Classifications

• • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • 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
  • 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
  • C07H21/04—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 deoxyribosyl as saccharide radical
• • 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 nucleic acid oligomer containing ribose, and more particularly to a method for deprotecting a hydroxyl protecting group of ribose contained in a nucleic acid oligomer.
• nucleic acid oligomers include antisense nucleic acids, aptamers, ribozymes, and nucleic acids that induce RNA interference (RNAi) such as siRNA, and these are called nucleic acid drugs.
• RNAi RNA interference
• Nucleic acid oligomers can be synthesized by solid phase synthesis, in which nucleoside phosphoramidites (hereinafter referred to as "amidites") are used as raw materials.
• a nucleic acid oligomer synthesized by elongating a nucleic acid on a solid phase carrier is excised from the solid phase carrier, and then the ribose-containing nucleic acid oligomer is removed by deprotecting the hydroxyl group at the 2' position of the ribose. , the desired nucleic acid oligomer has been produced.
• the purity of the nucleic acid oligomers synthesized in this way is determined by the multi-step process including the elongation reaction of the nucleic acid on a solid support, the cutting out process from the solid support, and the deprotection process of each protecting group. It was not always satisfactory and the synthesis was not efficient (Patent Documents 1 and 2).
• An object of the present invention is to provide an efficient method for producing nucleic acid oligomers.
• the present inventors have discovered that by contacting a nucleic acid oligomer with fluoride ions in the presence of a radical reaction inhibitor, the hydroxyl group of ribose contained in the nucleic acid oligomer can be removed.
• protecting groups can be efficiently deprotected.
• an efficient method for producing nucleic acid oligomers can be provided.
• the present invention has been completed based on these findings, and includes, but is not limited to, the following aspects.
• G 4 represents a hydrogen atom or a hydroxyl group protecting group
• G9 represents ammonium ion, alkylammonium ion, alkali metal ion, hydrogen ion or hydroxyalkylammonium ion
• B c each independently represent the same or different nucleobases
• R is each independently the same or different and represents a hydrogen atom, a fluorine atom or an OQ group
• Q is each independently the same or different and represents a tert-butyldimethylsilyl group, a methyl group, a 2-methoxyethyl group, a methylene group bonded to the 4'-position carbon atom of ribose, or a tert-butyldimethylsilyl group, a methyl group, a 2-methoxyethyl group, a methylene group bonded to the 4'-position carbon atom of ribose, or a tert-butyldimethyl
• ) represents a protecting group of Y is each independently the same or different and represents an oxygen atom or a sulfur atom, m represents any integer from 2 to 300, W and X are defined in either (a) or (b) below, (a) When W is a hydroxyl group, X has the same definition as the above R group. (b) When X is a hydroxyl group, W represents an OV group, V represents a tert-butyldimethylsilyl group or a group of the above formula (1). However, at least one group among R, W, and X represents a hydroxyl group protected with the protecting group of formula (1).
• nucleic acid oligomers represented by formula (3) between the nucleotides at the 5' and 3' ends (where p is the formula: m-1>p It is a nucleic acid oligomer in which a non-nucleotide linker may be incorporated in place of the nucleotide (a positive integer that satisfies the above).
• nucleic acid oligomers represented by formula (4) between the nucleotides at the 5' end and 3' end (where p is the formula: m-1>
• a positive integer satisfying p) is a nucleic acid oligomer in which a non-nucleotide linker may be incorporated in place of the nucleotide.
• a method for producing a nucleic acid oligomer represented by hereinafter referred to as "the production method of the present invention” or “the production method of the present embodiment” in this specification). 2. The manufacturing method according to item 1 above, wherein n in formula (1) is 0 or 1. 3. The manufacturing method according to item 1 above, wherein n in formula (1) is 0.
• n in formula (1) is 1. 5.
• the non-nucleotide linker is a linker consisting of an amino acid skeleton.
• the linker consisting of an amino acid skeleton is a linker having a structure of the following formula (A14-1), (A14-2) or (A14-3). (In the formula, 5' and 3' indicate the 5' end and 3' end of the nucleic acid oligomer, respectively.) 7. 7.
• the radical reaction inhibitor is a radical scavenger.
• the radical scavenger is a phenolic antioxidant or a hindered amine light stabilizer.
• the radical scavenger is a phenolic antioxidant.
• the phenolic antioxidant is a compound represented by the following formula (8).
• R 10 , R 11 , R 12 , and R 13 are the same or different and are a chain hydrocarbon group, a carbocyclyl group, a heterocyclyl group, an alkoxy group, an alkylsulfanyl group ⁇ the chain hydrocarbon group,
• the carbocyclyl group, the heterocyclyl group, the alkoxy group, and the alkylsulfanyl group may have one or more substituents ⁇ , SiR 51 R 52 R 53 , amide group, C(O)R 61 , OC (O) R 61 represents a hydroxyl group or a hydrogen atom;
• R 51 , R 52 and R 53 are the same or different and represent an alkyl group, an alkoxy group, or a hydrogen atom;
• R 61 is a chain hydrocarbon (indicates the group) 15.
• R 14 represents OC(O)R 20 , NHR 20 , or a hydrogen atom
• R 19 represents an alkyl group, an alkoxy group, an oxygen free radical, a hydroxyl group, or a hydrogen atom
• R 15 , R 16 , R 17 and R 18 are the same or different and represent an alkyl group or a hydrogen atom
• R 20 is a chain hydrocarbon group, carbocyclyl group, heterocyclyl group, alkoxy group, alkylsulfanyl group ⁇ the chain formula
• the hydrocarbon group, the carbocyclyl group, the heterocyclyl group, the alkoxy group, and the alkylsulfanyl group may have one or more substituents ⁇ , SiR 54 R 55 R 56 , amide group, hydroxyl group, or hydrogen
• R 54 , R 55 and R 56 are the same or different and represent an alkyl group, an alkoxy group, or a hydrogen atom.
• the radical reaction inhibitor is a peroxide decomposer. 18. 18. The manufacturing method according to item 17 above, wherein the peroxide decomposer is a phosphorus-based antioxidant or a sulfur-based antioxidant. 19. 18. The manufacturing method according to item 17, wherein the peroxide decomposer is a phosphorous antioxidant. 20. 18. The manufacturing method according to item 17 above, wherein the peroxide decomposer is a sulfur-based antioxidant. 21. 10. The production method according to any one of items 1 to 9 above, wherein the radical reaction inhibitor is a radical chain initiation inhibitor. 22. 22.
• the manufacturing method according to item 21 above, wherein the radical chain initiation inhibitor is a metal deactivator or an ultraviolet absorber. 23. 22. The manufacturing method according to item 21 above, wherein the radical chain initiation inhibitor is a metal deactivator. 24. 22. The manufacturing method according to item 21 above, wherein the radical chain initiation inhibitor is an ultraviolet absorber. 25.
• the amount of the radical reaction inhibitor to be used is the number of moles of the nucleic acid oligomer represented by formula (3) multiplied by the number in which R in formula (3) is a group represented by formula (1), 1 mole. 25.
• the manufacturing method according to any one of items 1 to 24 above, wherein the amount is 9 mol or less. 26.
• the present invention provides an efficient method for producing nucleic acid oligomers. It is expected that the purity of the produced nucleic acid oligomer will be improved.
• a halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
• a substituent is substituted with two or more halogen atoms or substituents, those halogen atoms or substituents may be the same or different.
• the chain hydrocarbon group refers to an alkyl group, an alkenyl group, or an alkynyl group.
• alkyl group examples include methyl group, ethyl group, propyl group, isopropyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 1-ethylpropyl group, butyl group, sec-butyl group, and isobutyl group. tert-butyl, pentyl, and hexyl groups.
• alkenyl group examples include vinyl group, 1-propenyl group, 2-propenyl group, 1-methyl-1-propenyl group, 1-methyl-2-propenyl group, 1,2-dimethyl-1-propenyl group, -ethyl-2-propenyl group, 3-butenyl group, 4-pentenyl group, and 5-hexenyl group.
• alkynyl group examples include ethynyl group, 1-propynyl group, 2-propynyl group, 1-methyl-2-propynyl group, 1,1-dimethyl-2-propynyl group, 1-ethyl-2-propynyl group, 2 -butynyl group, 4-pentynyl group, and 5-hexynyl group.
• alkoxy group examples include methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, tert-butoxy group, pentyloxy group, hexyloxy group, and octyloxy group.
• alkylsulfanyl group examples include a methylsulfinyl group, an ethylsulfinyl group, a propylsulfinyl group, an isopropylsulfinyl group, a butylsulfinyl group, a tert-butylsulfinyl group, a pentylsulfinyl group, a hexylsulfinyl group, and an octylsulfinyl group.
• alkenyloxy group examples include 2-propenyloxy group, 2-butenyloxy group, and 5-hexenyloxy group.
• alkynyloxy group examples include 2-propynyloxy group, 2-butynyloxy group, and 5-hexynyloxy group.
• the carbocyclyl group refers to an aromatic carbocyclic group and an alicyclic hydrocarbon group. Examples of aromatic carbocyclic groups include phenyl and naphthyl groups.
• the alicyclic hydrocarbon group refers to a cycloalkyl group, a cycloalkenyl group, and the like. Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and a cycloheptyl group.
• cycloalkenyl group examples include a cyclopropenyl group, a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and a cycloheptenyl group.
• heterocyclyl group means a cyclic group having one or more heteroatoms as ring constituent atoms, and includes an aromatic heterocyclic group and a non-aromatic heterocyclic group.
• heterocyclyl group examples include pyrrolyl group, furyl group, thienyl group, pyrazolyl group, imidazolyl group, triazolyl group, tetrazolyl group, oxazolyl group, isoxazolyl group, thiazolyl group, isothiazolyl group, oxadiazolyl group, thiadiazolyl group, pyridyl group, and pyridazinyl group.
• pyrimidinyl group pyrazinyl group, triazinyl group, tetrazinyl group, pyrrolidinyl group, imidazolinyl group, imidazolidinyl group, piperidinyl group, tetrahydropyrimidinyl group, hexahydropyrimidinyl group, piperazinyl group, oxazolidinyl group, isoxazolidinyl group, 1, Examples include 3-oxazinanyl group, morpholinyl group, thiazolidinyl group, isothiazolidinyl group, 1,3-thiazinanyl group, and thiomorpholinyl group.
• the nucleic acid oligomer represented by the formula (3) is brought into contact with fluoride ions to obtain the nucleic acid oligomer represented by the formula (4) from which the protecting group of the following formula (1) has been deprotected.
• the method of manufacturing will be explained.
• n is any integer greater than or equal to 0, more preferably an integer of 0 to 3, more preferably an integer of 0 to 2, still more preferably 0 or 1, particularly preferably 1. be.
• At least one group among R, W, and X in formula (3) represents a hydroxyl group protected with the protecting group of formula (1).
• the proportion of formula (1) may be 1% or more, more preferably 5% or more, more preferably 10% or more, more preferably 20% or more, and more Preferably it is 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more. It is more preferably 90% or more, and even more preferably 95% or more.
• the length of the nucleic acid chain to be synthesized is preferably 10 or more, more preferably 20 or more, more preferably 30 or more, and more preferably preferably has a chain length of 40 or more, and even more preferably has a chain length of 50 or more.
• tetraalkylammonium fluoride is typically used as a fluoride ion source.
• tetraalkylammonium fluoride include tetrabutylammonium fluoride and tetramethylammonium fluoride. Among them, tetrabutylammonium fluoride (TBAF) is more preferred.
• the amount of fluoride ion used is usually 1 to 1000 mol, preferably 1 to 500, more preferably 2 to 200 mol, more preferably 4 to 100 mol per mol of protecting group removed.
• radical reaction inhibitors examples include radical chain initiation inhibitors, radical scavengers, and peroxide decomposers.
• radical chain initiation inhibitor examples include metal deactivators, ultraviolet absorbers, and quenchers.
• metal deactivators include amide compounds, hydrazide compounds, etc. Specifically, n-octanohydrazide, succinic acid dihydrazide, 2-Hydroxy-N-1H-1,2,4-triazol- 3-ylbenzamide, N'1,N'12-Bis(2-hydroxybenzoyl)dodecanedihydrazide, N,N'-Bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionyl]hydrazine, or 1 , 3,5-Triazine-2,4,6-triamine, etc.
• Examples of the ultraviolet absorber include triazole compounds, triazine compounds, and benzophenone compounds. Specifically, benzophenone, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1- methyl-1-phenylethyl)phenol, 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol, 2,2'-Methylenebis[6-(2H-benzotriazol-2 -yl)-4-(1,1,3,3-tetramethylbutyl)phenol], 2-(2H-Benzotriazol-2-yl)-p-cresol, 2-(5-chloro-2H-benzotriazol-2-yl )-6-tert-butyl-4-methylphenol, 2-(4,6-Diphenyl-1,3,5-triazin-2-yl)-5-[2-(2-ethylhexanoyloxy)ethoxy]phenol, 2, Examples include 4,6-tri
• examples of the quencher include organic nickel compounds. Specifically, examples include bis(1,2-bis(2-methoxyphenyl)-1,2-ethylenedithiolat) nickel complex, Raney nickel, tetracarbonickel, NiX 2 (PR 3 ) [wherein, X is a halogen atom] and PR 3 represents a phosphine ligand (for example, a tertiary phosphine such as triphenylphosphine).
• examples include bis(1,2-bis(2-methoxyphenyl)-1,2-ethylenedithiolat) nickel complex, Raney nickel, tetracarbonickel, NiX 2 (PR 3 ) [wherein, X is a halogen atom] and PR 3 represents a phosphine ligand (for example, a tertiary phosphine such as triphenylphosphine).
• radical scavenger examples include phenolic antioxidants, hindered amine light stabilizers (HALS), and the like.
• phenolic antioxidants include hindered phenol compounds, semi-hindered phenol compounds, and unhindered phenol compounds.
• phenolic antioxidant examples include the following compounds, but any compound that is generally used as a phenolic antioxidant can be used in this embodiment.
• a compound in which R 12 is a hydrogen atom in formula (8) can also be represented by the following formula (8a).
• a compound in which R 11 is a hydrogen atom in formula (8) can also be represented by the following formula (8b).
• R 10 is preferably a tert-butyl group, a sec-butyl group, or a methyl group
• R 11 and R 12 are the same or different.
• R 13 is preferably a tert-butyl group, a sec-butyl group, a methyl group, or a hydrogen atom
• R 13 is preferably a tert-butyl group, a sec-butyl group, a methyl group, a hydroxyl group, or a hydrogen atom.
• R 10 , R 11 , and R 12 are the same as those in formula (8), and R 10' , R 11' , R 12' , R 21 , R 22 , and R 23 are The same or different, a chain hydrocarbon group, a carbocyclyl group, a heterocyclyl group, an alkoxy group, an alkylsulfanyl group ⁇ the chain hydrocarbon group, the carbocyclyl group, the heterocyclyl group, the alkoxy group, the alkylsulfanyl group, may have one or more substituents ⁇ , SiR 51 R 52 R 53 , amide group, C(O)R 61 , OC(O)R 61 , hydroxyl group, or hydrogen atom, and R 24 is oxygen atom, sulfur atom, S(O), or S(O) 2 , R 25 represents an oxygen atom or a sulfur atom, and n 1 , n 2 ,
• R 10 , R 11 , R 12 , R 10' , R 11' , R 12' , and n 1 are the same as those in the formula (13a)
• R 10'' , R 11 " , R 12" , R 21' , R 22' , R 21" , and R 22" are the same or different and each represents a chain hydrocarbon group, a carbocyclyl group, a heterocyclyl group, an alkoxy group, an alkylsulfanyl group ⁇ the The chain hydrocarbon group, the carbocyclyl group, the heterocyclyl group, the alkoxy group, and the alkylsulfanyl group may have one or more substituents ⁇ , SiR 51 R 52 R 53 , amide group, C( O)R 61 , OC(O)R 61 represents a hydroxyl group, or a hydrogen atom, and n 1' and n 1'' each
• 22'' , n 1 , n 1' , and n 1'' are the same as those in the formula (13c)
• R 10'' , R 11'' , R 12'' , R 21''' , and R 22''' are the same or different and are a chain hydrocarbon group, a carbocyclyl group, a heterocyclyl group, an alkoxy group, an alkylsulfanyl group ⁇ the chain hydrocarbon group, the carbocyclyl group, the heterocyclyl group, the alkoxy group , the alkylsulfanyl group may have one or more substituents ⁇ , SiR 51 R 52 R 53 ,
• 22'' , n 1 , n 1' , and n 1'' are the same as those in the formula (13c), and R 21'' , R 22'' , and R 23'' are the same or different.
• a chain hydrocarbon group, a carbocyclyl group, a heterocyclyl group, an alkoxy group, an alkylsulfanyl group may have a substituent ⁇ , SiR 51 R 52 R 53 , amide group, C(O)R 61 , OC(O)R 61 , hydroxyl group, or hydrogen atom
• substituents that the chain hydrocarbon group, carbocyclyl group, heterocyclyl group, alkoxy group, and alkylsulfanyl group may have include a halogen atom, a carbocyclyl group, a heterocyclyl group, a hydroxyl group, an amino group, Alkoxy group, sulfanyl group, alkylsulfanyl group, SiR 51 R 52 R 53 , O(SiR 51 R 52 R 53 ), C(O)R 61
• the phenolic antioxidants include 2,6-di-tert-butyl-p-cresol, 4-sec-butyl-2,6-di-tert-butylphenol, and 6-tert-butyl-2 ,4-xylenol, 4,6-di-tert-butyl-m-cresol, 1,4-dihydroxybenzene, 2,6-Di-tert-butyl-4-ethylphenol, 4,4'-Dihydroxy-3,3 ',5,5'-tetraisopropylbiphenyl, 3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-N'-[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl] propanehydrazide, Methyl 3-(3,5-Di-tert-butyl-4-hydroxyphenyl)propionate, 2,2'-Methylenebis(6-tert-butyl-4-
• HALS hindered amine light stabilizers
• R 15 , R 16 , R 17 and R 18 are preferably methyl groups.
• R 15 , R 16 , R 17 , R 18 , and R 19 are the same as those in formula (12) above, and R 19' is an alkyl group, an alkoxy group, an oxygen free radical, Represents a hydroxyl group or a hydrogen atom, and R 15' , R 16' , R 17' , R 18' , R 26 , R 27 , R 28 , and R 29 are the same or different and represent an alkyl group or a hydrogen atom.
• n5 indicates any 0 or positive integer.
• R 15 , R 16 , R 17 , R 18 , R 19 , R 15 ' , R 16' , R 17' , R 18' , R 19' , R 28 and R 29 are defined as above.
• the definition is the same as in formula (12a), and R 30 , R 31 , R 32 , and R 33 are the same or different and represent an alkyl group or a hydrogen atom.
• substituents that the chain hydrocarbon group, carbocyclyl group, heterocyclyl group, alkoxy group, and alkylsulfanyl group in R 20 may have include a halogen atom, a carbocyclyl group, a heterocyclyl group, a hydroxyl group, and an amino group.
• alkoxy group, sulfanyl group, alkylsulfanyl group, SiR 54 R 55 R 56 , O(SiR 51 R 52 R 53 ), C(O)R 63 , OC(O)R 63 , and P(O)(OR 64 ) 2 can be mentioned.
• R 63 represents a chain hydrocarbon group
• R 62 represents a chain hydrocarbon group.
• the alkyl groups in R 15 , R 16 , R 17 , R 18 , R 19 , R 15 ' , R 16' , R 17' , R 18' , R 26 , R 27 , R 28 , and R 29 are substituents.
• an alkyl group, a hydroxyl group may have a substituent such as a phenyl group.
• hindered amine light stabilizers include bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate, 2,2,6,6-tetramethyl-4 methacrylate, -Piperidyl, or 2,2,6,6-tetramethylpiperidin-1-oxyl-free radical (TEMPO free radical), N,N'-Bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane- 1,6-diamine, 2,2,6,6-tetramethylpiperidine 1-oxyl, Bis(2,2,6,6-tetramethyl-4-piperidyl-1-oxyl) Sebacate, Bis(1,2,2,6 ,6-pentamethyl-4-piperidyl) Sebacate, Bis(2,2,6,6-tetramethyl-4-piperidyl) Sebacate, 1,2,2,6,6-Pentamethyl-4-piperidyl Methacrylate, N 1 ,N 3 -Bis(2,2,6,6-tetramethyl-4-piperidy
• peroxide decomposers include phosphorus-based antioxidants and sulfur-based antioxidants.
• phosphorus antioxidants include phosphine compounds and phosphite compounds, and specifically, triphenylphosphine, triphenyl phosphite, 3,9-Bis(octadecyloxy)-2,4,8, 10-tetraoxa-3,9-diphosphaspiro[5.5]undecane, 3,9-Bis(2,6-di-tert-butyl-4-methylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro [5.5]undecane, 2,2'-Methylenebis(4,6-di-tert-butylphenyl) 2-ethylhexyl phosphite, Tris(2,4-ditert-butylphenyl) phosphite, Tris(nonylphenyl) phosphite, Tetra-C
• sulfur-based antioxidants include sulfide-based compounds, and specifically, dioctadecyl sulfide, 2,2-Bis ⁇ [3-(dodecylthio)-1-oxopropoxy]methyl ⁇ propane-1,3- Examples include diyl bis[3-(dodecylthio)propionate] and Di(tridecyl) 3,3'-thiodipropionate.
• the amount of the radical reaction inhibitor used is usually the number of moles obtained by multiplying the number of moles of the nucleic acid oligomer represented by formula (3) by the number in which R in formula (3) is a group represented by formula (1).
• R in formula (3) is a group represented by formula (1) relative to the number of moles of the nucleic acid oligomer represented by formula (3).
• R in formula (3) is a group represented by formula (1) relative to the number of moles of the nucleic acid oligomer represented by formula (3).
• R in formula (3) is a group represented by formula (1) relative to the number of moles of the nucleic acid oligomer represented by formula (3).
• organic solvents that are inert to the reaction are usually used, such as sulfoxide solvents, nitrile solvents, ether solvents, amide solvents, ketone solvents, aliphatic hydrocarbon solvents, ester solvents, aromatic Examples include solvents and mixed solvents of two or more of these, and among these solvents, sulfoxide solvents are preferred.
• the sulfoxide solvent include dimethyl sulfoxide.
• the nitrile solvent include acetonitrile and propionitrile.
• the ether solvent include tetrahydrofuran.
• the amide solvent include N-methyl-2-pyrrolidone and the like.
• Examples of the ketone solvent include acetone and methyl ethyl ketone.
• Examples of the aliphatic hydrocarbon solvent include hexane and heptane.
• Examples of the ester solvent include methyl acetate and ethyl acetate.
• Examples of the aromatic solvent include toluene and pyridine. Among these, dimethyl sulfoxide or a mixed solvent of dimethyl sulfoxide and acetonitrile is preferred.
• the fluoride ion source which is a reagent used in the step of deprotecting the protecting group represented by formula (1), is usually used after being dissolved in a solvent and dehydrated.
• the dehydrating agent include molecular sieves and sulfates, and preferably molecular sieve 4A is used.
• the amount of solvent used is usually 5 to 8,000 L, preferably 50 to 2,000 L, and more preferably 100 to 1,600 L per mole of nucleic acid oligomer to be subjected to the deprotection step.
• a compound that reacts with the compound represented by the following formula (2), which is a byproduct in this step, and captures the compound can be added.
• the compound to be captured include nitroalkanes, alkylamines, amidines, thiols, thiol derivatives, or mixtures of two or more thereof.
• nitroalkanes include nitromethane.
• alkylamine include linear alkylamines having 1 to 6 carbon atoms and cyclic amines having 1 to 8 carbon atoms.
• amidine include benzamidine and formamidine.
• thiol include linear thiols having 1 to 6 carbon atoms. Specific examples include methanethiol, ethanethiol, 1-propanethiol, 1-butanethiol, 1-pentanethiol, and 1-hexanethiol.
• the "thiol derivative” include alcohols or ethers having the same or different linear alkylthiol groups having 1 to 6 carbon atoms.
• 2-mercaptoethanol 4-mercapto-1-butanol, 6-mercapto-1-hexanol, mercaptomethyl ether, 2-mercaptoethyl ether, 3-mercaptopropyl ether, 4-mercaptobutyl ether, 5 -mercaptopentyl ether, 6-mercaptohexyl ether, and the like. More preferably nitromethane is used.
• the amount of the compound used to capture the by-product compound represented by formula (2) is 0.1 to 100.0 with respect to the fluoride ion source that deprotects the hydroxyl protecting group represented by formula (1). It can be used in mol%, preferably 1.0 to 50.0 mol%, more preferably 2.0 to 40.0 mol%.
• fluoride ions may be added to the nucleic acid oligomer represented by formula (3), or conversely, fluoride ions may be reacted with
• the nucleic acid oligomer shown in may be added, or both may be added at the same time.
• a method of adding fluoride ions to the nucleic acid oligomer represented by formula (3) is preferred.
• the time required to add the entire amount of fluoride ions to the nucleic acid oligomer represented by formula (3) is preferably 5 minutes or more, more preferably 10 minutes or more, more preferably 15 minutes or more, and even more preferably 30 minutes or more. More preferably, it is added dropwise over one hour or more.
• Such addition is preferably carried out dropwise over 5 minutes or more, more preferably 10 minutes or more, and added dropwise over 15 minutes or more into the surface or into the solution containing the nucleic acid oligomer represented by formula (3). It is more preferable to drop the solution over a period of 30 minutes or more, and still more preferably to add it dropwise over a period of 1 hour or more.
• the radical reaction inhibitor is preferably present in the reaction system before adding fluoride ions.
• the temperature of both or one of the solutions when adding fluoride ions to the nucleic acid oligomer represented by formula (3) may be 80°C or lower, preferably both are 40°C or lower, and preferably both are 35°C or lower. More preferably, both are 30°C or lower, more preferably both are 25°C or lower, more preferably both are 20°C or lower, more preferably both are 15°C or lower, and more preferably both are 15°C or lower. Both are below 10°C, even more preferably both are below 5°C.
• the mixture may be kept warm for 1 minute or more, preferably for 5 minutes or more, more preferably for 10 minutes or more, and more preferably for 15 minutes or more. It is kept warm for at least 30 minutes, more preferably for at least 30 minutes, and still more preferably for at least 1 hour.
• the temperature may be raised, and the temperature may be raised to 5°C or more and 80°C or less, preferably 10°C or more and 40°C or less, and preferably 10°C or more and 35°C or less. , preferably from 15°C to 35°C, more preferably from 20°C to 35°C, even more preferably from 25°C to 35°C.
• the time for the deprotection reaction varies depending on the type of deprotecting agent used and the reaction temperature, but is usually 1 hour to 100 hours, preferably 1 to 24 hours, more preferably 2 to 12 hours. More preferably, the time is 3 to 6 hours. Note that fluoride ions may be added at any timing.
• stirring is usually performed at a stirring power Pv of 0.0 to 0.5 kW/ m3 , and when Pv is 0.1 to 0.3 kW/m3. Stirring of m 3 is preferred.
• nucleic acid oligomers can be isolated by means such as precipitation of the nucleic acid oligomers used, dialysis, or ultrafiltration.
• the isolated nucleic acid oligomer is usually obtained as a nucleic acid oligomer whose 5'-terminal hydroxyl group is protected.
• the reaction for obtaining the nucleic acid oligomer represented by the following formula (4) by deprotecting the protecting group represented by the formula (1) from the nucleic acid oligomer represented by the formula (3) is as follows. (Scheme 1).
• G 4 in the formula represents a hydrogen atom or a hydroxyl group protecting group
• G9 represents ammonium ion, alkylammonium ion, alkali metal ion, hydrogen ion or hydroxyalkylammonium ion
• B c each independently represent the same or different nucleobases
• R is each independently the same or different and represents a hydrogen atom, a fluorine atom or an OQ group
• Q is each independently the same or different and represents a tert-butyldimethylsilyl group, a methyl group, a 2-methoxyethyl group, a methylene group bonded to the carbon atom at the 4' position of ribose, or a 4' position of rib
• Nucleosides (ribose and deoxyribose) contained in the nucleic acid oligomer used in this embodiment include DNA, RNA, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-Me , 2'-F RNA, or the above-mentioned LNA, but the nucleoside is not limited thereto.
• the nucleic acid oligomer represented by formula (3) can be obtained, for example, by cutting out the nucleic acid oligomer produced by solid phase synthesis represented by formula (5) from a solid phase carrier, as shown in Scheme 2 below.
• the nucleic acid oligomer of formula (5) synthesized on a solid phase carrier will be explained.
• the substituents B a each independently represent a nucleobase which may be the same or different protected.
• G 4 and Y are as defined in the above formula (3), G 2 each independently represent the same or different phosphoric acid protecting groups;
• X 1 represents OZ
• W 1 represents an OV group
• V represents a tert-butyldimethylsilyl group or a group of the above formula (1).
• X 1 represents an R group
• W 1 represents a group represented by OZ
• Z represents a group consisting of a solid phase carrier and a linking part that connects the solid phase carrier and the oxygen atom of the hydroxyl group at the 2' position or 3' position of the ribose at the 3' end of the nucleic acid oligomer.
• Z represents a structure schematically represented by the following formula (6).
• Sp represents a spacer.
• Examples of the spacer (Sp) include those having the structural formula shown in formula (7) below.
• Linker represents a structure that becomes a linker (junction structure).
• the structure of Linker is, for example, the following formula (8-1) (8-2) (8-3) (8-4) (8-5) (8-6) (8-7) or (8-8)
• the structure shown in may be used.
• Solid support indicates a structure that serves as a solid support.
• the solid support include inorganic porous carriers and organic resin carriers.
• Inorganic porous carriers include, for example, controlled pore glass (CPG) and zeolites.
• organic resin carriers include carriers made of polystyrene.
• A may each independently be a hydroxyl group, an alkoxy group, or an alkyl group.
• alkoxy group include a methoxy group and an ethoxy group.
• alkyl group include a methyl group, an ethyl group, Examples include isopropyl group and n-propyl group.Si indicates bonding with oxygen of hydroxyl group on the surface of the carrier.
• G 4 represents a protecting group for a hydrogen atom or a hydroxyl group, and when it represents a protecting group, it represents the same protecting group as G 1 .
• G 4 is a hydrogen atom when deprotected, and the nucleotide compound in that case is also subjected to a series of nucleic acid extension reaction steps.
• G9 represents ammonium ion, alkylammonium ion, alkali metal ion, hydrogen ion, hydroxyalkylammonium ion, or the like.
• alkyl moieties for the alkylammonium ion include methyl, ethyl, n-propyl, isopropyl, n-butyl, dibutyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl.
• More specific examples include diethylammonium ion, triethylammonium ion, tetrabutylammonium ion, hexylammonium ion, and dibutylammonium ion.
• examples of the alkali metal ions include sodium ions and lithium ions.
• examples of hydroxyalkylammonium ion specific examples of hydroxyalkyl moieties include hydroxymethyl, hydroxyethyl, hydroxy-n-propyl, hydroxyisopropyl, hydroxy-n-butyl, or trishydroxymethyl.
• more specific examples of hydroxyalkylammonium ions include trishydroxymethylammonium ions and the like.
• the compound of formula (5) is produced, for example, by an amidite method using an amidite compound of formula (A13) below.
• R represents a hydrogen atom, a fluorine atom, or an OQ group
• Q is a tert-butyldimethylsilyl group, a methyl group, a 2-methoxyethyl group, a methylene group bonded to the 4' carbon atom, an ethylene group bonded to the 4' carbon atom, an ethylidene group bonded to the 4' carbon atom
• B a represents an optionally protected nucleobase
• G 1 represents a hydroxyl protecting group
• G 2 represents a phosphoric acid protecting group
• G 3 represents an alkyl group or a cyclic structure by bonding to each other at their terminals.
• B a represents a nucleobase represented by B c or a nucleobase protected with a protecting group.
• the nucleobase in B a is not particularly limited. Examples of the nucleobase include adenine, cytosine, guanine, uracil, thymine, 5-methylcytosine, pseudouracil, 1-methylpseudouracil, and the like. Further, the nucleobase may be substituted with a substituent.
• substituents include halogen atoms such as fluoro, chloro, bromo, or iodo groups, acyl groups such as acetyl, alkyl groups such as methyl or ethyl, and arylalkyl such as benzyl. groups, alkoxy groups such as methoxy groups, alkoxyalkyl groups such as methoxyethyl groups, cyanoalkyl groups such as cyanoethyl groups, hydroxy groups, hydroxyalkyl groups, acyloxymethyl groups, amino groups, monoalkylamino groups, dialkylamino groups, carboxy group, a cyano group, a nitro group, and a combination of two or more of these substituents.
• substituents include halogen atoms such as fluoro, chloro, bromo, or iodo groups, acyl groups such as acetyl, alkyl groups such as methyl or ethyl, and arylalky
• the protecting group for the amino group is not particularly limited, and any protecting group used in known nucleic acid chemistry can be used, and such protecting groups include: For example, benzoyl group, 4-methoxybenzoyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, phenylacetyl group, phenoxyacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropylphenoxyacetyl group, or (dimethyl Examples include amino)methylene groups, and combinations of two or more of these protecting groups.
• R 4 represents a hydrogen atom, a methyl group, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group, or a benzoyl group
• R 5 represents a hydrogen atom, an acetyl group, an isobutyryl group, or a benzoyl group
• R 6 represents a hydrogen atom, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group, or an isobutyryl group
• R 7 represents a 2-cyanoethyl group
• R 8 represents a hydrogen atom, a methyl group, a benzoyl group, a 4-methoxybenzoyl group
• G 1 can be used without particular limitation as long as it can function as a protecting group, and a wide variety of known protecting groups used in amidite compounds can be used.
• G 1 is preferably the following group.
• R 1 , R 2 and R 3 are the same or different and represent hydrogen or an alkoxy group.
• R 1 , R 2 and R 3 is hydrogen, and the remaining two are the same or different (preferably the same) alkoxy groups, and the alkoxy group is particularly preferably a methoxy group.
• G 2 can be used without particular limitation as long as it can function as a protecting group, and a wide variety of known protecting groups used in amidite compounds can be used.
• Examples of G2 include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a haloalkyl group, an aryl group, a heteroaryl group, an arylalkyl group, a cycloalkenyl group, a cycloalkylalkyl group, a cyclylalkyl group, and a hydroxyalkyl group.
• silyl group aminoalkyl group, alkoxyalkyl group, heterocyclylalkenyl group, heterocyclylalkyl group, heteroarylalkyl group, silyl group, silyloxyalkyl group, mono, dialkylsilyl group or trialkylsilyl group, or monoalkylsilyloxyalkyl group , dialkylsilyloxyalkyl group, or trialkylsilyloxyalkyl group, which may be substituted with one or more electron-withdrawing groups.
• G 2 is preferably an alkyl group substituted with an electron-withdrawing group.
• the electron-withdrawing group include a cyano group, a nitro group, an alkylsulfonyl group, a halogen atom, an arylsulfonyl group, a trihalomethyl group, or a trialkylamino group, and preferably a cyano group.
• G 2 Particularly preferred as G 2 are the following groups.
• G 3s may be bonded to each other to form a cyclic structure.
• both are preferably isopropyl groups.
• the alkyl group in the above definitions of R 1 , R 2 , R 3 and G 2 may be linear or branched, preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms.
• Specific examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl.
• the alkyl group moiety constituting the alkoxy group in the above definition has the same definition as the alkyl group herein.
• nucleobase refers to a group having a natural or non-natural nucleobase skeleton.
• the nucleobases also include modified forms in which the backbone of a natural or non-natural nucleobase is modified. More specific examples of the nucleobase represented by B C include the following structures.
• R 4' represents a hydrogen atom or a methyl group
• R 5' represents a hydrogen atom or an acetyl group
• R 6' represents a hydrogen atom
• R 8' represents a hydrogen atom or a methyl group.
• non-nucleotide linkers that can be introduced in place of nucleotides.
• non-nucleotide linkers include linkers consisting of amino acid skeletons (for example, linkers consisting of amino acid skeletons described in Japanese Patent No. 5157168 or Japanese Patent No. 5554881).
• linkers consisting of amino acid skeletons for example, linkers consisting of amino acid skeletons described in Japanese Patent No. 5157168 or Japanese Patent No. 5554881.
• the following formula (A14-1), (A14-2) or (14-3) for example, described in Japanese Patent No.
• a linker represented by is exemplified.
• linkers described in International Publication No. 2012/005368, International Publication No. 2018/182008, or International Publication No. 2019/074110 are exemplified.
• Nucleotides and amidites in which the R group in formula (3) and the R' group in formula (4) are substituents other than hydroxyl groups can be prepared using known methods such as those described in Japanese Patent No. 3745226, and International Publication No. 2001/053528. It can also be produced from nucleosides synthesized by No. 2014-221817 or JP-A-2014-221817 and the known methods cited therein, and furthermore, by using commercially available nucleosides, It can be produced according to the methods described or by methods with appropriate modifications made to these methods.
• Excision of a nucleic acid oligomer (hereinafter also referred to as oligonucleotide) from a solid phase support
• the excision step was carried out using concentrated ammonia water as an excision agent for a nucleic acid oligomer of a desired chain length.
• each step of a deprotection step, a condensation step, and an oxidation step is repeated according to a generally known method (for example, the method described in the above-mentioned Japanese Patent No. 5157168 or Japanese Patent No. 5554881). By doing this, a nucleic acid elongation reaction is performed.
• nucleic acid elongation reaction refers to a reaction in which oligonucleotides are elongated by sequentially bonding nucleotides via phosphodiester bonds.
• the nucleic acid elongation reaction can be carried out according to the general phosphoramidite method.
• the nucleic acid elongation reaction may be performed using an automatic nucleic acid synthesizer or the like that employs the phosphoramidite method.
• the chain length of the nucleic acid oligomer may be, for example, 2 to 200 mer, 10 to 150 mer, or 15 to 110 mer.
• the 5' deprotection step is a step of deprotecting the protecting group of the 5' hydroxyl group at the end of the RNA chain supported on the solid phase support.
• general protecting groups 4,4'-dimethoxytrityl group (DMTr group), 4-monomethoxytrityl group, and 4,4',4''-trimethoxytrityl group are used.
• deprotecting acids include trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid, methanesulfonic acid, hydrochloric acid, acetic acid, and p-toluenesulfonic acid. .
• the condensation step is a reaction in which a nucleoside phosphoramidite represented by formula (A13) is bonded to the 5' hydroxyl group at the end of the oligonucleotide chain deprotected in the deprotection step.
• a nucleoside phosphoramidite represented by formula (A13) is bonded to the 5' hydroxyl group at the end of the oligonucleotide chain deprotected in the deprotection step.
• an amidite compound represented by formula (A13) or (A12) is used as the phosphoramidite used for nucleic acid elongation.
• nucleoside phosphoramidites include 2'-OMe, 2'-F, 2'-O-tert-butyldimethylsilyl group, 2'-O-methoxyethyl group, 2'-H, 2' -Fluoro-2'-deoxy- ⁇ -D-arabinofuranosyl and the like.
• a protecting group eg, DMTr group
• the condensation step can be carried out using an activator that activates the nucleoside phosphoramidite.
• Examples of the activator include 5-(benzylthio)-1H-tetrazole (BTT), 1H-tetrazole, 4,5-dicyanoimidazole (DCI), 5-(ethylthio)-1H-tetrazole (ETT), N- Methylbenzimidazolium triflate (N-MeBIT), benzimidazolium triflate (BIT), N-phenylimidazolium triflate (N-PhIMT), imidazolium triflate (IMT), 5-nitrobenzimidazolium triflate (NBT), or Examples include 1-hydroxybenzotriazole (HOBT) and 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole.
• Capping can be performed using a known capping solution such as an acetic anhydride-tetrahydrofuran solution or a phenoxyacetic anhydride/N-methylimidazole solution.
• the oxidation step is a step of converting the phosphorous acid group formed by the condensation step into a phosphoric acid group or a thiophosphoric acid group.
• This step is a reaction that converts trivalent phosphorus into pentavalent phosphorus using an oxidizing agent, and can be carried out by allowing an oxidizing agent to act on the oligonucleic acid derivative supported on a solid phase support. .
• the "oxidizing agent" is, for example, iodine, peracid such as tert-butyl hydroperoxide or hydrogen peroxide, or (1S)-(+)-( 10-camphorsulfonyl)-oxaziridine (CSO), or mixtures of two or more thereof can be used.
• the oxidizing agent can be used after being diluted with an appropriate solvent to a concentration of 0.005 to 2M.
• the solvent used in the reaction is not particularly limited as long as it does not participate in the reaction, but examples include pyridine, THF, water, acetonitrile, and any mixed solvent of two or more thereof.
• iodine/water/pyridine/acetonitrile or iodine/water/pyridine, or iodine/water/pyridine/acetonitrile/NMI, or iodine/water/pyridine/THF, or iodine/water/pyridine/THF/NMI, or CSO /acetonitrile, or iodine/pyridine-acetic acid or peracid (tert-butyl hydroperoxide/methylene chloride)
• iodine/pyridine-acetic acid or peracid tert-butyl hydroperoxide/methylene chloride
• examples of the "oxidizing agent" include 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-dimethylaminomethylidene) )-3H-1,2,4-dithiazoline-3-thione (DDTT), phenylacetyl disulfide (PADS), etc.
• sulfur 3H-1,2-benzodithiol-3-one-1,1-dioxide
• ADTT 3-amino-1,2,4-dithiazol-5-thione
• POS 5-phenyl-3H-1,2,4-dithiazol-3-one
• DDTT phenylacetyl disulfide
• PADS phenylacetyl disulfide
• the oxidizing agent can be used after being diluted with an appropriate solvent to a concentration of 0.01 to 2M.
• 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 any mixed solvent thereof.
• 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.
• amines are used to deprotect the protecting group of the phosphate moiety.
• examples of amines include diethylamine described in Japanese Patent No. 4705716.
• the protecting group for the 5' hydroxyl group of the nucleoside introduced at the end of elongation is used for column purification using the 5' protecting group as a tag after cleavage from the solid phase support and deprotection of the protecting group as described below. Alternatively, after column purification, the protecting group for the 5' hydroxyl group may be deprotected.
• oligonucleotide chain is cleaved from the solid support and recovered.
• amines include methylamine, ethylamine, propylamine, isopropylamine, ethylenediamine, and diethylamine.
• Nucleic acid oligomers that can be produced using the production method of this embodiment include RNA, DNA, 2'-O-MOE, 2'-O-Me, and 2'-F in which the nucleosides contained in the nucleic acid oligomer are Examples include, but are not limited to, nucleic acid oligomers that are RNA or LNA.
• nucleic acid oligomers that are RNA or LNA.
• nucleic acid oligomers that can be used in the production method of this embodiment are shown below in addition to the examples described in Examples, but are not limited thereto.
• U represents uridine
• C represents cytidine
• A represents adenosine
• G represents guanosine.
• nucleic acid oligomers having the following sequences (B) and (C) described in International Publication No. 2019/060442. Sequence (B): 5'-AUGGAAUmACUCUUGGUUmACdTdT-3' (Antisense) (SEQ ID NO: 3) 21mer Sequence (C): 5'-GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT-3' (Sense) (SEQ ID NO: 4) 21mer In sequences (B) and (C), Um represents 2'-O-methyluridine, Cm represents 2'-O-methylcytidine, and dT represents thymidine.
• nucleic acid oligomers examples include the nucleic acid oligomers described in Daniel O'Reilly et al., Nucleic Acids Research, 2019, Vol. 47, No. 2, 546-558 (see page 553).
• a typical example is a nucleic acid oligomer having the following sequence (D). Sequence (D): 5'-AGAGCCAGCCUUCUUAUUGUUUUAGAGCUAUGCUGU-3' (SEQ ID NO: 5) 36mer
• nucleic acid oligomers described in Japanese Patent No. 4965745.
• a typical example is a nucleic acid oligomer having the following sequence (E). Sequence (E): 5'-CCAUGAGAAGUAUGACAACAGCC-P-GGCUGUUGUCAUACUUCUCAUGGUU-3' 49mer. This sequence consists of CCAUGAGAAGUAUGACAACAGCC (SEQ ID NO: 6) and GGCUGUUGUCAUACUUCUCAUGGUU (SEQ ID NO: 7).
• "P” is represented by a partial structure separated by a wavy line in the following formula (A5).
• Nucleic acid oligomers having the following sequence (F) described in Nucleic Acids Research, 2019, Vol. 47, No. 2: 547 can be mentioned. Sequence (F): 5'-ACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 8) 67mer
• nucleic acid oligomer having the following sequence (G) described in JP 2015-523856, page 173. Sequence (G): 5'-GUUUUCCCUUUUCAAAGAAAUCUCCUGGGCACCUAUCUUCUUAGGUGCCCUCCCUUGUUUAAAACCUGACCAGUUAACCGGCUGGUUAGGUUUU-3' (SEQ ID NO: 9) 94mer
• nucleic acid oligomers described in JP 2017-537626 examples include nucleic acid oligomers having the following sequences (H), (J), (K), and (L). Sequence (H): 5'-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 10) 100mer sequence (J): 5'-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCC GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU-3' (SEQ ID NO: 11) 113mer Sequence (K): 5'-dAdGdTdCdCdTdCdTdCdCdTdCdCdAdGdAdG
• sequence (I) consists of AGCAGAGUAC ACACAGCAUA UACC (SEQ ID NO: 1) and GGUAUAUGCU GUGUGUACUC UGCUUC (SEQ ID NO: 2) linked by the above "P".
• CPG Controlled Pore Glass
• AKTA oligopilot plus 100 manufactured by GE Healthcare
• a nucleic acid synthesizer Using Controlled Pore Glass (CPG) as a solid phase carrier and AKTA oligopilot plus 100 (manufactured by GE Healthcare) as a nucleic acid synthesizer, the phosphoramidite solid phase synthesis method was used. An oligonucleotide consisting of the above sequence (I) was synthesized from the 3' side to the 5' side. Synthesis was performed on a 77.89 ⁇ mol scale.
• uridine EMM amidite (A11) described in Example 2 of International Publication No. 2013/027843 cytidine EMM amidite (A9) described in Example 3
• adenosine EMM amidite described in Example 4.
• oligonucleotide (nucleic acid oligomer) manufactured by the manufacturing method of this embodiment.
• the reaction was carried out under air (oxygen concentration 21%).
• the oligonucleotides produced by the production method of this embodiment are oligonucleotides having the sequences (I) shown in SEQ ID NOs: 1 and 2 above.
• the guanosine derivative described in the following Examples and Comparative Examples means a compound represented by the following structural formula.
• the circle illustrated in the structural formula below schematically represents CPG.
• Example 1 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• Example 2 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• Example 7 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite shown in formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• 6-tert-butyl-2,4-xylenol was added (the amount of 6-tert-butyl-2,4-xylenol was 0.9 mol per mol of protecting group), dissolved, and further molecular 0.76 g of a dimethyl sulfoxide solution of 1 M tetra-n-butylammonium fluoride (TBAF) (the amount of TBAF is 28.6 mol per 1 mol of protecting group), which has been dehydrated with sieve 4A, is introduced into the vortex mixer.
• the 2'-cyanoethoxymethoxy (CEM) protecting group was removed by stirring the mixture uniformly and then keeping the mixture warm at 30° C. for 4 hours.
• the crude product was obtained by a precipitation operation.
• the yield was 4.1 mg and the purity was 56%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• Example 9 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• the yield was 4.3 mg and the purity was 52%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• the yield was 4.3 mg and the purity was 54%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• Example 15 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• Example 16 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• Example 17 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• Example 18 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite shown in formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• Example 20 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• Example 21 of sequence (I) using CPG carrying 93.79 ⁇ mol of a guanosine derivative and an amidite represented by formula (A15), formula (A16), formula (A17), formula (A18), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.03 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.15 g of ammonia water and 3.07 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• the 2'-cyanoethoxymethoxy (CEM) protecting group was removed by stirring uniformly with a vortex mixer and then keeping the mixture at 30° C. for 4 hours.
• the crude product was obtained by a precipitation operation.
• the yield was 4.1 mg and the purity was 48%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• BHT 2,6-di-tert-butyl-p-cresol
• the crude product was obtained by a precipitation operation.
• the yield was 4.2 mg and the purity was 53%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• BHT 2,6-di-tert-butyl-p-cresol
• the crude product was obtained by a precipitation operation.
• the yield was 4.0 mg and the purity was 53%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• BHT 2,6-di-tert-butyl-p-cresol
• the crude product was obtained by a precipitation operation.
• the yield was 4.3 mg and the purity was 55%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• BHT 2,6-di-tert-butyl-p-cresol
• the crude product was obtained by a precipitation operation.
• the yield was 4.4 mg and the purity was 55%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• BHT 2,6-di-tert-butyl-p-cresol
• the crude product was obtained by a precipitation operation.
• the yield was 4.3 mg and the purity was 54%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• the yield was 4.2 mg and the purity was 50%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• Example 30 of sequence (I) using CPG carrying 93.86 ⁇ mol of a guanosine derivative and an amidite shown in formula (A8), formula (A9), formula (A10), formula (A11), or formula (A12).
• Solid phase synthesis was performed on an AKTA oligopilot plus100. Thereafter, the CPG carrier supporting 30.02 ⁇ mol of oligonucleotide was collected, and the oligonucleotide was released from the solid support using 10.18 g of ammonia water and 3.03 g of ethanol, and then the carrier was filtered to release the oligonucleotide. The filtrate containing the oligonucleotide was concentrated to dryness.
• the yield was 4.0 mg, and the purity was 52%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• the crude product was obtained by a precipitation operation.
• the yield was 4.1 mg and the purity was 49%.
• the purity of the oligonucleotide was measured using the method described in measurement method 1 above, and the yield of oligonucleotide was measured using the method described in measurement method 2 above.
• the crude product was obtained by a precipitation operation.
• the yield was 4.2 mg, and the purity was 49%.
• 4.7 mL of an aqueous solution containing the entire amount of the obtained crude product was purified by affinity chromatography.
• the entire amount of the nucleic acid aqueous solution was applied to a commercially available affinity column (SkillPak Toyopearl AF-Chelate-650M, 1 mL, Tosoh Corporation), water was used as the mobile phase, and the flow rate was 0. 20 mL of liquid was fed at a rate of 6 mL/min.
• the UV (260 nm) of the eluate could be confirmed by a monitor, and the entire amount of the portion in which eluted nucleic acid was observed was collected and concentrated by membrane filtration to obtain a purified nucleic acid.
• the amount of the radical reaction inhibitor used is the value obtained by multiplying 1 mol of the compound supported on the solid phase carrier by the number in which R in formula (3) is a group represented by formula (1). means the amount used for.
• nucleic acid oligomers can be efficiently produced.
• SEQ ID NOS: 1 to 13 in the sequence listing represent the base sequences of oligonucleotides produced according to the production method of the present invention.

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