Oligonucleotide precipitation method and production method

Abstract

The present invention provides a method for precipitating an oligonucleotide from a solution containing the oligonucleotide bearing a hydrophobic protective group and a non-polar solvent, the method comprising: mixing the solution with a first polar solvent; and evaporating off the first polar solvent from the mixture of the solution and the first polar solvent while maintaining the volume of the mixture at 1.00 times or more relative to the volume of the solution. The method according to the present invention enables reduction of the amount of polar solvent to be used for precipitation, and enables high-purity precipitation of an oligonucleotide from a solution containing the oligonucleotide bearing a hydrophobic protective group and a non-polar solvent.

Description

Methods for Precipitating and Preparing Oligonucleotides

[0001] The present invention relates to a method for depositing and preparing oligonucleotides.

[0002] Known methods for producing oligonucleotides include synthesizing an oligonucleotide having a hydrophobic protecting group in a solution containing a starting material having a hydrophobic protecting group (e.g., a monomer having a hydrophobic protecting group) and a non-polar solvent, and then adding a polar solvent to the reaction solution to precipitate the oligonucleotide (e.g., Patent Documents 1 and 2).

[0003] International Publication No. WO 2017 / 104836 International Publication No. WO 2022 / 172994

[0004] In the method of precipitating oligonucleotide by simply adding polar solvent to the solution containing the oligonucleotide having hydrophobic protecting group and non-polar solvent, it is necessary to use a large amount of polar solvent.If the amount of polar solvent used for precipitation is reduced by concentrating the solution and then adding polar solvent, the purity of the precipitated oligonucleotide may be reduced due to the side reaction that occurs during concentration.In the liquid phase synthesis of oligonucleotide using hydrophobic protecting group, the influence of impurities in the next synthesis cycle is greater than that in the solid phase synthesis of oligonucleotide using solid phase carrier, so it is necessary to isolate the oligonucleotide with as high purity as possible.

[0005] The present invention has been made in light of the above-mentioned circumstances, and an object of the present invention is to provide a method that can reduce the amount of polar solvent used for precipitation and can precipitate oligonucleotides with high purity from a solution containing an oligonucleotide having a hydrophobic protecting group and a non-polar solvent.

[0006] In order to achieve the above object, the present inventors have conducted extensive research and have found that by mixing a first polar solvent used for precipitation with an oligonucleotide having a hydrophobic protecting group (hereinafter sometimes abbreviated as "target oligonucleotide") and a solution containing a non-polar solvent (hereinafter sometimes abbreviated as "target solution"), and distilling off the solvent from the target mixture while maintaining the volume of the mixture of the first polar solvent and the target solution (hereinafter sometimes abbreviated as "target mixture") at least 1.00 times the volume of the target solution, it is possible to reduce the amount of polar solvent used for precipitation and to precipitate the target oligonucleotide with high purity. The present invention based on this finding is as follows.

[0007] [1] A method for precipitating an oligonucleotide having a hydrophobic protecting group from a solution containing the oligonucleotide and a non-polar solvent, the method comprising: mixing the solution with a first polar solvent; and distilling off the solvent from the mixture while maintaining the volume of the mixture of the solution and the first polar solvent at 1.00 times or more the volume of the solution.

[0008] [2] The method according to [1] above, wherein the solvent is distilled off from the mixture at a temperature of 5°C to 35°C and a pressure of 15 mmHg to 500 mmHg. [3] The method according to [1] above or [2] above, wherein the non-polar solvent comprises a halogenated solvent.

[0009] [4] The method according to any one of [1] to [3], wherein the first polar solvent comprises at least one selected from the group consisting of nitrile solvents and alcohol solvents. [5] The method according to any one of [1] to [4], wherein the total volume of the first polar solvent to be mixed with the solution is 1.0 to 3.5 times the volume of the nonpolar solvent.

[0010] [6] The method according to any one of [1] to [5], wherein the solution contains a second polar solvent. [7] The method according to [6], wherein the second polar solvent contains at least one selected from the group consisting of nitrile solvents and alcohol solvents. [8] The method according to [6] or [7], wherein the volume of the second polar solvent is 0.15 to 0.35 times the volume of the nonpolar solvent.

[0011] [9] The method according to any one of the above [1] to [8], wherein the molecular weight of the hydrophobic protecting group is 360 or more.

[10] The hydrophobic protecting group is represented by the following formula (Pg-5): **L-Y-Z (Pg-5) [wherein ** represents the bonding position to the protected group; L represents a single bond, or a group represented by formula (Pg-i-1) or (Pg-i-2):

[0012]

[0013] (wherein * indicates the bonding position to Y; ** has the same meaning as above; R １ｐ and R ２ｐ are each independently C １－２２ represents a hydrocarbon group; １ is an optionally substituted divalent C １－２２ represents a hydrocarbon group, and １－２２ -CH in hydrocarbon group ２ - may be replaced with a linker; L ２ indicates a single bond or ***C(═O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and R ４ｐ is C １－２２ represents an alkylene group, and R ３ｐ and R ５ｐ are each independently a hydrogen atom or C １－２２ represents an alkyl group or R ３ｐ and R ５ｐmay be taken together to form a ring. ) represents a group represented by the formula (Pg-ii-1); ) represents a group represented by the formula (Pg-ii-2); Y represents a single bond, an oxygen atom, or NR (R represents a hydrogen atom, an alkyl group, or an aralkyl group); and Z represents any of the formulas (Pg-ii-1) to (Pg-ii-5):

[0014]

[0015] [wherein * represents a bonding position; ring A' represents a benzene ring; ring B' represents a cyclohexane ring; ring D' represents a naphthalene ring or a bicyclic fused aromatic heterocycle; R ６ｐ is a hydrogen atom, or R ｂ is a group represented by the following formula (Pg-iii), R of ring A' or ring B' ６ｐ is R ８ｐ and may together represent a single bond or —O— to form a fused ring together with ring A′ or ring B′ and ring C′; k represents an integer of 1 to 4; k Qs each independently represent —O—, —C(═O)—, —C(═O)O—, —OC(═O)—, —NR′—, —C(═O)NR′—, or —NR′C(═O)— (wherein R′ each independently represents a hydrogen atom or C １－６ represents an alkyl group) (k Qs preferably each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-); k Q's each independently represent a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'- or -NR'C(=O)- (wherein R's each independently represent a hydrogen atom or C １－６ k R ７ｐ each independently represent a hydrocarbon group to which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker; ring A′ and ring B′ each independently represent k QR ７ｐ In addition to the above, a halogen atom, a C optionally substituted with a halogen atom １－６ C optionally substituted with an alkyl group or a halogen atom １－６R may have a substituent selected from the group consisting of alkoxy groups; ａ represents a hydrogen atom; and R ｂ is a hydrogen atom or a group represented by the formula (Pg-iii):

[0016]

[0017] (wherein * represents a bonding position; ring C′ represents a benzene ring; j represents an integer of 0 to 4; j Qs each independently have the same meaning as defined above; j R ９ｐ each independently represents a hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker; R ８ｐ represents a hydrogen atom, or R ６ｐ and may be taken together with j QR's to form a single bond or -O- to form a fused ring together with ring A' or ring B' and ring C'; ring C' may be taken together with j QR's ９ｐ In addition to the above, a halogen atom, a C optionally substituted with a halogen atom １－６ C optionally substituted with an alkyl group or a halogen atom １－６ R may have a substituent selected from the group consisting of alkoxy groups. ａ and R ｂ together form an oxo group; u represents 1 or 2; and R １０ｐ is C ２－２１ The method according to any one of the above [1] to [9], wherein the protecting group is a group represented by the formula:

[0018]

[11] A method for producing an oligonucleotide by one-pot synthesis, the method comprising: a step (1) of condensing, in a solution containing a nonpolar solvent, a nucleoside, nucleotide, or oligonucleotide (a) having a hydrophobic protecting group with a nucleoside, nucleotide, or oligonucleotide (b) whose hydroxyl group is protected by a temporary protecting group removable under acidic conditions and which has been phosphoramidized, to form an oligonucleotide precursor (c) having a phosphite bond and a hydrophobic protecting group and whose hydroxyl group is protected by a temporary protecting group removable under acidic conditions; a step (2) of mixing the solution obtained after the step (1) with a quenching agent (i) for the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b) to quench the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b); a step (3) of mixing the solution after step (2) with an oxidizing agent or a sulfurizing agent to oxidize or sulfurize the oligonucleotide precursor (c) to form an oligonucleotide (d) having a phosphate bond or a thiophosphate bond and a hydrophobic protecting group, and whose hydroxyl group is protected by a temporary protecting group removable under acidic conditions; a step (4) of mixing the solution after step (3) with an oxidizing agent (ii) to quench the oxidizing agent, if necessary, when an oxidizing agent is used in step (3); a step (5) of mixing the solution after step (3), or the solution after step (4) if step (4) is performed, with an acid to remove the temporary protecting group removable under acidic conditions from the oligonucleotide (d) to form an oligonucleotide (e) having an unprotected hydroxyl group and a hydrophobic protecting group; a step (6) of mixing the solution after step (5) with a base, if necessary; and a step (7) of precipitating the oligonucleotide (e) from a solution containing the oligonucleotide (e) and a nonpolar solvent by the method described in any one of [1] to

[10] above. Methods including:

[0019]

[12] The method according to the above

[11] , wherein the removal of the temporary protecting group in step (5) is carried out in the presence of a cation scavenger, or the solution obtained after the removal of the temporary protecting group is mixed with a cation scavenger.

[13] The method according to the above

[12] , wherein the cation scavenger is at least one selected from the group consisting of pyrrole, pyrrole derivatives, indole, indole derivatives, furan derivatives, and mercapto group-containing compounds.

[0020] According to the present invention, the amount of polar solvent used for precipitation can be reduced, and the oligonucleotide can be precipitated with high purity from a solution containing an oligonucleotide having a hydrophobic protecting group and a non-polar solvent.

[0021] First, the terms used in this specification will be explained. Unless otherwise specified, the terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0022] In the present specification, "C ａ－ｂ " means that the number of carbon atoms is from a to b (a and b are integers). In this specification, "halogen atom" means a fluorine atom, chlorine atom, bromine atom or iodine atom.

[0023] In this specification, examples of the "hydrocarbon group" include an aliphatic hydrocarbon group, an aromatic aliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group, and an aromatic hydrocarbon group, and specific examples thereof include monovalent and divalent groups such as an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an aralkyl group, and an alkylene group.

[0024] In this specification, the "alkyl (group)" may be either linear or branched. The "alkyl (group)" includes alkyl groups having one or more carbon atoms, and when there is no particular limitation on the range of the carbon number, it is preferably C １－１０ alkyl group, more preferably C １－６ It is an alkyl group, and specific examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0025] In the present specification, the "alkenyl (group)" may be either linear or branched. Examples of the "alkenyl (group)" include C ２－６ Examples of the alkyl group include alkenyl groups, such as vinyl, 1-propenyl, allyl, isopropenyl, butenyl, and isobutenyl.

[0026] In the present specification, the "alkynyl (group)" may be either linear or branched. Examples of the "alkynyl (group)" include C ２－６ Specific examples thereof include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, and 5-hexynyl.

[0027] As used herein, "cycloalkyl (group)" refers to a cyclic alkyl group, such as C ３－８ Cycloalkyl groups, preferably C ３－６ Specific examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0028] In the present specification, the term "aryl (group)" refers to a monocyclic or polycyclic (fused) aromatic hydrocarbon group, and examples thereof include C aryl groups such as phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, 1-anthryl, and 2-anthryl. ６－１４ Among them, C ６－１０ Aryl groups are more preferred, with phenyl being especially preferred.

[0029] As used herein, the term "heteroaryl (group)" refers to a monocyclic or polycyclic (fused) aromatic heterocyclic group containing, in addition to carbon atoms, a heteroatom selected from a nitrogen atom, a sulfur atom, and an oxygen atom as a ring-constituting atom. Examples of heteroaryl groups include 5- or 6-membered monocyclic heteroaryl groups and 8- to 14-membered fused polycyclic heteroaryl groups.

[0030]

[0033] In the present specification, examples of the "5- or 6-membered monocyclic heteroaryl (group)" include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl and the like.

[0031]

[0033] In the present specification, examples of the "8- to 14-membered fused polycyclic heteroaryl (group)" include benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl, and furopyrimidinyl. , pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like.

[0032] In the present specification, the term "aralkyl (group)" includes C ７－２０ An aralkyl group is exemplified, and preferably C ７－１６ Aralkyl group (C ６－１０ Aryl-C １－６ Specific examples thereof include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, and 1-naphthylpropyl.

[0033] In the present specification, the "alkylene (group)" may be either linear or branched. The "alkylene (group)" includes alkylene groups having one or more carbon atoms, and when there is no particular limitation on the range of the carbon number, it is preferably C １－１０ is an alkylene group, more preferably C １－６ It is an alkylene group, and specific examples thereof include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0034] In the present specification, the "alkoxy (group)" may be either linear or branched. The "alkoxy (group)" includes an alkoxy group having one or more carbon atoms, and when there is no particular limitation on the range of the carbon number, it is preferably an alkoxy group having one or more carbon atoms. １－１０ is an alkoxy group, more preferably C １－６ An alkoxy group, specific examples of which include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0035] In the present specification, the "acyl (group)" may be either linear or branched. Examples of the "acyl (group)" include C １－６ Alkanoyl group, C ７－１３ Examples of the "acyl (group)" include formyl, acetyl, n-propionyl, isopropionyl, n-butyryl, isobutyryl, pivaloyl, valeryl, hexanoyl, benzoyl, naphthoyl, and levulinyl. The "acyl (group)" may be substituted.

[0036] In the present specification, examples of the "bicyclic fused aromatic heterocycle" include a benzothiophene ring, a benzofuran ring, an isobenzofuran ring, a benzimidazole ring, a benzoxazole ring, a benzisoxazole ring, a benzothiazole ring, a benzisothiazole ring, a benzotriazole ring, an indole ring, an isoindole ring, a 1H-indazole ring, a purine ring, an isoquinoline ring, a 5,6,7,8-tetrahydroisoquinoline ring, a quinoline ring, a 5,6,7,8-tetrahydroquinoline ring, a phthalazine ring, a pteridine ring, a naphthyridine ring, a quinoxaline ring, a quinazoline ring, a cinnoline ring, etc. In addition, in the present specification, the "bicyclic fused aromatic heterocycle" also includes a bicyclic fused ring in which one ring is an aromatic heterocycle and the other ring is a non-aromatic heterocycle or a non-aromatic hydrocarbon ring (for example, a 5,6,7,8-tetrahydroisoquinoline ring).

[0037] In this specification, the term "perfluoroalkyl group" refers to an alkyl group in which all hydrogen atoms have been substituted with fluorine atoms.

[0038] In the present specification, examples of the "linker" include -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, -NR'C(=O)-, -S-, -SO-, and -SO ２ -, -Si(R')(R")O-, -Si(R')(R")- (R' and R" are each independently a hydrogen atom or C １－２２ The linker is preferably —O—, —C(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NH—, —NHC(═O)—, —S—, —SO—, or —SO ２ -, -Si(R')(R")O-, or -Si(R')(R")- (R' and R" are each independently a hydrogen atom or C １－２２ represents a hydrocarbon group).

[0039] In this specification, examples of the "substituent" include, in addition to the aforementioned halogen atoms, alkyl groups, aralkyl groups, alkoxy groups, acyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, and aryl groups, a hydroxyl group, a nitro group, a cyano group, a guanidyl group, a carboxy group, an alkoxycarbonyl group (the alkoxy moiety is the same as the aforementioned alkoxy group), a sulfo group, a phospho group, an alkylsulfanyl group (the alkyl moiety is the same as the aforementioned alkyl group), an alkylsulfinyl group (the alkyl moiety is the same as the aforementioned alkyl group), an alkylsulfonyl group (the alkyl moiety is the same as the aforementioned alkyl group), an amino group, a monoalkylamino group (the alkyl moiety is the same as the aforementioned alkyl group), a dialkylamino group (the alkyl moiety is the same as the aforementioned alkyl group), and an oxo group.

[0040] In the present specification, the "hydroxyl group-protecting group" is not particularly limited, and known protecting groups can be used. Examples of such protecting groups include methyl, benzyl, p-methoxybenzyl, tert-butyl, methoxymethyl, 2-methoxyethyl, 2-tetrahydropyranyl, 2-ethoxyethyl, 2-cyanoethyl, (2-cyanoethoxy)methyl, 1-(2-cyanoethoxy)ethyl, bis(2-acetoxyethoxy)methyl (ACE), (2-nitrobenzyl)oxymethyl (NBOM), (2-(trimethylsilyl)ethoxy)methyl (SEM), 1-(2-cyanoethoxy)ethyl, 2-((4-methylphenyl)sulfonyl)ethoxymethyl (TEM), tert-butyldithiomethyl (DTM), ((2-(methylthio)phenyl) )thio)methyl (MPTM) group, (N-dichloroacetyl-N-methyl)aminobenzyloxymethyl (DCMABOM) group, (2-acetylethyl)carbonyloxymethyl group (also referred to in the art as "acetallevunilyl ester (ALE) group"), phenylcarbamoyl group, 1,1-dioxothiomorpholine-4-thiocarbamoyl group, acetyl group, pivaloyl group, benzoyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group, [(triisopropylsilyl)oxy]methyl (Tom) group, 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group, and the like.

[0041] In the present specification, examples of the "temporary protecting group removable under acidic conditions for a hydroxyl group" include bis(C methyl) groups such as a trityl group, a 9-phenyl-9-xanthenyl group, a 9-phenyl-9-thioxanthenyl group, and a 1,1-bis(4-methoxyphenyl)-1-phenylmethyl group (sometimes referred to as a "4,4'-dimethoxytrityl group" in the present specification). １－６ mono(C alkoxy)trityl group, 1-(4-methoxyphenyl)-1,1-diphenylmethyl group (sometimes referred to as "4-monomethoxytrityl group" in this specification), etc. １－１８ From the viewpoint of ease of deprotection, the temporary protecting group is preferably a 4,4'-dimethoxytrityl group or a 4-monomethoxytrityl group, more preferably a 4,4'-dimethoxytrityl group.

[0042] In the present specification, the "protecting group for an amino group" is not particularly limited, and known protecting groups can be used. Examples of such protecting groups include pivaloyl, pivaloyloxymethyl, acetyl, trifluoroacetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, 4-tert-butylphenoxyacetyl, benzoyl, isobutyryl, (2-hexyl)decanoyl, dimethylformamidinyl, and 9-fluorenylmethyloxycarbonyl groups. Among these, acetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, benzoyl, isobutyryl, (2-hexyl)decanoyl, dimethylformamidinyl, and ═C(R １１ )-N(R １２ ) (R １３ ) (wherein R １１ represents a methyl group, and R １２ and R １３ are each independently C １－６ represents an alkyl group or R １１ and R １２ may be taken together to form a 5- or 6-membered nitrogen-containing heterocycle together with the carbon atom and nitrogen atom to which they are attached. １１ )-N(R１２ ) (R １３ ) includes, for example, a 1-(dimethylamino)ethylidene group.

[0043] In this specification, the "protecting group for the phosphate group" is not particularly limited, and known protecting groups can be used. Examples of the protecting group for the phosphate group include the following: a 2-cyanoethyl group represented by the following formula (Pg-1), a 2-[2-(4,4'-dimethoxytrityloxy)ethylsulfonyl]ethyl group represented by the following formula (Pg-2), a [3-(4,4'-dimethoxytrityloxy)-2,2-di(ethoxycarbonyl)]propyl group represented by the following formula (Pg-3), and a [3-(4,4'-dimethoxytrityloxy)-2,2-di(N-methylcarbamoyl)]propyl group represented by the following formula (Pg-4). The protecting group for the phosphate group is preferably a 2-cyanoethyl group.

[0044]

[0045] [In the formula, Me represents a methyl group, Et represents an ethyl group, DMTr represents a 4,4′-dimethoxytrityl group, and * represents the bonding position with the phosphate group.]

[0046] In the present specification, examples of the "hydrophobic protecting group" include those described in International Publication Nos. WO 2017 / 038650, WO 2017 / 086397, WO 2017 / 104836, WO 2018 / 203574, WO 2018 / 212236, WO 2019 / 131719, WO 2020 / 017085, WO 2020 / 175472, WO 2020 / 227618, WO 2021 / 198883, WO 2022 / 103842, and WO 2022 / 214692.

[0047] From the viewpoint of dissolving the target oligonucleotide in the target solution, the molecular weight of the hydrophobic protecting group is preferably 360 or more, more preferably 600 or more. From the viewpoint of dissolving the target oligonucleotide in the target solution and precipitating the target oligonucleotide from the target solution when a polar solvent is added, the molecular weight of the hydrophobic protecting group is preferably 2,000 or less, more preferably 1,200 or less.

[0048] The hydrophobic protecting group is preferably a protecting group represented by the following formula (Pg-5): **LYZ (Pg-5) [wherein ** represents the bonding position to the protected group; L represents a single bond, or a group represented by formula (Pg-i-1) or (Pg-i-2):

[0049]

[0050] (wherein * indicates the bonding position to Y; ** has the same meaning as above; R １ｐ and R ２ｐ are each independently C １－２２ represents a hydrocarbon group; １ is an optionally substituted divalent C １－２２ represents a hydrocarbon group, and １－２２ -CH in hydrocarbon group ２ - may be replaced with a linker; L ２ indicates a single bond or ***C(═O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and R ４ｐ is C １－２２ represents an alkylene group, and R ３ｐ and R ５ｐ are each independently a hydrogen atom or C １－２２ represents an alkyl group or R ３ｐ and R ５ｐmay be taken together to form a ring. ) represents a group represented by the formula (Pg-ii-1); ) represents a group represented by the formula (Pg-ii-2); Y represents a single bond, an oxygen atom, or NR (R represents a hydrogen atom, an alkyl group, or an aralkyl group); and Z represents any of the formulas (Pg-ii-1) to (Pg-ii-5):

[0051]

[0052] [wherein * represents a bonding position; ring A' represents a benzene ring; ring B' represents a cyclohexane ring; ring D' represents a naphthalene ring or a bicyclic fused aromatic heterocycle; R ６ｐ is a hydrogen atom, or R ｂ is a group represented by the following formula (Pg-iii), R of ring A' or ring B' ６ｐ is R ８ｐ and may together represent a single bond or —O— to form a fused ring together with ring A′ or ring B′ and ring C′; k represents an integer of 1 to 4; k Qs each independently represent —O—, —C(═O)—, —C(═O)O—, —OC(═O)—, —NR′—, —C(═O)NR′—, or —NR′C(═O)— (wherein R′ each independently represents a hydrogen atom or C １－６ represents an alkyl group) (k Qs preferably each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-); k Q's each independently represent a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'- or -NR'C(=O)- (wherein R's each independently represent a hydrogen atom or C １－６ k R ７ｐ each independently represent a hydrocarbon group to which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker; ring A′ and ring B′ each independently represent k QR ７ｐ In addition to the above, a halogen atom, a C optionally substituted with a halogen atom １－６ C optionally substituted with an alkyl group or a halogen atom １－６R may have a substituent selected from the group consisting of alkoxy groups; ａ represents a hydrogen atom; and R ｂ is a hydrogen atom or a group represented by the formula (Pg-iii):

[0053]

[0054] (wherein * represents a bonding position; ring C′ represents a benzene ring; j represents an integer of 0 to 4; j Qs each independently have the same meaning as defined above; j R ９ｐ each independently represents a hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker; R ８ｐ represents a hydrogen atom, or R ６ｐ and may be taken together with j QR's to form a single bond or -O- to form a fused ring together with ring A' or ring B' and ring C'; ring C' may be taken together with j QR's ９ｐ In addition to the above, a halogen atom, a C optionally substituted with a halogen atom １－６ C optionally substituted with an alkyl group or a halogen atom １－６ R may have a substituent selected from the group consisting of alkoxy groups. ａ and R ｂ together form an oxo group; u represents 1 or 2; and R １０ｐ is C ２－２１ represents a perfluoroalkyl group.] represents a group represented by the formula:

[0055] Hereinafter, the "protecting group represented by formula (Pg-5)" may be abbreviated as "protecting group (Pg-5)." Compounds represented by other formulae, monomers represented by other formulae, etc. may also be abbreviated in the same manner as the protecting group represented by formula (Pg-5).

[0056] Ring A', Ring C', R ６ｐ and R ８ｐ When R forms a fused ring, ring A' and ring C' each represent a benzene ring portion in the fused ring. ６ｐ and R ８ｐform a fused ring, ring C' represents the benzene ring portion in the fused ring, and ring B' represents the cyclohexane ring portion in the fused ring.

[0057] The linear aliphatic hydrocarbon group having 10 or more carbon atoms is preferably a linear C １０－４０ Alkyl groups and linear C １０－４０ Alkenyl groups are more preferably straight-chain C １０－４０ alkyl group, more preferably a straight-chain C １０－３０ The alkyl group is preferably a linear C １２－２８ alkyl group, most preferably a straight-chain C １４－２６ It is an alkyl group.

[0058] The linker is preferably —O—, —C(═O)—, —C(═O)O—, —OC(═O)—, —NR′—, —C(═O)NR′— or —NR′C(═O)— (wherein R′ is a hydrogen atom or C １－２２ represents a hydrocarbon group), more preferably —O—, —C(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NH— or —NHC(═O)—, and still more preferably —O—.

[0059] The "hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker" is preferably a linear C １０－４０ Alkyl group, 1 to 3 straight-chain C １０－４０ A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０ It is a cyclohexylmethyl group in which the alkyl group is bonded via —O—.

[0060] Q is preferably —O—, —C(═O)NH— or —NHC(═O)—, more preferably —O—.

[0061] Q' is preferably a single bond, --O--, --C(.dbd.O)NH-- or --NHC(.dbd.O)--, more preferably a single bond or --O--.

[0062] In formula (Pg-5), preferred embodiments of L represented by formula (Pg-i-1) include: １ But divalent C１－２２ Hydrocarbon group, or CH ２ —O-1,4-phenylene-O—CH ２ and L ２ is a single bond or ***C(═O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and R ４ｐ is C １－６ represents an alkylene group, and R ３ｐ and R ５ｐ are each independently a hydrogen atom or an optionally substituted C １－６ represents an alkyl group or R ３ｐ and R ５ｐ are taken together to form an optionally substituted C １－６ It is a group represented by the formula (I) which may form an alkylene group.

[0063] Another preferred embodiment of L represented by formula (Pg-i-1) is １ But divalent C １－２２ is a hydrocarbon group; and L ２ is a group where R is a single bond.

[0064] Another preferred embodiment of L represented by formula (Pg-i-1) is １ is an ethylene group; and L ２ However, ***C(=O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and R ４ｐ is C １－２２ represents an alkylene group, and R ３ｐ and R ５ｐ are each independently a hydrogen atom or C １－２２ represents an alkyl group or R ３ｐ and R ５ｐ may be taken together to form a ring.

[0065] Another preferred embodiment of L represented by formula (Pg-i-1) is １ is an ethylene group; and L ２ However, ***C(=O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) portion forms a 1,4-piperazinediyl group.

[0066] Another preferred embodiment of L represented by formula (Pg-i-1) is １ is an ethylene group; and L ２ However, ***C(=O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and R ４ｐ represents a pentylene group or a hexylene group, and R ３ｐ and R ５ｐ are each independently a hydrogen atom or a methyl group.

[0067] Another preferred embodiment of L represented by formula (Pg-i-1) is １ is an optionally substituted phenylene group; and L ２ However, ***C(=O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) portion forms a 1,4-piperazinediyl group.

[0068] L represented by formula (Pg-i-1) is particularly preferably a succinyl group.

[0069] Next, L represented by formula (Pg-i-2) in formula (Pg-5) will be described. １ is preferably a divalent C ６－１０ It is an aromatic hydrocarbon group, and more preferably a phenylene group. ２ is preferably a single bond. １ and L ２ A preferred combination of １ is divalent C ６－１０ is an aromatic hydrocarbon group, and L ２ is a single bond. １ and L ２ A more preferred combination of １ is a phenylene group, and L ２ is a single bond. １ｐ and R ２ｐ are each independently preferably C １－２２ alkyl group, more preferably C １－１０ It is an alkyl group.

[0070] A preferred embodiment of L represented by formula (Pg-i-2) is R １ｐ and R ２ｐ are each independently, C １－２２ is an alkyl group; １ But divalent C ６－１０ an aromatic hydrocarbon group; and L ２ is a group where R is a single bond.

[0071] Another preferred embodiment of L represented by formula (Pg-i-2) is R １ｐ and R ２ｐ are each independently, C １－１０ is an alkyl group; １ is a phenylene group; and L ２ is a group where R is a single bond.

[0072] When Y in formula (Pg-5) is NR, the R is preferably a hydrogen atom, C １－６ Alkyl group or C７－１６ Y is an aralkyl group, more preferably a hydrogen atom, methyl, ethyl or benzyl, and even more preferably a hydrogen atom. Y is preferably a single bond, an oxygen atom or NR, and more preferably a single bond or an oxygen atom.

[0073] In formula (Pg-ii-1), R ６ｐ is preferably a hydrogen atom. ａ and R ｂ are preferably hydrogen atoms or together form an oxo group.

[0074] A preferred embodiment of Z represented by formula (Pg-ii-1) is R ａ and R ｂ is a hydrogen atom; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ is an alkyl group;

[0075] Another preferred embodiment of Z represented by formula (Pg-ii-1) is R ａ and R ｂ is a hydrogen atom; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ each independently represents 1 to 3 straight-chain C １０－４０ A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０ A group in which the alkyl group is a cyclohexylmethyl group bonded via —O—.

[0076] Another preferred embodiment of Z represented by formula (Pg-ii-1) is R ａ is a hydrogen atom; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ is an alkyl group; and R ｂis represented by formula (Pg-iii) (wherein * represents a bonding position, j represents an integer of 0 to 3, j Qs are -O-, and j R ９ｐ are each independently a linear C １０－４０ is an alkyl group, and R ８ｐ is a hydrogen atom.) is a group represented by

[0077] Another preferred embodiment of Z represented by formula (Pg-ii-1) is R ａ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ is an alkyl group; and R ｂ is represented by formula (Pg-iii) (wherein * represents a bonding position, j represents an integer of 0 to 3, j Qs are -O-, and j R ９ｐ are each independently a linear C １０－４０ is an alkyl group, and R ８ｐ is R ６ｐ and together with the ring A' and the ring C' represent a single bond or -O- to form a fused ring together with the ring A' and the ring C'.

[0078] Another preferred embodiment of Z represented by formula (Pg-ii-1) is R ａ and R ｂ together form an oxo group; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ is an alkyl group;

[0079] Another preferred embodiment of Z represented by formula (Pg-ii-1) is R ａ and R ｂ together form an oxo group; R ６ｐ is a hydrogen atom; k is an integer of 1 to 3; k Qs are —O—; k R ７ｐ each independently represents 1 to 3 straight-chain C １０－４０A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０ A group in which the alkyl group is a cyclohexylmethyl group bonded via —O—.

[0080] A preferred embodiment of Z represented by formula (Pg-ii-2) is R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ Alkyl group, 1 to 3 straight-chain C １０－４０ A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０ A group in which the alkyl group is a cyclohexylmethyl group bonded via —O—.

[0081] In formula (Pg-ii-3), R ６ｐ is preferably a hydrogen atom. ａ and R ｂ are preferably hydrogen atoms or together form an oxo group.

[0082] A preferred embodiment of Z represented by formula (Pg-ii-3) is R ａ and R ｂ is a hydrogen atom; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ is an alkyl group;

[0083] Another preferred embodiment of Z represented by formula (Pg-ii-3) is R ａ and R ｂ is a hydrogen atom; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ each independently represents 1 to 3 straight-chain C １０－４０ A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０A group in which the alkyl group is a cyclohexylmethyl group bonded via —O—.

[0084] Another preferred embodiment of Z represented by formula (Pg-ii-3) is R ａ is a hydrogen atom; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ is an alkyl group; and R ｂ is represented by formula (Pg-iii) (wherein * represents a bonding position, j represents an integer of 0 to 3, j Qs are -O-, and j R ９ｐ are each independently a linear C １０－４０ is an alkyl group, and R ８ｐ is a hydrogen atom.) is a group represented by

[0085] Another preferred embodiment of Z represented by formula (Pg-ii-3) is R ａ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０ is an alkyl group; and R ｂ is represented by formula (Pg-iii) (wherein * represents a bonding position, j represents an integer of 0 to 3, j Qs are -O-, and j R ９ｐ are each independently a linear C １０－４０ is an alkyl group, and R ８ｐ is R ６ｐ and together with the ring B' and the ring C' represent a single bond or -O- to form a fused ring together with the ring B' and the ring C'.

[0086] Another preferred embodiment of Z represented by formula (Pg-ii-3) is R ａ and R ｂ together form an oxo group; R ６ｐ is a hydrogen atom; k is an integer from 1 to 3; k Qs are —O—; and k R ７ｐ are each independently a linear C １０－４０is an alkyl group;

[0087] Another preferred embodiment of Z represented by formula (Pg-ii-3) is R ａ and R ｂ together form an oxo group; R ６ｐ is a hydrogen atom; k is an integer of 1 to 3; k Qs are —O—; k R ７ｐ each independently represents 1 to 3 straight-chain C １０－４０ A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０ A group in which the alkyl group is a cyclohexylmethyl group bonded via —O—.

[0088] A preferred embodiment of Z represented by formula (Pg-ii-4) is a ring D' in which ring D is a naphthalene ring; ａ and R ｂ is a hydrogen atom; k is an integer from 1 to 3; k Q' are -O-; and k R ７ｐ are each independently a linear C １０－４０ Alkyl group, 1 to 3 straight-chain C １０－４０ A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０ A group in which the alkyl group is a cyclohexylmethyl group bonded via —O—.

[0089] Another preferred embodiment of Z represented by formula (Pg-ii-4) is a group in which ring D' is an indole ring; ａ and R ｂ is a hydrogen atom; k is 1; Q' is a single bond; and R ７ｐ But linear C １０－４０ Alkyl group, 1 to 3 straight-chain C １０－４０ A benzyl group in which an alkyl group is bonded via —O—, or a linear C １０－４０ is a cyclohexylmethyl group to which the alkyl group is bonded via —O—; and R ７ｐ is a group attached to the nitrogen atom of the indole ring.

[0090] A preferred embodiment of Z represented by formula (Pg-ii-5) is where u is 1 or 2; and R １０ｐ But C ４－１０ It is a group which is a perfluoroalkyl group.

[0091] Z is preferably a group represented by formula (Pg-ii-1), formula (Pg-ii-2) or formula (Pg-ii-3).

[0092] The protecting group (Pg-5) is preferably a protecting group represented by formula (Pg-i-2) in which L is a succinyl group or a group represented by formula (Pg-i-2) in which R １ｐ and R ２ｐ are each independently, C １－１０ is an alkyl group, and L １ is a divalent phenylene group, and L ２is a single bond, and Y-Z is a 3,4,5-tris(octadecyloxy)benzyloxy group, a 3,5-bis(docosyloxy)benzyloxy group, a 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, a 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, a 3,4,5-tris(octadecyloxy)benzylamino group, a 2,4-bis(docosyloxy)benzylamino group, a 3,5-bis(docosyloxy)benzylamino group, a bis(4 -docosyloxyphenyl)methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzylamino group, 2,4-bis(dodecyloxy)benzylamino group, phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino group, 3,5-bis[3',4 ',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, decyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylamino group, bis(4-docosyloxycyclohexyl)methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 2,4-bis(dodecyloxy)cyclohexylmethylamino group, phenyl(2,3,4-tris(octadecyloxy)cyclohexyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)cyclohexyl]methylamino group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or L-Y is a single bond or a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group, a 3,5-bis(docosyloxy)benzoyl group, a 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group.

[0093] The protecting group (Pg-5) is more preferably selected from the group consisting of: L is a succinyl group; and Y-Z is selected from the group consisting of 3,4,5-tris(octadecyloxy)benzyloxy group, 3,5-bis(docosyloxy)benzyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy group, 3,4,5-tris(octadecyloxy)benzylamino group, 2,4-bis(docosyloxy)benzylamino group, 3,5-bis(docosyloxy)benzylamino group, bis(4 -docosyloxyphenyl)methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]benzylamino group, 2,4-bis(dodecyloxy)benzylamino group, phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)phenyl]methylamino group, 3,5-bis[3',4 ',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino group, 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, decyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylamino group, bis(4-docosyloxycyclohexyl)methylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 2,4-bis(dodecyloxy)cyclohexylmethylamino group, phenyl(2,3,4-tris(octadecyloxy)cyclohexyl)methylamino group, bis[4-(12-docosyloxydodecyloxy)cyclohexyl]methylamino group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or L-Y is a single bond or a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group, a 3,5-bis(docosyloxy)benzoyl group, a 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group, or a 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl group.

[0094] The protecting group (Pg-5) is more preferably a protecting group selected from the group consisting of: L is a succinyl group; and Y-Z is a protecting group selected from the group consisting of 3,4,5-tris(octadecyloxy)benzyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, 3,5-bis(docosyloxy)cyclohexylmethyloxy group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylamino group, 3,5-bis(docosyloxy)cyclohexylmethylamino group, 3,4,5-tris(octadecyloxy)benzyloxy group, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 2,4 ... and 3,4,5-tris(octadecyloxy)cyclohexylmethylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, 2,4-bis(dodecyloxy)cyclohexylmethylamino group, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethyloxy]cyclohexylmethylamino group. or LY is a single bond or a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group.

[0095] The protecting group (Pg-5) is particularly preferably a group in which L is a succinyl group, and Y-Z is a 3,4,5-tris(octadecyloxy)benzyloxy group, a 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, or a phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino group, or a group in which L-Y is a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group.

[0096] The protecting group (Pg-5) is most preferably a group in which L is a succinyl group, and Y-Z is a 3,4,5-tris(octadecyloxy)benzyloxy group or a 3,4,5-tris(octadecyloxy)cyclohexylmethyloxy group, or a group in which L-Y is a succinyl-1,4-piperazinediyl group, and Z is a 3,4,5-tris(octadecyloxy)benzoyl group.

[0097] The protecting group (Pg-5) and the compound used to form it are known methods (for example, the methods described in WO 2017 / 104836, WO 2019 / 131719, WO 2020 / 235658, WO 2021 / 039935, or WO 2021 / 198883) or methods similar thereto. It can be formed or produced.

[0098] Terminology Related to Oligonucleotide Synthesis In this specification, the term "nucleoside," which is a structural unit of an oligonucleotide, refers to a compound in which a nucleic acid base is bound to the 1-position of a sugar (e.g., 2-deoxyribose, ribose, 2-deoxyribose or ribose in which the 2nd and 4th carbon atoms are bound via a divalent organic group, 2-deoxyribose or ribose in which the 3rd and 5th carbon atoms are bound via a divalent organic group, 2-deoxyribose or ribose in which the 3rd and 4th carbon atoms are bound via a divalent organic group, etc.) by N-glycosidation.

[0099] As used herein, the term "nucleotide" refers to a compound in which a phosphate group is bound to a nucleoside.

[0100] As used herein, the terms "nucleoside" and "nucleotide" also include morpholino nucleosides and morpholino nucleotides, respectively, which have a morpholine residue in place of a sugar residue (e.g., a 2-deoxyribose residue, a ribose residue). As used herein, a "morpholino nucleoside" is a compound represented by the following formula (1) (in the formula, Base represents an optionally protected nucleic acid base):

[0101]

[0102] In this specification, the term "nucleobase" is not particularly limited as long as it is used in the synthesis of nucleic acid, and examples thereof include pyrimidine bases such as cytosyl group (cytosine base), uracil group (uracil base), and thyminyl group (thymine base), and purine bases such as adenyl group (adenine base) and guanyl group (guanine base).In addition, "nucleobase that may be protected" means, for example, that the amino group, carbonyl group, etc. in the nucleic acid base may be protected.The nucleic acid base that may be protected is preferably a nucleic acid base whose amino group may be protected with the above-mentioned amino group protecting group, and more preferably a nucleic acid base that does not have an amino group, or a nucleic acid base whose amino group is protected with the above-mentioned amino group protecting group.

[0103] The carbonyl group can be protected by reacting it with, for example, phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamic acid chloride, diethylcarbamic acid chloride, ethylphenylcarbamic acid chloride, 1-pyrrolidinecarboxylic acid chloride, 4-morpholinecarboxylic acid chloride, diphenylcarbamic acid chloride, or the like.

[0104] As used herein, the term "nucleobase" also encompasses modified nucleobases (e.g., 8-bromoadenyl group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, 5-methylcytosyl group, 8-oxoguanyl group, hypoxanthinyl group, etc.) in which the nucleobase is substituted with a substituent (e.g., a halogen atom, an alkyl group, an aralkyl group, an alkoxy group, an acyl group, an alkoxyalkyl group, a hydroxyl group, an amino group, a monoalkylamino, a dialkylamino, a carboxy, a cyano, a nitro, etc.).

[0105] The term "sugar" as used herein also includes amino sugars in which the hydroxyl group is replaced with an amino group, and ribose in which the hydroxyl group at the 2-position is replaced with a halogen atom.

[0106] Examples of amino sugars include 2-deoxyribose in which the hydroxyl group at the 3-position is replaced with an amino group, ribose in which the hydroxyl group at the 3-position is replaced with an amino group, and ribose in which the hydroxyl group at the 3-position is replaced with an amino group and the hydroxyl group at the 2-position is replaced with a halogen (in the following formula, X ｓ indicates a halogen atom).

[0107]

[0108] Examples of 2-deoxyribose or ribose in which the 2- and 4-carbon atoms are bonded via a divalent organic group, 2-deoxyribose or ribose in which the 3- and 5-carbon atoms are bonded via a divalent organic group, or 2-deoxyribose or ribose in which the 3- and 4-carbon atoms are bonded via a divalent organic group include the following compounds:

[0109]

[0110] [In the formula, R represents a hydrogen atom, an optionally substituted hydrocarbon group, an optionally substituted hydroxyl group, or an optionally substituted amino group, and R′ represents a hydrogen atom or a hydroxyl group.]

[0111] As used herein, the term "phosphate group" refers to -O-P(=O)(OH). ２as well as groups in which an oxygen atom is replaced by a sulfur atom or NH (for example, —O—P(═S)(OH) ２ , -NH-P(=O)(OH) ２ , -NH-P(=S)(OH) ２ ) is also included. ｐ (In the formula, R ｐ indicates an organic group (e.g., a protecting group for a phosphate group)) is also encompassed by the term "phosphate group."

[0112] In this specification, the term "phosphite ester bond" refers to a bond represented by the following formula (P1-1), or a bond represented by the following formula (P1-2) in which the oxygen atom in the above bond is replaced with NH (in the following formulas, * indicates the bond position):

[0113]

[0114] As used herein, the term "phosphate ester bond" refers to a bond represented by the following formula (P3-1), or a bond represented by the following formula (P3-2) in which the oxygen atom in the above bond is replaced with NH (in the following formulas, * indicates the bond position):

[0115]

[0116] As used herein, the term "thiophosphate bond" refers to a bond represented by the following formula (P4-1), or a bond represented by the following formula (P4-2) in which the oxygen atom in the above bond is replaced with NH (in the following formulas, * indicates the bond position):

[0117]

[0118] As used herein, the term "oligonucleotide" refers to a compound in which one or more nucleotides are linked to a nucleoside. The term "oligonucleotide" encompasses not only oligonucleotides having a phosphate ester bond represented by formula (P3-1) but also oligonucleotides having a phosphate ester bond represented by formula (P3-2), oligonucleosides having a thiophosphate ester bond represented by formula (P4-1), and oligonucleosides having a thiophosphate ester bond represented by formula (P4-2). The number of nucleosides in the oligonucleotide of the present invention is not particularly limited, but is preferably 2 to 50, more preferably 2 to 30.

[0119] As used herein, the term "phosphoramidite" refers to a monoamide of a phosphorous diester (P(OR) ２ (NR ２ ), four R's each independently represent an optionally substituted alkyl group, and the NR ２ means that two R may be bonded to each other to form a cyclic amino group.

[0120] As used herein, a "phosphoramidite-modified nucleoside" refers to a nucleoside that has -X ｎ -P(OR)(NR ２ ) (wherein X ｎ represents an oxygen atom or NH, three R's each independently represent an optionally substituted alkyl group, and the NR ２ means a compound obtained by introducing a group (two R may be bonded to each other to form a cyclic amino group).

[0121] As used herein, a "phosphoramidized nucleotide" refers to a nucleotide that has -X ｎ -P(OR)(NR ２ ) (wherein X ｎ represents an oxygen atom or NH, three R's each independently represent an optionally substituted alkyl group, and the NR ２ is obtained by introducing —X ｎ -P(OR)(NR２ ) and a phosphate group. The phosphate group may be substituted.

[0122] As used herein, a "phosphoramidized oligonucleotide" refers to an oligonucleotide having -X ｎ -P(OR)(NR ２ ) (wherein X ｎ represents an oxygen atom or NH, three R's each independently represent an optionally substituted alkyl group, and the NR ２ means a compound obtained by introducing a group (two R may be bonded to each other to form a cyclic amino group).

[0123] As used herein, the term "oligonucleotide precursor having a phosphite ester bond" refers to a precursor in which the phosphate ester bond in an oligonucleotide has been replaced with a phosphite ester bond.

[0124] The oligonucleotide precursor having a phosphite bond is preferably (1) an oligonucleotide precursor having a phosphite bond obtained by condensing a nucleoside, nucleotide, or oligonucleotide with a phosphoramidite-modified nucleoside, nucleotide, or oligonucleotide, or (2) an oligonucleotide precursor having a phosphite bond obtained by condensing an oligonucleotide with a phosphoramidite.

[0125] The oligonucleotide precursor having a phosphite bond is more preferably an oligonucleotide precursor having a phosphite bond obtained by condensing a nucleoside, nucleotide, or oligonucleotide with a phosphoramidite-modified nucleoside, nucleotide, or oligonucleotide.

[0126] The oligonucleotide precursor having a phosphite ester bond is more preferably an oligonucleotide precursor having a phosphite ester bond obtained by condensing a nucleoside, nucleotide or oligonucleotide (a) having a hydrophobic protecting group with a nucleoside, nucleotide or oligonucleotide (b) whose hydroxyl group is protected with a temporary protecting group removable under acidic conditions and which has been phosphoramidized.

[0127] The condensation may be either condensation in which an oligonucleotide chain is elongated in the direction from the 3' end to the 5' end (hereinafter referred to as "3'-5' condensation") or condensation in which an oligonucleotide chain is elongated in the direction from the 5' end to the 3' end (hereinafter referred to as "5'-3' condensation"). Hereinafter, the nucleoside, nucleotide, or oligonucleotide (a) and the phosphoramidite-modified nucleoside, nucleotide, or oligonucleotide (b) that are preferred for each of the 3'-5' condensation and 5'-3' condensation will be described in order.

[0128] In the 3'-5' condensation, the nucleoside, nucleotide or oligonucleotide (a) may be, for example, a nucleotide represented by formula (a-I):

[0129]

[0130] [Wherein, r represents an integer of 0 or more; r+1 Base １ each independently represent an optionally protected nucleobase; r+1 X ｎ１ each independently represents an oxygen atom or NH; r+1 X ｎ２ each independently represents a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4; １０ each independently represents an oxygen atom or a sulfur atom; r R ｐ１ each independently represents a protecting group for a phosphate group; Pg represents **LYZ (wherein ** represents X ｎ１and the definitions and explanations of L, Y and Z are the same as those of L, Y and Z in the protecting group (Pg-5) above.]

[0131] When r is 0, compound (a-I) is a nucleoside, and when r is 1 or more, compound (a-I) is an oligonucleotide. r is preferably 49 or less, more preferably 29 or less, even more preferably 19 or less, particularly preferably 4 or less, and most preferably 2 or less.

[0132] Base, a nucleic acid base １ The amino group is preferably protected with a protecting group. Examples of the protecting group for the amino group include the above-mentioned protecting groups for the amino group. １ The protecting group for the amino group in the formula (I) is preferably an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a benzoyl group, an isobutyryl group, a (2-hexyl)decanoyl group, a dimethylformamidinyl group, or a ═C(R １１ )-N(R １２ ) (R １３ ) (wherein R １１ represents a methyl group, and R １２ and R １３ are each independently C １－６ represents an alkyl group or R １１ and R １２ may be bonded together with the carbon atom and nitrogen atom to form a 5- or 6-membered nitrogen-containing heterocycle. When compound (a-I) has multiple amino groups, the number of types of protecting groups for the amino groups may be one or more.

[0133] X ｎ２ As the halogen atom, a fluorine atom or a chlorine atom is preferred, and a fluorine atom is more preferred.

[0134] X ｎ２Examples of the protecting group for the optionally protected hydroxyl group include the aforementioned protecting groups for the hydroxyl group, and preferred are a methyl group, a 2-methoxyethyl group, a triethylsilyl group, a triisopropylsilyl group, a tert-butyldimethylsilyl group, a (2-cyanoethoxy)methyl group, and a 1-(2-cyanoethoxy)ethyl group.

[0135] X ｎ２ The "divalent organic group bonded to the carbon atoms at positions 2 and 4" is not particularly limited as long as it is bonded to the carbon atoms at positions 2 and 4 of the nucleoside. Examples of this divalent organic group include optionally substituted C ２－７ Alkylene groups, as well as —O—, —NR ３３ - (R ３３ is a hydrogen atom or C １－６ alkyl group), -S-, -CO-, -COO-, -OCONR ３４ - (R ３４ is a hydrogen atom or C １－６ represents an alkyl group) and -CONR ３５ - (R ３５ is a hydrogen atom or C １－６ a divalent linker selected from the group consisting of an alkyl group and an optionally substituted C １－７ and a divalent organic group composed of an alkylene group. １－７ Alkylene group and C ２－７ Examples of the substituent that the alkylene group may have include a methylidene group (CH ２ =) are examples.

[0136] The "divalent organic group bonded to the carbon atom at the 2nd position and the carbon atom at the 4th position" includes an optionally substituted C ２－７ Alkylene group, —OR ｉ - (R ｉ C is bonded to the 4th carbon atom １－６ an alkylene group), —O—NR ３３ -R ｊ - (R ｊ C is bonded to the 4th carbon atom １－６ represents an alkylene group, and R ３３ has the same meaning as above), -O-R ｋ -O-R ｌ - (Rｋ is C １－６ represents an alkylene group, and R ｌ C is bonded to the 4-position carbon atom and bridged １－６ alkylene group), and -OR ｉ - (R ｉ has the same meaning as above), —O—NR ３３ -R ｊ - (R ｊ and R ３３ has the same meaning as above), -O-R ｋ -O-R ｌ - (R ｋ and R ｌ is more preferably the same as defined above. ｉ , R ｊ , R ｋ and R ｌ C shown by １－６ The alkylene groups are each independently preferably a methylene group or an ethylene group.

[0137] Examples of the "divalent organic group bonded to the carbon atom at the 2nd position and the carbon atom at the 4th position" include -O-CH ２ --, --O-CH ２ -CH ２ -, -O-NR ３３ -CH ２ - (R ３３ has the same meaning as above), —O—CH ２ -O-CH ２ - is more preferred, and -O-CH ２ --, --O-CH ２ -CH ２ -, -O-NH-CH ２ -, -O-N(CH ３ )-CH ２ --, --O-CH ２ -O-CH ２ - (the left side is bonded to the 2-position carbon atom, and the right side is bonded to the 4-position carbon atom) is more preferred.

[0138] r+1 Xs ｎ２ are each independently preferably a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group, more preferably a hydrogen atom, a fluorine atom, or an optionally protected hydroxyl group. ｐ１Examples of the protecting group include the aforementioned phosphate protecting group, and preferably the 2-cyanoethyl group.

[0139] In the 3'-5' condensation, the phosphoramidite-modified nucleoside, nucleotide or oligonucleotide (b) may be, for example, a nucleoside represented by the formula (b-I):

[0140]

[0141] [Wherein, s represents an integer of 0 or more; s+1 Base ２ each independently represent an optionally protected nucleobase; s+1 X ｎ３ each independently represents an oxygen atom or NH; s+1 X ｎ４ each independently represents a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4; Q" represents a temporary protecting group of a hydroxyl group that can be removed under acidic conditions; s R １４ each independently represents an oxygen atom or a sulfur atom; s+1 R ｐ２ each independently represents a protecting group for a phosphate group; and R １５ and R １６ each independently represents an alkyl group, or represents a 5- or 6-membered saturated cyclic amino group formed together with the adjacent nitrogen atom, and such saturated cyclic amino group may have one oxygen atom or one sulfur atom as a ring-constituting atom in addition to the nitrogen atom.]

[0142] When s is 0, compound (b-I) is a phosphoramidite-modified nucleoside, and when s is 1 or more, compound (b-I) is a phosphoramidite-modified oligonucleotide. In compound (b-I), s is preferably 49 or less, more preferably 29 or less, even more preferably 19 or less, particularly preferably 4 or less, and most preferably 2 or less.

[0143] Base, a nucleic acid base ２is preferably protected with a protecting group. In addition to the above-mentioned amino-protecting groups, the above-mentioned -LYZ group can be used as the protecting group. The protecting group is preferably the above-mentioned amino-protecting group, and more preferably an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a benzoyl group, an isobutyryl group, a (2-hexyl)decanoyl group, a dimethylformamidinyl group, or a ═C(R １１ )-N(R １２ ) (R １３ ) (wherein R １１ represents a methyl group, and R １２ and R １３ are each independently C １－６ represents an alkyl group or R １１ and R １２ may be bonded together with the carbon atom and nitrogen atom to form a 5- or 6-membered nitrogen-containing heterocycle. When compound (b-I) has multiple amino groups, the number of protecting groups for the amino groups may be one or more.

[0144] X in the formula (b-I) ｎ４ The explanation of X in the formula (a-I) is as follows: ｎ２ The explanation is the same as for s+1 X ｎ４ are each independently preferably a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group, more preferably a hydrogen atom or an optionally protected hydroxyl group.

[0145] R in the formula (b-I) ｐ２ The explanation of R in the formula (a-I) is as follows: ｐ１ The same as the explanation for s+1 R ｐ２ Examples of the protecting group include the aforementioned phosphate protecting group, and preferably the 2-cyanoethyl group.

[0146] R in the formula (b-I) １５ and R １６ are each independently preferably C １－１０ It is an alkyl group or a 5- or 6-membered saturated cyclic amino group formed together with the adjacent nitrogen atom, more preferably C １－１０alkyl group, more preferably C １－６ It is an alkyl group.

[0147] Compound (a-I) and compound (b-I) can be produced by a known method (for example, the method described in WO 2017 / 104836) or a method analogous thereto.

[0148] The oligonucleotide precursor having a phosphite ester bond obtained by condensation of compound (a-I) and compound (b-I) is a compound represented by the following formula (c-I) (the definitions and explanations of the symbols in the following formula (c-I) are as described above).

[0149]

[0150] In the 5'-3' condensation, the nucleoside, nucleotide or oligonucleotide (a) may be, for example, a nucleotide represented by the formula (a-I'):

[0151]

[0152] [Wherein, r represents an integer of 0 or more; r+1 Base １ each independently represent an optionally protected nucleobase; r X's ｎ１ each independently represents an oxygen atom or NH; r+1 X ｎ２ each independently represents a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the 2- and 4-position carbon atoms; X ｎ５ represents a hydroxyl group or an amino group; r R １０ each independently represents an oxygen atom or a sulfur atom; r R ｐ１ each independently represent a protecting group for a phosphate group; Pg represents **L-Y-Z (wherein ** represents the bonding position to the oxygen atom); and the definitions and explanations of L, Y, and Z are the same as those of L, Y, and Z in the protecting group (Pg-5) described above.]

[0153] X in formula (a-I') ｎ５ is preferably a hydroxyl group.ｎ５ The explanations of the symbols other than are the same as those of the symbols in formula (aI) above.

[0154] In the 5'-3' condensation, the phosphoramidite-modified nucleoside, nucleotide or oligonucleotide (b) may be, for example, a nucleoside represented by the formula (b-I'):

[0155]

[0156] [Wherein, s represents an integer of 0 or more; s+1 Base ２ each independently represent an optionally protected nucleobase; s X's ｎ３ each independently represents an oxygen atom or NH; s+1 X ｎ４ each independently represents a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the 2- and 4-position carbon atoms; X ｎ６ represents a hydroxyl group protected by a temporary protecting group removable under acidic conditions, or an amino group protected by a temporary protecting group removable under acidic conditions; １４ each independently represents an oxygen atom or a sulfur atom; s+1 R ｐ２ each independently represents a protecting group for a phosphate group; and R １５ and R １６ each independently represents an alkyl group, or represents a 5- or 6-membered saturated cyclic amino group formed together with the adjacent nitrogen atom, and such saturated cyclic amino group may have one oxygen atom or one sulfur atom as a ring-constituting atom in addition to the nitrogen atom.

[0157] X ｎ６ is preferably a hydroxyl group protected with a temporary protecting group that can be removed under acidic conditions. ｎ６ The explanations of the symbols other than are the same as those of the symbols in formula (bI) above.

[0158] Compound (a-I') and compound (b-I') can be produced by a known method (for example, the method described in WO 2017 / 104836) or a method similar thereto.

[0159] The oligonucleotide precursor having a phosphite bond obtained by condensation of compound (a-I') and compound (b-I') is represented by the following formula (c-I'):

[0160]

[0161] [Wherein, r represents an integer of 0 or more; r+1 Base １ each independently represent an optionally protected nucleobase; r+1 X ｎ１ each independently represents an oxygen atom or NH; r+1 X ｎ２ each independently represents a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4; １０ each independently represents an oxygen atom or a sulfur atom; r R ｐ１ each independently represents a protecting group for a phosphate group; s represents an integer of 0 or more; s+1 Base ２ each independently represent an optionally protected nucleobase; s X's ｎ３ each independently represents an oxygen atom or NH; s+1 X ｎ４ each independently represents a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the 2- and 4-position carbon atoms; X ｎ６ represents a hydroxyl group protected by a temporary protecting group removable under acidic conditions, or an amino group protected by a temporary protecting group removable under acidic conditions; １４ each independently represents an oxygen atom or a sulfur atom; s+1 R ｐ２ each independently represent a protecting group for a phosphate group; Pg represents **L-Y-Z (wherein ** represents the bonding position to the oxygen atom); and the definitions and explanations of L, Y, and Z are the same as those of L, Y, and Z in the protecting group (Pg-5) described above.] (The explanations of the symbols in the formula are as described above.)

[0162] In the formula (c-I′), r R １０ and s R１４ At least one of these is preferably a sulfur atom.

[0163] Next, an "oligonucleotide precursor having a phosphite bond obtained by condensing an oligonucleotide with a phosphoramidite" will be described. Examples of the precursor include those represented by formula (γ-I) or (γ-I'):

[0164]

[0165] [Wherein, t represents an integer of 1 or more, t+1 Base １ each independently represent an optionally protected nucleobase; t+1 X ｎ１ each independently represents an oxygen atom or NH; t+1 X ｎ２ each independently represents a hydrogen atom, a halogen atom, an optionally protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4; １０ each independently represents an oxygen atom or a sulfur atom; t R ｐ１ and R ｐ３ each independently represents a protecting group for a phosphate group; R ｐ４ represents an alkyl group having a protected amino group or a protecting group for a phosphate group; Pg represents **LYZ (wherein ** represents X ｎ１ and the definitions and explanations of L, Y and Z are the same as those of L, Y and Z in the protecting group (Pg-5) described above.

[0166] The symbols in the formula (γ-I) and formula (γ-I') are explained below. t is preferably 49 or less, more preferably 29 or less, even more preferably 19 or less, particularly preferably 4 or less, and most preferably 2 or less. R ｐ３ and R ｐ４ Examples of the "protecting group for a phosphate group" include the above-mentioned protecting groups for a phosphate group. ｐ３ is preferably a 2-cyanoethyl group. ｐ４Examples of the "protected amino group" in the "alkyl group having a protected amino group" include the amino group protected by the above-mentioned amino-protecting group. ｐ４ The "alkyl group having a protected amino group" is preferably a C １－６ R is an alkyl group, and more preferably a 6-(trifluoroacetylamino)hexyl group. ｐ４ is preferably a 2-cyanoethyl group or a 6-(trifluoroacetylamino)hexyl group. The explanations for the other symbols are as described above.

[0167] In this specification, the term "one-pot synthesis" refers to a synthesis consisting of multiple steps including a step of synthesizing an intermediate and a step of synthesizing a target final product, in which the intermediate is not isolated. The "step of synthesizing an intermediate" may be one or more.

[0168] The present invention provides a method for precipitating a target oligonucleotide (i.e., an oligonucleotide having a hydrophobic protecting group) from a target solution (i.e., a solution containing a target oligonucleotide and a non-polar solvent), the method comprising: mixing the target solution with a first polar solvent; and distilling off the solvent from the target mixture (i.e., a mixture of the target solution and the first polar solvent) while maintaining the volume of the target mixture (i.e., a mixture of the target solution and the first polar solvent) at least 1.00 times the volume of the target solution (hereinafter sometimes abbreviated as the "precipitation method of the present invention"). The first polar solvent may be used alone or in combination of two or more types.

[0169] As described above, the precipitation method of the present invention can precipitate target oligonucleotides with high purity by distilling off the solvent without concentrating the target mixture more than the target solution, while reducing the amount of polar solvent used for precipitation.As shown in the following examples and comparative examples, the precipitation method of the present invention can obtain target oligonucleotides with higher purity than the method of precipitation by concentration and adding polar solvent.Surprisingly, as shown in the following examples and comparative examples, the precipitation method of the present invention can obtain target oligonucleotides with higher purity than the method of precipitation by only adding polar solvent.

[0170] From the viewpoint of the purity of the target oligonucleotide, the volume of the target mixture upon distillation of the solvent is 1.00 times or more, preferably 1.05 times or more, more preferably 1.10 times or more, and preferably 3.00 times or less, more preferably 2.70 times or less, the volume of the target solution.

[0171] From the viewpoint of the purity of the target oligonucleotide, the temperature during the solvent removal is preferably 5°C or higher, more preferably 7°C or higher, even more preferably 10°C or higher, and preferably 35°C or lower, more preferably 30°C or lower, even more preferably 25°C or lower.

[0172] The pressure during distillation of the solvent is not particularly limited as long as the solvent can be distilled off, but is preferably 15 mmHg or more, more preferably 20 mmHg or more, even more preferably 25 mmHg or more, and is preferably 500 mmHg or less, more preferably 450 mmHg or less, even more preferably 400 mmHg or less.

[0173] The target solution contains a nonpolar solvent. Examples of nonpolar solvents include halogen-based solvents such as chloroform, dichloromethane, and 1,2-dichloroethane; aromatic solvents such as benzene, toluene, xylene, and mesitylene; ester-based solvents such as ethyl acetate and isopropyl acetate; aliphatic solvents such as hexane, pentane, heptane, octane, nonane, and cyclohexane; and nonpolar ether-based solvents such as diethyl ether, cyclopentyl methyl ether, and tert-butyl methyl ether. Only one type of nonpolar solvent may be used, or two or more types may be used in combination. In this specification, dichloromethane is classified as a nonpolar solvent.

[0174] In one embodiment of the present invention, the non-polar solvent contained in the target solution preferably includes a halogenated solvent. The halogenated solvent may be used alone or in combination of two or more. The non-polar solvent is more preferably a halogenated solvent, even more preferably at least one selected from the group consisting of dichloromethane and chloroform, and particularly preferably dichloromethane.

[0175] In another embodiment of the present invention, the nonpolar solvent contained in the target solution preferably includes at least one selected from the group consisting of halogenated solvents and toluene. Only one halogenated solvent may be used, or two or more halogenated solvents may be used in combination. The nonpolar solvent is more preferably at least one selected from the group consisting of halogenated solvents and toluene, even more preferably at least one selected from the group consisting of dichloromethane, chloroform, and toluene, and particularly preferably at least one selected from the group consisting of dichloromethane and toluene.

[0176] From the viewpoint of precipitation of the target oligonucleotide, the first polar solvent preferably includes at least one selected from the group consisting of nitrile solvents and alcohol solvents. The nitrile solvents and alcohol solvents may be used singly or in combination of two or more. Examples of nitrile solvents include acetonitrile and propionitrile. Examples of alcohol solvents include methanol, ethanol, and isopropanol (2-propanol). In one aspect of the present invention, the first polar solvent is more preferably a nitrile solvent, and even more preferably acetonitrile. In another aspect of the present invention, the first polar solvent is more preferably a mixed solvent of a nitrile solvent and an alcohol solvent, and even more preferably a mixed solvent of acetonitrile and isopropanol.

[0177] From the viewpoint of reducing the amount of polar solvent used and precipitating the target oligonucleotide, the total volume of the first polar solvent mixed with the target solution is preferably 1.0 times or more, more preferably 1.5 times or more, even more preferably 1.7 times or more, and particularly preferably 2 times or more, and is preferably 3.5 times or less, more preferably 3.2 times or less, and even more preferably 3.0 times or less, relative to the volume of the nonpolar solvent contained in the target solution.

[0178] The target solution may contain a second polar solvent. The second polar solvent may be used alone or in combination of two or more. In the present invention, the first polar solvent is the polar solvent used for precipitation, and the second polar solvent is the polar solvent contained in the target solution before mixing with the first polar solvent.

[0179] The second polar solvent preferably includes at least one selected from the group consisting of nitrile solvents and alcohol solvents. The nitrile solvents and alcohol solvents may each be used alone or in combination of two or more. Examples of nitrile solvents include acetonitrile and propionitrile. Examples of alcohol solvents include methanol, ethanol, 2,2,2-trifluoroethanol, and isopropanol. The second polar solvent is more preferably at least one selected from the group consisting of nitrile solvents and alcohol solvents, even more preferably at least one selected from the group consisting of acetonitrile, methanol, and 2,2,2-trifluoroethanol, and particularly preferably a mixed solvent of acetonitrile, methanol, and 2,2,2-trifluoroethanol.

[0180] From the viewpoint of dissolution of the target oligonucleotide in the target solution and precipitation of the target oligonucleotide from the target solution when a polar solvent is added, the volume of the second polar solvent is preferably 0.15 times or more, more preferably 0.17 times or more, even more preferably 0.20 times or more, and preferably 0.35 times or less, more preferably 0.32 times or less, even more preferably 0.30 times or less, relative to the volume of the nonpolar solvent contained in the target solution.

[0181] One-Pot Synthesis The present invention provides a method for producing an oligonucleotide by one-pot synthesis, the method comprising the steps of: (1) condensing, in a solution containing a non-polar solvent, a nucleoside, nucleotide, or oligonucleotide (a) having a hydrophobic protecting group with a nucleoside, nucleotide, or oligonucleotide (b) whose hydroxyl group is protected by a temporary protecting group removable under acidic conditions and which has been phosphoramidized, to form an oligonucleotide precursor (c) having a phosphite bond and a hydrophobic protecting group, and whose hydroxyl group is protected by a temporary protecting group removable under acidic conditions; (2) mixing the solution obtained after the step (1) with a quenching agent (i) for the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b), to quench the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b); Step (3) of mixing the solution after step (2) with an oxidizing agent or a sulfurizing agent to oxidize or sulfurize the oligonucleotide precursor (c) to form an oligonucleotide (d) having a phosphate bond or a thiophosphate bond and a hydrophobic protecting group, and whose hydroxyl group is protected by a temporary protecting group removable under acidic conditions; Step (4) of mixing the solution after step (3) with an oxidizing agent (ii) to quench the oxidizing agent, if necessary, when an oxidizing agent is used in step (3); Step (5) of mixing the solution after step (3) or, if step (4) is performed, the solution after step (4) with an acid to remove the temporary protecting group removable under acidic conditions from the oligonucleotide (d) to form an oligonucleotide (e) having an unprotected hydroxyl group and a hydrophobic protecting group; Step (6) of mixing the solution after step (5) with a base, if necessary; and Step (7) of precipitating the oligonucleotide (e) from a solution containing the oligonucleotide (e) and a nonpolar solvent by the precipitation method of the present invention. The present invention also provides a method for producing a hydroxybenzoate comprising the steps of:

[0182] (Step (1) (Condensation)) In step (1), a nucleoside, nucleotide, or oligonucleotide (a) having a hydrophobic protecting group is condensed with a nucleoside, nucleotide, or oligonucleotide (b) whose hydroxyl group is protected with a temporary protecting group removable under acidic conditions and which has been phosphoramidized, in a solution containing a non-polar solvent to form an oligonucleotide precursor (c) having a phosphite bond and a hydrophobic protecting group and whose hydroxyl group is protected with a temporary protecting group removable under acidic conditions.

[0183] The hydrophobic protecting group possessed by the nucleoside, nucleotide or oligonucleotide (a) is preferably the protecting group (Pg-5). The protecting group (Pg-5) is as described above.

[0184] The temporary protecting group for the hydroxyl group in the phosphoramidite-modified nucleoside, nucleotide or oligonucleotide (b) is preferably a 4,4'-dimethoxytrityl group or a 4-monomethoxytrityl group, more preferably a 4,4'-dimethoxytrityl group, from the viewpoint of ease of deprotection and the like.

[0185] The combination of the nucleoside, nucleotide or oligonucleotide (a) and the phosphoramidite-modified nucleoside, nucleotide or oligonucleotide (b) used in step (1) is preferably a combination of compound (a-I) and compound (b-I), or a combination of compound (a-I') and compound (b-I'), and more preferably a combination of compound (a-I) in which Pg is **L-Y-Z and compound (b-I), or a combination of compound (a-I') in which Pg is **L-Y-Z and compound (b-I').

[0186] Step (1) is carried out in a solution containing a nonpolar solvent. Examples of nonpolar solvents include halogenated solvents such as chloroform, dichloromethane, and 1,2-dichloroethane; aromatic solvents such as benzene, toluene, xylene, and mesitylene; ester solvents such as ethyl acetate and isopropyl acetate; aliphatic solvents such as hexane, pentane, heptane, octane, nonane, and cyclohexane; and nonpolar ether solvents such as diethyl ether, cyclopentyl methyl ether, and tert-butyl methyl ether. The nonpolar solvents may be used alone or in combination of two or more.

[0187] In one embodiment of the present invention, the nonpolar solvent is preferably at least one selected from the group consisting of halogenated solvents, aromatic solvents, ester solvents, and aliphatic solvents, more preferably at least one selected from the group consisting of halogenated solvents and toluene, even more preferably at least one selected from the group consisting of chloroform, dichloromethane, and toluene, and particularly preferably dichloromethane and / or toluene. In another embodiment of the present invention, the nonpolar solvent is preferably a halogenated solvent, more preferably at least one selected from the group consisting of dichloromethane and chloroform, and even more preferably dichloromethane. Steps after step (1) are also similarly carried out in a solution containing a nonpolar solvent.

[0188] The solution containing a nonpolar solvent may further contain an aprotic polar solvent. Examples of the aprotic polar solvent include nitrile-based solvents such as acetonitrile and propionitrile; and polar ether-based solvents such as tetrahydrofuran. Among these, nitrile-based solvents are preferred, and acetonitrile is more preferred. The amount of the aprotic polar solvent is preferably 10 to 100 mL, more preferably 10 to 50 mL, per 100 mL of the nonpolar solvent.

[0189] The ratio of the phosphoramidite-modified nucleoside, nucleotide, or oligonucleotide (b) to 1 mol of the nucleoside, nucleotide, or oligonucleotide (a) is, for example, 1 to 10 mol, preferably 1 to 5 mol. The concentration of the nucleoside, nucleotide, or oligonucleotide (a) in the solution is preferably 1 to 30% by weight.

[0190] The reaction temperature in step (1) is not particularly limited as long as the condensation proceeds, but is, for example, 0 to 100° C., preferably 20 to 50° C. The reaction time varies depending on the types of raw materials used, the reaction temperature, etc., but is, for example, 5 minutes to 24 hours.

[0191] A known activator may be used to promote condensation. Examples of the activator include pyridine trifluoroacetate, tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole, and 4,5-dicyanoimidazole. One type of activator may be used alone, or two or more types may be used in combination. When an activator is used, the amount thereof is preferably 0.5 to 10 mol, more preferably 1 to 5 mol, per 1 mol of the phosphoramidite-modified nucleoside, nucleotide, or oligonucleotide (b).

[0192] (Step (2) (Quenching of phosphoramidite-conjugated nucleoside, nucleotide, or oligonucleotide (b))) In step (2), the solution obtained after step (1) is mixed with a quenching agent (i) for the phosphoramidite-conjugated nucleoside, nucleotide, or oligonucleotide (b) to quench the phosphoramidite-conjugated nucleoside, nucleotide, or oligonucleotide (b).

[0193] The quenching agent (i) may be used alone or in combination of two or more thereof. Examples of the quenching agent (i) include water, alcohols, phenols, and amines.

[0194] Examples of alcohols that can be used as the quenching agent (i) include optionally halogenated monohydric alcohols such as methanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol, tetrahydrofurfuryl alcohol, furfuryl alcohol, 2,3-O-isopropylidene-D-ribofuranose, and 3'-O-triisopropylsilyl-thymidine, and optionally halogenated polyhydric alcohols such as ethylene glycol and diethylene glycol.

[0195] Examples of phenols that can be used as the quenching agent (i) include 4-nitrophenol and pentafluorophenol, and examples of amines that can be used as the quenching agent (i) include morpholine.

[0196] The amount of the quenching agent (i) is preferably 1 to 20 mol, more preferably 1 to 10 mol, and even more preferably 1 to 5 mol, relative to 1 mol of the phosphoramidite-modified nucleoside, nucleotide, or oligonucleotide (b) used in step (1).

[0197] The temperature of the solution after mixing with the quenching agent (i) is not particularly limited as long as it can quench the phosphoramidite-modified nucleoside, nucleotide, or oligonucleotide (b), but is preferably 5 to 40° C., more preferably 15 to 30° C. The stirring time of the solution after mixing with the quenching agent (i) varies depending on the type of quenching agent (i) used, the temperature, etc., but is, for example, 10 minutes to 3 hours.

[0198] (Step (3) (Oxidation or Sulfurization)) In step (3), the solution obtained after step (2) is mixed with an oxidizing agent or a sulfurizing agent to oxidize or sulfurize the oligonucleotide precursor (c) to form an oligonucleotide (d) having a phosphate ester bond or a thiophosphate ester bond and a hydrophobic protecting group, and whose hydroxyl group is protected with a temporary protecting group that can be removed under acidic conditions. Either one of the oxidizing agent or the sulfurizing agent may be used alone, or two or more of them may be used in combination.

[0199] The oxidizing agent is not particularly limited, and known oxidizing agents can be used. Examples of the oxidizing agent include iodine, (1S)-(+)-(10-camphanylsulfonyl)oxaziridine, tert-butyl hydroperoxide (TBHP), 2-butanone peroxide, 1,1-dihydroperoxycyclododecane, bis(trimethylsilyl)peroxide, and m-chloroperbenzoic acid. In addition, the oxidizing agents described in WO 2022 / 172994 can be used.

[0200] The sulfurizing agent is not particularly limited, and known sulfurizing agents can be used. Examples of the sulfurizing agent include 5-phenyl-3H-1,2,4-dithiazol-3-one (POS), 5-[(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazol-3-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide (PADS), tetraethylthiuram disulfide (TETD), and 3-amino-1,2,4-dithiazole-5-thione (ADTT).

[0201] The amount of the oxidizing agent or sulfurizing agent is, for example, 1 to 50 moles, preferably 1 to 5 moles, per mole of the oligonucleotide precursor (c).

[0202] The reaction temperature is not particularly limited as long as the reaction proceeds, but is preferably 0° C. to 100° C., more preferably 20° C. to 50° C. The reaction time varies depending on the type of the oligonucleotide precursor (c), the type of oxidizing agent or sulfurizing agent used, the reaction temperature, etc., but is, for example, 1 minute to 3 hours.

[0203] (Use of aromatic amine) It is preferable to mix an aromatic amine with the solution before, during, or after oxidation or sulfurization. That is, it is preferable to carry out oxidation or sulfurization in the presence of an aromatic amine and / or mix the solution after oxidation or sulfurization with a cation scavenger. Only one type of aromatic amine may be used, or two or more types may be used in combination. The use of an aromatic amine can suppress the generation of by-products.

[0204] Examples of aromatic amines include aniline, 2-chloroaniline, 3-chloroaniline, 2,4-dichloroaniline, 2-fluoroaniline, 4-methoxyaniline, 4-nitroaniline, 2,6-dichloroaniline, and 2,6-dimethylaniline (also known as 2,6-xylidine).

[0205] From the viewpoint of suppressing by-products, the ratio of the aromatic amine to 1 mol of the oxidizing agent or sulfurizing agent used in step (3) is preferably 1 to 20 mol, more preferably 1 to 10 mol, and even more preferably 1 to 5 mol.

[0206] (Step (4) (Quenching of Oxidizing Agent)) When an oxidizing agent is used in step (3), step (4) is carried out as necessary. In step (4), the solution obtained after step (3) is mixed with a quenching agent (ii) for the oxidizing agent to quench the oxidizing agent. The quenching agent (ii) may be used alone or in combination of two or more.

[0207] The quenching agent (ii) is preferably an organic phosphorus compound. The organic phosphorus compounds may be used alone or in combination of two or more.

[0208] The organic phosphorus compound is preferably at least one selected from the group consisting of phosphines, phosphite triesters, phosphinite esters, phosphonite diesters, and phosphinate esters, and more preferably phosphines. The phosphines and the like may be used alone or in combination of two or more.

[0209] The phosphines have the formula: P(R) ３ (wherein the three R's each independently represent a hydrogen atom, an alkyl group, or an aryl group). Examples of phosphines include triphenylphosphine and methyldiphenylphosphine.

[0210] The phosphite triester has the formula: P(OR') ３(wherein the three R's each independently represent an alkyl group or an aryl group) Examples of the phosphite triester include triethyl phosphite.

[0211] The phosphinite ester has the formula: P(OR')(R) ２ (wherein two R's each independently represent a hydrogen atom, an alkyl group, or an aryl group, and R' represents an alkyl group or an aryl group). Examples of phosphinous acid esters include ethoxydiphenylphosphine.

[0212] The phosphonite diester has the formula: P(R)(OR') ２、 (wherein R represents a hydrogen atom, an alkyl group, or an aryl group, and two R' each independently represents an alkyl group or an aryl group). Examples of phosphonous acid diesters include diethoxyphenylphosphine.

[0213] The phosphinate ester has the formula: P(=O)(R) ２ (OR') (wherein two R's each independently represent a hydrogen atom, an alkyl group, or an aryl group, R' represents an alkyl group or an aryl group, and one of R and R' may form a heterocycle together with the phosphorus atom and oxygen atom to which they are bonded). Examples of phosphinic acid esters include 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide represented by the following formula:

[0214]

[0215] The amount of the quenching agent (ii) should be such that it can quench the excess of the oxidizing agent used in step (3), and is preferably 1 to 10 mol, more preferably 1 to 5 mol, per 1 mol of the excess of the oxidizing agent (i.e., "the amount of the oxidizing agent used in step (3)" - "the amount of the phosphoramidite-modified nucleoside, nucleotide or oligonucleotide (b) used in step (1)").

[0216] The temperature of the solution after mixing with the quenching agent (ii) is not particularly limited as long as it can quench the oxidizing agent, but is preferably 0° C. to 50° C., more preferably 10° C. to 40° C. The stirring time of the solution after mixing with the quenching agent (ii) varies depending on the type of quenching agent (ii) used, the temperature, etc., but is preferably 5 minutes to 5 hours, more preferably 5 minutes to 2 hours.

[0217] (Step (5) (Removal of Temporary Protecting Groups)) In step (5), the solution obtained after step (3), or the solution obtained after step (4) if step (4) is performed, is mixed with an acid to remove the temporary protecting groups of the oligonucleotide (d) that are removable under acidic conditions, thereby forming an oligonucleotide (e) having unprotected hydroxyl groups and hydrophobic protecting groups. Only one type of acid may be used, or two or more types may be used in combination.

[0218] The acid is not particularly limited as long as it can satisfactorily remove the temporary protecting group, and examples thereof include trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid, methanesulfonic acid, hydrochloric acid, acetic acid, p-toluenesulfonic acid, etc. From the viewpoint of satisfactorily removing the temporary protecting group, trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, and trichloroacetic acid are more preferred, trifluoroacetic acid, dichloroacetic acid, and trifluoromethanesulfonic acid are even more preferred, and trifluoroacetic acid is particularly preferred.

[0219] The amount of the acid is, for example, 1 to 100 mol, preferably 1 to 40 mol, per 1 mol of the oligonucleotide (d).

[0220] The reaction temperature in step (5) is not particularly limited as long as the reaction proceeds, but is, for example, −10° C. to 50° C., more preferably 0° C. to 40° C. The reaction time varies depending on the oligonucleotide (d) used, the type of acid and the type of nonpolar solvent, the reaction temperature, etc., but is, for example, 5 minutes to 5 hours.

[0221] (Use of cation scavenger) It is preferable to mix a solution with a cation scavenger before, during, or after the removal of the temporary protecting group of the oligonucleotide (d). That is, it is preferable to perform the removal of the temporary protecting group in the presence of a cation scavenger, or to mix the solution after the removal of the temporary protecting group with a cation scavenger. Only one type of cation scavenger may be used, or two or more types may be used in combination.

[0222] The cation scavenger is not particularly limited as long as it does not cause a side reaction of the removed temporary protecting group with the reprotected or deprotected functional group. In one embodiment of the present invention, the cation scavenger is at least one selected from the group consisting of pyrrole, pyrrole derivatives, indole, indole derivatives, furan derivatives, and mercapto group-containing compounds. Examples of pyrrole derivatives include 2-methylpyrrole, 3-methylpyrrole, 2,3-dimethylpyrrole, and 2,4-dimethylpyrrole. Examples of indole derivatives include 3-methylindole, 4-methylindole, 5-methylindole, 6-methylindole, 7-methylindole, 5,6-dimethylindole, 6,7-dimethylindole, and 5-methoxyindole. Examples of furan derivatives include 2-methylfuran, 2,3-dimethylfuran, 2-methyl-3-(methylthio)furan, and menthofuran. Examples of the mercapto group-containing compound include thiomalic acid, 3-mercaptopropionic acid, cysteine, cysteinylglutamic acid, 1-octanethiol, 1-dodecanethiol, cyclohexanethiol, 2,2'-(ethylenedioxy)diethanethiol, etc. The amount of the cation scavenger is preferably 1 to 50 mol, more preferably 5 to 20 mol, relative to 1 mol of the oligonucleotide (d).

[0223] (Step (6) (Neutralization)) Step (6) is performed as necessary to neutralize the acid used in step (5). In step (6), the solution after step (5) is mixed with a base. Note that, since the acid used in step (5) can be removed from oligonucleotide (e) by performing solid-liquid separation and washing, step (6) (neutralization) is not essential.

[0224] The base may be used alone or in combination of two or more. The base is preferably an organic base. Examples of the organic base include pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-triazole, N-phenylimidazole, 2-amino-4,6-dimethylpyrimidine, 1,10-phenanthroline, imidazole, N-methylimidazole, 2-chlorobenzimidazole, 2-bromobenzimidazole, 2-methylimidazole, 2-phenylbenzimidazole, N-phenylbenzimidazole, and 5-nitrobenzimidazole. Among these, pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-triazole, N-phenylimidazole, N-methylimidazole, 2-amino-4,6-dimethylpyrimidine, and 1,10-phenanthroline are preferred, pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-triazole, and N-phenylimidazole are more preferred, pyridine, 2,4,6-trimethylpyridine, benzimidazole, and 1,2,4-triazole are still more preferred, and pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, and benzimidazole are particularly preferred.

[0225] The ratio of the base in step (6) is preferably 1 to 10 mol, more preferably 1 to 3 mol, per 1 mol of the acid used in step (5).

[0226] (Step (7) (Precipitation)) In step (7), the oligonucleotide (e) is precipitated from a solution containing the oligonucleotide (e) and a non-polar solvent (i.e., the solution after step (5), or the solution after step (6) as needed) by the precipitation method of the present invention. The precipitation method of the present invention has been described above.

[0227] After the precipitation method of the present invention, protecting groups (e.g., phosphate protecting groups, hydrophobic protecting groups) of the obtained target oligonucleotide may be removed by known methods. For example, the 2-cyanoethyl group, which is a phosphate protecting group, and the aforementioned protecting group (Pg-5), etc., can be removed by treatment with ammonia water, an ammonia water / ethanol solution, or a mixture of ammonia water and an aqueous methylamine solution.

[0228] The present invention will be described in more detail below with reference to examples, etc. However, the present invention is not limited to the following examples, etc., and can be practiced with appropriate modifications within the scope that can comply with the above and below aims, and all such modifications are included in the technical scope of the present invention.

[0229] "Room temperature" in the following examples means "20°C to 30°C." Abbreviations used in the following examples have the following meanings. Monomer (1m): Thymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate 5'-DMT-dG(iBu)-3'-phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-deoxyguanosine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite 5'-DMT-dA(dma)-3'-phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N6-[1-(dimethylamino)ethylidene]-deoxyadenosine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite 5'-DMT-dmeC(Bz)-3'-phosphoramidite: 5'-O-(4,4'-dimethoxytrityl)-N4-benzoyl-2'-deoxy-5-methylcytidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite DMTr: 4,4'-dimethoxytrityl POS: 5-phenyl-3H-1,2,4-dithiazol-3-one

[0230] Example 1: Synthesis of dimer and solid-liquid separation

[0231]

[0232] Under an argon atmosphere, monomer (1m) (2.5 g, 2.0 mmol) and triphenylphosphine (0.26 g, 1.0 mmol) were dissolved in a mixed solvent of acetonitrile (30.3 mL) and chloroform (100.1 mL). Molecular sieves 3A (6.1 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dG(iBu)-3'-phosphoramidite (3.4 g, 4.0 mmol) was then added, and the mixture was stirred at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.53 g, 4.0 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. To the reaction mixture, 2,2,2-trifluoroethanol (1.5 mL, 20.2 mmol) was added and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.9 mL, 15.2 mmol) and POS (0.99 g, 5.1 mmol) were added and stirred at room temperature for 19 hours. 2,3-Dimethylfuran (2.1 mL, 20.2 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (5.2 mL, 67.5 mmol) and 2,6-dimethylaniline (0.083 mL, 0.67 mmol) dissolved in dehydrated chloroform (10.4 mL). The mixture was stirred at room temperature for 1.5 hours. 2,6-Dimethylpyridine (19.7 mL, 171.7 mmol) and methanol (0.66 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was filtered, and the funnel and other components used for the filtration were washed with chloroform (10 mL). This gave a target solution, which was a mixture of the chloroform used for washing and the filtrate (volume of target solution: 190 mL, volume of nonpolar solvent (chloroform): 120.5 mL, volume of second polar solvent (acetonitrile, methanol, and 2,2,2-trifluoroethanol): 32.46 mL, volume of second polar solvent / volume of nonpolar solvent: 0.27).

[0233] Acetonitrile, the first polar solvent, was added dropwise to the resulting target solution, and the volume of the mixture of the target solution and the first polar solvent was adjusted to 240-500 mL (volume of the mixture of the target solution and the first polar solvent / volume of the target solution: 1.26-2.63). The solvent was then distilled off at a temperature of 14.8-23.4 °C and a pressure of 28-254 mmHg (total volume of the added first polar solvent: 285 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.37). The precipitated solid was collected by filtration and dried to obtain the dimer (2m) as a white solid (3.2 g, yield: 92.6%). m / z (TOF-MS): Calcd. 1705.08, Found 1706.0773 [M+H] +

[0234] Example 2: Trimer synthesis and solid-liquid separation

[0235]

[0236] Under an argon atmosphere, the dimer (2m) (3.0 g, 1.8 mmol) obtained in Example 1 and triphenylphosphine (0.23 g, 0.90 mmol) were dissolved in a mixed solvent of acetonitrile (26.6 mL) and dichloromethane (88.5 mL). Molecular sieves 3A (5.3 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dA(dma)-3'-phosphoramidite (2.9 g, 3.6 mmol) was then added, followed by stirring at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.46 g, 3.6 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. To the reaction mixture, 2,2,2-trifluoroethanol (1.3 mL, 1.8 mmol) was added and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.6 mL, 13.3 mmol) and POS (0.86 g, 4.4 mmol) were added and stirred at room temperature for 19 hours. 2,3-Dimethylfuran (1.9 mL, 17.7 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (4.5 mL, 59.1 mmol) and 2,6-dimethylaniline (0.073 mL, 0.59 mmol) dissolved in anhydrous dichloromethane (9.0 mL), and the mixture was stirred at room temperature for 2 hours. 2,6-Dimethylpyridine (17.2 mL, 147.8 mmol) and methanol (0.58 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was filtered, and the funnel and other parts used for the filtration were washed with dichloromethane (12 mL). A target solution was obtained, which was a mixture of the dichloromethane used for washing and the filtrate (volume of target solution: 167.3 mL, volume of nonpolar solvent (dichloromethane): 109.5 mL, volume of second polar solvent (acetonitrile, methanol, and 2,2,2-trifluoroethanol): 28.48 mL, volume of second polar solvent / volume of nonpolar solvent: 0.26).

[0237] Acetonitrile, the first polar solvent, was added dropwise to the resulting target solution, and the volume of the mixture of the target solution and the first polar solvent was adjusted to 210-300 mL (volume of the mixture of the target solution and the first polar solvent / volume of the target solution: 1.26-1.79). The solvent was distilled off at a temperature of 17.3-24.0 ° C. and a pressure of 74-360 mmHg (total volume of the added first polar solvent: 251 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.29). The precipitated solid was collected by filtration and dried to obtain the trimer (3m) as a white solid (3.7 g, yield: 97.8%). m / z (TOF-MS): Calcd. 2156.20, Found 2157.20 [M+H] +

[0238] Example 3: Synthesis of tetramer and solid-liquid separation

[0239]

[0240] Under an argon atmosphere, the trimer (3m) (3.4 g, 1.6 mmol) obtained in Example 2 and triphenylphosphine (0.21 g, 0.81 mmol) were dissolved in a mixed solvent of acetonitrile (24.1 mL) and dichloromethane (80.4 mL). Molecular sieves 3A (4.8 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dA(dma)-3'-phosphoramidite (2.6 g, 3.2 mmol) was then added, followed by stirring at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.42 g, 3.2 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. To the reaction mixture, 2,2,2-trifluoroethanol (1.2 mL, 1.6 mmol) was added and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.5 mL, 12.2 mmol) and POS (0.78 g, 4.0 mmol) were added and stirred at room temperature for 19 hours. After adding 2,3-dimethylfuran (1.7 mL, 16.1 mmol), a mixed solution of trifluoroacetic acid (4.1 mL, 53.9 mmol) and 2,6-dimethylaniline (0.067 mL, 0.54 mmol) dissolved in anhydrous dichloromethane (8.3 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. To the reaction mixture, 2,6-dimethylpyridine (15.7 mL, 134.6 mmol) and methanol (0.52 mL) were added and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was filtered, and the funnel and other parts used for the filtration were washed with dichloromethane (10 mL). A target solution was obtained, which was a mixture of the dichloromethane used for washing and the filtrate (volume of target solution: 153 mL, volume of nonpolar solvent (dichloromethane): 98.7 mL, volume of second polar solvent (acetonitrile, methanol, and 2,2,2-trifluoroethanol): 25.82 mL, volume of second polar solvent / volume of nonpolar solvent: 0.26).

[0241] Acetonitrile, the first polar solvent, was added dropwise to the resulting target solution, and the volume of the mixture of the target solution and the first polar solvent was adjusted to 190-260 mL (volume of the mixture of the target solution and the first polar solvent / volume of the target solution: 1.24-1.69). The solvent was then distilled off at a temperature of 13.7-21.2 °C and a pressure of 36-400 mmHg (total volume of the added first polar solvent: 230 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.33). The precipitated solid was collected by filtration and dried to obtain the tetramer (4m) as a white solid (4.1 g, yield: 97.9%). m / z (TOF-MS): Calcd. 2608.32, Found 2608.32 [M+H] +

[0242] Example 4: Synthesis of pentamer and solid-liquid separation

[0243]

[0244] Under an argon atmosphere, the tetramer (4m) (3.8 g, 1.5 mmol) obtained in Example 3 and triphenylphosphine (0.19 g, 0.74 mmol) were dissolved in a mixed solvent of acetonitrile (22.1 mL) and dichloromethane (73.7 mL). Molecular sieves 3A (4.4 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dG(iBu)-3'-phosphoramidite (2.5 g, 3.0 mmol) was then added, followed by stirring at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.38 g, 2.9 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. To the reaction mixture, 2,2,2-trifluoroethanol (1.1 mL, 1.5 mmol) was added and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.4 mL, 11.1 mmol) and POS (0.72 g, 4.0 mmol) were added and stirred at room temperature for 17 hours. 2,3-Dimethylfuran (1.5 mL, 14.7 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (3.8 mL, 49.2 mmol) and 2,6-dimethylaniline (0.061 mL, 0.49 mmol) dissolved in anhydrous dichloromethane (7.5 mL). The mixture was stirred at room temperature for 2 hours. 2,6-Dimethylpyridine (14.3 mL, 122.8 mmol) and methanol (0.48 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was filtered, and the funnel and other parts used for the filtration were washed with dichloromethane (9 mL). A target solution was obtained, which was a mixture of the dichloromethane used for washing and the filtrate (volume of target solution: 140 mL, volume of nonpolar solvent (dichloromethane): 90.2 mL, volume of second polar solvent (acetonitrile, methanol, and 2,2,2-trifluoroethanol): 23.68 mL, volume of second polar solvent / volume of nonpolar solvent: 0.26).

[0245] Acetonitrile, the first polar solvent, was added dropwise to the resulting target solution, and the volume of the mixture of the target solution and the first polar solvent was adjusted to 175-230 mL (volume of the mixture of the target solution and the first polar solvent / volume of the target solution: 1.25-1.64). The solvent was then distilled off at a temperature of 10.3-23.9 °C and a pressure of 43-400 mmHg (total volume of the added first polar solvent: 211 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.34). The precipitated solid was collected by filtration and dried to obtain the pentamer (5m) as a white solid (4.5 g, yield: 100.0%). m / z (TOF-MS): Calcd. 3075.42, Found 1538.71 [M+2H] +

[0246] Example 5: Synthesis of hexamer and solid-liquid separation

[0247]

[0248] Under an argon atmosphere, the pentamer (5m) (4.3 g, 1.4 mmol) obtained in Example 4 and triphenylphosphine (0.18 g, 0.71 mmol) were dissolved in a mixed solvent of acetonitrile (20.9 mL) and dichloromethane (69.8 mL). Molecular sieves 3A (4.2 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dmeC(Bz)-3'-phosphoramidite (2.4 g, 2.8 mmol) was then added, followed by stirring at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.36 g, 2.8 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. To the reaction mixture, 2,2,2-trifluoroethanol (1.0 mL, 1.4 mmol) was added and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.3 mL, 10.5 mmol) and POS (0.68 g, 3.5 mmol) were added and stirred at room temperature for 18 hours. 2,3-Dimethylfuran (1.5 mL, 13.9 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (3.4 mL, 43.8 mmol) and 2,6-dimethylaniline (0.054 mL, 0.44 mmol) dissolved in anhydrous dichloromethane (6.7 mL), and the mixture was stirred at room temperature for 2 hours. 2,6-Dimethylpyridine (13.6 mL, 116.4 mmol) and methanol (0.45 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was filtered, and the funnel and other parts used for the filtration were washed with dichloromethane (9 mL). A target solution was obtained, which was a mixture of the dichloromethane used for washing and the filtrate (volume of target solution: 134 mL, volume of nonpolar solvent (dichloromethane): 85.5 mL, volume of second polar solvent (acetonitrile, methanol, and 2,2,2-trifluoroethanol): 22.35 mL, volume of second polar solvent / volume of nonpolar solvent: 0.26).

[0249] Acetonitrile, the first polar solvent, was added dropwise to the resulting target solution, and the volume of the mixture of the target solution and the first polar solvent was adjusted to 165-220 mL (volume of the mixture of the target solution and the first polar solvent / volume of the target solution: 1.23-1.64). The solvent was distilled off at a temperature of 10.5-21.2 ° C. and a pressure of 48-400 mmHg (total volume of the added first polar solvent: 201 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.35). The precipitated solid was collected by filtration and dried to obtain the hexamer (6m) as a white solid (4.8 g, yield: 98.6%). m / z (TOF-MS): Calcd. 3551.51, Found 1776.77 [M+2H] +

[0250] <Deprotection Reaction> The hexamer (6m) (5 mg) obtained in Example 5 and 28% by mass aqueous ammonia (5 mL) were mixed, and the mixture was heated at 65°C for 4 hours and then cooled to room temperature. After removing unnecessary substances from the reaction mixture using a syringe filter, the mixture was concentrated under reduced pressure using a centrifugal evaporator to obtain a deprotected hexamer.

[0251] Comparative Example 1-1: Synthesis of dimer and solid-liquid separation (precipitation by addition of polar solvent only) Under an argon atmosphere, monomer (1m) (1.5 g, 1.2 mmol) and triphenylphosphine (0.16 g, 0.63 mmol) were dissolved in a mixed solvent of acetonitrile (18.7 mL) and chloroform (62.4 mL). Molecular sieves 3A (3.7 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dG(iBu)-3'-phosphoramidite (2.1 g, 2.5 mmol) was then added, and the mixture was stirred at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.31 g, 2.4 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. To the reaction mixture, 2,2,2-trifluoroethanol (0.91 mL, 12.5 mmol) was added and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.2 mL, 9.4 mmol) and POS (0.61 g, 3.1 mmol) were added and stirred at room temperature for 1 hour. 2,3-Dimethylfuran (1.3 mL, 12.4 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (3.0 mL, 39.1 mmol) and 2,6-dimethylaniline (0.048 mL, 0.42 mmol) dissolved in dehydrated chloroform (6.4 mL). The mixture was stirred at room temperature for 1.5 hours. 2,6-Dimethylpyridine (12.1 mL, 104.0 mmol) and methanol (0.41 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was filtered, and acetonitrile (525 mL) was added to the filtrate. The precipitated solid was collected by filtration and dried to give the dimer (2m) as a white solid (2.0 g, yield: 94.3%). m / z (TOF-MS): Calcd. 1705.08, Found 1706.0773 [M+H] +

[0252] Comparative Example 1-2: Trimer Synthesis and Solid-Liquid Separation Under an argon atmosphere, the dimer (2m) (1.8 g, 1.1 mmol) obtained in Comparative Example 1-1 and triphenylphosphine (0.14 g, 0.53 mmol) were dissolved in a mixed solvent of acetonitrile (15.8 mL) and dichloromethane (52.7 mL). Molecular sieves 3A (3.2 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dA(dma)-3'-phosphoramidite (1.7 g, 2.1 mmol) was then added, followed by stirring at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.26 g, 2.0 mmol) was then added to the mixture, which was then stirred at room temperature for 4 hours. To the reaction mixture, 2,2,2-trifluoroethanol (0.77 mL, 10.5 mmol) was added and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (0.98 mL, 7.9 mmol) and POS (0.51 g, 2.6 mmol) were added and stirred at room temperature for 1 hour. 2,3-Dimethylfuran (1.1 mL, 10.5 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (2.5 mL, 33.0 mmol) and 2,6-dimethylaniline (0.048 mL, 0.35 mmol) dissolved in dehydrated chloroform (5.4 mL). The mixture was stirred at room temperature for 1.5 hours. 2,6-Dimethylpyridine (10.2 mL, 87.7 mmol) and methanol (0.34 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was filtered, and acetonitrile (443 mL) was added to the filtrate. The precipitated solid was collected by filtration and dried to give the trimer (3m) as a white solid (2.2 g, yield: 98.4%). m / z (TOF-MS): Calcd. 2156.20, Found 2157.20 [M+H] +

[0253] Comparative Example 1-3: Synthesis of tetramer and solid-liquid separation

[0073] The procedure of Comparative Example 1-2 was repeated except that the trimer (3m) (2.0 g, 0.94 mmol) obtained in Comparative Example 1-2 and 5'-DMT-dA(dma)-3'-phosphoramidite (1.5 g, 1.8 mmol) were used, and tetramer (4m) (2.3 g, yield: 97.1%) was obtained as a white solid. m / z (TOF-MS): Calcd. 2608.32, Found 2608.32 [M+H] +

[0254] Comparative Example 1-4: Synthesis of pentamer and solid-liquid separation

[0079] Pentamer (5m) (2.1 g, yield: 96.7%) was obtained as a white solid in the same manner as in Comparative Example 1-2, except that the tetramer (4m) (1.9 g, 0.72 mmol) obtained in Comparative Example 1-3 and 5'-DMT-dG(iBu)-3'-phosphoramidite (1.2 g, 1.4 mmol) were used. m / z (TOF-MS): Calcd. 3075.42, Found 1538.71 [M+2H] +

[0255] Comparative Example 1-5: Synthesis of hexamer and solid-liquid separation

[0043] The hexamer (6m) (1.6 g, yield: 83.9%) was obtained as a white solid in the same manner as in Comparative Example 1-2, except that the pentamer (5m) (1.7 g, 0.55 mmol) obtained in Comparative Example 1-4 and 5'-DMT-dmeC(Bz)-3'-phosphoramidite (0.94 g, 1.1 mmol) were used. m / z (TOF-MS): Calcd. 3551.51, Found 1776.76 [M+2H] +

[0256] <Deprotection Reaction> The hexamer (6m) (5 mg) obtained in Comparative Example 1-5 and 28% by mass aqueous ammonia (5 mL) were mixed, and the mixture was heated at 65°C for 4 hours and then cooled to room temperature. After removing unnecessary substances from the reaction mixture using a syringe filter, the mixture was concentrated under reduced pressure using a centrifugal evaporator to obtain a deprotected hexamer.

[0257] Comparative Example 2-1: Synthesis of dimer and solid-liquid separation (concentration and precipitation by addition of polar solvent) Under an argon atmosphere, monomer (1m) (2.5 g, 2.0 mmol) and triphenylphosphine (0.27 g, 1.0 mmol) were dissolved in a mixed solvent of acetonitrile (30.3 mL) and chloroform (101.1 mL). Molecular sieves 3A (6.1 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dG(iBu)-3'-phosphoramidite (3.4 g, 4.0 mmol) was then added, and the mixture was stirred at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.53 g, 4.0 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. 2,2,2-Trifluoroethanol (1.5 mL, 20.2 mmol) was added to the reaction mixture and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.9 mL, 15.2 mmol) and POS (0.99 g, 5.1 mmol) were added and stirred at room temperature for 1 hour. 2,3-Dimethylfuran (2.1 mL, 20.2 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (5.2 mL, 67.5 mmol) and 2,6-dimethylaniline (0.083 mL, 0.68 mmol) dissolved in dehydrated chloroform (10.4 mL). The mixture was stirred at room temperature for 1.5 hours. 2,6-Dimethylpyridine (19.7 mL, 169.1 mmol) and methanol (0.66 mL) were added to the reaction mixture and stirred at room temperature for 45 minutes. The reaction mixture was filtered, and the filtrate was concentrated to 63 mL. Acetonitrile (444 mL) was added to the concentrate, and the precipitated solid was collected by filtration and dried to obtain the dimer (2m) as a white solid (3.1 g, yield: 92.2%). m / z (TOF-MS): Calcd. 1705.08, Found 1706.0773 [M+H] +

[0258] Comparative Example 2-2: Trimer Synthesis and Solid-Liquid Separation Under an argon atmosphere, the dimer (2m) (3.0 g, 2.0 mmol) obtained in Comparative Example 2-1 and triphenylphosphine (0.23 g, 0.89 mmol) were dissolved in a mixed solvent of acetonitrile (26.4 mL) and dichloromethane (87.8 mL). Molecular sieves 3A (5.3 g) were added to the mixture, which was then stirred at room temperature for 30 minutes. 5'-DMT-dA(dma)-3'-phosphoramidite (2.9 g, 3.5 mmol) was then added, followed by stirring at room temperature for 15 minutes. 5-Ethylthio-1H-tetrazole (0.46 g, 3.5 mmol) was then added to the mixture, which was then stirred at room temperature for 3 hours. 2,2,2-Trifluoroethanol (1.3 mL, 17.6 mmol) was added to the reaction mixture and stirred at room temperature for 30 minutes. Then, 2,6-dimethylaniline (1.6 mL, 13.3 mmol) and POS (0.86 g, 4.4 mmol) were added and stirred at room temperature for 1 hour. 2,3-Dimethylfuran (1.8 mL, 17.6 mmol) was added, followed by the dropwise addition of a mixture of trifluoroacetic acid (4.2 mL, 54.9 mmol) and 2,6-dimethylaniline (0.068 mL, 0.59 mmol) dissolved in dehydrated chloroform (8.5 mL). The mixture was stirred at room temperature for 1.5 hours. 2,6-Dimethylpyridine (17.1 mL, 146.7 mmol) and methanol (0.57 mL) were added to the reaction mixture and stirred at room temperature for 45 minutes. The reaction mixture was filtered, and the filtrate was concentrated to 55 mL. Acetonitrile (387 mL) was added to the concentrate, and the precipitated solid was collected by filtration and dried to give the trimer (3m) as a white solid (3.6 g, yield: 96.9%). m / z (TOF-MS): Calcd. 2156.20, Found 2157.20 [M+H] +

[0259] Comparative Example 2-3: Synthesis of tetramer and solid-liquid separation

[0073] The procedure of Comparative Example 2-2 was repeated except that the trimer (3m) (3.4 g, 1.5 mmol) obtained in Comparative Example 2-2 and 5'-DMT-dA(dma)-3'-phosphoramidite (2.6 g, 3.1 mmol) were used, and tetramer (4m) (4.0 g, yield: 99.6%) was obtained as a white solid. m / z (TOF-MS): Calcd. 2608.32, Found 2608.32 [M+H] +

[0260] Comparative Example 2-4: Synthesis of pentamer and solid-liquid separation

[0111] Pentamer (5m) (4.2 g, yield: 97.7%) was obtained as a white solid in the same manner as in Comparative Example 2-2, except that the tetramer (4m) (3.7 g, 1.4 mmol) obtained in Comparative Example 2-3 and 5'-DMT-dG(iBu)-3'-phosphoramidite (2.4 g, 2.9 mmol) were used. m / z (TOF-MS): Calcd. 3075.42, Found 1538.71 [M+2H] +

[0261] Comparative Example 2-5: Synthesis of hexamer and solid-liquid separation

[0111] The hexamer (6m) (4.5 g, yield: 97.5%) was obtained as a white solid in the same manner as in Comparative Example 2-2, except that the pentamer (5m) (4.1 g, 1.3 mmol) obtained in Comparative Example 2-4 and 5'-DMT-dmeC(Bz)-3'-phosphoramidite (2.3 g, 2.7 mmol) were used. m / z (TOF-MS): Calcd. 3551.51, Found 1776.77 [M+2H] +

[0262] <Deprotection Reaction> The hexamer (6m) (5 mg) obtained in Comparative Example 2-5 and 28% by mass aqueous ammonia (5 mL) were mixed, and the mixture was heated at 65°C for 4 hours and then cooled to room temperature. After removing unnecessary substances from the reaction mixture using a syringe filter, the mixture was concentrated under reduced pressure using a centrifugal evaporator to obtain a deprotected hexamer.

[0263] The purity (area %) of the deprotected hexamer obtained as described above was calculated from the peak area of ​​the chromatogram obtained by high performance liquid chromatography (HPLC). The results are shown in Table 1 below.

[0264]

[0265] As shown in Table 1, the difference in purity between Example 5 and Comparative Examples 1-5 was 2.31 area %, and the difference in purity between Example 5 and Comparative Examples 2-5 was 3.27 area %. As mentioned above, in liquid-phase synthesis of oligonucleotides using hydrophobic protecting groups, the influence of impurities in the next synthesis cycle is greater than in solid-phase synthesis of oligonucleotides using a solid support. Therefore, the above-mentioned difference in purity can be said to be significant in liquid-phase synthesis of oligonucleotides using hydrophobic protecting groups. In this way, the precipitation method of the present invention can achieve excellent effects (high purity).

[0266] The oligonucleotides obtained by the precipitation method of the present invention can be used for various purposes, such as human or animal pharmaceuticals (RNA, DNA, oligonucleic acid drugs, etc.), functional foods, foods for specified health uses, food, chemical products, and polymeric materials for biological or industrial use.

[0267] This application is based on Japanese Patent Application No. 2024-056447, the contents of which are incorporated in their entirety herein.

Claims

1. A method for precipitating an oligonucleotide having a hydrophobic protecting group from a solution containing the oligonucleotide and a non-polar solvent, the method comprising: mixing the solution with a first polar solvent; and distilling off the solvent from the mixture while maintaining the volume of the mixture of the solution and the first polar solvent at a volume equal to or greater than 1.00 times the volume of the solution.

2. The method according to claim 1, wherein the solvent is removed from the mixture by distillation at a temperature of 5°C to 35°C and a pressure of 15 mmHg to 500 mmHg.

3. The method according to claim 1 or 2, wherein the non-polar solvent comprises a halogenated solvent.

4. The method according to claim 1 or 2, wherein the first polar solvent comprises at least one selected from the group consisting of nitrile solvents and alcohol solvents.

5. The method according to claim 1 or 2, wherein the total volume of the first polar solvent mixed with the solution is 1.0 to 3.5 times the volume of the nonpolar solvent.

6. The method of claim 1 or 2, wherein the solution comprises a second polar solvent.

7. The method according to claim 6, wherein the second polar solvent comprises at least one selected from the group consisting of nitrile solvents and alcohol solvents.

8. The method according to claim 6, wherein the volume of the second polar solvent is 0.15 to 0.35 times the volume of the nonpolar solvent.

9. The method according to claim 1 or 2, wherein the molecular weight of the hydrophobic protecting group is 360 or more.

10. The hydrophobic protecting group is represented by the following formula (Pg-5): **L-Y-Z (Pg-5) [wherein ** represents the bonding position to the protected group; L represents a single bond, or a group represented by the formula (Pg-i-1) or (Pg-i-2): (wherein * indicates the bonding position to Y; ** has the same meaning as above; R １ｐ and R ２ｐ are each independently C １－２２ represents a hydrocarbon group; １ is an optionally substituted divalent C １－２２ represents a hydrocarbon group, and １－２２ -CH in hydrocarbon group ２ - may be replaced with a linker; L ２ indicates a single bond or ***C(═O)N(R ３ｐ )-R ４ｐ -N(R ５ｐ ) **** (wherein **** represents L １ indicates the bonding position with C═O, and R ４ｐ is C １－２２ represents an alkylene group, and R ３ｐ and R ５ｐ are each independently a hydrogen atom or C １－２２ represents an alkyl group or R ３ｐ and R ５ｐ may be taken together to form a ring. ) represents a group represented by the formula (Pg-ii-1); ) represents a group represented by the formula (Pg-ii-2); Y represents a single bond, an oxygen atom, or NR (R represents a hydrogen atom, an alkyl group, or an aralkyl group); and Z represents any of the formulas (Pg-ii-1) to (Pg-ii-5): [wherein * represents a bonding position; ring A' represents a benzene ring; ring B' represents a cyclohexane ring; ring D' represents a naphthalene ring or a bicyclic fused aromatic heterocycle; R ６ｐ is a hydrogen atom, or R ｂ is a group represented by the following formula (Pg-iii), R of ring A' or ring B' ６ｐ is R ８ｐ and may together represent a single bond or —O— to form a fused ring together with ring A′ or ring B′ and ring C′; k represents an integer of 1 to 4; k Qs each independently represent —O—, —C(═O)—, —C(═O)O—, —OC(═O)—, —NR′—, —C(═O)NR′—, or —NR′C(═O)— (wherein R′ each independently represents a hydrogen atom or C １－６ represents an alkyl group) (k Qs preferably each independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-); k Q's each independently represent a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'- or -NR'C(=O)- (wherein R's each independently represent a hydrogen atom or C １－６ k R ７ｐ each independently represent a hydrocarbon group to which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker; ring A′ and ring B′ each independently represent k QR ７ｐ In addition to the above, a halogen atom, a C optionally substituted with a halogen atom １－６ C optionally substituted with an alkyl group or a halogen atom １－６ R may have a substituent selected from the group consisting of alkoxy groups; ａ represents a hydrogen atom; and R ｂ is a hydrogen atom or a group represented by the formula (Pg-iii): (wherein * represents a bonding position; ring C′ represents a benzene ring; j represents an integer of 0 to 4; j Qs each independently have the same meaning as defined above; j R ９ｐ each independently represents a hydrocarbon group in which a linear aliphatic hydrocarbon group having 10 or more carbon atoms is bonded via a single bond or a linker; R ８ｐ represents a hydrogen atom, or R ６ｐ and may be taken together with j QR's to form a single bond or -O- to form a fused ring together with ring A' or ring B' and ring C'; ring C' may be taken together with j QR's ９ｐ In addition to the above, a halogen atom, a C optionally substituted with a halogen atom １－６ C optionally substituted with an alkyl group or a halogen atom １－６ R may have a substituent selected from the group consisting of alkoxy groups. ａ and R ｂ together form an oxo group; u represents 1 or 2; and R １０ｐ is C ２－２１ The method according to claim 1 or 2, wherein the protecting group is a group represented by the formula:

11. A method for producing an oligonucleotide by one-pot synthesis, the method comprising: (1) condensing, in a solution containing a nonpolar solvent, a nucleoside, nucleotide, or oligonucleotide (a) having a hydrophobic protecting group with a nucleoside, nucleotide, or oligonucleotide (b) whose hydroxyl group is protected with a temporary protecting group removable under acidic conditions and which has been phosphoramidized, to form an oligonucleotide precursor (c) having a phosphite bond and a hydrophobic protecting group, and whose hydroxyl group is protected with a temporary protecting group removable under acidic conditions; (2) mixing the solution obtained after step (1) with a quenching agent (i) for the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b), to quench the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b); a step (3) of mixing the solution after step (2) with an oxidizing agent or a sulfurizing agent to oxidize or sulfurize the oligonucleotide precursor (c) to form an oligonucleotide (d) having a phosphate bond or a thiophosphate bond and a hydrophobic protecting group, and whose hydroxyl groups are protected by temporary protecting groups removable under acidic conditions; a step (4) of mixing the solution after step (3) with an oxidizing agent (ii) to quench the oxidizing agent, if an oxidizing agent is used in step (3); a step (5) of mixing the solution after step (3) or, if step (4) is performed, the solution after step (4) with an acid to remove the temporary protecting groups removable under acidic conditions from the oligonucleotide (d) to form an oligonucleotide (e) having unprotected hydroxyl groups and hydrophobic protecting groups; a step (6) of mixing the solution after step (5) with a base, if necessary; and a step (7) of precipitating the oligonucleotide (e) from a solution containing the oligonucleotide (e) and a nonpolar solvent by the method of claim 1 or 2.

12. The method according to claim 11, wherein the removal of the temporary protecting group in step (5) is carried out in the presence of a cation scavenger, or the solution obtained after the removal of the temporary protecting group is mixed with a cation scavenger.

13. The method according to claim 12, wherein the cation scavenger is at least one selected from the group consisting of pyrrole, pyrrole derivatives, indole, indole derivatives, furan derivatives, and mercapto group-containing compounds.

Images