WO2023282120A1 - 核酸オリゴマーを含む組成物 - Google Patents

核酸オリゴマーを含む組成物 Download PDF

Info

Publication number
WO2023282120A1
WO2023282120A1 PCT/JP2022/025692 JP2022025692W WO2023282120A1 WO 2023282120 A1 WO2023282120 A1 WO 2023282120A1 JP 2022025692 W JP2022025692 W JP 2022025692W WO 2023282120 A1 WO2023282120 A1 WO 2023282120A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
formula
nucleic acid
solution
independently
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/025692
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
悠也 森山
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sumitomo Chemical Co Ltd
Original Assignee
Sumitomo Chemical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Chemical Co Ltd filed Critical Sumitomo Chemical Co Ltd
Priority to KR1020247000264A priority Critical patent/KR20240028416A/ko
Priority to CN202280040815.5A priority patent/CN117425663B/zh
Priority to EP22837536.6A priority patent/EP4368627A4/en
Priority to JP2023533547A priority patent/JP7806051B2/ja
Priority to US18/574,655 priority patent/US20240300993A1/en
Publication of WO2023282120A1 publication Critical patent/WO2023282120A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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 compositions containing nucleic acid oligomers.
  • the invention more particularly relates to compositions comprising phosphorothioate-containing nucleic acid oligomers.
  • nucleic acid oligomers include nucleic acids that induce RNA interference (RNAi) such as antisense nucleic acids, aptamers, ribozymes, and siRNA, and these are called nucleic acid drugs.
  • RNAi RNA interference
  • Nucleic acid oligomers are known to be synthesized by solid-phase synthesis, and nucleic acid oligomers having phosphorothioate bonds are also known as useful compounds synthesized by solid-phase synthesis (Patent Document 1).
  • Nucleic acid oligomers with phosphorothioate bonds may have problems with stability during the manufacturing process.
  • An object of the present invention is to provide a stable composition comprising a nucleic acid oligomer having phosphorothioate linkages, a method for producing the same, and an efficient method for producing the nucleic acid oligomer from the composition.
  • the present inventors have found that a crude nucleic acid oligomer having phosphorothioate bonds produced by the phosphoramidite method in the solid-phase synthesis method can be obtained by reversed-phase chromatography. It has been found that compositions obtained by mixing nucleic acid oligomers with alkylammonium salts, nitrile organic solvents, water, and certain additives can be stabilized. Accordingly, the present invention provides such compositions, methods for their production, and efficient methods for producing nucleic acid oligomers from such compositions.
  • the present invention includes the following aspects, but is not limited to them.
  • B C each independently represent the same or different nucleobases, R are each independently the same or different and represent a hydrogen atom, a fluorine atom, or an OQ group; Q are each independently the same or different, a hydrogen atom, a methyl group, a 2-methoxyethyl group, a methylene group bonded to the carbon atom at the 4'-position of ribose, and a carbon atom at the 4'-position of ribose Representing a bonded ethylene group or an ethylidene group bonded to the carbon atom at the 4'-position of ribose, each X is independently the same or different and represents an oxygen atom or a sulfur atom (provided that at least one X represents a sulfur atom); Y represents a hydrogen atom or a hydroxyl-protecting group, G represents an ammonium ion, an alkylammonium ion, an alkali metal ion
  • L 2 is each independently the same or different C6-14 arylene optionally substituted with 1 to 3 substituents selected from the group consisting of a C1-6 alkyl group and a C1-6 alkoxy group group, or represents a C1-6 alkylene group
  • L 3 represents a C6-14 arylene group optionally substituted with 1 to 3 substituents selected from the group consisting of a C1-6 alkyl group and a C1-6 alkoxy group.
  • the group wherein the additive consists of triphenylphosphine, diethylphosphite, triethylphosphite, diethoxyphenylphosphine, ethoxydiphenylphosphine, and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide 2.
  • each R is independently a hydroxy group or a methoxy group.
  • a method for producing a nucleic acid oligomer comprising:
  • a nucleic acid oligomer represented by formula (1) obtained by subjecting a crude nucleic acid oligomer of formula (1) synthesized by a solid-phase synthesis method to reversed-phase column chromatography, an alkylammonium salt, and a water-soluble organic solvent. 6.
  • the present invention provides a stable composition containing a nucleic acid oligomer having a phosphorothioate bond and an efficient method for producing the nucleic acid oligomer using the composition.
  • FIG. 1 shows an example of nucleic acid oligomer synthesis by the phosphoramidite method.
  • a composition comprising a nucleic acid oligomer having a phosphorothioate bond represented by formula (1), an alkylammonium salt, a nitrile organic solvent, water, and an additive, wherein the additive comprises a compound having a disulfide bond and a sulfide bond.
  • a composition characterized by being at least one compound selected from the group consisting of compounds having In the above formula (1), the nucleic acid base represented by B C (hereinafter also referred to as "base”) may be a natural or non-natural nucleic acid base. Examples of such non-natural nucleobases are modified analogs of natural or non-natural nucleobases. Nucleic acid bases are typically exemplified by purine compounds and pyrimidine compounds.
  • purine bases such as adenine, isoguanine, xanthine, hypoxanthine and guanine
  • pyrimidine bases such as cytosine, uracil and thymine.
  • nucleobases represented by B C include, for example, amino derivatives such as 2-aminoadenine, 2-aminopurine, 2,6-diaminopurine; 5-methyluracil, 5-methylcytosine, 7-methylguanine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6-azauracil, 6-azacytosine and 6-azathymine; 5-uracil (pseudo uracil), 4-thiouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil; 8-halogenated, aminated, thiolated, thioalkylated, hydroxylated and other 8-substituted purines; fluoromethylated and other 5-substituted pyrimidines; 6-azapyrimidines; N-2, N-6 and O-6 substituted purines (including 2-aminopropyladenine); dihydrour
  • R represents an OQ group and Q is a methylene group bonded to the carbon atom at the 4'-position of ribose, an ethylene group bonded to the carbon atom at the 4'-position of ribose, or a carbon atom at the 4'-position of ribose
  • the structure is represented by the structures of LNA-1, LNA-2 and LNA-3 shown in formula (3) below.
  • B c represents a nucleobase as defined above.
  • the hydroxyl-protecting group represented by Y can be used without particular limitation as long as it can function as a protecting group in the amidite method.
  • known protecting groups used for amidite compounds are widely used. can be used.
  • the hydroxyl-protecting group represented by Y is preferably the following groups.
  • R 1 , R 2 and R 3 are each independently the same or different and represent hydrogen or an alkoxy group.
  • alkoxy group examples include a methoxy group.
  • the chain length of the nucleic acid oligomer of formula (1) is n ⁇ 15.
  • the upper limit of the chain length is, for example, n ⁇ 200.
  • the nucleic acid oligomer of formula (1) may be, for example, a DNA or RNA oligomer, or one containing non-natural nucleic acid bases in these oligomers. Said nucleic acid oligomer is typically a single-stranded DNA or RNA oligomer.
  • each substituent R is preferably independently a hydroxy group or a methoxy group.
  • the nucleic acid oligomer is preferably an RNA that is a nucleic acid oligomer of formula (1) in which each substituent R is independently a hydroxy group or a methoxy group. More particularly, nucleic acid oligomers containing both nucleotides in which the substituent R is a hydroxy group and nucleotides in which a methoxy group are preferred.
  • the concentration of the nucleic acid oligomer in the composition is generally 0.05 mg/mL to 5 mg/mL, preferably 0.05 mg/mL to 1 mg/mL, more preferably 0.1 mg/mL to 0.1 mg/mL. 5 mg/mL.
  • alkylammonium salts monoalkylammonium salts, dialkylammonium salts and trialkylammonium salts are usually used, preferably monoalkylammonium salts and dialkylammonium salts, more preferably dialkylammonium salts.
  • the monoalkylamine that forms the monoalkylammonium salt preferably has 3 to 10 carbon atoms, more preferably 4 to 6 carbon atoms, and still more preferably hexylamine.
  • the dialkylamine that forms the dialkylammonium salt preferably has 4 to 10 carbon atoms, more preferably 5 to 9 carbon atoms.
  • a preferred dialkylamine is di-n-butylamine.
  • the trialkylamine that forms the trialkylammonium salt preferably has 6 to 12 carbon atoms, more preferably 6 to 9 carbon atoms, and is specifically exemplified by triethylamine.
  • acids that form the monoalkylammonium salts, dialkylammonium salts and trialkylammonium salts include carbonic acid, acetic acid, formic acid, trifluoroacetic acid and propionic acid.
  • the concentration of the ammonium salt is usually 1-200 mM, preferably 5-150 mM, more preferably 20-100 mM.
  • Acetonitrile is exemplified as the nitrile-based organic solvent.
  • the amount of the nitrile-based organic solvent in the composition is generally 10-70%, preferably 20-60%, more preferably 30-50%, based on the total mass of the composition. % represents mass %).
  • the composition may further contain an alcoholic organic solvent. Examples of alcoholic organic solvents include C1-4 alcohols, preferably C1-3 alcohols, more preferably C1-2 alcohols, and still more preferably methanol.
  • the amount of the alcoholic organic solvent in the composition is generally 0-20%, preferably 0-15%, more preferably 0-10%, based on the total weight of the composition ( All percentages above represent % by mass).
  • the amount of water may be an amount that balances to satisfy the concentration ranges of the above components, and is usually 90% to 30%, preferably 80% to 40%, based on the total weight of the composition. and more preferably 70% to 40% (% above represents % by mass).
  • At least one compound selected from the group consisting of compounds represented by formulas (3a) to (3h) will be described.
  • a C1-6 alkyl group in a C6-10 aryl group optionally substituted with 1 to 3 substituents selected from the group consisting of a C1-6 alkyl group and a C1-6 alkoxy group represented by L 1 is exemplified by a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group and a hexyl group.
  • a C1-4 alkyl group is more preferable, and a C1-2 alkyl group is still more preferable.
  • C1-6 alkoxy groups include methoxy, ethoxy, propoxy, butoxy, pentyloxy and hexyloxy groups.
  • the C6-10 aryl group is exemplified by a phenyl group, a 1-naphthyl group and a 2-naphthyl group, with a phenyl group being preferred.
  • Examples of the C6-10 aryl group substituted with 1 to 3 substituents selected from the group consisting of a C1-6 alkyl group and a C1-6 alkoxy group include a tolyl group and a methoxyphenyl group.
  • a specific example of the compound represented by formula (3a) is triphenylphosphine.
  • the C1-6 alkyl group represented by L 1 is exemplified by the same groups as those mentioned above.
  • a specific example of the compound represented by formula (3b) is triethyl phosphite.
  • Specific examples of the compound represented by formula (3c) include ethoxydiphenylphosphine.
  • Specific examples of the compound represented by formula (3d) include diethoxyphenylphosphine.
  • a specific example of the compound represented by formula (3e) is diethyl phosphite.
  • Specific examples of the compound represented by formula (3f) include ethyl phenylphosphinate.
  • the C6-14 arylene group optionally substituted with 1 to 3 substituents selected from the group consisting of a C1-6 alkyl group and a C1-6 alkoxy group represented by L 2 includes 1,2 -phenylene group, 1,8-naphthylene group, and 1,6-biphenylene group are exemplified.
  • Examples of the C1-6 alkylene group represented by L 2 include methylene group, ethylene group, propylene group, butylene group, pentylene group and hexylene group.
  • the C6-14 arylene group optionally substituted with 1 to 3 substituents selected from the group consisting of a C1-6 alkyl group and a C1-6 alkoxy group represented by L 3 includes the above L 2 are exemplified.
  • Specific compounds represented by formula (3g) include, for example, 1,2-bis(diphenylphosphino)benzene, 1,8-bis((diphenylphosphino)naphthalene, and 2,2′-bis( Diphenylphosphino)biphenyl is exemplified.
  • Specific examples of the compound represented by formula (3h) include 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide.
  • the concentration of the additive is usually 0.1 ⁇ M to 100 mM, preferably 1 mM to 10 mM.
  • the composition of the present invention is generally prepared by reverse digestion of the crude nucleic acid oligomer of formula (1) synthesized by solid-phase synthesis using a mobile phase containing an alkylammonium salt, a water-soluble organic solvent, and water. It is obtained by adding the above additives to the column eluate obtained by phase column chromatography.
  • the composition of the present invention may be prepared as an elution fraction of reversed-phase column chromatography by using a mobile phase containing the additive in advance.
  • the water-soluble organic solvent means an organic solvent including the nitrile-based water-soluble organic solvent and optionally a hydrophilic organic solvent commonly known in the organic field (for example, the alcohol-based organic solvent).
  • the eluted fractions obtained by reversed-phase column chromatography are analyzed for composition with UV absorption at a wavelength of 260 nm under chromatographic conditions generally used for separation and analysis of nucleic acids, and are selected and collected.
  • a purified target nucleic acid oligomer having a predetermined amount of phosphorothioate linkages is obtained from the collected fractions.
  • the analysis method for example, the method described in Non-Patent Literature (Handbook of Analysis of Oligonucleotides and Related Products, CRC Press) can be used.
  • silica or a polymer serving as a hydrophobic stationary phase for example, any one or more selected from a phenyl group, an alkyl group having 1 to 20 carbon atoms, and a cyanopropyl group.
  • immobilized silica or polymer Silica or polymer used as such a filler has a particle size of, for example, 2 ⁇ m or more, or 5 ⁇ m or more.
  • the mobile phase for reversed-phase column chromatography includes, for example, a mobile phase containing an aqueous solution of an ammonium salt having the concentration and pH as described above and the water-soluble organic solvent described above, and is used with a gradient whose concentration is gradually increased.
  • a mobile phase that The temperature for reverse phase column chromatography is generally 20-100°C, preferably 30-80°C, more preferably 40-70°C.
  • the compositions of the invention are typically obtained as eluate fractions of reversed-phase column chromatography as described above.
  • compositions of the invention are selected from post-treatment steps such as reprecipitation, liquid separation, ultrafiltration, deprotection, and lyophilization to isolate nucleic acid oligomers, e.g., after storage steps. It may be subjected to single or multiple steps.
  • the atmosphere in the storage container may be replaced by using an inert gas.
  • inert gases include nitrogen gas, argon gas, and helium gas.
  • the stabilized solution can be contacted with a poor solvent to precipitate and isolate nucleic acid oligomers. If necessary, the liquid portion may be removed from the solid-liquid separated state, and the precipitated nucleic acid oligomer may be collected and isolated by filtration or the like. Poor solvents for the reprecipitation step include C1-C4 organic solvents having at least one oxygen atom (eg, C1-C4 alcohols, tetrahydrofuran, dioxane). Ethanol or isopropanol is preferred as such a solvent.
  • the stabilized solution is mixed with at least one of an acidic aqueous solution such as an acetic acid aqueous solution, water, and a saline solution, and then an organic solvent immiscible with water is added to separate the aqueous layer and the organic layer. It is possible to obtain an aqueous layer containing the desired nucleic acid oligomer.
  • an acidic aqueous solution such as an acetic acid aqueous solution, water, and a saline solution
  • an ultrafiltration membrane can be used to separate nucleic acid oligomers present in the solution after the storage step from low-molecular-weight components having a desired molecular weight or less.
  • the solution after the storage step is mixed with an acidic aqueous solution such as an aqueous acetic acid solution, or a solution obtained by dissolving an acidic substance such as acetic acid in an organic solvent.
  • an acidic aqueous solution such as an aqueous acetic acid solution, or a solution obtained by dissolving an acidic substance such as acetic acid in an organic solvent.
  • the water is sublimated by decompressing the frozen aqueous solution of nucleic acid oligomers, and the nucleic acid oligomers and water can be separated.
  • nucleic acid elongation reactions can be carried out according to known methods (for example, the method described in Japanese Patent No. 5157168 or Japanese Patent No. 5554881).
  • the production of nucleic acid oligomers by the phosphoramidite method the synthesis of RNA in the scheme shown in FIG. A manufacturing method will be described.
  • Ba is an optionally protected nucleobase
  • Tr is a protecting group
  • X is as defined above
  • SP is a portion other than the nucleoside structure of the inorganic porous carrier.
  • the inorganic porous support (Sp-Nu) having a nucleoside structure and the nucleobase constituting the nucleoside of the amidite monomer (Am-1) are the nucleobase as described above or the nucleobase protected with a protecting group.
  • Suitable amidite monomers (Am-1) include compounds represented by the following chemical formula (Am-1′), when R represents a protected hydroxyl group, specific protecting groups include tert- Butyldimethylsilyl (TBDMS) group, bis(2-acetoxy)methyl (ACE) group, (triisopropylsilyloxy)methyl (TOM) group, (2-cyanoethoxy)ethyl (CEE) group, (2-cyanoethoxy) TBDMS amidites (TBDMS RNA Amidites, trade name, ChemGenes Corporation), ACE protected with methyl (CEM) group, para-toluylsulfonylethoxymethyl (TEM) group, (2-cyanoethoxy) methoxymethyl (EMM) group, etc.
  • TDMS tert- Butyldimethylsilyl
  • ACE bis(2-acetoxy)methyl
  • TOM triisopropylsilyloxy)methyl
  • CEE (2-cyanoethoxy)e
  • R represents a group as described above, and Ba represents an optionally protected nucleobase.
  • a solid phase carrier (Am-2) is obtained by deprotecting the Tr group of the inorganic porous carrier (Sp-Nu). Thereafter, the amidite monomer (Am-1) and the solid phase carrier (Am-2) are subjected to a condensation reaction to obtain a reaction product (Am-3). After that, the reaction product (Am-3) is oxidized to obtain the product (Am-4). After this, the product (Am-4) is deprotected (-Tr) to give the product (Am-5). Next, the amidite monomer (Am-1) and the product (Am-5) are further condensed to extend the phosphodiester bond.
  • a nucleic acid molecule with a desired sequence can be produced by excising from the . Such synthesis may be performed using an automatic nucleic acid synthesizer or the like that employs the phosphoramidite method.
  • RNA is taken as an example for explanation, but it can also be applied to nucleic acid compounds containing nucleotides other than ribonucleotides.
  • the protecting group of the hydroxyl group at the 5'-position of the RNA chain end carried on the solid phase carrier is deprotected.
  • a protective group a trityl-based protective group (typically, a 4,4'-dimethoxytrityl group (DMTr group)) is used.
  • Deprotection can be performed with an acid. Acids for deprotection include, for example, trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, hydrochloric acid, acetic acid, p-toluenesulfonic acid, and the like.
  • a phosphite is generated by binding a nucleoside phosphoramidite to the hydroxyl group at the 5'-position of the RNA chain end deprotected in the deprotection step.
  • a nucleoside phosphoramidite one in which the hydroxyl group at the 5'-position is protected with a protecting group (for example, DMTr group) is used.
  • the condensation step can be performed using an activating agent that activates the nucleoside phosphoramidite.
  • activating agents include 5-benzylthio-1H-tetrazole (BTT), 1H-tetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), N-methylbenzimidazolium.
  • N-MeBIT benzimidazolium triflate
  • BIT N-phenylimidazolium triflate
  • IMT imidazolium triflate
  • NBT 5-nitrobenzimidazolium triflate
  • HOBT 1-hydroxybenzotriazole
  • Activator-42 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole
  • the unreacted 5' hydroxyl group may be capped as appropriate.
  • Capping can be carried out using known capping solutions such as acetic anhydride-tetrahydrofuran solution and phenoxyacetic anhydride/N-methylimidazole solution.
  • the oxidation step is a step of oxidizing the phosphite formed by the condensation step.
  • the oxidation step can be performed using an oxidizing agent.
  • Oxidizing agents include iodine, m-chloroperbenzoic acid, tert-butyl hydroperoxide, 2-butanone peroxide, bis(trimethylsilyl) peroxide, 1,1-dihydroperoxycyclododecane, hydrogen peroxide and the like.
  • the "oxidizing agent" may be, for example, sulfur, 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent ), 3-amino-1,2,4-dithiazol-5-thione (ADTT), 5-phenyl-3H-1,2,4-dithiazol-3-one (POS), [(N,N-dimethylamino Methylidene)amino]-3H-1,2,4-dithiazoline-3-thione (DDTT), and phenylacetyl disulfide (PADS) can be used.
  • STT 3-amino-1,2,4-dithiazol-5-thione
  • POS 5-phenyl-3H-1,2,4-dithiazol-3-one
  • DDTT [(N,N-dimethylamino Methylidene)amino]-3H-1,2,4-dithiazoline-3-thione
  • PADS phenylacetyl
  • the oxidizing agent can be diluted with an appropriate solvent to a concentration of 0.001 to 2M before use.
  • the solvent used in the reaction is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, pyridine, or a mixed solvent of two or more of these at any ratio.
  • the oxidation step may be performed after the capping operation, or conversely, the capping operation may be performed after the oxidation step, and the order is not limited.
  • ammonia or an amine compound is used to cleave and recover the RNA strand from the solid phase carrier.
  • amine compounds examples include methylamine, ethylamine, isopropylamine, ethylenediamine, diethylamine, and triethylamine.
  • the chain length of the nucleic acid oligomer thus obtained is, for example, n ⁇ 60, n ⁇ 80, or n ⁇ 100 and n ⁇ 200.
  • n ⁇ 60 Preferably, n ⁇ 60.
  • n 67, 100 or 120, for example.
  • an amine compound is allowed to act to deprotect the protecting group of the phosphate moiety.
  • Amine compounds include, for example, the diethylamine described.
  • RNA purity measurement method The purity of RNA in the post-collection solution was measured by HPLC. The fractionated RNA was separated into each component by HPLC (wavelength 260 nm, column DNAPac TM PA200, 4.0 mm ⁇ 250 mm, 8.0 ⁇ m), and the peak area of the main product in the total peak area value of the obtained chromatogram was calculated. The purity of RNA was calculated from the area value. HPLC measurement conditions are shown in Table 1 below.
  • Reference example 1 Solid Phase Synthesis of RNA by Amidite Method RNA having the nucleic acid sequence of strand I shown below was synthesized. The chain consists of 103 bases long.
  • RNA was synthesized from the 3' side to the 5' side using a nucleic acid synthesizer (AKTA oligopilot plus100, GE Healthcare) based on the phosphoramidite method. Synthesis was performed on a 63 ⁇ mol scale.
  • RNA amidites for the synthesis uridine EMM amidite of the following formula (described in Example 2 of WO 2013/027843), cytidine EMM amidite (described in Example 3 of the same), adenosine EMM amidite (described in Example 3), (described in Example 4) and guanosine EMM amidite (described in Example 5), porous glass was used as the solid support, a toluene solution of dichloroacetate was used as the deblocking solution, and the condensing agent was Using 5-benzylthio-1H-tetrazole, using iodine solution as oxidizing agent, using 3-amino-1,2,4-dithiazole-5-thione as sulfurizing agent, and phenoxyacetic anhydride solution as capping solution.
  • N-methylimidazole solution was used. After completion of the nucleic acid elongation, the nucleic acid on the carrier was treated with a diethylamine solution to selectively deprotect the cyanoethyl protecting group of the phosphoric acid moiety.
  • EMM is an abbreviation for a (2-cyanoethoxy)methoxymethyl group.
  • RNA column chromatography purification was performed under the conditions shown in Table 2 below. However, before purification, mobile phase A was passed through the column at a flow rate of 4.7 mL/min for 12.5 minutes, and then the sample was added. The retention time of 94.2 minutes to 95.8 minutes was fractionated, and the obtained solution was analyzed by HPLC. The purity was calculated by the method described in Measurement Method 1 above. As a result, the purity was 87.7%. Using this fractionated and purified RNA solution, experiments of the following examples and comparative examples were carried out.
  • Example 1 99 ⁇ L of the RNA solution preparatively purified by reversed-phase column chromatography in Reference Example 1 was placed in a 300 mL polypropylene vial (Thermo Fisher), and 1 ⁇ L of an acetonitrile solution of triphenylphosphine was mixed as an additive solution. Concentration samples were prepared. The vial containing the mixed solution was placed in an incubator (Kennis Co.) temperature-controlled at 60° C. and allowed to stand for 8 hours. After standing, the headspace vial was removed from the incubator and cooled to room temperature, and the purity was calculated by the method described in Measurement Method 1 above. Table 3 shows the results.
  • the composition prepared to have a triphenylphosphine concentration of 3 mM has the following composition. Water: 63.79%, acetonitrile: 34.87%, dibutylamine: 0.84%, acetic acid: 0.39% (1.23% as dibutylammonium acetic acid), nucleic acid concentration: 0.21 mg/mL (0. 02%).
  • Comparative example 1 100 ⁇ L of the RNA solution preparatively purified by reversed-phase column chromatography in Reference Example 1 was placed in a 300 mL polypropylene vial (Thermo Fisher), and the vial containing the solution was placed in an incubator (Kennis) temperature-controlled at 60°C. and allowed to stand for 8 hours. After standing, the headspace vial was removed from the incubator and cooled to room temperature, and the purity was calculated by the method described in Measurement Method 1 above. Table 3 shows the results.
  • RNA was synthesized from the 3' side to the 5' side using a nucleic acid synthesizer (AKTA oligopilot plus100, GE Healthcare) based on the phosphoramidite method. Synthesis was performed on a 53 ⁇ mol scale.
  • RNA amidites for the synthesis uridine EMM amidite (described in Example 2 of WO 2013/027843), cytidine EMM amidite (described in Example 3), adenosine EMM amidite (described in Example 4) described), and guanosine EMM amidites (described in Example 5 of the same) with uridine 2′OMe amidite, cytidine 2′OMe amidite, adenosine 2′OMe amidite, and guanosine 2′OMe amidite, respectively, of the following formulas: A porous glass was used as a solid support, a toluene solution of dichloroacetic acid was used as a deblocking solution, 5-benzylthio-1H-tetrazole was used as a condensing agent, an iodine solution was used as an oxidizing agent, and 3 was used as a sulfiding agent.
  • EMM is an abbreviation for a (2-cyanoethoxy)methoxymethyl group.
  • RNA column chromatography purification was performed under the conditions shown in Table 4 below. However, before purification, mobile phase A was passed through the column at a flow rate of 4.7 mL/min for 12.5 minutes, and then the sample was added. The retention time of 66.7 minutes to 70.9 minutes was fractionated, and the obtained solution was analyzed by HPLC. The purity was calculated by the method described in Measurement Method 1 above. As a result, the purity was 94.2%. Using this fractionated and purified RNA solution, experiments of the following examples and comparative examples were carried out.
  • Example 2 Place 99 ⁇ L of the RNA solution preparatively purified by reversed-phase column chromatography in Reference Example 2 in a 300-mL polypropylene vial (Thermo Fisher), and mix 1 ⁇ L of an acetonitrile solution of triphenylphosphine as an additive solution. A sample with a phosphine concentration of 3 mM was prepared. The vial containing the mixed solution was placed in an incubator (Kennis Co.) temperature-controlled at 60° C. and allowed to stand for 8 hours. After standing, the polypropylene vial was removed from the incubator and cooled to room temperature, and the purity was calculated by the method described in Measurement Method 1 above. Table 5 shows the results.
  • the composition prepared to have a triphenylphosphine concentration of 3 mM has the following composition. Water: 62.02%, acetonitrile: 33.06%, methanol: 3.60%, dibutylamine: 0.82%, acetic acid: 0.38% (1.20% as dibutylammonium acetic acid), nucleic acid concentration: 0 .31 mg/mL (0.03%)
  • Example 3 In the experiment of Example 2, instead of the acetonitrile solution of triphenylphosphine, the acetonitrile solution of diethyl phosphite was used as the additive solution, and the concentration of diethyl phosphite was 3 mM (0.05%). Experiments were conducted under the same conditions except for preparation, and the purity of RNA after the experiment was measured. Table 5 shows the results.
  • Example 4 In the experiment of Example 2, an acetonitrile solution of triethyl phosphite was used as the additive solution instead of the acetonitrile solution of triphenylphosphine, and the concentration of triethyl phosphite was 3 mM (0.05%). Experiments were conducted under the same conditions except for preparation, and the purity of RNA after the experiment was measured. Table 5 shows the results.
  • Example 5 In the experiment of Example 2, instead of the acetonitrile solution of triphenylphosphine, the acetonitrile solution of diethoxyphenylphosphine was used as the additive solution, and the concentration of diethoxyphenylphosphine was adjusted to 3 mM (0.07%). Experiments were conducted under the same conditions except for preparation, and the purity of RNA after the experiment was measured. Table 5 shows the results.
  • Example 6 In the experiment of Example 2, instead of the acetonitrile solution of triphenylphosphine, an acetonitrile solution of ethoxydiphenylphosphine was used as the additive solution to prepare a solution with an ethoxydiphenylphosphine concentration of 3 mM (0.08%). The experiment was performed under the same conditions except for the above, and the purity of RNA was measured after the experiment. Table 5 shows the results.
  • Example 7 In the experiment of Example 2, an acetonitrile solution of 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide was used as the additive solution instead of the acetonitrile solution of triphenylphosphine9, The experiment was conducted under the same conditions except that the concentration of 10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide was prepared as a solution of 3 mM (0.07%), and the purity of RNA was measured after the experiment. bottom. Table 5 shows the results.
  • RNA obtained in Reference Example 2 from which the hydroxyl groups had been deprotected using tetrabutylammonium fluoride, was purified by column chromatography under the conditions shown in Table 6 below. However, before purification, mobile phase A was passed through the column at a flow rate of 4.7 mL/min for 12.5 minutes, and then the sample was added. The retention time of 91.7 minutes to 94.2 minutes was fractionated, and the obtained solution was analyzed by HPLC. The purity was calculated by the method described in Measurement Method 1 above. As a result, the purity was 95.1%. Using this fractionated and purified RNA solution, experiments of the following examples and comparative examples were carried out.
  • Example 8 99 ⁇ L of the RNA solution preparatively purified by reversed-phase column chromatography in Reference Example 3 was placed in a 300 mL polypropylene vial (Thermo Fisher), and 1 ⁇ L of an acetonitrile solution of triphenylphosphine was mixed as an additive solution. A sample with a phosphine concentration of 3 mM was prepared. The vial containing the mixed solution was placed in an incubator (Kennis Co.) temperature-controlled at 60° C. and allowed to stand for 14 hours. After standing, the polypropylene vial was removed from the incubator and cooled to room temperature, and the purity was calculated by the method described in Measurement Method 1 above. Table 7 shows the results.
  • the composition prepared to have a triphenylphosphine concentration of 3.0 mM (0.09%) has the following composition. Water: 59.70%, acetonitrile: 35.36%, methanol: 3.84%, hexylamine: 0.61%, acetic acid: 0.36% (0.97% as hexylammonium acetic acid), nucleic acid concentration: 0 .35 mg/mL (0.04%).
  • Example 9 Column of RNA from preparatively purified RNA solution
  • the following treatment was performed using the solution obtained by mixing the triphenylphosphine in Example 8 so that the concentration thereof was 3 mM, and allowing it to stand at 60° C. for 14 hours.
  • the resulting slurry solution was centrifuged at 3000 g and 25° C. for 10 minutes and the supernatant was removed.
  • nucleic acid oligomers having phosphorothioate bonds can be stabilized and produced efficiently.
  • SEQ ID NOS: 1 and 2 in the sequence listing represent the nucleotide sequences of oligonucleotides produced according to the production method of the present invention.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Saccharide Compounds (AREA)
PCT/JP2022/025692 2021-07-06 2022-06-28 核酸オリゴマーを含む組成物 Ceased WO2023282120A1 (ja)

Priority Applications (5)

Application Number Priority Date Filing Date Title
KR1020247000264A KR20240028416A (ko) 2021-07-06 2022-06-28 핵산 올리고머를 함유하는 조성물
CN202280040815.5A CN117425663B (zh) 2021-07-06 2022-06-28 包含核酸寡聚物的组合物
EP22837536.6A EP4368627A4 (en) 2021-07-06 2022-06-28 COMPOSITION CONTAINING A NUCLEIC ACID OLIGOMER
JP2023533547A JP7806051B2 (ja) 2021-07-06 2022-06-28 核酸オリゴマーを含む組成物
US18/574,655 US20240300993A1 (en) 2021-07-06 2022-06-28 Composition containing nucleic acid oligomer

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021112316 2021-07-06
JP2021-112316 2021-07-06

Publications (1)

Publication Number Publication Date
WO2023282120A1 true WO2023282120A1 (ja) 2023-01-12

Family

ID=84801563

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/025692 Ceased WO2023282120A1 (ja) 2021-07-06 2022-06-28 核酸オリゴマーを含む組成物

Country Status (6)

Country Link
US (1) US20240300993A1 (https=)
EP (1) EP4368627A4 (https=)
JP (1) JP7806051B2 (https=)
KR (1) KR20240028416A (https=)
CN (1) CN117425663B (https=)
WO (1) WO2023282120A1 (https=)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4675778A1 (en) 2023-02-28 2026-01-07 SBTL Advanced Materials Co. Ltd. Stainless steel pouch film for secondary battery

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
WO2006022323A1 (ja) 2004-08-26 2006-03-02 Nippon Shinyaku Co., Ltd. ホスホロアミダイト化合物及びオリゴｒｎａの製法
WO2013027843A1 (ja) 2011-08-25 2013-02-28 株式会社ボナック 配糖体化合物、チオエーテルの製造方法、エーテル、エーテルの製造方法、配糖体化合物の製造方法、核酸の製造方法
WO2017068377A1 (en) 2015-10-23 2017-04-27 Silence Therapeutics (London) Ltd Modified guide rnas, methods and uses
WO2017115655A1 (ja) * 2015-12-28 2017-07-06 昭和電工株式会社 分離分析方法
WO2018212236A1 (ja) * 2017-05-16 2018-11-22 日産化学株式会社 オリゴヌクレオチドの製造方法
WO2019208571A1 (ja) 2018-04-24 2019-10-31 住友化学株式会社 アミダイト化合物及び該化合物を用いたポリヌクレオチドの製造方法
JP2020515525A (ja) * 2017-02-03 2020-05-28 ジーン シグナル インターナショナル ソシエテ アノニム 安定なホスホロチオエートオリゴヌクレオチドを含む無菌製剤
WO2020196890A1 (ja) * 2019-03-28 2020-10-01 味の素株式会社 ホスホロチオエート化部位を有するオリゴヌクレオチドの製造方法
WO2021024467A1 (ja) * 2019-08-08 2021-02-11 住友化学株式会社 一本鎖ｒｎａの製造方法
WO2021024465A1 (ja) * 2019-08-08 2021-02-11 住友化学株式会社 核酸分子の製造方法
WO2021210409A1 (ja) * 2020-04-14 2021-10-21 住友化学株式会社 核酸オリゴマーを含む組成物

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07170981A (ja) * 1993-12-17 1995-07-11 Res Dev Corp Of Japan オリゴヌクレオチドの製造法
US8138330B2 (en) * 2006-09-11 2012-03-20 Sigma-Aldrich Co. Llc Process for the synthesis of oligonucleotides
TWI729028B (zh) * 2015-11-17 2021-06-01 日商日產化學工業股份有限公司 寡核苷酸的製造方法
CN116063365A (zh) * 2015-12-16 2023-05-05 味之素株式会社 寡核苷酸的制备方法以及核苷、核苷酸或寡核苷酸

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
WO2006022323A1 (ja) 2004-08-26 2006-03-02 Nippon Shinyaku Co., Ltd. ホスホロアミダイト化合物及びオリゴｒｎａの製法
JP5157168B2 (ja) 2004-08-26 2013-03-06 日本新薬株式会社 ホスホロアミダイト化合物及びオリゴｒｎａの製法
WO2013027843A1 (ja) 2011-08-25 2013-02-28 株式会社ボナック 配糖体化合物、チオエーテルの製造方法、エーテル、エーテルの製造方法、配糖体化合物の製造方法、核酸の製造方法
JP5554881B2 (ja) 2011-08-25 2014-07-23 株式会社ボナック 配糖体化合物、チオエーテルの製造方法、エーテル、エーテルの製造方法、配糖体化合物の製造方法、核酸の製造方法
WO2017068377A1 (en) 2015-10-23 2017-04-27 Silence Therapeutics (London) Ltd Modified guide rnas, methods and uses
WO2017115655A1 (ja) * 2015-12-28 2017-07-06 昭和電工株式会社 分離分析方法
JP2020515525A (ja) * 2017-02-03 2020-05-28 ジーン シグナル インターナショナル ソシエテ アノニム 安定なホスホロチオエートオリゴヌクレオチドを含む無菌製剤
WO2018212236A1 (ja) * 2017-05-16 2018-11-22 日産化学株式会社 オリゴヌクレオチドの製造方法
WO2019208571A1 (ja) 2018-04-24 2019-10-31 住友化学株式会社 アミダイト化合物及び該化合物を用いたポリヌクレオチドの製造方法
WO2020196890A1 (ja) * 2019-03-28 2020-10-01 味の素株式会社 ホスホロチオエート化部位を有するオリゴヌクレオチドの製造方法
WO2021024467A1 (ja) * 2019-08-08 2021-02-11 住友化学株式会社 一本鎖ｒｎａの製造方法
WO2021024465A1 (ja) * 2019-08-08 2021-02-11 住友化学株式会社 核酸分子の製造方法
WO2021210409A1 (ja) * 2020-04-14 2021-10-21 住友化学株式会社 核酸オリゴマーを含む組成物

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Concise Encyclopedia of Polymer Science and Engineering", 1990, JOHN WILEY & SONS, pages: 858 - 859
"Reviews by Chakhmakhcheva: Protective Groups in the Chemical Synthesis of Oligoribonucleotides", RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY, vol. 39, no. 1, 2013, pages 1 - 21
ENGLISCH ET AL.: "Angewandte Chemie", vol. 30, 1991, pages: 613
See also references of EP4368627A4

Also Published As

Publication number Publication date
CN117425663A (zh) 2024-01-19
EP4368627A4 (en) 2025-10-08
US20240300993A1 (en) 2024-09-12
JP7806051B2 (ja) 2026-01-26
CN117425663B (zh) 2026-04-17
EP4368627A1 (en) 2024-05-15
KR20240028416A (ko) 2024-03-05
JPWO2023282120A1 (https=) 2023-01-12

Similar Documents

Publication Publication Date Title
EP2331558B1 (en) Protected monomers and methods of deprotection for rna synthesis
KR102190852B1 (ko) 변형된 커플링 프로토콜을 이용하는 올리고머 화합물의 제조 방법
CN111684073B (zh) 核酸分子的制造方法
EP1119578A4 (en) IMPROVED TEA METHOD FOR OLIGONUCLEOTID SYNTHESIS
JP7719788B2 (ja) 核酸オリゴマーの製造方法
Manoharan et al. Allyl group as a protecting group for internucleotide phosphate and thiophosphate linkages in oligonucleotide synthesis: Facile oxidation and deprotection conditions
JP7699581B2 (ja) 核酸オリゴマーを含む組成物
JP7806051B2 (ja) 核酸オリゴマーを含む組成物
JP7649779B2 (ja) 核酸オリゴマーの製造方法
KR20230034969A (ko) 핵산 올리고머의 제조 방법
KR20220160010A (ko) 핵산 올리고머의 제조 방법
KR20240082343A (ko) 정제 디클로로아세트산의 제조 방법
US20230322842A1 (en) Composition containing nucleic acid oligomer
JP7522099B2 (ja) Ｒｎａの製造方法
CN115315430A (zh) 使用了链段型亚磷酰胺化合物的核酸合成法
WO2026071145A1 (ja) 核酸オリゴマーの製造方法
CN120418262A (zh) 寡核苷酸的制造方法
PL181682B1 (pl) Sposób wytwarzania modyfikowanych P-chiralnych analogów nukleotydów

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22837536

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023533547

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 202280040815.5

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 202417006728

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 2022837536

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022837536

Country of ref document: EP

Effective date: 20240206