Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
  • C07F9/572—Five-membered rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the present invention relates to a method for producing a monomer for producing nucleic acid.
• Non-Patent Document 1 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.
• 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.
• 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.
• R 1 and R 2 each independently represent a protecting group
• R 3 and R 4 each independently represent a optionally substituted hydrocarbon group.
• the present invention includes the following.
• R 1 is synonymous with the above definition
• R 2 represents a protecting group
• R 3 and R 4 each independently represent a optionally substituted hydrocarbon group.
• alkali metal salt alkaline earth metal salt or quaternary ammonium salt of 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ⁇ -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.
• 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-.
• 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
• R 3 is a group.
• R 4 is an ethyl group or an isopropyl group, a process according to any one of [1] to [5].
• 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.
• 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.
• 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.
• 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.
• a 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.
• R 1 is preferably a trityl group, a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group.
• 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.
• 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.
• 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.
• 6-hydroxyhexanoate a commercially available product is used, or 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ⁇ -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.
• 6-hydroxycaproic acid, 6-hydroxycaproic acid ester or ⁇ -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.
• methyl 6-hydroxyhexanoate ethyl 6-hydroxyhexanoate, benzyl 6-hydroxyhexanoate, 6-hydroxyhexanoic acid (5-carboxypentyl), phenyl 6-hydroxyhexanoate and the like can be mentioned.
• alkali metal salt of 6-hydroxyhexanoic acid examples include lithium 6-hydroxyhexanoate, sodium 6-hydroxyhexanoate, potassium 6-hydroxyhexanoate, rubidium 6-hydroxyhexanoate, cesium 6-hydroxyhexanoate or 6-hydroxy.
• Francium hexanoate can be mentioned.
• alkaline earth metal salt of 6-hydroxyhexanoic acid examples 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.
• 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.
• 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.
• a desorbable functional group such as a quaternary ammonium or a halogen atom (for example, a free form or a salt).
• a desorbable functional group such as a quaternary ammonium or a halogen atom (for example, a free form or a salt).
• 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]-.
• 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- Methyl
• 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.),
• 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).
• benzotriazolyluronium type dehydration condensate examples 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.
• the benzotriazolyluronium-type dehydration condensing agent is a benzotriazolyluronium, for example, O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyl.
• azabenzotriazolyluronium for example, O- (7-azabenzotriazo
• 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.
• 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.
• 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
• 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.
• 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.
• HOBt 1-hydroxybenzotriazole
• HOAt 1-hydroxy-7-azabenzotriazole
• 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.
• 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.).
• 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 simultaneously mixing the 6-hydroxyhexanoate mixed with and the dehydration condensing agent mixed with the solvent can be mentioned.
• 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.
• 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
• EDCI hydrochloride and HOAt 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.
• 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.
• 6-hydroxyhexanoate 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ⁇ -caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal hydrogen carbonate, alkali metal alkoxide, alkaline earth
• a base selected from the group consisting of metal hydroxide and quaternary ammonium hydroxide in an aqueous solvent
• 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.
• step 1 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ⁇ -caprolactone, alkali metal hydroxide, alkali metal carbonate, alkali metal bicarbonate, alkali metal alkoxide, alkaline earth metal water.
• 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.
• 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ⁇ -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.
• 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.
• each reagent is not particularly limited.
• 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid ester or ⁇ -caprolactone, alkali metal hydroxide, alkali metal carbonate are added to the 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 ⁇ .
• 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 ⁇ -caprolactone is preferable.
• 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 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.
• 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.
• alkali metal hydroxide examples include lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide or francium hydroxide.
• alkali metal carbonate examples include lithium carbonate, sodium carbonate, potassium carbonate, rubidium carbonate, cesium carbonate or franchium carbonate.
• alkali metal bicarbonate examples include lithium hydrogencarbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, rubidium hydrogencarbonate, cesium hydrogencarbonate, and franchium hydrogencarbonate.
• alkali metal alkoxide examples include, but are not limited to, sodium methoxide, sodium ethoxide, lithium tert-butoxide, sodium tert-butoxide or potassium tert-butoxide.
• alkaline earth metal hydroxides examples 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 Tributylmethylammonium oxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetraoctylammonium hydroxide, benzylcinconidinium hydroxide, benzylcinconinium hydroxide, hydroxide Benzylkinidinium or benzylkininium hydroxide can be mentioned.
• 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.
• 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 ⁇ -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 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.
• 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 ⁇ -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.).
• 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 ⁇ -caprolactone, an alkali metal hydroxide and an aqueous solvent, a combination of 6-hydroxyhexanoic acid, sodium hydroxide and water, 6-.
• 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.
• R 1 and R 2 each independently represent a protecting group
• 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.
• R 1 in formula (I) can be incorporated into the description of R 1 in 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.
• a 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
• 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.
• 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).
• the hydrocarbon group means a functional group composed of a carbon atom and a hydrogen atom.
• 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.
• 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.
• R 1 is preferably a trityl group, a 4-methoxytrityl group or a 4,4′-dimethoxytrityl group.
• R 2 is preferably a 2-cyanoethyl group, a benzyl group or a 2-chlorophenyl group.
• R 3 and R 4 are preferably ethyl groups or isopropyl groups independently of each other.
• 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
• R 4 is an ethyl group or an isopropyl group
• R 1 is 4 , 4'-dimethoxytrityl group
• R 2 is a 2-cyanoethyl group
• R 3 and R 4 are both isopropyl groups
• 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.
• 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'-tetrais
• chlorophosphoroamidite examples 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).
• 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.
• 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.
• each reagent is not particularly limited.
• 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.
• 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.
• 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.
• 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.
• 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.
• oligonucleic acid examples include, but are not limited to, the ssTbRNA molecule, strand 1 or strand 2 described in Example 1 of WO2019 / 189722.
• 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.
• 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.
• 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.
• DMTr is a 4,4'-dimethoxytrityl group.
• ⁇ -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.
• the solvent was distilled off from the obtained filtrate under reduced pressure.
• 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.
• 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.
• amino silica gel as a filler
• DMTr-diamide-L-proline amidite was obtained.
• the above purity value represents the peak area ratio based on the chromatogram measured under the following HPLC analysis conditions.
• Example 2 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.
• RRT 1.64 impurities 1.8% of individual maximum impurities
• 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.
• 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
• 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.
• a compound represented by the formula (III) or a salt thereof, which can produce a high-purity oligonucleic acid can be produced.

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