04-7.97 (1H, m, HC (6)); 7.40-6.80 (13H, m, DMTr) 5.96-5.93 (1H, m, HC (1 ')); 5.27-5.06 (2H, m, MeCH (O) ₂ , HC (5)); 4.60-3.41 (12H, m, HC (2') HC (3 ') ) HC (4 ') HC (5'), MeO, CH ₂ CH ₂ O, CH ₂ CH ₂ OP); 2.69-2.55 (4H, m, CH ₂ CH ₂ O, CH ₂ CH ₂ OP); 1.44- 1.35 (3H, m, MeCH (O) ₂ ); 1.188-1.13 (12H, m, (CH ₃ ) ₂ CH); 1.04-1.00 (2H, m, (CH ₃ ) ₂ CH));
³¹ P NMR (CDCl ₃ ) δ150.78, 150.22, 149.54, 148.97

[Example 4] Synthesis of protected uridylic acid dimer by liquid phase method The synthesis process of this example is shown in FIG.
2 ′, 3′-O-bisphenoxyacetyluridine (7) (24.4 mg, 48 μmol) and 2′-O- [1- (2-cyanoethoxy) ethyl]]-5′-O dried in a desiccator -(4,4'-dimethoxytrityl) -uridine-3 '-(2-cyanoethyl-N, N-diisopropylphosphoramidite) (5) (47.4 mg, 56 μmol) was dissolved in acetonitrile and 0.5 M 1 A solution of -H tetrazole in acetonitrile (114 μmol / 228 μl) was added and reacted at room temperature for 3 hours. An aqueous solution (80 wt%) of t-butyl alcohol peroxide was added to 35.7 μl of the reaction solution and stirred for 30 minutes. After the reaction solution was diluted with dichloromethane, the organic layer was washed twice with a saturated aqueous sodium hydrogen carbonate solution and dried over sodium sulfate. Sodium sulfate was removed by filtration and concentrated to dryness. The reaction mixture was purified by silica gel column chromatography (dichloromethane, methanol containing 1% pyridine) to obtain the desired protected uridine dimer (8) (60.5 mg, 48 μmol) as a mixture of four diastereomers. Obtained quantitatively.
¹ H NMR (CDCl ₃ ) δ9.88-9.70 (2H, br.m, HN (3) (up and low)); 7.85-7.69 (2H, m, HC (6) (up and low)); 7.38 -6.78 (23H, m, DMTr, PheO) 606-5.91 (2H, m, HC (5) (up and low)); 5.76-5.48 (3H, m, MeCH (O) ₂ , HC (1 ') ( up and low)); 4.60-3.41 (24H, m, HC (2 ') HC (3')) HC (4 ') HC (5') (up and low), MeO, CH ₂ CH ₂ O, CH ₂ CH ₂ OP, PheOAc); 2.69-2.55 (4H, m, CH ₂ CH ₂ O, CH ₂ CH ₂ OP); 1.42-1.25 (3H, m, MeCH (O) ₂ );
³¹ P NMR (CDCl ₃ ) δ-1.73, -1.99, -2.10, -2.13

[Example 5] Deprotection of uridylic acid dimer The deprotection step of this example is shown in FIG.
The protected uridine dimer was dissolved in dichloromethane, dichloroacetic acid (final concentration: 3%) was added thereto, and the reaction was performed for 1 minute. The reaction was stopped with a saturated aqueous sodium hydrogen carbonate solution, and the dichloromethane layer was washed twice with an aqueous sodium hydrogen carbonate solution, and then the organic layer was dried over anhydrous sodium sulfate. The organic layer was concentrated to dryness, a mixed solvent of 25% aqueous ammonia and ethanol (mixing ratio 3: 1) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated and lyophilized. Thereto was added a tetrahydrofuran solution of 1M tetrabutylammonium fluoride, and the reaction was performed at room temperature for 2 hours. An ammonium acetate buffer solution was added to the reaction solution to terminate the reaction. After the reaction mixture was diluted with water, the aqueous phase was washed 5 times with dichloromethane. The aqueous phase was concentrated and dried, and the residue was dissolved in a small amount of ammonium acetate buffer solution and purified by reverse phase silica gel column chromatography to obtain the desired uridine dimer.

[Example 6] Synthesis of oligoribonucleic acid by solid phase method Oligoribonucleic acid was synthesized according to the synthesis method of oligomer by general phosphoramidite method. Of the hydroxyl groups at the 2'-position or 3'-position, one is supported on a solid phase carrier (CPG) via a succinyl linker and the other is protected with an acetyl group. 0.2 ml of acetonitrile solution and 0.2 ml of 0.5M tetrazole in acetonitrile as an activator were added and condensed under the condition of a reaction time of 5 minutes. Furthermore, (i) oxidation of iodine (2%), water (2%), pyridine (20%) with a tetrahydrofuran solution, (ii) acetic anhydride, a solution of 2,6-lutidine (10%) in tetrahydrofuran, and 1- The cycle of capping methyl-1-H-imidazole (20%) with a tetrahydrofuran solution and (iii) deprotection of the 5 ′ hydroxyl group with a dichloromethane solution of dichloroacetic acid (3% (v / v)) was repeated nine times. The average condensation reaction yield was 99.5% as determined by quantification with 4,4′-dimethoxytrityl cation. Cleavage from the solid support and deprotection on the phosphorus atom were performed with a mixed solution of 28% aqueous ammonia and ethanol (3: 1). The solution of the target product was lyophilized, and 1M tetrabutylammonium fluoride in tetrahydrofuran was added to deprotect the 2 ′ hydroxyl group. Purification by HPLC allowed deprotected uridine decamer to be obtained in 95% yield.

[Example 7] Deprotection conditions and stability of 2'-O-1- (2-cyanoethoxy) ethyl group (CEE group)
Deprotection conditions and stability of CEE group in 2'-O- [1- (2-cyanoethoxy) ethyl]]-5'-O- (4,4'-dimethoxytrityl) -uridine (4) were investigated .

The deprotection conditions and stability of the CEE group were as follows: acetonitrile solution (0.5M DBU / MeCN) containing 0.5M 1,8-diazabicyclo [5.4.0] -7-undecene (DBU) as a reagent (base), 0.5M A solution of acetonitrile (0.5M DBU, 0.5M BSA / MeCN) containing M 1,8-diazabicyclo [5.4.0] -7-undecene (DBU) and 0.5MN, O-bis (trimethylsilyl) acetamide (BSA), 0.5 M Tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (0.5M TBAF / THF), 25% NH ₃ aqueous solution, 25% NH ₃ aqueous solution and ethanol mixed solution (3: 1, v / v) (25% NH ₃ aqueous -EtOH (3: 1, v / v)), using an ethanol solution of _{2M NH 3 (2M NH 3 /} EtOH), it was measured by determining the half-life at room temperature or 55 ° C. (min). The reaction mixture was spotted on a thin-layer silica gel plate (Merck F254) using a capillary, developed with dichloromethane-methanol (9: 1, v / v), and then the starting material and product spots were spotted under ultraviolet irradiation. The ultraviolet absorption intensity was compared, and the time when both were equal was defined as the half-life.
The results are shown in Table 1.

As shown in Table 1, the CEE group is a 25% NH ₃ aqueous solution used for removal of a protecting group of phosphodiester, removal of a protecting group of a nucleobase, excision of a nucleotide chain from a solid phase carrier, 25 % NH ₃ aqueous solution-EtOH (3: 1, v / v), stable to 2M NH ₃ / EtOH, but easily removed by 0.5M DBU / MeCN, 0.5M TBAF / THF. In particular, 0.5M TBAF / THF could be removed in a short time.

  The protecting group for the 2 ′ hydroxyl group in the ribonucleotide derivative (II) or (III) can be efficiently introduced into the 2 ′ hydroxyl group, does not cause steric hindrance in the condensation reaction, and further removes the protecting group for the 5 ′ hydroxyl group. , Stable removal under conditions such as removal of protecting group of phosphodiester, removal of protecting group of nucleobase, excision of nucleotide chain from solid phase carrier, but short time under conditions different from these conditions Therefore, the ribonucleotide derivative (I) can be efficiently produced by using the ribonucleotide derivative (II) or (III).

Claims (8)

Formula (II):

[Wherein B is a nucleobase or or

[Wherein R ⁷ represents a phenoxyacetyl group, a phenylacetyl group, an acetyl group or a benzoyl group, R ⁸ represents an isobutyryl group or a benzoyl group, and R ⁹ represents a phenoxyacetyl group, a phenylacetyl group, an acetyl group or an isobutyryl group. R ¹⁰ represents a 2-cyanoethyl group, R ¹¹ represents a benzoyl group, a 4-methoxybenzoyl group or a 4-methylbenzoyl group, and R ¹² represents a dimethylaminomethylene group. ]
In represents a nucleobase having a protective group represented, X represents oxo anions or thioanion, Y and Z independently of one another are hydrogen atom, a solid phase support or a hydroxyl protecting group, R ¹ represents a hydrogen atom or Represents a trityl group, a dimethoxytrityl group, a monomethoxytrityl group, or a 9-phenylxanthenyl group, and R ² , R ³ and R ⁴ may each independently 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, alkoxythiocarbonyl group, arylthiocarbonyl Boniru group, aralkylthio group, an aryloxy thiocarbonyl group or an aralkyloxycarbonyl thiocarbonyl group, R ⁵ is a cyano group, a nitro group, p- nitrophenyl group, an alkylsulfonyl group, an arylsulfonyl group, a trifluoromethyl group, An electron withdrawing group selected from the group consisting of a halogen atom, an alkoxycarbonyl group, a hydroxycarbonyl group, and an aryloxycarbonyl group is represented, and n represents an integer of 1 or more. ]
By subjecting the ribonucleotide derivative (II) represented by the following formula to a tertiary amine treatment or phosphazene base treatment in the presence of a silylating agent in an anhydrous solvent, and then hydrolyzing under neutral or acidic conditions,
Formula (I):

[Wherein, B, X, Y, Z, R ¹ and n are as defined above. ]
The manufacturing method of ribonucleotide or ribonucleotide derivative (I) including the process of manufacturing the ribonucleotide or ribonucleotide derivative (I) represented by these. The method according to claim 1, wherein 1,8-diazabicyclo [5.4.0] -7-undecene is used as the tertiary amine. Formula (III):

[ Wherein , B, Y, Z, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and n are as defined in claim 1, W represents an oxygen atom or a sulfur atom, R ⁶ represents a phosphate protecting group. ]
The manufacturing method of Claim 1 or 2 including the process of manufacturing the said ribonucleotide derivative (II) by carrying out the ammonia process or the primary amine process of the ribonucleotide derivative (III) represented by these. Manufacturing method of the claim 3, wherein the nucleobase acid base represented by B in formula (III) according to claim 3 having a protecting group. The manufacturing method in any one of Claims 1-4 including the process of removing the protecting group of 5 'hydroxyl group represented by R < ¹ > by acid treatment. The ribonucleotide derivative (III) represented by the formula (III) according to claim 3 is treated with a tertiary amine or phosphazene base in an anhydrous solvent in the presence of a silylating agent, and then neutral or acidic conditions. A method for producing a ribonucleotide or ribonucleotide derivative (I), comprising a step of producing a ribonucleotide or ribonucleotide derivative (I) represented by formula (I) according to claim 1 by hydrolysis under the following conditions. The method according to claim 6, wherein 1,8-diazabicyclo [5.4.0] -7-undecene is used as the tertiary amine. The manufacturing method of Claim 6 or 7 including the process of removing the protecting group of 5 'hydroxyl group represented by R < ¹ > by acid treatment.

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