WO2021162070A1 - Method for producing monomer for nucleic acid production - Google Patents

Abstract

The purpose of the present invention is to provide a method for producing a monomer for nucleic acid production by which an oligonucleic acid having a high purity can be produced. Provided is a method for producing a compound represented by formula (III) or a salt thereof, said method comprising a step for, in the presence of a dehydration condensation agent, reacting a compound represented by formula (I) or a salt thereof with an alkali metal salt, alkaline earth metal salt or quaternary ammonium salt of 6-hydroxyhexanoic acid in a solvent to give a compound represented by formula (II), and a step for, in the presence of a coupling agent, reacting the compound represented by formula (II) with an amiditation reagent to give the compound represented by formula (III) or a salt thereof.　

Description

Method for producing monomer for nucleic acid production

The present invention relates to a method for producing a monomer for producing nucleic acid.

Examples of the method for synthesizing oligonucleic acid include a phosphoric acid triester method, an H-phosphonate method, and a phosphoramidite method, but the phosphoramidite method is most used as a general-purpose method (Non-Patent Document 1).

Patent Document 1 describes a method for producing a single-stranded nucleic acid molecule capable of suppressing the expression of a target gene, and is represented by the following formula (III') in Example A3 as a monomer used for the production. A method for producing a compound is described.

Further, Patent Document 2 describes a method for producing a compound represented by the above formula (III'), which can produce a single-stranded nucleic acid molecule in a high yield.

US2012 / 0035246 International release WO2017 / 188042

An object of the present invention is to provide a method for producing a compound represented by the following formula (III) or a salt thereof, which can produce a high-purity oligonucleic acid.

[In the formula, R ¹ and R ² each independently represent a protecting group, and R ³ and R ⁴ each independently represent a optionally substituted hydrocarbon group. ]

As a result of diligent studies to solve the above problems, the present inventors have replaced the 6-hydroxycaproic acid used in Patent Documents 1 and 2 in the process of synthesizing the compound represented by the above formula (III). Therefore, it was found that the compound represented by the above formula (III) or a salt thereof can be obtained with high purity by using a salt of 6-hydroxycaproic acid. Then, they have found that a high-purity oligonucleic acid can be produced by using the obtained compound represented by the formula (III) or a salt thereof as a monomer, and have completed the present invention.

In the amidation reaction, it is generally known that there is no difference in the reaction result between the case where the carboxylic acid as a substrate is used in the form of a free acid and the case where the carboxylic acid is used in the form of a salt. Considering this, the above findings found by the present inventors were surprising.

That is, the present invention includes the following.

[1] In the presence of a dehydration condensing agent, a compound represented by the following formula (I) or a salt thereof and an alkali metal salt of 6-hydroxyhexanoic acid, an alkaline earth metal salt or a quaternary ammonium salt are mixed in a solvent. The step of reacting to obtain a compound represented by the following formula (II) and

[In the formula, R ¹ represents a protecting group. ]

[In the formula, R ¹ is synonymous with the above definition. ]
In the presence of a coupling agent, the compound represented by the above formula (II) is reacted with the amidite-forming reagent to obtain the compound represented by the following formula (III) or a salt thereof.

[In the formula, R ¹ is synonymous with the above definition, R ² represents a protecting group, and R ³ and R ⁴ each independently represent a optionally substituted hydrocarbon group. ]
The method for producing a compound represented by the above formula (III) or a salt thereof.

[2] The production method according to [1], wherein the solvent is a water-containing solvent.

[3] The production method according to [1] or [2], wherein the dehydration condensing agent is a triazine-type dehydration condensation agent.

[4] As the above-mentioned alkali metal salt, alkaline earth metal salt or quaternary ammonium salt of 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone and alkali metal hydroxide , Alkali metal carbonate, alkali metal hydrogen carbonate, alkali metal alkoxide, alkali earth metal hydroxide, and base selected from the group consisting of quaternary ammonium hydroxide. The production method according to any one of [1] to [3] to be used.

[5] The above-mentioned alkali metal salt, alkaline earth metal salt or quaternary ammonium salt of 6-hydroxyhexanoic acid is lithium 6-hydroxyhexanoate, sodium 6-hydroxyhexanoate, potassium 6-hydroxyhexanoate or 6-. The production method according to any one of [1] to [4], which is cesium hydroxyhexanoate.

[6] R ¹ is a trityl group, 4-methoxytrityl group or 4,4′-dimethoxytrityl group, R ² is a 2-cyanoethyl group, a benzyl group or a 2-chlorophenyl group, and R ³ is a group. an ethyl group or an isopropyl group, R ⁴ is an ethyl group or an isopropyl group, a process according to any one of [1] to [5].

[7] Production of oligonucleic acid comprising a step of carrying out a nucleic acid synthesis reaction using the compound represented by the following formula (III) or a salt thereof obtained by the production method according to any one of [1] to [6]. Method.

[In the formula, R ¹ and R ² each independently represent a protecting group, and R ³ and R ⁴ each independently represent a optionally substituted hydrocarbon group. ]

According to the present invention, it is possible to produce a compound represented by the formula (III) or a salt thereof, which can produce a high-purity oligonucleic acid.

Hereinafter, the present invention will be described in detail.

1. 1. Method for producing the compound represented by the formula (III) or a salt thereof:
The present invention provides a method for producing a compound represented by the formula (III) or a salt thereof. The process of the manufacturing method is shown below.

[In the formula, R ¹ and R ² each independently represent a protecting group, and R ³ and R ⁴ each independently represent a optionally substituted hydrocarbon group. ]

(Step 1: Amidation reaction)
The compound represented by the formula (II) is a compound represented by the formula (I) or a salt thereof in the presence of a dehydration condensing agent, and an alkali metal salt, an alkaline earth metal salt or a quaternary ammonium salt of 6-hydroxyhexanoic acid. And can be obtained by reacting in a solvent.

[In the formula, R ¹ represents a protecting group. ]

The compound represented by the formula (I) or a salt thereof may be not only a single stereoisomer but also a mixture of stereoisomers such as racemic (for example, a mixture of enantiomers).

A stereoisomer refers to a compound having the same chemical structure but different arrangement in three-dimensional space, for example, a conformer isomer, a rotational isomer, a metamutant, a mirror image isomer, a diastereomer, or the like. Can be mentioned.

The compound represented by the formula (I) or a salt thereof can be produced by a synthetic method known to those skilled in the art. In one embodiment, for example, the compound represented by the formula (I') described later can be obtained by the method described in US2012 / 0035246.

The salt of the compound represented by the formula (I) is not particularly limited as long as it does not inhibit the amidation reaction, and is, for example, hydrochloride, bromate, iodate, perchlorate, carbonate, etc. Examples include trifluoromethanesulfonate, tetrafluoroborate or hexafluorophosphate.

In the formulas (I) and (II), R ¹ represents a protecting group, which may be, for example, a functional group that converts a hydroxyl group into an inactive state, and a known protecting group for a hydroxyl group can be used. As for the protecting group for the hydroxyl group, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. R ¹ is, for example, tert-butyldimethylsilyl group, bis (2-acetoxyethyloxy) methyl group, triisopropylsilyloxymethyl group, 1- (2-cyanoethoxy) ethyl group, 2-cyanoethoxymethyl group, 2 Examples include, but are not limited to, a cyanoethyl group, a trilsulfonylethoxymethyl group, a trityl group, a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group.

In formulas (I) and (II), R ¹ is preferably a trityl group, a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group.

In a preferred embodiment, the compound represented by the formula (I) or a salt thereof may be a compound represented by the following formula (I') or a salt thereof.

The compound represented by the formula (I') or a salt thereof may be an optically active substance represented by the following formula (I'-1) or the following formula (I'-2) or a salt thereof.

In a preferred embodiment, the compound represented by the formula (II) may be a compound represented by the following formula (II').

The compound represented by the formula (II') may be an optically active substance represented by the following formula (II'-1) or the following formula (II'-2).

The alkali metal salt, alkaline earth metal salt or quaternary ammonium salt of 6-hydroxyhexanoic acid is an alkali metal salt of 6-hydroxyhexanoic acid, alkaline earth metal salt of 6-hydroxyhexanoic acid or 6-hydroxyhexanoic acid. It means the quaternary ammonium salt of. In the present specification, the alkali metal salt of 6-hydroxyhexanoic acid, the alkaline earth metal salt of 6-hydroxyhexanoic acid, or the quaternary ammonium salt of 6-hydroxyhexanoic acid are collectively referred to as 6-hydroxyhexanoate. In some cases.

As the 6-hydroxyhexanoate, a commercially available product is used, or 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal hydrogen carbonate. , Alkali metal alkoxide, alkaline earth metal hydroxide and quaternary ammonium hydroxide can be obtained by reacting with a base selected from the group in an aqueous solvent. Here, as 6-hydroxycaproic acid, 6-hydroxycaproic acid ester or ε-caprolactone, commercially available products can be used. The 6-hydroxyhexanoic acid ester is not limited to the following, and for example, 6-hydroxyhexanoic acid alkyl ester, 6-hydroxyhexanoic acid aryl ester, 6-hydroxyhexanoic acid alkylaryl ester, 6-hydroxyhexanoic acid arylalkyl ester, Examples thereof include 6-hydroxyhexanoic acid cycloalkyl ester, 6-hydroxyhexanoic acid alkoxyalkyl ester, 6-hydroxyhexanoic acid silyloxyalkyl ester, 6-hydroxyhexanoic acid carboxylalkyl ester and 6-hydroxyhexanoic acid silyl ester. More specifically, methyl 6-hydroxyhexanoate, ethyl 6-hydroxyhexanoate, benzyl 6-hydroxyhexanoate, 6-hydroxyhexanoic acid (5-carboxypentyl), phenyl 6-hydroxyhexanoate and the like can be mentioned.

Examples of the alkali metal salt of 6-hydroxyhexanoic acid include lithium 6-hydroxyhexanoate, sodium 6-hydroxyhexanoate, potassium 6-hydroxyhexanoate, rubidium 6-hydroxyhexanoate, cesium 6-hydroxyhexanoate or 6-hydroxy. Francium hexanoate can be mentioned.

Examples of the alkaline earth metal salt of 6-hydroxyhexanoic acid include magnesium 6-hydroxyhexanoate, calcium 6-hydroxyhexanoate, strontium 6-hydroxyhexanoate, barium 6-hydroxyhexanoate or radium 6-hydroxyhexanoate. Be done.

The quaternary ammonium salt of 6-hydroxyhexanoic acid is not limited to the following, and includes, for example, benzyltrimethylammonium 6-hydroxyhexanoate, benzyldimethyltetradecylammonium 6-hydroxyhexanoate, benzyltriethylammonium 6-hydroxyhexanoate, and the like. Benzyltributylammonium 6-hydroxyhexanoate, triethylmethylammonium 6-hydroxyhexanoate, tributylmethylammonium 6-hydroxyhexanoate, tetramethylammonium 6-hydroxyhexanoate, tetraethylammonium 6-hydroxyhexanoate, tetra-hydroxyhexanoate 6-hydroxyhexanoate Propylammonium, tetrabutylammonium 6-hydroxyhexanoate, tetraoctylammonium 6-hydroxyhexanoate, benzylsinconidinium 6-hydroxyhexanoate, benzylcinconinium 6-hydroxyhexanoate, benzylkinidinium 6-hydroxyhexanoate Alternatively, benzylkininium 6-hydroxyhexanoate can be mentioned.

In a preferred embodiment, the 6-hydroxycaproate may be an alkali metal salt of 6-hydroxycaproic acid, eg, lithium 6-hydroxycaproate, sodium 6-hydroxycaproate, 6-hydroxycaproic acid. It may be potassium or cesium 6-hydroxyhexanoate.

The molar equivalent of 6-hydroxyhexanoate is preferably 1 to 10 molar equivalents, more preferably 1 to 2 molar equivalents, relative to the compound represented by the formula (I) or a salt thereof.

The dehydration condensate used in the amidation reaction is not particularly limited, and examples thereof include a triazine type dehydration condensate, a uronium type dehydration condensate, and a carbodiimide type dehydration condensate. When a carbodiimide type dehydration condensate is used as the dehydration condensate, it may be used in combination with an additive. The dehydration condensate may be in the form of a free form or a salt, preferably in the form of a salt.

A triazine-type dehydration condensing agent is a compound having a structure in which at least one hydrogen atom on a carbon atom of triazine is replaced with a desorbable functional group such as a quaternary ammonium or a halogen atom (for example, a free form or a salt). ) Means. Examples of the triazine-type dehydration condensing agent include the literature (Kunishima et al., Tetrahedron, 2001, Vol. 57, p.1551-1558; Kunishima et al., Chemistry A European Journal, 2012, Vol. 18, p.1556-15867. The dehydration condensing agent described in Japanese Patent No. 4349479, Japanese Patent Application Laid-Open No. 2008-214473, Japanese Patent Application Laid-Open No. 2016-141618, Japanese Patent Application Laid-Open No. 2016-141619, Japanese Patent Application Laid-Open No. 2017-149876, etc.) Can be used. More specifically, the triazine-type dehydration-condensant is a triazine-type quaternary morpholinium, for example, 4- [4,6-bis (2,6-kisilyl) -1,3,5-triazine-2-yl]-. 4-Methylmorpholinium, 4- [4-methoxy-6- (2,6-kisilyl) -1,3,5-triazine-2-yl] -4-methylmorpholinium, 4- (4-t) -Butyl-6-methoxy-1,3,5-triazine-2-yl) 4-methoxymorpholinium, 4- (4,6-di-t-butyl-1,3,5-triazine-2-yl) ) -4-Methylmorpholinium, N- [4-methoxy-6- (N'-phenylacetamide) -1,3,5-triazine-2-yl] -4-methylmorpholinium, N- [4 -Methoxy-6- (N'-methylacetamide) -1,3,5-triazine-2-yl] -4-methylmorpholinium, N- [4-methoxy-6- (N'-phenylbenzamide)- 1,3,5-Triazine-2-yl] -4-methylmorpholinium, N- [4-methoxy-6- (N'-methylbenzamide) -1,3,5-triazine-2-yl]- 4-Methylmorpholinium, N- [4-methoxy-6- (2,5-dioxopyrrolidyl) -1,3,5-triazine-2-yl] -4-methylmorpholinium, N- [ 4-methoxy-6- (2-piperidone-1-yl) -1,3,5-triazine-2-yl] -4-methylmorpholinium, 4- (4,6-dimethoxy-1,3,5 -Triazine-2-yl) -4-methylmorpholinium or 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium or a derivative thereof can be mentioned. Examples of derivatives include salts such as perchlorate, trifluoromethanesulfonate, chloride or hexafluorophosphate. The triazine-type dehydration condensing agent may be, for example, a 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium salt.

Examples of the derivative of triazine-type quaternary morpholinium, which is a triazine-type dehydration condensant, include 4- [4,6-bis (2,6-kisilyl) -1,3,5-triazine-2-yl] -4- Methylmorpholinium perchlorate, 4- [4-methoxy-6- (2,6-kisilyl) -1,3,5-triazine-2-yl] -4-methylmorpholinium perchlorate, 4- (4-t-butyl-6-methoxy-1,3,5-triazine-2-yl) 4-methoxymorpholinium trifluoromethanesulfonate, 4- (4,6-di-t-butyl-1,3) , 5-Triazine-2-yl) -4-methylmorpholinium chloride, N- [4-methoxy-6- (N'-phenylacetamide) -1,3,5-triazine-2-yl] -4- Methylmorpholinium perchlorate, N- [4-methoxy-6- (N'-methylacetamide) -1,3,5-triazine-2-yl] -4-methylmorpholinium perchlorate, N- [4-methoxy-6- (N'-phenylbenzamide) -1,3,5-triazine-2-yl] -4-methylmorpholinium chloride, N- [4-methoxy-6- (N'-methyl) Benzamide) -1,3,5-triazine-2-yl] -4-methylmorpholinium chloride, N- [4-methoxy-6- (2,5-dioxopyrrolidyl) -1,3,5- Triazine-2-yl] -4-methylmorpholinium chloride, N- [4-methoxy-6- (2-piperidone-1-yl) -1,3,5-triazine-2-yl] -4-methyl Morholinium chloride, 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride or 4- (4,6-dimethoxy-1,3,5- Triazine-2-yl) -4-methylmorpholinium trifluoromethanesulfonate is preferred, 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride (4,6-dimethoxy-1,3,5-triazine-2-yl). Hereinafter, DMT-MM) is more preferable.

An example of a uronium-type dehydration-condensing agent is a benzotriazolyl-uronium-type dehydration-condensation agent. The benzotriazolyluronium-type dehydration condensing agent means a compound in which a tetraalkylamidinium structure is added to a 1-hydroxybenzotriazole structure. The four substituents on the two nitrogen atoms of the amidinium structure are not particularly limited as long as they do not inhibit the amidation reaction, and may form a ring together with the nitrogen atom, for example. The 1-hydroxybenzotriazole structure is not particularly limited as long as it does not inhibit the amidation reaction. For example, it may have a substituent on the benzene ring, or a part of carbon atoms in the benzene ring. May be replaced with a nitrogen atom.

The benzotriazolyl uronium type dehydration condensing agent generally takes two forms, an O-acyl type (uronium type) and an N-acyl type (aminium type) (Carpino et al., Angewandte Chemie International Edition, 2002). , Vol. 41, p. 441-445.), In the present specification, the benzotriazolyl uronium type dehydration condensate is an O-acyl type (uronium type) and an N-acyl type (Uronium type). Includes both forms of (aminium type).

Examples of the benzotriazolyluronium type dehydration condensate include the literature (Knorr et al., Tetrahedron Letters, 1989, Vol. 30, p. 1927-1930; Carpino et al., Organic Letters, 2001, Vol. 3, p. .2793-2795; EL-Faham et al., The Journal of Organic Chemistry, 2008, Vol. 73, p.2731-2737; International Publication No. 1994/007910, International Publication No. 2002/094822, etc.) A dehydration condensing agent can be used. More specifically, the benzotriazolyluronium-type dehydration condensing agent is a benzotriazolyluronium, for example, O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyl. Uronium or [(benzotriazole-1-yloxy) piperidine-1-ylmethylene] piperidinium or a derivative thereof, or azabenzotriazolyluronium, for example, O- (7-azabenzotriazole-1-yl) -N , N, N', N'-tetramethyluronium, O- (7-azabenzotriazole-1-yl) -N, N, N', N'-tetraethyluronium, [(7-azabenzotriazole- 1-Iloxy) -4-morpholinomethylene] dimethylammonium, [(7-azabenzotriazole-1-yloxy) piperidine-1-ylmethylene] piperidinium, [(7-azabenzotriazole-1-yloxy) pyrrolidine-1-ylmethylene ] Pyrrolidineium or a derivative thereof can be mentioned. Examples of derivatives include salts such as perchlorate, trifluoromethanesulfonate, chloride or hexafluorophosphate. Benzotriazolyluronium-type dehydration condensing agents include, for example, O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium salt and O- (7-azabenzotriazole-). 1-yl) -N, N, N', N'-tetramethyluronium salt may be used.

Preferred examples of the benzotriazolyluronium type dehydration condensing agent are O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate or O- (7). -Azabenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (hereinafter referred to as HATU) can be mentioned.

Another example of the uronium-type dehydration condensing agent is a compound in which a tetraalkylamidinium structure is added to a leaving group. The four substituents on the two nitrogen atoms of the amidinium structure are not particularly limited as long as they do not inhibit the amidation reaction, and may form a ring together with the nitrogen atom, for example. The leaving group is not particularly limited as long as it does not inhibit the amidation reaction. For example, N-hydroxysuccinimide, ethyl (hydroxyimino) cyanoacetate, N-hydroxyphthalimide, 3-hydroxy-4-oxo-3. , 4-Dihydro-1,2,3-benzotriazine, N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide, 1-hydroxy-1,2,3-triazole-4-carboxylate ethyl, 2- Hydroxypyridine-N-oxide or halogen atom can be mentioned. Examples of the uronium-type dehydration condensing agent include O- (1,2-dihydro-2-oxo-1-pyridyl) -N, N, N', N'-tetramethyluronium tetrafluoroborate, O- ( 3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl) -N, N, N', N'-tetramethyluronium tetrafluoroborate, chloro-N, N, N' , N'-Tetramethylform Amidinium Hexafluoroborate, Fluoro-N, N, N', N'-Tetramethylform Amidinium Hexafluoroborate, (1-cyano-2-ethoxy-2-oxoethylideneamino Oxy) dimethylaminomorpholinocarbenium hexafluoroborate or N, N, N', N'-tetramethyl-O- (N-succinimidyl) uronium hexafluoroborate can be mentioned.

The carbodiimide type dehydration condensing agent means a compound having a carbodiimide structure represented by N = C = N. The substituent on N is not particularly limited as long as it does not inhibit the amidation reaction. Examples of the carbodiimide-type dehydration condensing agent include N, N'-dimethylcarbodiimide, N, N'-diethylcarbodiimide, N, N'-dipropylcarbodiimide, N, N'-diisopropylcarbodiimide, and N-tert-butyl-N. '-Methylcarbodiimide, N, N'-dicyclohexylcarbodiimide, N-benzyl-N'-methylcarbodiimide, N-benzyl-N'-ethylcarbodiimide, N-benzyl-N'-propylcarbodiimide, N- (3'-dimethyl Aminopropyl) -N'-ethylcarbodiimide, N, N'-bis (3'-dimethylaminopropyl) carbodiimide, 3- (ethyliminomethylideneamino) propyltrimethylammonium or 3- (isopropyliminomethylideneamino) propyltrimethyl A carbodiimide derivative such as ammonium or a salt thereof (including an acid addition salt) can be used. Examples of salts include perchlorate, trifluoromethanesulfonate, chloride, hexafluorophosphate, hydrochloride, bromate, trifluoromethanesulfonate, perchlorate and the like. A carbodiimide derivative containing a tertiary amine structure or a quaternary ammonium structure is more preferable, for example, a salt such as N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide or an acid addition salt thereof, N, N'-bis. Examples thereof include salts such as (3'-dimethylaminopropyl) carbodiimide or an acid addition salt thereof, 3- (ethyliminomethylideneamino) propyltrimethylammonium salt or 3- (isopropyliminomethylideneamino) propyltrimethylammonium salt. A particularly preferred example of a carbodiimide-type dehydration condensing agent is N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (hereinafter, EDCI hydrochloride).

Examples of the additive used in combination with the carbodiimide type dehydration condensing agent include 1-hydroxybenzotriazole (hereinafter, HOBt), 1-hydroxy-7-azabenzotriazole (hereinafter, HOAt), N-hydroxysuccinimide, and the like. Ethyl (hydroxyimino) cyanoacetate, N-hydroxyphthalimide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, N-hydroxy-5-norbornene-2,3-dicarboxylic acidimide , 1-Hydroxy-1,2,3-triazole-4-carboxylate ethyl or 2-hydroxypyridine-N-oxide.

The dehydration condensation agent and the additive may be commercially available products, or may be synthesized by a known method or a method similar thereto.

The molar equivalent of the dehydration condensing agent is preferably 1 to 10 molar equivalents, more preferably 1 to 2 molar equivalents, relative to the compound represented by the formula (I) or a salt thereof. The molar equivalent of the additive is preferably 0.05 to 10 molar equivalents, more preferably 0.2 to 2 molar equivalents, relative to the compound represented by the formula (I) or a salt thereof.

The solvent used for the amidation reaction is appropriately selected depending on the type of reagent used and the like, but is not particularly limited as long as it does not inhibit the reaction. For example, a non-hydrophilic solvent, a hydrophilic solvent or a mixture thereof. However, a water-containing solvent is preferable. One type of solvent may be used, or two or more types may be used in combination. When two or more kinds of solvents are used in combination, they may not be mixed with each other and may be non-uniform, or they may be mixed and become uniform. The amount of the solvent used is preferably 1 to 100 times by weight with respect to the compound represented by the formula (I) or a salt thereof.

The non-hydrophilic solvent means a solvent that is immiscible with water at an arbitrary ratio. Non-hydrophilic solvents are not limited to, for example, hexane, heptane, toluene, xylene, dichloromethane, chloroform, dichloroethane, methyl tert-butyl ether, cyclopentyl methyl ether, diethyl ether, methyl ethyl ketone, methyl isobutyl ketone, methyl acetate, acetic acid. Examples include ethyl or propionitrile.

The hydrophilic solvent means a solvent and water that are mixed with water at an arbitrary ratio. The hydrophilic solvent is not limited to the following, but for example, water, methanol, ethanol, tetrahydrofuran, 1,4-dioxane, acetone, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, 1, Examples thereof include 3-dimethyl-2-imidazolidinone, N, N'-dimethylpropyleneurea, acetonitrile, dimethyl sulfoxide or sulfolane.

The hydrous solvent means a solvent in which water and an organic solvent (a non-hydrophilic solvent or a hydrophilic solvent other than water) are combined, and they may not be mixed with each other and may be non-uniform, or they may be mixed with each other. It may be uniform. One kind of organic solvent may be used, or two or more kinds may be used in combination. The water-containing solvent is not limited to the following, and examples thereof include a combination of water and a hydrophilic solvent other than water, for example, a combination of water and methanol, a combination of water and ethanol, and water and N, N-dimethylformamide. Or a combination of water and dimethyl sulfoxide is preferred. The composition ratio of the solvent can be appropriately selected according to the reaction.

A tertiary amine may be added as an optional component to the amidation reaction, and the tertiary amine is not limited to the following, and examples thereof include triethylamine and diisopropylethylamine. The molar equivalent of the tertiary amine is preferably 0.05 to 10 molar equivalents, more preferably 1 to 2 molar equivalents, relative to the compound represented by the formula (I) or a salt thereof.

The reaction time of the amidation reaction can be set in a timely manner, but is typically 10 minutes or more, for example, 10 minutes to 24 hours.

The reaction temperature of the amidation reaction can be set in a timely manner, and may be in the range of 0 to 100 ° C., and is usually preferably room temperature (about 10 ° C. to about 35 ° C.).

In the amidation reaction, the order of addition of each reagent is not particularly limited, and for example, a method of sequentially adding a compound represented by the formula (I) or a salt thereof, a 6-hydroxyhexanoate and a dehydration condensing agent to a solvent, a formula ( A method of sequentially adding a solvent, 6-hydroxyhexanoate and a dehydration condensing agent to the compound represented by I) or a salt thereof, and to the solvent the compound represented by the formula (I) or a salt thereof, 6-hydroxyhexanelate and A method of adding a dehydration condensing agent at the same time, a method of adding a solvent to a mixture of a compound represented by the formula (I) or a salt thereof, a 6-hydroxyhexanoate and a dehydration condensing agent, and a method represented by a formula (I) mixed with a solvent. A method of sequentially mixing a 6-hydroxyhexanoate mixed with a solvent and a dehydration condensing agent mixed with a solvent with the compound or a salt thereof, or a solvent mixed with a compound represented by the formula (I) represented by the formula (I) or a salt thereof. A method of simultaneously mixing the 6-hydroxyhexanoate mixed with and the dehydration condensing agent mixed with the solvent can be mentioned. Usually, a method of sequentially adding a solvent, a 6-hydroxycaproate and a dehydration condensing agent to the compound represented by the formula (I) or a salt thereof is preferable.

The preferred embodiment of the dehydration condensing agent and the preferred embodiment of the solvent can be appropriately combined. For example, a combination of a triazine type dehydration condensing agent and a hydrophilic solvent, a combination of a triazine type dehydration condensing agent and a water-containing solvent, a combination of a uronium type dehydration condensing agent and a hydrophilic solvent, and a combination of a uronium type dehydration condensing agent and a water-containing solvent. Examples thereof include a combination, a combination of a carbodiimide type dehydration condensing agent and a hydrophilic solvent, or a combination of a carbodiimide type dehydration condensing agent and an additive and a hydrophilic solvent, and examples of a particularly preferable combination include, for example, DMT-MM and methanol. , DMT-MM with dimethyl sulfoxide, DMT-MM with water and methanol, DMT-MM with water and dimethyl sulfoxide, HATU with dimethyl sulfoxide, HATU with water and dimethyl sulfoxide The combination with, the combination of EDCI hydrochloride and dimethyl sulfoxide, or the combination of EDCI hydrochloride and HOAt and dimethyl sulfoxide can be mentioned.

After completion of the amidation reaction, for example, the organic layer can be washed with an aqueous sodium hydrogen carbonate solution or the like, and then the organic layer can be concentrated to isolate the compound represented by the formula (II).

The compound represented by the formula (II) can be purified by appropriately using a known purification method such as chromatography.

As 6-hydroxyhexanoate, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal hydrogen carbonate, alkali metal alkoxide, alkaline earth When a mixture obtained by reacting a base selected from the group consisting of metal hydroxide and quaternary ammonium hydroxide in an aqueous solvent is used, the mixture may be post-treated or isolated and purified. , It may be used as it is for the reaction with the compound represented by the formula (I) or a salt thereof without post-treatment or isolation and purification. That is, in step 1, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal bicarbonate, alkali metal alkoxide, alkaline earth metal water. A step of reacting a base selected from the group consisting of an oxide and a quaternary ammonium hydroxide in an aqueous solvent to obtain a mixture (hereinafter, also referred to as step 1'), and in the presence of a dehydration condensing agent. A step of reacting the compound represented by the formula (I) or a salt thereof with the above mixture in a solvent to obtain a compound represented by the formula (II) (hereinafter, also referred to as step 1 ″). May be good. Alternatively, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal hydrogen carbonate, alkali metal alkoxide, alkaline earth metal water are added to the aqueous solvent. The amidation reaction may be carried out by simultaneously adding a base selected from the group consisting of an oxide and a quaternary ammonium hydroxide, a compound represented by the formula (I) or a salt thereof, and a dehydration condensing agent.

In the above step 1', the order of addition of each reagent is not particularly limited. For example, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal carbonate are added to the aqueous solvent. , A method of sequentially adding a base selected from the group consisting of alkali metal hydrogen carbonate, alkali metal alkoxide, alkaline earth metal hydroxide and quaternary ammonium hydroxide, alkali metal hydroxide, alkali metal to an aqueous solvent. A base selected from the group consisting of carbonates, alkali metal hydrogen carbonates, alkali metal alkoxides, alkaline earth metal hydroxides and quaternary ammonium hydroxides, as well as 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid esters or ε. -Method of adding caprolactone sequentially, to aqueous solvent, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal hydrogen carbonate, alkali metal alkoxide, alkali A method of simultaneously adding a base selected from the group consisting of earth metal hydroxides and quaternary ammonium hydroxides, or to 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone mixed with an aqueous solvent. , A base selected from the group consisting of alkali metal hydroxides mixed with aqueous solvents, alkali metal carbonates, alkali metal hydrogen carbonates, alkali metal alkoxides, alkaline earth metal hydroxides and quaternary ammonium hydroxides. There is a method of mixing. Usually, an aqueous solvent contains a base selected from the group consisting of alkali metal hydroxide, alkali metal carbonate, alkali metal hydrogen carbonate, alkali metal alkoxide, alkaline earth metal hydroxide and quaternary ammonium hydroxide. A method of sequentially adding 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone is preferable.

In the above step 1 ″, the order of addition of each reagent is not particularly limited, and for example, a method of sequentially adding a dehydration condensing agent, a compound represented by the formula (I) or a salt thereof, and the above mixture to a solvent, the formula (I). A method of sequentially adding a solvent, the above mixture and a dehydration condensing agent to the compound represented by (I) or a salt thereof, a method of simultaneously adding a dehydration condensing agent, a compound represented by the formula (I) or a salt thereof and the above mixture to the solvent, and a solvent. A method of sequentially adding the compound represented by the formula (I) mixed with a solvent or a salt thereof and the above mixture mixed with the solvent to the mixed dehydration condensing agent, or a formula of mixing the dehydration condensing agent mixed with the solvent with the solvent ( Examples thereof include a method of simultaneously adding the compound represented by I) or a salt thereof and the above-mentioned mixture mixed with a solvent. Usually, a method of sequentially adding a solvent, the above mixture and a dehydration condensing agent to the compound represented by the formula (I) or a salt thereof is preferable.

Examples of the alkali metal hydroxide include lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide or francium hydroxide.

Examples of the alkali metal carbonate include lithium carbonate, sodium carbonate, potassium carbonate, rubidium carbonate, cesium carbonate or franchium carbonate.

Examples of the alkali metal bicarbonate include lithium hydrogencarbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, rubidium hydrogencarbonate, cesium hydrogencarbonate, and franchium hydrogencarbonate.

Examples of the alkali metal alkoxide include, but are not limited to, sodium methoxide, sodium ethoxide, lithium tert-butoxide, sodium tert-butoxide or potassium tert-butoxide.

Examples of alkaline earth metal hydroxides include magnesium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, and radium hydroxide.

The quaternary ammonium hydroxide is not limited to the following, and includes, for example, benzyltrimethylammonium hydroxide, benzyldimethyltetradecylammonium hydroxide, benzyltriethylammonium hydroxide, benzyltributylammonium hydroxide, triethylmethylammonium hydroxide, and water. Tributylmethylammonium oxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetraoctylammonium hydroxide, benzylcinconidinium hydroxide, benzylcinconinium hydroxide, hydroxide Benzylkinidinium or benzylkininium hydroxide can be mentioned.

In step 1', as a base selected from the group consisting of alkali metal hydroxides, alkali metal carbonates, alkali metal hydrogen carbonates, alkali metal alkoxides, alkaline earth metal hydroxides and quaternary ammonium hydroxides. Is preferably an alkali metal hydroxide, and more preferably lithium hydroxide, sodium hydroxide, potassium hydroxide or cesium hydroxide.

The molar equivalent of 6-hydroxycaproic acid, 6-hydroxycaproic acid ester or ε-caprolactone is preferably 1 to 10 molar equivalents with respect to the compound represented by the formula (I) or a salt thereof, preferably 1 to 2 molar equivalents. More preferred.

The molar equivalent of a base selected from the group consisting of alkali metal hydroxides, alkali metal carbonates, alkali metal hydrogen carbonates, alkali metal alkoxides, alkaline earth metal hydroxides and quaternary ammonium hydroxides , 1 to 10 molar equivalents are preferable with respect to the compound represented by the formula (I) or a salt thereof, and 1 to 2 molar equivalents are more preferable.

The aqueous solvent means a single water or a solvent in which water and an organic solvent are mixed and become uniform. The organic solvent to be mixed with water may be one kind, or two or more kinds may be used in combination. Examples of the aqueous solvent include water, a combination of water and methanol, or a combination of water and dimethyl sulfoxide. When two or more kinds of solvents are mixed, the composition ratio of the solvent can be appropriately selected according to the reaction. The amount of the aqueous solvent used is preferably 1 to 100 times by weight with respect to 6-hydroxycaproic acid, 6-hydroxycaproic acid ester or ε-caprolactone.

The reaction time of the above step 1'can be set in a timely manner, but is typically 10 minutes or more, for example, 10 minutes to 24 hours.

The reaction temperature in the above step 1'can be set in a timely manner, for example, it may be in the range of 0 to 100 ° C., and is usually preferably room temperature (about 10 ° C. to about 35 ° C.).

The reaction time of the above step 1 ″ can be set in a timely manner, but is typically 10 minutes or more, for example, 10 minutes to 24 hours.

The reaction temperature in the above step 1 ″ can be set in a timely manner, for example, it may be in the range of 0 to 100 ° C., and is usually preferably room temperature (about 10 ° C. to about 35 ° C.).

6-Hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal hydrogen carbonate, alkali metal alkoxide, alkaline earth metal hydroxide and quaternary ammonium The mode of the combination of the base and the aqueous solvent selected from the group consisting of hydroxide is not particularly limited, and for example, a combination of 6-hydroxyhexanoic acid, an alkali metal hydroxide and an aqueous solvent, 6-hydroxyhexanoic acid. Examples include a combination of an ester, an alkali metal hydroxide and an aqueous solvent, a combination of ε-caprolactone, an alkali metal hydroxide and an aqueous solvent, a combination of 6-hydroxyhexanoic acid, sodium hydroxide and water, 6-. Combination of methyl hydroxyhexanoate, sodium hydroxide and water, combination of ε-caprolactone, lithium hydroxide and water, combination of ε-caprolactone, sodium hydroxide and water, ε-caprolactone, potassium hydroxide and water , A combination of ε-caprolactone, sodium hydroxide, water and methanol, or a combination of ε-caprolactone, sodium hydroxide, water and dimethylsulfoxide is suitable.

(Step 2: Amidite conversion reaction)
The compound represented by the formula (III) or a salt thereof is obtained by reacting the compound represented by the formula (II) with an amidite-forming reagent in the presence of a coupling agent.

[In the formula, R ¹ and R ² each independently represent a protecting group, and R ³ and R ⁴ each independently represent a optionally substituted hydrocarbon group. ]

The salt of the compound represented by the formula (III) is not particularly limited, and is, for example, hydrochloride, bromate, iodate, perchlorate, carbonate, trifluoromethanesulfonate, tetrafluoroborate. , Hexafluorophosphate or tetrazole salt.

^{The description of R 1} in formula (I) can be incorporated into the description ^{of R 1 in} formula (III).

In the formula (III), R ² represents a protecting group, for example, it may be a functional group that converts the phosphoric acid group into inactive, and a known protecting group of the phosphoric acid group can be used. For the protecting group of the phosphoric acid group, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. The R ^2, for example, substitution such as a methyl group, an ethyl group, an isopropyl group, tert- butyl group, an allyl group, a 2-cyanoethyl group, 2-trimethylsilylethyl group, 2,2,2-trichloroethyl group, a benzyl group Examples thereof include, but are not limited to, an alkyl group which may be used, a cycloalkyl group such as a cyclohexyl group, or an aryl group which may be substituted such as a phenyl group and a 2-chlorophenyl group.

In formula (III), R ³ and R ⁴ represent hydrocarbon groups that may be substituted independently, respectively, and R ³ and R ⁴ are rings (eg, for example) with the nitrogen atom to which they are attached. It may form a pyrrolidine ring). Here, the hydrocarbon group means a functional group composed of a carbon atom and a hydrogen atom. Examples of R ³ and R ⁴ independently include an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an arylalkyl group, an alkylaryl group, a cycloalkyl group, a cycloalkenyl group, and a cycloalkylalkyl group. .. Examples of the functional group which may be substituted in the above-mentioned hydrocarbon group include a halogen atom, an alkoxy group, an amino group, a silyloxy group, a carbonyl group and a carboxyl group. Preferred embodiments of R ³ and R ⁴ include, but are not limited to, a methyl group, an ethyl group, an isopropyl group, a tert-butyl group, a phenyl group or a benzyl group, respectively.

In formula (III), R ² and R ³ and / or R ⁴ may be combined to form a ring (eg, an oxazaphosphoridine ring), and the ring may be monocyclic or bicyclic.

In formula (III), R ¹ is preferably a trityl group, a 4-methoxytrityl group or a 4,4′-dimethoxytrityl group.

In formula (III), R ² is preferably a 2-cyanoethyl group, a benzyl group or a 2-chlorophenyl group.

In formula (III), R ³ and R ⁴ are preferably ethyl groups or isopropyl groups independently of each other.

In the preferred combination of ^{R 1} , R ² , R ³ and R ⁴ in formula (III) ^{, R 1} is a trityl group, a 4-methoxytrityl group or a 4,4′-dimethoxytrityl group, and R ² is a 2-cyanoethyl group, a benzyl group or a 2-chlorophenyl group, R ³ is an ethyl group or an isopropyl group, R ⁴ is an ethyl group or an isopropyl group, and R ¹ is 4 , 4'-dimethoxytrityl group, R ² is a 2-cyanoethyl group, R ³ and R ⁴ are both isopropyl groups, or R ¹ is a 4,4'-dimethoxytrityl group and R ^{A combination in which 2} is a 2-chlorophenyl group and R ³ and R ⁴ are both isopropyl groups is suitable.

In a preferred embodiment, the compound represented by the formula (III) or a salt thereof may be a compound represented by the following formula (III') or a salt thereof.

The compound represented by the formula (III') or a salt thereof may be an optically active substance represented by the following formula (III'-1) or the following formula (III'-2) or a salt thereof.

The coupling agent used for the amidite reaction is not particularly limited, and examples thereof include a coupling activator used for oligonucleotide synthesis by a tertiary amine or a phosphoramidite method.

The tertiary amine is not limited to the following, and examples thereof include triethylamine and diisopropylethylamine. The molar equivalent of the tertiary amine is preferably 1 to 10 molar equivalents, more preferably 1 to 2 molar equivalents, relative to the compound represented by formula (II).

Coupling activators used for oligonucleotide synthesis by the phosphoramidite method are described, for example, in Tetrazole, 69, 2013, 3615-3637, for example, tetrazole, 5-benzylthiotetrazole, 5-ethylthiotetrazole, Azol coupling activators such as 4,5-dicyanoimidazole, 1-hydroxy-benztriazole or 3-nitro-1,2,4-triazole, diisopropylammonium tetrazolide, benzimidazole trifluoroacetate, N- Examples thereof include salt complex coupling activators such as phenylimidazole trifluoroacetate or 1- (cyanomethyl) piperidinium tetrafluoroborate.

The molar equivalent of the coupling agent is preferably 0.1 to 10 molar equivalents, more preferably 1 to 2 molar equivalents, relative to the compound represented by formula (II).

The amidite-forming reagent used in the amidite-forming reaction is not particularly limited, and examples thereof include phosphoramidite and chlorophosphoroamidite.

Examples of phosphoramidite include 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphomidite, methyl-N, N, N', N'-tetraisopropylphosphoromidite, tert-butyl-. N, N, N', N'-tetraisopropylphosphorodiamidite, allyl-N, N, N', N'-tetraisopropylphosphorodiamidite, benzyl-N, N, N', N'-tetraisopropylphosphorologite Amidite, 2-chlorophenyl-N, N, N', N'-tetraisopropylphosphorodite amidite, 2-cyanoethyl-N, N, N', N'-tetraethyl phosphoramidite, methyl-N, N, N', N'-Tetraethyl phosphoramidite, tert-butyl-N, N, N', N'-tetraethyl phosphoramidite, allyl-N, N, N', N'-tetraethyl phosphoramidite, benzyl-N, N, Examples thereof include N', N'-tetraisopropyl phosphoramidite or 2-chlorophenyl-N, N, N', N'-tetraethyl phosphoramidite.

Examples of chlorophosphoroamidite include 2-cyanoethyl-N, N, -diisopropylchlorophosphoroamidite, methyl-N, N, -diisopropylchlorophosphoroamidite or 2-chlorophenyl-N, N, -diisopropylchlorophosphoro. Amidite can be mentioned.

The molar equivalent of the amidite-forming reagent is preferably 1 to 10 molar equivalents, more preferably 1 to 2 molar equivalents, relative to the compound represented by the formula (II).

The coupling agent and the amidite-forming reagent may be commercially available products, or may be synthesized by a known method or a method similar thereto.

The amidite reaction may be carried out in an aprotic solvent. The amount of the aprotic solvent used is preferably 1 to 100 times by weight with respect to the compound represented by the formula (II).

Here, the aprotic solvent means an organic solvent having no proton donating property. The aprotic solvent is not particularly limited, but for example, toluene, xylene, dichloromethane, chloroform, dichloroethane, methyl tert-butyl ether, cyclopentyl methyl ether, diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl ethyl ketone, methyl isobutyl ketone, acetone. , Methyl acetate, ethyl acetate, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, 1,3-dimethyl-2-imidazolidinone, N, N'-dimethylpropyleneurea, acetonitrile, propionitrile, dimethyl sulfoxide or sulfolane Can be mentioned.

The reaction time of the amidite reaction can be set in a timely manner, but is typically 2 hours or more, for example, 2 to 24 hours.

The reaction temperature of the amidite reaction can be set in a timely manner, for example, it may be in the range of 10 to 100 ° C., and usually room temperature (about 10 ° C. to about 35 ° C.) is preferable.

In the amidation reaction, the order of addition of each reagent is not particularly limited. For example, a method of sequentially adding a compound represented by the formula (II), a coupling agent and an amidite reagent to a solvent, a compound represented by the formula (II). A method of sequentially adding a solvent, a coupling agent and an amidation reagent to the solvent, a method of simultaneously adding a compound represented by the formula (II), a coupling agent and an amidite reagent to the solvent, a compound represented by the formula (II), and a cup. A method of adding a solvent to a mixture of a ring agent and an amidite-forming reagent, a method of sequentially mixing a coupling agent mixed with a solvent and an amidite-forming reagent mixed with a solvent with a compound represented by the formula (II) mixed with the solvent. Alternatively, a method of simultaneously mixing the coupling agent mixed with the solvent and the amidite-forming reagent mixed with the solvent to the compound represented by the formula (II) mixed with the solvent can be mentioned. Usually, a method of sequentially adding a solvent, a coupling agent and an amidite-forming reagent to the compound represented by the formula (II) is preferable.

After completion of the amidite reaction, for example, the organic layer can be washed with an aqueous sodium hydrogen carbonate solution or the like, and then the organic layer can be concentrated to isolate the compound represented by the formula (III) or a salt thereof.

The compound represented by the formula (III) or a salt thereof can be purified by appropriately using a known purification method such as chromatography.

The preferred embodiment of the coupling agent and the preferred embodiment of the amidite-forming reagent can be appropriately combined. The mode of the combination of the coupling agent and the amidite-forming reagent is not limited to the following, but is a combination of a tertiary amine and chlorophosphoroamidite, or a coupling activator and phospho used for oligonucleotide synthesis by the phosphoramidite method. A combination with a logiamidite can be mentioned. Preferred combinations of the coupling agent and the amidite-forming reagent include a combination of triethylamine and 2-cyanoethyl-N, N, -diisopropylchlorophosphoroamidite or a combination of diisopropylammonium tetrazolide and 2-cyanoethyl-N, N, N. A combination with', N'-tetraisopropylphosphorodiamidite can be mentioned.

The compound represented by the formula (III) or a salt thereof can be used as a monomer for nucleic acid synthesis.

2. Method for producing oligonucleic acid:
The present invention also provides a method for producing an oligonucleic acid, which comprises a step of carrying out a nucleic acid synthesis reaction using the compound represented by the formula (III) obtained by the above production method or a salt thereof.

The above nucleic acid synthesis reaction can be carried out based on the phosphoramidite method.

The obtained oligonucleic acid can be purified by appropriately using a known purification method such as chromatography.

Examples of the oligonucleic acid include, but are not limited to, the ssTbRNA molecule, strand 1 or strand 2 described in Example 1 of WO2019 / 189722.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the technical scope of the present invention is not limited to these examples.

(Reference example 1)
Synthesis of DMTr-amide-L-proline:
DMTr-amide-L-proline can be synthesized, for example, as described in WO2012 / 017919. Specific examples of synthesis are shown below, but the synthesis method is not limited thereto.

(1) Fmoc-hydroxyamide-L-proline Fmoc-L-proline (10.0 g, 29.6 mmol), 4-amino-1-butanol (3.18 g, 35.6 mol) and 1-hydroxybenzotriazole (10). .9 g, 70.7 mmol) was mixed, and the mixture was degassed under reduced pressure and charged with argon gas. Anhydrous acetonitrile (140 mL) was added to the obtained mixture at room temperature, and an anhydrous acetonitrile solution (70.0 mL) of dicyclohexylcarbodiimide (7.34 g, 35.6 mmol) was added, and then 15 at room temperature under an argon atmosphere. Stirred for hours. After completion of the reaction, the generated precipitate was filtered off, and the solvent was distilled off from the recovered filtrate under reduced pressure. The obtained residue was diluted with dichloromethane (200 mL), washed with saturated aqueous sodium hydrogen carbonate (200 mL), and then the organic layer was separated. The organic layer was dried over magnesium sulfate and then filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure, and diethyl ether (200 mL) was added to the residue to make a powder. The resulting powder was collected by filtration to give Fmoc-hydroxyamide-L-proline as a colorless powdery substance. Here, Fmoc is a 9-fluorenylmethyloxycarbonyl group.

(2) DMTr-amide-L-proline Fmoc-hydroxyamide-L-proline (7.80 g, 19.1 mmol) was mixed with anhydrous pyridine (5.00 mL) and azeotropically dried twice at room temperature. To the obtained residue was added 4,4'-dimethoxytritylchloride (8.20 g, 24.2 mmol), 4-dimethylaminopyridine (23.3 mg, 0.191 mmol) and anhydrous pyridine (39.0 mL). .. The mixture was stirred at room temperature for 1 hour, methanol (7.80 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was diluted with dichloromethane (100 mL), washed with saturated aqueous sodium hydrogen carbonate (150 mL), and then the organic layer was separated. The organic layer was dried over sodium sulfate and then filtered. The solvent was distilled off from the obtained filtrate under reduced pressure. Anhydrous N, N-dimethylformamide (39.0 mL) and piperidine (18.7 mL, 189 mmol) were added to the obtained unpurified residue, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off from the mixed solution under reduced pressure at room temperature. The obtained residue was subjected to silica gel column chromatography (trade name: Wakogel C-300, developing solvent: dichloromethane / methanol = 9/1, containing 0.05% pyridine) to obtain DMTr-amide-L as a pale yellow oily substance. -I got proline. Here, DMTr is a 4,4'-dimethoxytrityl group.

(Example 1)
Synthesis of DMTr-Hydroxydiamide-L-Proline:

Ε-Caprolactone (0.280 g, 2.46 mmol) was added to a 1 M aqueous sodium hydroxide solution (2.66 mL) cooled in an ice bath. Then, the ice bath was removed, and the mixture was stirred at room temperature for 2 hours to prepare an aqueous sodium 6-hydroxyhexanoate solution. Methanol (10.0 mL) was added to DMTr-amide-L-proline (1.00 g, 2.05 mmol) synthesized in Reference Example 1 at room temperature, and the previously prepared sodium 6-hydroxyhexanoate aqueous solution and 4- ( 4,6-Dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride (0.849 g, 3.07 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate (20.0 mL), washed with 5% aqueous sodium hydrogen carbonate solution, and then the organic layer was separated. The organic layer was dried over sodium sulfate and then filtered. The solvent was distilled off from the obtained filtrate under reduced pressure. By subjecting the obtained residue to column chromatography (developing solvent: ethyl acetate / methanol = 100/0 to 97/3) using amino silica gel as a filler, DMTr-hydroxydiamid-L-proline (developing solvent: ethyl acetate / methanol = 100/0 to 97/3) was used as an oily substance. 1.00 g, yield 81%) was obtained.

(Comparative Example 1)
Synthesis of DMTr-Hydroxydiamide-L-Proline:

DMTr-amide-L-proline (6.01 g, 12.3 mmol), N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide (2.83 g, 14.7 mmol) synthesized in Reference Example 1, 1 -Hydroxybenzotriazole (3.98 g, 29.5 mmol) and triethylamine (4.47 g, 44.2 mmol) in anhydrous dichloromethane (120 mL) were mixed. 6-Hydroxycaproic acid (1.95 g, 14.5 mmol) was further added to this mixed solution under an argon atmosphere at room temperature, and then the mixture was stirred at room temperature for 1 hour. The mixture was diluted with dichloromethane (600 mL), washed 3 times with saturated brine (800 mL), and then the organic layer was separated. The organic layer was dried over sodium sulfate and then filtered. The solvent was distilled off from the obtained filtrate under reduced pressure. As a result, DMTr-hydroxydiamide-L-proline (6.29 g) was obtained as a pale yellow foamy substance.

(Example 2)
Synthesis of DMTr-diamide-L-proline amidite:

DMTr-hydroxydiamide-L-proline (1.00 g, 1.66 mmol) synthesized in Example 1 was mixed with anhydrous acetonitrile and azeotropically dried 3 times at room temperature. Anhydrous acetonitrile (10.0 mL) and diisopropylammonium tetrazolide (0.341 g, 1.99 mmol) were added to the obtained residue, degassed under reduced pressure, and filled with argon gas. To the mixture was added 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite (0.603 g, 1.99 mmol). The mixture was stirred at room temperature for 3 hours. The mixture was diluted with dichloromethane, washed with saturated aqueous sodium hydrogen carbonate (20.0 mL), and further washed with saturated brine (20.0 mL) to separate the organic layer. The organic layer was dried over sodium sulfate and then filtered. The solvent was distilled off from the obtained filtrate under reduced pressure. By subjecting the obtained residue to column chromatography (developing solvent: n-hexane / ethyl acetate = 1/3) using amino silica gel as a filler, DMTr-diamide-L-proline amidite as a colorless syrup-like substance. (1.13 g, yield 85%, purity 99%) was obtained. The above purity value represents the peak area ratio based on the chromatogram measured under the following HPLC analysis conditions.

The conditions for the purity analysis of DMTr-diamide-L-proline amidite are as follows.
-Column: L-column2 ODS (CERI) 4.6 x 150 mm, 3 μm
-Column temperature: 30 ° C
-Mobile phase: 20 mM potassium phosphate aqueous solution / acetonitrile = 30/70
・ Analysis time: 50 min
・ Flow velocity: 1.0 mL / min
-Detection: PDA detector (210 nm)
20 mM Potassium Phosphate Aqueous Solution Preparation Method Potassium dihydrogen phosphate (2.70 g) and dipotassium hydrogen phosphate (3.45 g) are added to distilled water (1980 mL) to dissolve them.

(Comparative Example 2)
Synthesis of DMTr-diamide-L-proline amidite:

DMTr-diamide-L- by the same method as in Example 2 except that DMTr-hydroxydiamide-L-proline synthesized in Comparative Example 1 was used instead of DMTr-hydroxydiamide-L-proline synthesized in Example 1. Proline amidite (2-step yield 72%, purity 97%) was obtained.

Here, comparing Example 2 and Comparative Example 2, the DMTr-diamide-L-proline amidite synthesized in Comparative Example 2 was converted to RRT 1.64 based on DMTr-diamide-L-proline amidite, and that of Example 2. While 1.8% of individual maximum impurities (hereinafter referred to as RRT 1.64 impurities) were detected in the peak area ratio based on the chromatogram measured under HPLC analysis conditions, DMTr-diamide-L synthesized in Example 2 was detected. -No impurities of RRT1.64 were detected in proline amidite. Impurities of RRT1.64 are difficult to purify by column chromatography using amino silica gel as a filler, and DMTr-diamide-L-proline amidite, which is a syrup-like substance, cannot be purified by recrystallization. Therefore, the impurities of RRT1.64 were impurities that were difficult to separate by the purification operation.

(Example 3)
An oligonucleic acid having the structure shown below was synthesized.

Oligonucleic acid was synthesized by the phosphoramidite method using an automatic nucleic acid synthesizer. TBDMS amidite was used as the RNA amidite for this synthesis. Polymer beads for nucleic acid synthesis to which protected guanosine was bound were used at the 3'end of the oligonucleic acid, and DMTr-diamide-L-proline amidite synthesized in Example 2 was used as a special amidite for ligation of Ly. In addition, solid-phase synthesis of nucleic acid and deprotection reaction after synthesis were carried out according to a conventional method.

(Comparative Example 3)
Oligonucleic acid was synthesized by the same method as in Example 3 except that DMTr-diamide-L-proline amidite synthesized in Comparative Example 2 was used instead of DMTr-diamide-L-proline amidite synthesized in Example 2. rice field.

When the solutions of the oligonucleic acids synthesized in Example 3 and Comparative Example 3 were purified by reverse phase chromatography, the oligonucleic acid synthesized in Comparative Example 3 contained all the fractions in which the target oligonucleic acid was eluted. RRT1.05 impurities (hereinafter, RRT1.05 impurities) were detected at a peak area ratio of 2.0% or more based on the chromatogram measured under the following UPLC analysis conditions, and separated from the target oligonucleic acid. The result was that it contained difficult impurities. On the other hand, the oligonucleic acid synthesized in Example 3 did not contain impurities of RRT1.05 at the stage before purification, and a high-purity oligonucleic acid was obtained by the purification operation.

The purified oligonucleic acids of Example 3 and Comparative Example 3 were analyzed by mass spectrometry after desalting treatment, respectively, and it was confirmed that they matched the molecular weight of the target product. From the above results, it was shown that a high-purity oligonucleic acid can be produced by using the compound represented by the formula (III) obtained by the production method of the present invention.

The conditions for the purity analysis of oligonucleic acid are as follows.
-Column: AQUITY UPLC C18 (Waters), 2.1 x 50 mm, 1.7 μm
-Column temperature: 60 ° C
-Mobile phase A: 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol-8 mM triethylamine aqueous solution-Mobile phase B: methanol-Development conditions: A / B = 95/5 → 80/20 ( 0-20min, linear gradient)
・ Flow velocity: 0.2 mL / min
-Detection: PDA detector (260 nm)

According to the present invention, a compound represented by the formula (III) or a salt thereof, which can produce a high-purity oligonucleic acid, can be produced.

Claims (7)

1. In the presence of a dehydration condensing agent, the compound represented by the following formula (I) or a salt thereof is reacted with an alkali metal salt of 6-hydroxyhexanoic acid, an alkaline earth metal salt or a quaternary ammonium salt in a solvent. The step of obtaining the compound represented by the following formula (II) and
   

   

   [In the formula, R ¹ represents a protecting group. ]
   

   

   [In the formula, R ¹ is synonymous with the above definition. ]
   In the presence of a coupling agent, the compound represented by the above formula (II) is reacted with the amidite-forming reagent to obtain the compound represented by the following formula (III) or a salt thereof.
   

   

   [In the formula, R ¹ is synonymous with the above definition, R ² represents a protecting group, and R ³ and R ⁴ each independently represent a optionally substituted hydrocarbon group. ]
   The method for producing a compound represented by the above formula (III) or a salt thereof.
2. The production method according to claim 1, wherein the solvent is a water-containing solvent.
3. The production method according to claim 1 or 2, wherein the dehydration condensate is a triazine-type dehydration condensate.
4. As the alkali metal salt, alkaline earth metal salt or quaternary ammonium salt of 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ε-caprolactone, alkali metal hydroxide, alkali metal Claimed to use a mixture of a base selected from the group consisting of carbonates, alkali metal hydrogen carbonates, alkali metal alkoxides, alkaline earth metal hydroxides and quaternary ammonium hydroxides reacted in an aqueous solvent. Item 3. The production method according to any one of Items 1 to 3.
5. The alkali metal salt, alkaline earth metal salt or quaternary ammonium salt of 6-hydroxyhexanoic acid is lithium 6-hydroxyhexanoate, sodium 6-hydroxyhexanoate, potassium 6-hydroxyhexanoate or 6-hydroxyhexanoic acid. The production method according to any one of claims 1 to 4, which is cesium.
6. R ¹ is a trityl group, a 4-methoxytrityl group or a 4,4′-dimethoxytrityl group.
   R ² is a 2-cyanoethyl group, a benzyl group or a 2-chlorophenyl group.
   R ³ is an ethyl group or an isopropyl group,
   The production method according to any one of claims 1 to 5, wherein R ^{4 is an ethyl group or an isopropyl group.}
7. A method for producing an oligonucleic acid, comprising a step of carrying out a nucleic acid synthesis reaction using the compound represented by the following formula (III) or a salt thereof obtained by the production method according to any one of claims 1 to 6.
   

   

   [In the formula, R ¹ and R ² each independently represent a protecting group, and R ³ and R ⁴ each independently represent a optionally substituted hydrocarbon group. ]