Method for elution and production of oligonucleotides

Abstract

The present invention provides a method for precipitating an oligonucleotide having a hydrophobic protecting group from a solution containing the oligonucleotide and a non-polar solvent, wherein the method comprises: 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 relative to the volume of the solution. The method of the present invention can reduce the amount of polar solvent used for precipitation, and enables the precipitation of the oligonucleotide having a hydrophobic protecting group from a solution containing the oligonucleotide and a non-polar solvent with high purity.

Description

Technical Field

[0001] This invention relates to methods for precipitating and manufacturing oligonucleotides. Background Technology

[0002] As a method for manufacturing oligonucleotides, it is known to synthesize oligonucleotides with hydrophobic protecting groups in a solution containing a starting material (e.g., a monomer with a hydrophobic protecting group) and a nonpolar solvent, and then add a polar solvent to the reaction solution to precipitate the oligonucleotides (e.g., Patent Documents 1 and 2).

[0003] Existing technical documents Patent documents Patent Document 1: International Publication No. 2017 / 104836 Patent Document 2: International Publication No. 2022 / 172994. Summary of the Invention

[0004] The technical problem that the invention aims to solve Methods that precipitate oligonucleotides by adding a polar solvent to a solution containing an oligonucleotide with a hydrophobic protecting group and a nonpolar solvent require the use of a large amount of polar solvent. If the solution is concentrated before adding the polar solvent to reduce the amount of polar solvent used for precipitation, the purity of the precipitated oligonucleotides may decrease due to side reactions that occur during concentration. In liquid-phase synthesis of oligonucleotides using hydrophobic protecting groups, compared to solid-phase synthesis of oligonucleotides using a solid support, the influence of impurities in the next synthesis cycle is greater, thus requiring the separation of oligonucleotides with the highest possible purity.

[0005] The present invention was made with regard to the situation described above, and its object is to provide a method for reducing the amount of polar solvent used for precipitation and for precipitating the oligonucleotide in high purity from a solution containing the oligonucleotide having a hydrophobic protecting group and a nonpolar solvent.

[0006] Technical means used to solve technical problems To achieve the above objectives, the inventors, through repeated and careful investigation, discovered that by mixing a first polar solvent used for precipitation with a solution containing an oligonucleotide with a hydrophobic protecting group (hereinafter referred to as the "target oligonucleotide") and a nonpolar solvent (hereinafter referred to as the "target solution"), and by maintaining the volume of the mixture of the first polar solvent and the target solution (hereinafter referred to as the "target mixture") at more than 1.00 times the volume of the target solution while removing the solvent by distillation, the amount of polar solvent used for precipitation can be reduced, and the target oligonucleotide can be precipitated with high purity. Based on this insight, the present invention is as follows.

[0007] [1] A method for precipitating an oligonucleotide from a solution containing an oligonucleotide having a hydrophobic protecting group and a nonpolar solvent, wherein the method comprises: Mix the solution with a first polar solvent; and While maintaining the volume of the mixture of the solution and the first polar solvent at more than 1.00 times the volume of the solution, the solvent is removed from the mixture by distillation.

[0008] [2] According to the method described in [1] above, the solvent is removed by distillation from the mixture under the conditions of a temperature of 5°C to 35°C and a pressure of 15 mmHg to 500 mmHg.

[0009] [3] According to the method described in [1] or [2] above, the nonpolar solvent includes halogen solvents.

[0010] [4] The method according to any one of [1] to [3] above, wherein the first polar solvent comprises at least one selected from nitrile solvents and alcohol solvents.

[0011] [5] According to any one of [1] to [4] above, 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.

[0012] [6] The method according to any one of [1] to [5] above, wherein the solution contains a second polar solvent.

[0013] [7] According to the method described in [6] above, the second polar solvent comprises at least one selected from nitrile solvents and alcohol solvents.

[0014] [8] According to the method described in [6] or [7] above, the volume of the second polar solvent is 0.15 to 0.35 times that of the nonpolar solvent.

[0015] [9] The method according to any one of [1] to [8] above, wherein the molecular weight of the hydrophobic protecting group is 360 or more.

[0016]

[10] According to any one of [1] to [9] above, wherein the hydrophobic protecting group is a protecting group represented by the following formula (Pg-5), **LYZ (Pg-5) [In the formula, **Indicates the binding site with the protected group; L represents a single bond, or a group represented by the formula (Pg-i-1) or (Pg-i-2). [Chemical Formula 1] (In the formula, * indicates the position where it is associated with Y. **Same as the meaning above, R 1p and R 2p Represent C independently. 1-22 hydrocarbon group, L 1 Indicates divalent C with optional substitution 1-22 Hydrocarbon group, the divalent C 1-22 The -CH2- in the hydrocarbon group may be optionally replaced by a linking group. L 2 Represents a single bond, or represents a bond with ***C(=O)N(R) 3p )-R 4p -N(R 5p **** represents the group (where *** represents the group with L). 1 The binding position, **** indicates the binding position with C=O, R 4p C represents 1-22 Alkylene, R 3p and R 5p Each can be used independently to represent a hydrogen atom or a carbon atom. 1-22 Alkyl, or R 3p and R 5p (Optionally, they can be combined to form a ring) 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 one of the groups represented by formulas (Pg-ii-1) to (Pg-ii-5). [Chemical Formula 2] [In the formula, * indicates the binding position. Ring A' represents the benzene ring. Ring B' represents the cyclohexane ring. Ring D' represents a naphthalene ring or a bicyclic fused aromatic heterocycle. R 6p For hydrogen atoms, or when R b In the case of a group represented by the following formula (Pg-iii), R of ring A' or ring B' 6p Optional with R 8p Together they represent a single bond or -O-, forming a fused ring together with ring A' or ring B' and ring C'. k represents an integer from 1 to 4; k Q's independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, or -NR'C(=O)- (where R' independently represents a hydrogen atom or a C atom). 1-6 Alkyl groups (preferably the k Qs represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-, respectively). Each of the k Q's independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, or -NR'C(=O)- (where R' independently represents a hydrogen atom or a C atom). 1-6 alkyl), k R 7p Each can be independently represented as a hydrocarbon group with a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms bonded together via a single bond or a linking group. Rings A' and B' are independent, except that they have k QRs. 7p In addition, it may optionally have substituents selected from halogen atoms, C atoms optionally substituted with halogen atoms. 1-6 Alkyl groups, and C atoms optionally substituted with halogen atoms 1-6 Alkoxy R a Represents hydrogen atoms, and R b Represents a hydrogen atom, or a group represented by formula (Pg-iii), [Chemical Formula 3] (In the formula, * indicates the binding position. The ring C' represents the benzene ring. j represents an integer from 0 to 4. Each of the j Q's independently has the same meaning as described above. j R 9p Each can be independently represented as a hydrocarbon group with a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms bonded together via a single bond or a linking group. R 8p Represents a hydrogen atom, or optionally a R atom of ring A' or ring B'. 6p Together they represent a single bond or -O-, forming a fused ring together with ring A' or ring B' and ring C'. In addition to having j QRs 9p In addition, it may optionally have substituents selected from halogen atoms, C atoms optionally substituted with halogen atoms. 1-6 Alkyl groups, and C atoms optionally substituted with halogen atoms 1-6alkoxy), or R a and R b Together they form an oxo group. u represents 1 or 2, and R 10p C represents 2-21 Perfluoroalkyl]).

[0017]

[11] A method for producing oligonucleotides by one-pot synthesis, wherein the method comprises: Step (1): In a solution containing a nonpolar solvent, 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 by a temporary protecting group that can be removed under acidic conditions, and which is 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 that can be removed under acidic conditions; Step (2) involves mixing the solution from step (1) with the quencher (i) of the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b) to quench the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b). Step (3) involves mixing the solution from step (2) with an oxidizing agent or a sulfiding agent to oxidize or sulfidate 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 the hydroxyl group being protected by a temporary protecting group that can be removed under acidic conditions. In step (4), if an oxidant is used in step (3), the solution after step (3) is mixed with an oxidant quencher (ii) as needed to quench the oxidant; In step (5), the solution after step (3) or the solution after step (4) in the case of step (4) is mixed with acid to remove the temporary protecting group of the oligonucleotide (d) that can be removed under acidic conditions, forming an oligonucleotide (e) with unprotected hydroxyl groups and hydrophobic protecting groups. Step (6): As needed, mix the solution from step (5) with alkali; and Step (7) involves precipitating the oligonucleotide (e) from a solution containing the oligonucleotide (e) and a nonpolar solvent using any one of the methods described in [1] to

[10] .

[0018]

[12] According to the method described in

[11] above, the temporary protecting group of step (5) is removed in the presence of a cation scavenger, or the solution after the temporary protecting group has been removed is mixed with a cation scavenger.

[0019]

[13] According to the method described in

[12] above, the cation scavenger is selected from at least one of pyrrole, pyrrole derivatives, indole, indole derivatives, furan derivatives, and thiol-containing compounds.

[0020] The effects of the invention 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 nonpolar solvent. Detailed Implementation

[0021] [the term] First, the terminology used in this specification will be explained. It should be noted that, 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 pertains.

[0022] Terminology related to groups In this instruction manual, "C" a-b "Halogen atom" refers to a carbon number of a or more and b or less (where a and b represent integers). In this specification, "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0023] In this specification, the term "hydrocarbon group" can be exemplified by aliphatic hydrocarbon groups, aromatic aliphatic hydrocarbon groups, monocyclic saturated hydrocarbon groups, and aromatic hydrocarbon groups. Specific examples include monovalent and divalent groups such as alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, and alkylene groups.

[0024] In this invention, "alkyl" can be either straight-chain or branched. Examples of "alkyl" include alkyl groups having one or more carbon atoms; where there is no particular limitation on the range of carbon atoms, C10 is preferred. 1-10 Alkyl, preferably C 1-6 Alkyl groups. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

[0025] In this invention, "alkenyl" can be either linear or branched. Examples of "alkenyl" include C1. 2-6 Alkenyl, etc. Specifically, examples include vinyl, 1-propenyl, allyl, isopropenyl, butenyl, isobutenyl, etc.

[0026] In this invention, "alkynyl group" can be either linear or branched. Examples of "alkynyl group" include C1. 2-6Alkyne, etc. Specific examples 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, 5-hexynyl, etc.

[0027] In this specification, "cycloalkyl" refers to a cyclic alkyl group, such as C10. 3-8 Cycloalkyl, preferably C 3-6 Cycloalkyl groups. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0028] In this specification, "aryl" refers to a monocyclic or polycyclic (fused) aromatic hydrocarbon group, such as phenyl, 1-naphthyl, 2-naphthyl, biphenyl, 1-anthrayl, 2-anthrayl, etc. 6-14 Aryl. Among them, C is preferred. 6-10 Aryl, especially phenyl.

[0029] In this specification, "heteroaryl" refers to a monocyclic or polycyclic (fused) aromatic heterocyclic group that contains, in addition to a carbon atom, heteroatoms selected from nitrogen, sulfur, and oxygen atoms as cyclic atoms. Examples of "heteroaryl" include 5- or 6-membered monocyclic heteroaryl groups and 8- to 14-membered fused polycyclic heteroaryl groups.

[0030] In this specification, examples of "5- or 6-membered monocyclic heteroaryl groups" include thienyl, furanyl, pyrroleyl, 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, etc.

[0031] In this specification, examples of "8- to 14-membered fused polycyclic heteroaryl groups" include, for example, benzothiophene, benzofuran, benzimidazol, benzoxazol, benzoisoxazol, benzothiazolyl, benzoisothiazolyl, benzotriazol, imidazopyridyl, thienopyridyl, furanopyridyl, pyrrolopyridyl, pyrazolopyridyl, oxazolopyridyl, thiazopyridyl, imidazopyrazinyl, imidazopyrimidinyl, and thienopyrimidinyl. The following are listed: [List of compounds, including] furanylpyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thiopheneyl, phenoxazinyl, indoleyl, isoindoleyl, 1H-indazoleyl, purineyl, isoquinolinyl, quinolinyl, phthalazinyl, naphridinyl, quinoxolinyl, quinazolinyl, cyclolinyl, carbazoyl, β-carolinyl, phenanthidyl, acridineyl, phenazinyl, phenothiazinyl, phenotoxazinyl, etc.

[0032] In this specification, C can be cited as an example of "aryl group". 7-20 Aryl groups, preferably C4 7-16 Arane (C 6-10 Aryl-C 1-6 Alkyl groups. Specific examples include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, 1-naphthylpropyl, etc.

[0033] In this invention, "alkylene" can be either linear or branched. Examples of "alkylene" include alkylene with one or more carbon atoms; where there is no particular limitation on the range of carbon atoms, C10 is preferred. 1-10 Alkylene, preferably C 1-6 Alkylenes. Specific examples include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0034] In this invention, "alkoxy group" can be either linear or branched. Examples of "alkoxy group" include alkoxy groups with 1 or more carbon atoms; where there is no particular limitation on the range of carbon atoms, C10 is preferred. 1-10 Alkoxy, preferably C 1-6 Alkyl groups. Specific examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and hexoxy.

[0035] In this invention, the "acyl group" can be either linear or branched. Examples of "acyl groups" include C1. 1-6 Alkyl group, C 7-13 Aromatic acyl groups, etc. Specific examples of "acyl groups" include formyl, acetyl, n-propionyl, isopropionyl, n-butyryl, isobutyryl, neopentanoyl, pentanoyl, hexanoyl, benzoyl, naphthoyl, levulinyl, etc. Acyl groups can be substituted.

[0036] In this specification, examples of "bicyclic fused aromatic heterocycles" include, for instance, benzothiophene rings, benzofuran rings, isobenzofuran rings, benzimidazole rings, benzoxazole rings, benzoisoxazole rings, benzothiazole rings, benzoisothiazole rings, benzotriazole rings, indole rings, isoindole rings, 1H-indazole rings, purine rings, isoquinoline rings, 5,6,7,8-tetrahydroisoquinoline rings, quinoline rings, 5,6,7,8-tetrahydroquinoline rings, phthalazine rings, pteridine rings, naphthidine rings, quinoxaline rings, quinazoline rings, and zoline rings. It should be noted that "bicyclic fused aromatic heterocycles" in this specification also includes bicyclic fused rings where one ring is an aromatic heterocycle and the other ring is a non-aromatic heterocycle or a non-aromatic hydrocarbon ring (e.g., 5,6,7,8-tetrahydroisoquinoline ring).

[0037] In this specification, "perfluoroalkyl" refers to an alkyl group in which all hydrogen atoms are replaced by fluorine atoms.

[0038] In this specification, examples of "linking groups" include -O-, -C(=O)-, -C(=O)O-, -OC(=O-), -NR'-, -C(=O)NR'-, -NR'C(=O-), -S-, -SO-, -SO2-, -Si(R')(R'')O-, and -Si(R')(R'')- (where R' and R'' independently represent a hydrogen atom or a C atom). 1-22 Hydrocarbon groups, etc. Preferred linking groups are -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, -NHC(=O)-, -S-, -SO-, -SO2-, -Si(R')(R'')O-, -Si(R')(R'')- (R' and R'' independently represent a hydrogen atom or a C atom). 1-22 (hydrocarbon group).

[0039] In this specification, in addition to the aforementioned halogen atoms, alkyl, aralkyl, alkoxy, acyl, alkenyl, alkynyl, cycloalkyl, and aryl groups, the term "substituent" may also include hydroxyl, nitro, cyano, guanidinyl, carboxyl, alkoxycarbonyl (alkoxy moiety has the same meaning as the aforementioned alkoxy group), sulfo group, phospho group, alkylthio group (alkyl moiety has the same meaning as the aforementioned alkyl group), alkylsulfinyl (alkyl moiety has the same meaning as the aforementioned alkyl group), alkylsulfonyl (alkyl moiety has the same meaning as the aforementioned alkyl group), amino, monoalkylamino (alkyl moiety has the same meaning as the aforementioned alkyl group), dialkylamino (alkyl moiety has the same meaning as the aforementioned alkyl group), and oxo group.

[0040] In this specification, there is no particular limitation on the "protecting group of hydroxyl groups", and any known protecting group may be used. Examples of 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-butyl dithiomethyl (DTM), ((2-(methylthio)phenyl)thio)methyl (MPTM), (N-dichloroacetyl-N-methyl)aminobenzyloxymethyl (DCMABOM), and (2-acetylethyl)carbonyloxymethyl (also known in this field as "acetal levulinyl"). ester)(ALE) group, phenylcarbamoyl group, 1,1-dioxothiomorpholine-4-thiocarbamoyl group, acetyl group, neopentyl group, benzoyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group, [(triisopropylsilyl)oxy]methyl (Tom), 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep), etc.

[0041] In this specification, examples of "temporary protecting groups of the hydroxyl group that can be removed under acidic conditions" include: triphenylmethyl, 9-phenyl-9-xanthenyl, 9-phenyl-9-thiazenyl, 1,1-bis(4-methoxyphenyl)-1-phenylmethyl (also referred to as "4,4'-dimethoxytriphenylmethyl" in this specification), etc. 1-6 Alkoxy)triphenylmethyl, 1-(4-methoxyphenyl)-1,1-diphenylmethyl (also referred to as "4-monomethoxytriphenylmethyl" in this specification) and other mono(C 1-18 Alkoxy)triphenylmethyl, etc. From the viewpoint of ease of deprotection, the temporary protecting group is preferably 4,4'-dimethoxytriphenylmethyl or 4-monomethoxytriphenylmethyl, and more preferably 4,4'-dimethoxytriphenylmethyl.

[0042] In this specification, there is no particular limitation on the "protecting group of the amino group," and known protecting groups may be used. Examples of such protecting groups include neopentanoyl, neopentanoyloxymethyl, acetyl, trifluoroacetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, 4-tert-butylphenoxyacetyl, benzoyl, isobutyryl, (2-hexyl)decanoyl, dimethylformamidinyl, and 9-fluorenylmethyloxycarbonyl. Among these, examples include acetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, benzoyl, isobutyryl, (2-hexyl)decanoyl, dimethylformamidinyl, and groups with =C(R 11 )-N(R 12 (R) 13 The group represented by ) (where R is a group in the formula) 11 R represents methyl. 12 and R 13 Represent C independently. 1-6 Alkyl, or R 11 and R 12 They can combine with the carbon and nitrogen atoms they bond with to form 5- or 6-membered nitrogen-containing heterocycles, etc. As described, =C(R 11 )-N(R 12 (R) 13 The group represented by ) can be exemplified by, for example, 1-(dimethylamino)ethylene.

[0043] In this specification, there is no particular limitation on the "protecting group of the phosphate group," and known protecting groups may be used. Examples of protecting groups for the phosphate group include the following: 2-Cyanoethyl, represented by the following formula (Pg-1) 2-[2-(4,4'-dimethoxytriphenylmethyloxy)ethylsulfonyl]ethyl, represented by the following formula (Pg-2) [3-(4,4'-dimethoxytriphenylmethyloxy)-2,2-di(ethoxycarbonyl)]propyl, represented by the following formula (Pg-3) [3-(4,4'-dimethoxytriphenylmethyloxy)-2,2-di(N-methylcarbamoyl)]propyl, represented by the following formula (Pg-4).

[0044] The best protecting group for phosphate groups is 2-cyanoethyl.

[0045] [Chemical Formula 4] .

[0046] In the formula, Me represents methyl, Et represents ethyl, DMTr represents 4,4'-dimethoxytriphenylmethyl, and * indicates the binding position with the phosphate group.

[0047] In this specification, examples of "hydrophobic protective groups" include those described in International Publication Nos. 2017 / 038650, 2017 / 086397, 2017 / 104836, 2018 / 203574, 2018 / 212236, 2019 / 131719, 2020 / 017085, 2020 / 175472, 2020 / 227618, 2021 / 198883, 2022 / 103842, and 2022 / 214692.

[0048] From the perspective of the solubility of the target oligonucleotide in the target solution, the molecular weight of the hydrophobic protecting group is preferably 360 or higher, and more preferably 600 or higher. From the perspective of the solubility of the target oligonucleotide in the target solution and the precipitation of the target oligonucleotide from the target solution upon the addition of a polar solvent, the molecular weight of the hydrophobic protecting group is preferably 2000 or lower, and more preferably 1200 or lower.

[0049] The hydrophobic protecting group is preferably represented by the following formula (Pg-5): **LYZ (Pg-5) In the formula, **Indicates the binding site with the protected group; L represents a single bond, or a group represented by the formula (Pg-i-1) or (Pg-i-2).

[0050] [Chemical Formula 5] .

[0051] In the formula, * indicates the position where it is associated with Y. **Same as the meaning above, R 1p and R 2p Represent C independently. 1-22 hydrocarbon group, L 1 Indicates divalent C with optional substitution 1-22 Hydrocarbon group, the divalent C 1-22 The -CH2- in the hydrocarbon group may be optionally replaced by a linking group. L 2 Represents a single bond, or represents a bond with ***C(=O)N(R) 3p )-R 4p -N(R 5p **** represents the group (where *** represents the group with L). 1The binding position, **** indicates the binding position with C=O, R 4p Indicate C 1-22 Alkylene, R 3p and R 5p Each can be used independently to represent a hydrogen atom or a carbon atom. 1-22 Alkyl, or R 3p and R 5p (Optionally, they can be grouped together to form a ring). 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 a group represented by any one of the formulas (Pg-ii-1) to (Pg-ii-5).

[0052] [Chemical Formula 6] .

[0053] In the formula, * indicates the binding position. Ring A' represents the benzene ring. Ring B' represents the cyclohexane ring. Ring D' represents a naphthalene ring or a bicyclic fused aromatic heterocycle. R 6p It is a hydrogen atom, or R b In the case of a group represented by the following formula (Pg-iii), R of ring A' or ring B' 6p Optional with R 8p Together they represent a single bond or -O-, forming a fused ring together with ring A' or ring B' and ring C'. k represents an integer from 1 to 4; k Q's independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, or -NR'C(=O)- (where R' independently represents a hydrogen atom or a C atom). 1-6 Alkyl groups), preferably the k Qs independently represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, or -NHC(=O)-. Each of the k Q's independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, or -NR'C(=O)- (where R' independently represents a hydrogen atom or a C atom). 1-6 alkyl), k R 7p Each can be independently represented as a hydrocarbon group with a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms bonded together via a single bond or a linking group. Rings A' and B' are independently, except that they have k QRs.7p In addition, it may optionally have substituents selected from halogen atoms, C atoms optionally substituted with halogen atoms. 1-6 Alkyl groups, and C atoms optionally substituted with halogen atoms 1-6 Alkoxy R a Represents hydrogen atoms, and R b It represents a hydrogen atom or a group represented by formula (Pg-iii).

[0054] [Chemical Formula 7] .

[0055] (In the formula, * indicates the binding position. The ring C' represents the benzene ring. j represents an integer from 0 to 4. Each of the j Q's is independent and has the same meaning as described above. j R 9p Each can be independently represented as a hydrocarbon group with a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms bonded together via a single bond or a linking group. R 8p Represents a hydrogen atom, or optionally an R atom of ring A' or ring B'. 6p Together they represent a single bond or -O-, forming a fused ring together with ring A' or ring B' and ring C'. Ring C' has j QRs 9p In addition, it may optionally have substituents selected from halogen atoms, C atoms optionally substituted with halogen atoms. 1-6 Alkyl groups, and C atoms optionally substituted with halogen atoms 1-6 alkoxy), or R a and R b Together they form an oxygen group. u represents 1 or 2, and R 10p Indicate C 2-21 Perfluoroalkyl.

[0056] Hereinafter, "protecting group represented by formula (Pg-5)" will sometimes be abbreviated as "protecting group (Pg-5)". Compounds represented by other formulas, monomers represented by other formulas, etc., will sometimes be abbreviated similarly to protecting group represented by formula (Pg-5).

[0057] Ring A', Ring C', R 6p and R 8p In the case of a fused ring, ring A' and ring C' represent the benzene ring portion of the fused ring, respectively. Ring B', ring C', and R... 6p and R 8pIn the case of a fused ring, ring C' represents the benzene ring portion of the fused ring, and ring B' represents the cyclohexane ring portion of the fused ring.

[0058] The aforementioned straight-chain aliphatic hydrocarbon groups with 10 or more carbon atoms are preferably selected from straight-chain C-shaped hydrocarbons. 10-40 Alkyl and straight-chain C 10-40 The groups in the alkenyl group are preferably straight-chain C groups. 10-40 Alkyl groups, more preferably straight-chain C4 groups 10-30 Alkyl groups, especially straight-chain C4 groups. 12-28 Alkyl groups, preferably straight-chain C4 groups 14-26 alkyl.

[0059] The aforementioned linking groups are preferably -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, or -NR'C(=O)- (R' represents a hydrogen atom or C). 1-22 (Hydrocarbon group), preferably -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-, and even more preferably -O-.

[0060] The aforementioned "hydrocarbon group with a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms bonded by a single bond or linking group" is preferably a straight-chain C 10-40 Alkyl groups, with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl.

[0061] Q is preferably -O-, -C(=O)NH-, or -NHC(=O)-, and even better is -O-.

[0062] Q' is preferably a single bond, -O-, -C(=O)NH-, or -NHC(=O)-, and even better is a single bond or -O-.

[0063] In formula (Pg-5), the preferred form of L represented by formula (Pg-i-1) is the following group: wherein, L 1 divalent C 1-22 Hydrocarbon group, or CH2-O-1,4-phenylene-O-CH2, and L 2 It is a single bond, or it is a ***C(=O)N(R) bond. 3p )-R 4p -N(R 5p **** represents the group (where *** represents the group with L). 1 The binding position, **** indicates the binding position with C=O, R 4p Indicate C1-6 Alkylene, R 3p and R 5p Each C atom can be independently represented by a hydrogen atom or by an optional substitution. 1-6 Alkyl, or R 3p and R 5p Together they can form optional substitutions of C 1-6 Alkylene).

[0064] Another preferred form of L represented by formula (Pg-i-1) is the following group: wherein, L 1 divalent C 1-22 hydrocarbon groups, and L 2 It is a single key.

[0065] Another preferred form of L represented by formula (Pg-i-1) is the following group: wherein, L 1 Ethylene, and L 2 For ***C(=O)N(R) 3p )-R 4p -N(R 5p **** represents the group (where *** represents the group with L). 1 The binding position, **** indicates the binding position with C=O, R 4p Indicate C 1-22 Alkylene, R 3p and R 5p Each can be used independently to represent a hydrogen atom or a carbon atom. 1-22 Alkyl, or R 3p and R 5p They can form a ring together.

[0066] Another preferred form of L represented by formula (Pg-i-1) is the following group: wherein, L 1 Ethylene, and L 2 For ***C(=O)N(R) 3p )-R 4p -N(R 5p **** represents the group (where *** represents the group with L). 1 The binding position, **** indicates the binding position with C=O, N(R) 3p )-R 4p -N(R 5p (Partially forms 1,4-piperazinediyl).

[0067] Another preferred form of L represented by formula (Pg-i-1) is the following group: wherein, L 1 Ethylene, and L 2 For ***C(=O)N(R) 3p )-R 4p -N(R 5p **** represents the group (where *** represents the group with L). 1 The binding position, **** indicates the binding position with C=O, R 4p R represents pentylene or hexylene. 3p and R 5p Each can be used independently to represent a hydrogen atom or a methyl group.

[0068] Another preferred form of L represented by formula (Pg-i-1) is the following group: wherein, L 1 For optional phenylene with substituents, and L 2 For ***C(=O)N(R) 3p )-R 4p -N(R 5p **** represents the group (where *** represents the group with L). 1 The binding position, **** indicates the binding position with C=O, N(R) 3p )-R 4p -N(R 5p (Partially forms 1,4-piperazinediyl).

[0069] L represented by formula (Pg-i-1) is preferably succinyl.

[0070] Next, we will explain L in equation (Pg-i-2) as represented by equation (Pg-5): L in equation (Pg-i-2) 1 It is better to have divalent C 6-10 Aromatic hydrocarbon group, preferably phenylene. L in formula (Pg-i-2) 2 It is better to have a single bond. As for L in equation (Pg-i-2) 1 and L 2 The optimal combination is L 1 divalent C 6-10 Aromatic hydrocarbon groups, L 2 This is a combination of single bonds. As L in equation (Pg-i-2) 1 and L 2 A better combination is L 1 It is phenylene, L 2 For combinations of single bonds. R in equation (Pg-i-2) 1p and R 2p C is better when they are independent.1-22 Alkyl, preferably C 1-10 alkyl.

[0071] The preferred form of L represented by formula (Pg-i-2) is the following group: wherein, R 1p and R 2p C, each independently 1-22 alkyl, L 1 divalent C 6-10 Aromatic hydrocarbon groups, and L 2 It is a single key.

[0072] Another preferred form of L represented by formula (Pg-i-2) is the following group: wherein, R 1p and R 2p C, each independently 1-10 alkyl, L 1 It is a phenylene, and L 2 It is a single key.

[0073] In formula (Pg-5), when Y is NR, R is preferably a hydrogen atom or C. 1-6 Alkyl or C 7-16 Aryl group, more preferably hydrogen atom, methyl, ethyl or benzyl, and even more preferably hydrogen atom. Y is preferably single bond, oxygen atom or NR, and more preferably single bond or oxygen atom.

[0074] In formula (Pg-ii-1), R 6p Preferably, it is a hydrogen atom. Furthermore, in formula (Pg-ii-1), R... a and R b It is better to have hydrogen atoms, or to form an oxo group together.

[0075] The preferred form of Z represented by formula (Pg-ii-1) is the following group: wherein, R a and R b It is a hydrogen atom. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 alkyl.

[0076] Another preferred form of Z, represented by formula (Pg-ii-1), is the following group: wherein, R aand R b It is a hydrogen atom. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C-chain linked by -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl.

[0077] Another preferred form of Z, represented by formula (Pg-ii-1), is the following group: wherein, R a It is a hydrogen atom. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 Alkyl groups, and R b The group represented by formula (Pg-iii) (where * represents the binding position, j is an integer from 0 to 3, j Qs are -O-, j Rs are -O-) 9p Each is independently a linear C 10-40 Alkyl groups, and R 8p (For hydrogen atoms).

[0078] Another preferred form of Z, represented by formula (Pg-ii-1), is the following group: wherein, R a It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, k R 7p Each is independently a linear C 10-40 Alkyl groups, and R b The group represented by formula (Pg-iii) (where * represents the binding position, j is an integer from 0 to 3, j Qs are -O-, j Rs are -O-) 9p Each is independently a linear C 10-40 Alkyl, and R 8p With R 6p Together they represent a single bond or -O-, to form a fused ring together with ring A' and ring C'.

[0079] Another preferred form of Z, represented by formula (Pg-ii-1), is the following group: wherein, Ra and R b Together they form an oxygen group. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 alkyl.

[0080] Another preferred form of Z, represented by formula (Pg-ii-1), is the following group: wherein, R a and R b Together they form an oxygen group. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, k R 7p Each is independently a linear C-chain linked by -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl.

[0081] The preferred form of Z, represented by formula (Pg-ii-2), is the following group: wherein, R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 Alkyl groups, with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl.

[0082] In formula (Pg-ii-3), R 6p Preferably, it is a hydrogen atom. Furthermore, in formula (Pg-ii-3), R... a and R b It is better to have hydrogen atoms, or to form an oxo group together.

[0083] The preferred form of Z, represented by formula (Pg-ii-3), is the following group: wherein, R a and R b It is a hydrogen atom. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 alkyl.

[0084] Another preferred form of Z, represented by formula (Pg-ii-3), is the following group: wherein, R a and R b It is a hydrogen atom. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C-chain linked by -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl.

[0085] Another preferred form of Z, represented by formula (Pg-ii-3), is the following group: wherein, R a It is a hydrogen atom. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 Alkyl groups, and R b The group represented by formula (Pg-iii) (where * represents the binding position, j is an integer from 0 to 3, j Qs are -O-, j Rs are -O-) 9p Each is independently a linear C 10-40 Alkyl, and R 8p (For hydrogen atoms).

[0086] Another preferred form of Z, represented by formula (Pg-ii-3), is the following group: wherein, R a It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, k R 7p Each is independently a linear C 10-40 Alkyl groups, and R b The group represented by formula (Pg-iii) (where * represents the binding position, j is an integer from 0 to 3, j Qs are -O-, j Rs are -O-) 9pEach is independently a linear C 10-40 Alkyl, and R 8p With R 6p Together they represent a single bond or -O-, to form a fused ring together with ring B' and ring C'.

[0087] Another preferred form of Z, represented by formula (Pg-ii-3), is the following group: wherein, R a and R b Together they form an oxygen group. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 alkyl.

[0088] Another preferred form of Z, represented by formula (Pg-ii-3), is the following group: wherein, R a and R b Together they form an oxygen group. R 6p It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, k R 7p Each is independently a linear C-chain linked by -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl.

[0089] The preferred form of Z, represented by formula (Pg-ii-4), is the following group: wherein, Ring D' is a naphthalene ring. R a and R b It is a hydrogen atom. k is an integer from 1 to 3. k Q's are -O-, and k R 7p Each is independently a linear C 10-40 Alkyl groups, with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl.

[0090] Another preferred form of Z, represented by formula (Pg-ii-4), is the following group: wherein, Ring D' is an indole ring. R a and R b It is a hydrogen atom. k is 1, Q' is a single bond, and R 7p It is a linear C 10-40 Alkyl groups, with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl benzyl groups, or those with 1 to 3 straight-chain carbon atoms bonded via -O-. 10-40 Alkyl cyclohexylmethyl, and R 7p It binds to the nitrogen atom of the indole ring.

[0091] The preferred form of Z, represented by formula (Pg-ii-5), is the following group: wherein, u is 1 or 2; and R 10p C 4-10 Perfluoroalkyl.

[0092] Z is preferably a group represented by formula (Pg-ii-1), formula (Pg-ii-2), or formula (Pg-ii-3).

[0093] The protecting group (Pg-5) is preferably the following group: Wherein, L is a succinyl group or a group represented by formula (Pg-i-2) (in formula (Pg-i-2), R... 1p and R 2p C, each independently 1-10 Alkyl, L 1 It is a divalent phenylene, L 2 (for single keys), and YZ represents 3,4,5-tris(octadecyloxy)benzyloxy, 3,5-bis(eicosyloxy)benzyloxy, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tris(octadecyloxy)benzylamino, 2,4-bis(eicosyloxy)benzylamino, 3,5-bis(eicosyloxy)benzylamino, bis(4-eicosyloxyphenyl)methylamino, and 4-methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy] Benzylamino, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]benzylamino, 2,4-bis(dodecyloxy)benzylamino, phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino, bis[4-(12-dococoyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino, 3,4,5-tris(octadecyloxy)cyclohexylmethoxy, 3,5-bis (dococoyloxy)cyclohexylmethoxy, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino, 2,4-bis(dococoyloxy)cyclohexylmethylamino, 3,5-bis(dococoyloxy)cyclohexylmethylamino, bis(4-dococoyloxycyclohexyl)methylamino, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy [3',4',5'-tris(octadecyloxy)cyclohexylmethylamino], 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, 2,4-bis(dodecyloxy)cyclohexylmethylamino, phenyl(2,3,4-tris(octadecyloxy)cyclohexyl)methylamino, bis[4-(12-docosahexyloxydodecyloxy)cyclohexyl]methylamino, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino; or Wherein, LY is a single bond or succinyl-1,4-piperazine dimethyl, and Z is 3,4,5-tris(octadecyloxy)benzoyl, 3,5-bis(docodecyloxy)benzoyl, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl.

[0094] The protecting group (Pg-5) is preferably the following group: Where L is succinyl, and YZ represents 3,4,5-tris(octadecyloxy)benzyloxy, 3,5-bis(eicosyloxy)benzyloxy, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzyloxy, 3,4,5-tris(octadecyloxy)benzylamino, 2,4-bis(eicosyloxy)benzylamino, 3,5-bis(eicosyloxy)benzylamino, bis(4-eicosyloxyphenyl)methylamino, and 4-methoxy-2-[3',4',5'-tris(octadecyloxy)benzyloxy] Benzylamino, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]benzylamino, 2,4-bis(dodecyloxy)benzylamino, phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino, bis[4-(12-dococoyloxydodecyloxy)phenyl]methylamino, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino, 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzylamino, 3,4,5-tris(octadecyloxy)cyclohexylmethoxy, 3,5-bis (dococoyloxy)cyclohexylmethoxy, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino, 2,4-bis(dococoyloxy)cyclohexylmethylamino, 3,5-bis(dococoyloxy)cyclohexylmethylamino, bis(4-dococoyloxycyclohexyl)methylamino, 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy [3',4',5'-tris(octadecyloxy)cyclohexylmethylamino], 4-methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, 2,4-bis(dodecyloxy)cyclohexylmethylamino, phenyl(2,3,4-tris(octadecyloxy)cyclohexyl)methylamino, bis[4-(12-docosahexyloxydodecyloxy)cyclohexyl]methylamino, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino; or Wherein, LY is a single bond or succinyl-1,4-piperazine dimethyl, and Z is 3,4,5-tris(octadecyloxy)benzoyl, 3,5-bis(docodecyloxy)benzoyl, 3,5-bis[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)benzyloxy]benzoyl.

[0095] The protecting group (Pg-5) is further preferably the following group: Where L is succinyl, and YZ represents 3,4,5-tris(octadecyloxy)benzyloxy, 3,4,5-tris(octadecyloxy)cyclohexylmethoxy, 3,5-bis(dococosyloxy)cyclohexylmethoxy, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethoxy, 3,4,5-tris(octadecyloxy)cyclohexylmethylamino, 2,4-bis(dococosyloxy)cyclohexylmethylamino, 3,5-bis(dococosyloxy)cyclohexylmethoxy 4-Methoxy-2-[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, 2,4-bis(dodecyloxy)cyclohexylmethylamino, 3,5-bis[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino, or 3,4,5-tris[3',4',5'-tris(octadecyloxy)cyclohexylmethoxy]cyclohexylmethylamino; or Wherein, LY is a single bond or succinyl-1,4-piperazine dimethyl, and Z is 3,4,5-tris(octadecyloxy)benzoyl.

[0096] The protecting group (Pg-5) is particularly preferably the following group: Where L is succinyl, and YZ is 3,4,5-tris(octadecyloxy)benzyloxy, 3,4,5-tris(octadecyloxy)cyclohexylmethoxy, or phenyl(2,3,4-tris(octadecyloxy)phenyl)methylamino; or Wherein, LY is succinoyl-1,4-piperazinediyl, and Z is 3,4,5-tris(octadecyloxy)benzoyl.

[0097] The protecting group (Pg-5) is preferably one of the following groups: Where L is succinyl, and YZ is 3,4,5-tris(octadecyloxy)benzyloxy or 3,4,5-tris(octadecyloxy)cyclohexylmethoxy; or Wherein, LY is succinoyl-1,4-piperazinediyl, and Z is 3,4,5-tris(octadecyloxy)benzoyl.

[0098] As a protecting group (Pg-5), and the compounds used to form it, it can be formed or manufactured by known methods (e.g., the methods described in International Publication No. 2017 / 104836, International Publication No. 2019 / 131719, International Publication No. 2020 / 235658, International Publication No. 2021 / 039935 or International Publication No. 2021 / 198883) or methods based thereon.

[0099] Terminology related to oligonucleotide synthesis In this specification, "nucleoside" as a structural unit of oligonucleotide refers to a compound in which a nucleic acid base is bonded to the 1 position of a sugar (e.g., 2-deoxyribose, ribose, 2-deoxyribose or ribose with carbon atoms at positions 2 and 4 bonded by a divalent organic group, 2-deoxyribose or ribose with carbon atoms at positions 3 and 5 bonded by a divalent organic group, 2-deoxyribose or ribose with carbon atoms at positions 3 and 4 bonded by a divalent organic group, etc.) via N-glycosylation.

[0100] In this specification, "nucleotide" refers to a compound with a phosphate group attached to a nucleoside.

[0101] In this specification, "nucleoside" and "nucleotide" also include morpholine nucleosides and morpholine nucleotides having morpholine residues as sugar residues (e.g., 2-deoxyribose residues, ribose residues). In this specification, "morpholine nucleoside" refers to a compound represented by the following formula (1) (in the following formula, Base represents a protected nucleic acid base).

[0102] [Chemical Formula 8] .

[0103] In this specification, "nucleic acid base" refers to any base used in nucleic acid synthesis without special limitations. Examples include pyrimidine bases such as cytosine (cytosine base), uracil (uracil base), and thymine (thymine base), and purine bases such as adenine (adenine base) and guanine (guanine base). Furthermore, "protectable nucleic acid base" refers to, for example, amino or carbonyl groups in nucleic acid bases that can be protected. Preferably, a protectable nucleic acid base is one whose amino group can be protected by the aforementioned amino group protecting groups; more preferably, a nucleic acid base without an amino group, or a nucleic acid base with an amino group protected by the aforementioned amino group protecting groups.

[0104] The carbonyl group can be protected by reacting it with substances such as phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetaminophenol, pentafluorophenol, 4-neopentyloxybenzyl alcohol, 4-nitrophenylethanol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbamoyl chloride, 4-morpholinocarbamoyl chloride, diphenylcarbamoyl chloride, etc.

[0105] The term "nucleic acid base" in this specification also includes modified nucleic acid bases that have been substituted by substituents (e.g., halogen atom, alkyl, aralkyl, alkoxy, acyl, alkoxyalkyl, hydroxyl, amino, monoalkylamino, dialkylamino, carboxyl, cyano, nitro, etc.). These modified nucleic acid bases are substituted with substituents (e.g., halogen atom, alkyl, aralkyl, alkoxy, acyl, alkoxyalkyl, hydroxyl, amino, monoalkylamino, dialkylamino, carboxyl, cyano, nitro, etc.).

[0106] The term "sugar" in this specification also includes amino sugars with hydroxyl groups replaced by amino groups, and ribose with 2-hydroxyl groups replaced by halogen atoms.

[0107] Examples of amino sugars include, for example, 2-deoxyribose with the 3-hydroxyl group replaced by an amino group, ribose with the 3-hydroxyl group replaced by an amino group, and ribose with the 3-hydroxyl group replaced by an amino group and the 2-hydroxyl group replaced by a halogen (in the following formula, X...). s (Represents halogen atoms).

[0108] [Chemical Formula 9] .

[0109] Examples of compounds that are 2-deoxyribose or ribose with carbon atoms at positions 2 and 4 bonded by divalent organic groups, 2-deoxyribose or ribose with carbon atoms at positions 3 and 5 bonded by divalent organic groups, or 2-deoxyribose or ribose with carbon atoms at positions 3 and 4 bonded by divalent organic groups include the following compounds.

[0110] [Chemical Formula 10] .

[0111] 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.

[0112] The term "phosphate group" in this specification includes not only -OP(=O)(OH)2, but also groups in which the oxygen atom is replaced by a sulfur atom or NH atom (e.g., -OP(=S)(OH)2, -NH-P(=O)(OH)2, -NH-P(=S)(OH)2). Furthermore, the hydroxyl group (-OH) in the phosphate group is replaced by -OR. p (where R is in the formula) p Groups that replace organic groups (e.g., protecting groups of phosphate groups) are also included in "phosphate group".

[0113] In this specification, "phosphite bond" means 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 by NH (in the following formula, * indicates the bonding position).

[0114] [Chemical Formula 11] .

[0115] In this specification, "phosphate bond" means a bond represented by the following formula (P3-1), or a bond in which the oxygen atom is replaced by NH and represented by the following formula (P3-2) (in the following formula, * indicates the bonding position).

[0116] [Chemical Formula 12] .

[0117] In this specification, "thiophosphate bond" means a bond represented by the following formula (P4-1), or a bond in which the oxygen atom is replaced by NH and represented by the following formula (P4-2) (in the following formula, * indicates the bonding position).

[0118] [Chemical Formula 13] .

[0119] In this specification, "oligonucleotide" refers to a compound having one or more nucleotides attached to a nucleoside. It should be noted that "oligonucleotide" includes not only oligonucleotides having the phosphate ester bond shown in formula (P3-1), but also oligonucleotides having the phosphate ester bond shown in formula (P3-2), oligonucleotides having the thiophosphate ester bond shown in formula (P4-1), and oligonucleotides having the thiophosphate ester bond shown in formula (P4-2). The number of nucleosides in the oligonucleotides of this invention is not particularly limited, but is preferably 2 to 50, more preferably 2 to 30.

[0120] In this specification, "phosphite" refers to a monoamide of a phosphite diester (P(OR)2(NR2), where the four Rs each independently represent an optionally substituted alkyl group, and the two Rs of NR2 can combine with each other to form a cyclic amino group).

[0121] In this specification, "phosphoramiditized nucleoside" refers to nucleoside produced by introducing -X into it. n -P(OR)(NR2) represents the group (where X is a group that represents ... n The compound obtained by having an oxygen atom or NH, and three Rs each independently representing an optionally substituted alkyl group, wherein the two Rs of NR2 can combine with each other to form a cyclic amino group.

[0122] In this specification, "phosphoramiditized nucleotide" refers to a nucleotide that has been phosphoramiditized by introducing a -X group into it. n -P(OR)(NR2) represents the group (where X is a group that represents ... n The NR2 represents an oxygen atom or NH, and the three Rs each independently represent an optionally substituted alkyl group. The two Rs of NR2 can combine with each other to form a cyclic amino group, resulting in a compound with -X. n Compounds containing a group represented by -P(OR)(NR2) and a phosphate group. It should be noted that the phosphate group may optionally be substituted.

[0123] In this specification, "phosphoramidized oligonucleotide" refers to oligonucleotides that have undergone phosphoramidization by introducing -X into them. n -P(OR)(NR2) represents the group (where X is a group that represents ... n The compound obtained by having an oxygen atom or NH, and three Rs each independently representing an optionally substituted alkyl group, wherein the two Rs of NR2 can combine with each other to form a cyclic amino group.

[0124] In this specification, "oligonucleotide precursors with phosphite bonds" refers to precursors in which the phosphate bonds in the oligonucleotide are replaced by phosphite bonds.

[0125] Oligonucleotide precursors with phosphite bonds are preferred. (1) An oligonucleotide precursor with a phosphite bond obtained by condensation of a nucleoside, nucleotide, or oligonucleotide with a phosphoramidated nucleoside, nucleotide, or oligonucleotide, or (2) Oligonucleotide precursors with phosphite bonds obtained by condensation of oligonucleotides with phosphorous amide.

[0126] Oligonucleotide precursors with phosphite bonds are preferably oligonucleotide precursors with phosphite bonds obtained by condensation of a nucleoside, nucleotide, or oligonucleotide with a phosphoramidated nucleoside, nucleotide, or oligonucleotide.

[0127] The oligonucleotide precursor with a phosphite bond is further preferably obtained by condensation of a nucleoside, nucleotide or oligonucleotide (a) with a hydrophobic protecting group and a hydroxyl group protected by a temporary protecting group that can be removed under acidic conditions, and by phosphoramidation of the nucleoside, nucleotide or oligonucleotide (b).

[0128] The condensation can be either a condensation in which the oligonucleotide chain extends from the 3' end to the 5' end (hereinafter referred to as "3'-5' condensation") or a condensation in which the oligonucleotide chain extends from the 5' end to the 3' end (hereinafter referred to as "5'-3' condensation"). Hereinafter, the nucleoside, nucleotide or oligonucleotide (a) preferred in "3'-5' condensation" and "5'-3' condensation" respectively, and the phosphoramidized nucleoside, nucleotide or oligonucleotide (b) will be described in turn.

[0129] In the 3'-5' condensation, the nucleoside, nucleotide, or oligonucleotide (a) can be exemplified by compounds represented by formula (aI) (i.e., nucleoside or oligonucleotide). [Chemical Formula 14] .

[0130] In the formula, r represents an integer greater than or equal to 0. r+1 Bases 1 Each base can be independently represented as a protected nucleic acid base. r+1 X n1 Each can be used independently to represent an oxygen atom or NH. r+1 X n2 Each can independently represent a hydrogen atom, a halogen atom, a protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4. r R 10 Each can be used independently to represent an oxygen atom or a sulfur atom. r R p1 Each protecting group represents a phosphate group independently. Pg represents **LYZ (where ** indicates that it is related to X). n1 (the binding position), and The definitions and descriptions of L, Y, and Z are the same as those in the aforementioned protecting base (Pg-5).

[0131] When r is 0, the compound (aI) is a nucleoside; when r is 1 or higher, the compound (aI) is an oligonucleotide. Preferably r is 49 or lower, more preferably 29 or lower, even more preferably 19 or lower, particularly preferably 4 or lower, and most preferably 2 or lower.

[0132] Base as nucleic acid bases 1 The amino group is preferably protected by a protecting group. Examples of protecting groups for amino groups include the aforementioned protecting groups for amino groups, such as Base. 1 The preferred protecting group for the amino group is acetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, benzoyl, isobutyryl, (2-hexyl)decanoyl, dimethylformamidinyl, or in the form of =C(R 11 )-N(R 12 (R) 13 The group represented by ) (where R is a group in the formula) 11 R represents methyl. 12 and R 13 Represent C independently. 1-6 Alkyl, or R 11 and R 12 They can combine with carbon and nitrogen atoms to form 5- or 6-membered nitrogen-containing heterocycles. When a compound (aI) has multiple amino groups, the protecting groups of the amino groups can be only one type or two or more types.

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

[0134] As X n2 The protecting group of the protected hydroxyl group can be, for example, the aforementioned protecting groups of the hydroxyl group, preferably methyl, 2-methoxyethyl, triethylsilyl, triisopropylsilyl, or tert-butyldimethylsilyl, (2-cyanoethoxy)methyl, 1-(2-cyanoethoxy)ethyl.

[0135] As X n2 The term "divalent organic group bonded to carbon atoms at positions 2 and 4" is not particularly limited in its application, as long as it bonds to carbon atoms at positions 2 and 4 of a nucleoside. Examples of such divalent organic groups include, for instance, optionally substituted C atoms. 2-7 Alkylenes, and those selected from -O-, -NR 33 -(R) 33 Represents a hydrogen atom or C1-6 Alkyl groups, -S-, -CO-, -COO-, -OCONR 34 -(R) 34 Represents a hydrogen atom or C 1-6 Alkyl) and -CONR 35 -(R) 35 Represents a hydrogen atom or C 1-6 The divalent linking group in the alkyl group and the optionally substituted C 1-7 Divalent organic groups composed of alkylene groups, etc. As C 1-7 Alkylene and C 2-7 Alkyl groups may have substituents, for example, methylene (methylidene) (CH2=).

[0136] As a "divalent organic group bonded to carbon atoms at positions 2 and 4", it is preferably an optionally substituted C group. 2-7 Alkylene, -OR i -(R) i This indicates the C atom bonded to the 4th carbon atom. 1-6 alkylene), -O-NR 33 -R j -(R) j This indicates the C atom bonded to the 4th carbon atom. 1-6 Alkylene, R 33 -OR (meaning the same as above) k -OR l -(R) k Indicate C 1-6 Alkylene, R l This indicates that the C atoms are cross-linked with the carbon atom at position 4. 1-6 Alkylene), or better yet -OR i -(R) i (Indicates the same meaning as above), -O-NR 33 -R j -(R) j and R 33 -OR (meaning the same as above) k -OR l -(R) k and R l (This indicates the same meaning as above). As R i R j R k and R l C represents 1-6 Alkylene, preferably methylene or ethylene, respectively.

[0137] As a "divalent organic group bonded to carbon atoms at positions 2 and 4", it is more preferably -O-CH2-, -O-CH2-CH2-, or -O-NR.33 -CH2-(R 33 (meaning the same as above), -O-CH2-O-CH2-, and even better -O-CH2-, -O-CH2-CH2-, -O-NH-CH2-, -ON(CH3)-CH2-, -O-CH2-O-CH2- (which are respectively bonded to the carbon atom at position 2 on the left and to the carbon atom at position 4 on the right).

[0138] r+1 X n2 Each and every one of the R atoms is preferably a hydrogen atom, a halogen atom, or a protected hydroxyl group, and more preferably a hydrogen atom, a fluorine atom, or a protected hydroxyl group. As r R p1 Examples of protecting groups include the aforementioned phosphate group, preferably 2-cyanoethyl.

[0139] In the 3'-5' condensation, the phosphoramidized nucleoside, nucleotide or oligonucleotide (b) can be exemplified by compounds represented by formula (bI) (i.e., nucleoside or oligonucleotide).

[0140] [Chemical Formula 15] .

[0141] In the formula, s represents an integer greater than or equal to 0. s+1 Bases 2 Each base can be independently represented as a protected nucleic acid base. s+1 X n3 Each can be used independently to represent an oxygen atom or NH. s+1 X n4 Each can independently represent a hydrogen atom, a halogen atom, a protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4. "Q" is a temporary protecting group for the hydroxyl group that can be removed under acidic conditions. s R 14 Each can be used independently to represent an oxygen atom or a sulfur atom. s+1 R p2 Each protecting group independently represents a phosphate group, and R 15 and R 16 Each can be independently represented as an alkyl group, or as a 5- or 6-membered saturated cyclic amino group formed together with an adjacent nitrogen atom, wherein the saturated cyclic amino group may have one oxygen or sulfur atom as a cyclic atom in addition to the nitrogen atom.

[0142] When s is 0, compound (bI) is a phosphoramidized nucleoside; when s is 1 or more, compound (bI) is a phosphoramidized oligonucleotide. In compound (bI), 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 as nucleic acid bases 2 The amino group is preferably protected by a protecting group. In addition to the aforementioned amino group, the aforementioned -LYZ group can also be used as the protecting group. The protecting group is preferably the aforementioned amino group, and more preferably acetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, benzoyl, isobutyryl, (2-hexyl)decanoyl, dimethylformamidinyl, or in the form of =C(R 11 )-N(R 12 (R) 13 The group represented by ) (where R is a group in the formula) 11 R represents methyl. 12 and R 13 Represent C independently. 1-6 Alkyl, or R 11 and R 12 They can form 5- or 6-membered nitrogen-containing heterocycles together with the carbon and nitrogen atoms they are bonded to. When the compound (bI) has multiple amino groups, the protecting groups of the amino groups can be only one type or two or more types.

[0144] X in the above formula (bI) n4 The explanation is related to X in the aforementioned formula (aI). n2 The explanation is the same. s+1 X n4 It is preferable that each is a hydrogen atom, a halogen atom, or a protected hydroxyl group, and more preferably a hydrogen atom or a protected hydroxyl group.

[0145] R in the above formula (bI) p2 The explanation is the same as R in the aforementioned formula (aI). p1 The explanation is the same. As s+1 R p2 Examples of protecting groups include the aforementioned phosphate group, preferably 2-cyanoethyl.

[0146] R in the above formula (bI) 15 and R 16 C is better when they are separate and independent. 1-10 Alkyl groups, or 5- or 6-membered saturated cyclic amino groups formed together with the adjacent nitrogen atom, preferably C14. 1-10 Alkyl, and even better, C 1-6 alkyl.

[0147] Compound (aI) and compound (bI) can be manufactured by known methods (e.g., the methods described in International Publication No. 2017 / 104836) or methods based thereon.

[0148] The oligonucleotide precursor with phosphite bonds obtained by condensation of compound (aI) and compound (bI) is a compound represented by the following formula (cI) (the definitions and explanations of the symbols in the following formula (cI) are as described above).

[0149] [Chemical Formula 16] .

[0150] In the 5'-3' condensation, the aforementioned nucleoside, nucleotide, or oligonucleotide (a) can be exemplified by compounds represented by formula (a-I') (i.e., nucleoside or oligonucleotide). [Chemical Formula 17] .

[0151] In the formula, r represents an integer greater than or equal to 0. r+1 Bases 1 Each base can be independently represented as a protected nucleic acid base. r X n1 Each can be used independently to represent an oxygen atom or NH. r+1 X n2 Each can independently represent a hydrogen atom, a halogen atom, a protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4. X n5 Indicates hydroxyl or amino. r R 10 Each can be used independently to represent an oxygen atom or a sulfur atom. r R p1 Each protecting group represents a phosphate group independently. Pg represents **LYZ (where ** indicates the bonding position with an oxygen atom), and The definitions and descriptions of L, Y, and Z are the same as those in the aforementioned protecting base (Pg-5).

[0152] X in equation (a-I') n5 The hydroxyl group is preferred. X n5 The explanations of the symbols other than those in the aforementioned equation (aI) are the same.

[0153] In the 5'-3' condensation, the aforementioned phosphoramidized nucleoside, nucleotide, or oligonucleotide (b) can be exemplified by compounds represented by formula (b-I'); [Chemical Formula 18] .

[0154] In the formula, s represents an integer greater than or equal to 0. s+1 Bases 2 Each base can be independently represented as a protected nucleic acid base. s X n3 Each can be used independently to represent an oxygen atom or NH. s+1 X n4 Each can independently represent a hydrogen atom, a halogen atom, a protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4. X n6 This refers to a hydroxyl group protected by a temporary protecting group that can be removed under acidic conditions, or an amino group protected by a temporary protecting group that can be removed under acidic conditions. s R 14 Each can be used independently to represent an oxygen atom or a sulfur atom. s+1 R p2 Each protecting group independently represents a phosphate group, and R 15 and R 16 Each can be independently represented as an alkyl group, or as a 5- or 6-membered saturated cyclic amino group formed together with an adjacent nitrogen atom, wherein the saturated cyclic amino group may have one oxygen or sulfur atom as a cyclic atom in addition to the nitrogen atom.

[0155] X n6 Preferably, the hydroxyl group is protected by a temporary protecting group that can be removed under acidic conditions. X n6 The explanations of the symbols other than those in the previous equation (bI) are the same.

[0156] Compounds (a-I') and (b-I') can be manufactured by known methods (e.g., the methods described in International Publication No. 2017 / 104836) or methods based thereon.

[0157] The oligonucleotide precursor with phosphite bonds obtained by condensation of compounds (a-I') and (b-I') is a compound represented by the following formula (c-I') (the symbols in the formula are explained as previously).

[0158] [Chemical Formula 19] .

[0159] In the formula, r represents an integer greater than or equal to 0. r+1 Bases 1 Each base can be independently represented as a protected nucleic acid base. r+1 X n1 Each can be used independently to represent an oxygen atom or NH. r+1 X n2 Each can independently represent a hydrogen atom, a halogen atom, a protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4. r R 10 Each can be used independently to represent an oxygen atom or a sulfur atom. r R p1 Each protecting group represents a phosphate group independently. s represents an integer greater than or equal to 0. s+1 Bases 2 Each base can be independently represented as a protected nucleic acid base. s X n3 Each can be used independently to represent an oxygen atom or NH. s+1 X n4 Each can independently represent a hydrogen atom, a halogen atom, a protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4. X n6 This refers to a hydroxyl group protected by a temporary protecting group that can be removed under acidic conditions, or an amino group protected by a temporary protecting group that can be removed under acidic conditions. s R 14 Each can be used independently to represent an oxygen atom or a sulfur atom. s+1 R p2 Each protecting group represents a phosphate group independently. Pg represents **LYZ (where ** indicates the bonding position with an oxygen atom), and The definitions and descriptions of L, Y, and Z are the same as those in the aforementioned protecting base (Pg-5).

[0160] In the above formula (c-I'), it is preferable to have r R's. 10 and s R 14 At least one of them is a sulfur atom.

[0161] Next, an "oligonucleotide precursor having a phosphite bond obtained by condensation of an oligonucleotide with phosphorous amide" will be described. Examples of such precursors include compounds of formula (γ-I) or (γ-I').

[0162] [Chemical Formula 20] .

[0163] In the formula, t represents an integer greater than or equal to 1. t+1 Base 1Each base can be independently represented as a protected nucleic acid base. t+1 X n1 Each can be used independently to represent an oxygen atom or NH. t+1 X n2 Each can independently represent a hydrogen atom, a halogen atom, a protected hydroxyl group, or a divalent organic group bonded to the carbon atoms at positions 2 and 4. t R 10 Each can be used independently to represent an oxygen atom or a sulfur atom. t R p1 and R p3 Each protecting group represents a phosphate group independently. R p4 A protecting group indicating an alkyl group containing a protected amino group or a phosphate group. Pg represents **LYZ (where ** indicates that it is related to X). n1 (the binding position of the oxygen atom or the binding position of the oxygen atom), and The definitions and descriptions of L, Y, and Z are the same as those in the aforementioned protecting base (Pg-5).

[0164] The symbols in the above equations (γ-I) and (γ-I') are explained. 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. As R p3 and R p4 The "protecting group of phosphate group" can be exemplified by the aforementioned phosphate group protecting group. R p3 2-Cyanoethyl is preferred. As R p4 The "protected amino group" in "alkyl group having a protected amino group" can be exemplified by amino groups protected by the aforementioned amino groups. p4 The "alkyl group having a protected amino group" is preferably a C group having a protected amino group. 1-6 Alkyl, preferably 6-(trifluoroacetylamino)hexyl. R p4 Preferably, it is 2-cyanoethyl or 6-(trifluoroacetylamino)hexyl. Other symbols are explained as previously stated.

[0165] In this specification, "one-pot synthesis" refers to a synthesis consisting of multiple stages, including steps for synthesizing intermediates and steps for synthesizing the target final product, without separating the intermediates. It should be noted that the "step for synthesizing intermediates" may be one or more.

[0166] Precipitation methods This invention provides a method for precipitating a target oligonucleotide (i.e., an oligonucleotide with a hydrophobic protecting group) from a target solution (i.e., a solution containing the target oligonucleotide and a nonpolar solvent), wherein, The method includes: Mix the target solution with the first polar solvent, and While maintaining the volume of the target mixture (i.e., the mixture of the target solution and the first polar solvent) at more than 1.00 times the volume of the target solution, the solvent is removed from the target mixture by distillation (hereinafter also referred to as "the precipitation method of the present invention"). The first polar solvent can be a single type or a combination of two or more types.

[0167] As described above, the precipitation method of the present invention, by distilling away the solvent without concentrating the target mixture compared to the target solution, allows for the precipitation of the target oligonucleotide with high purity 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 yields target oligonucleotides with high purity compared to precipitation methods employing concentration and the addition of polar solvents. Surprisingly, as shown in the following examples and comparative examples, the precipitation method of the present invention also yields target oligonucleotides with high purity compared to precipitation methods employing only the addition of polar solvents.

[0168] From the viewpoint of the purity of the target oligonucleotide, the volume of the target mixture when the solvent is removed by distillation is more than 1.00 times the volume of the target solution, preferably more than 1.05 times, more preferably more than 1.10 times, preferably less than 3.00 times, and more preferably less than 2.70 times.

[0169] From the perspective of the purity of the target oligonucleotide, the temperature for removing the solvent by distillation is preferably above 5°C, more preferably above 7°C, even more preferably above 10°C, preferably below 35°C, more preferably below 30°C, and even more preferably below 25°C.

[0170] As long as the solvent can be removed by distillation, there is no particular limitation on the pressure during distillation to remove the solvent, but it is preferable to be 15 mmHg or more, more preferably 20 mmHg or more, even more preferably 25 mmHg or more, preferably 500 mmHg or less, more preferably 450 mmHg or less, and even more preferably 400 mmHg or less.

[0171] The target solution contains a nonpolar solvent. Examples of nonpolar solvents include: halogen 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, cyclopentylmethyl ether, and tert-butylmethyl ether. Only one nonpolar solvent may be used, or two or more may be used in combination. It should be noted that dichloromethane is classified as a nonpolar solvent in this specification.

[0172] In one embodiment of the invention, the nonpolar solvent contained in the target solution preferably comprises a halogen solvent. Only one halogen solvent may be used, or two or more may be used in combination. The nonpolar solvent is more preferably a halogen solvent, further preferably at least one selected from dichloromethane and chloroform, and particularly preferably dichloromethane.

[0173] In another embodiment of the invention, the nonpolar solvent contained in the target solution preferably comprises at least one selected from halogenated solvents and toluene. Only one halogenated solvent may be used, or two or more may be used in combination. The nonpolar solvent is more preferably selected from at least one selected from halogenated solvents and toluene, further preferably selected from at least one selected from dichloromethane, chloroform, and toluene, and particularly preferably selected from at least one selected from dichloromethane and toluene.

[0174] From the viewpoint of precipitating the target oligonucleotide, the first polar solvent preferably comprises at least one selected from nitrile solvents and alcohol solvents. Only one nitrile solvent or one or more alcohol solvents may be used. Examples of nitrile solvents include acetonitrile and propionitrile. Examples of alcohol solvents include methanol, ethanol, and isopropanol (2-propanol). In one embodiment of the invention, the first polar solvent is preferably a nitrile solvent, and more preferably acetonitrile. In another embodiment of the invention, the first polar solvent is preferably a mixture of nitrile and alcohol solvents, and more preferably a mixture of acetonitrile and isopropanol.

[0175] From the viewpoint of reducing the amount of polar solvent used and the precipitation of the target oligonucleotide, the total volume of the first polar solvent mixed with the target solution is preferably more than 1.0 times, more preferably more than 1.5 times, even more preferably more than 1.7 times, particularly preferably more than 2 times, preferably less than 3.5 times, more preferably less than 3.2 times, and even more preferably less than 3.0 times.

[0176] The target solution may contain a second polar solvent. Only one second polar solvent may be used, or two or more may be used together. It should be noted that in this 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.

[0177] The second polar solvent preferably comprises at least one selected from nitrile solvents and alcohol solvents. Only one nitrile solvent or one or more alcohol solvents may be used. Examples of nitrile solvents include acetonitrile and propionitrile. Examples of alcohol solvents include methanol, ethanol, 2,2,2-trifluoroethanol, and isopropanol. More preferably, the second polar solvent is selected from at least one selected from nitrile solvents and alcohol solvents; more preferably, it is selected from at least one selected from acetonitrile, methanol, and 2,2,2-trifluoroethanol; and particularly preferably, it is a mixture of acetonitrile, methanol, and 2,2,2-trifluoroethanol.

[0178] From the viewpoint of the dissolution of the target oligonucleotide in the target solution and the precipitation of the target oligonucleotide from the target solution when a polar solvent is added, the volume of the second polar solvent relative to the volume of the nonpolar solvent contained in the target solution is preferably 0.15 times or more, more preferably 0.17 times or more, even more preferably 0.20 times or more, preferably 0.35 times or less, more preferably 0.32 times or less, and even more preferably 0.30 times or less.

[0179] One-pot synthesis This invention also provides a method for producing oligonucleotides via a one-pot synthesis, wherein, The method includes: Step (1): In a solution containing a nonpolar solvent, 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 by a temporary protecting group that can be removed under acidic conditions, and which is 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 that can be removed under acidic conditions; Step (2) involves mixing the solution from step (1) with the quencher (i) of the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b) to quench the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b). Step (3) involves mixing the solution from step (2) with an oxidizing agent or a sulfiding agent to oxidize or sulfidate 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 the hydroxyl group being protected by a temporary protecting group that can be removed under acidic conditions. In step (4), if an oxidant is used in step (3), the solution after step (3) is mixed with an oxidant quencher (ii) as needed to quench the oxidant; In step (5), the solution after step (3) or the solution after step (4) in the case of step (4) is mixed with acid to remove the temporary protecting group of the oligonucleotide (d) that can be removed under acidic conditions, forming an oligonucleotide (e) with unprotected hydroxyl groups and hydrophobic protecting groups. Step (6): As needed, mix the solution from step (5) with alkali; and Step (7) involves precipitating the oligonucleotide (e) from a solution containing the oligonucleotide (e) and a nonpolar solvent using the precipitation method of the present invention (hereinafter also referred to as "the manufacturing method of the present invention").

[0180] (Process (1) (Contraction)) In step (1), in a solution containing a nonpolar solvent, 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 by a temporary protecting group that can be removed under acidic conditions, and which is 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 that can be removed under acidic conditions.

[0181] The hydrophobic protecting group of the nucleoside, nucleotide, or oligonucleotide (a) is preferably the aforementioned protecting group (Pg-5). The protecting group (Pg-5) is described above.

[0182] From the viewpoint of ease of deprotection, the temporary protecting group of the hydroxyl group of the phosphoramidated nucleoside, nucleotide or oligonucleotide (b) is preferably 4,4'-dimethoxytriphenylmethyl or 4-monomethoxytriphenylmethyl, and more preferably 4,4'-dimethoxytriphenylmethyl.

[0183] The combination of the aforementioned nucleoside, nucleotide or oligonucleotide (a) and the aforementioned phosphoramidized nucleoside, nucleotide or oligonucleotide (b) used in step (1) is preferably a combination of the aforementioned compound (aI) and the aforementioned compound (bI), or a combination of the aforementioned compound (a-I') and the aforementioned compound (b-I'), more preferably a combination of the aforementioned compound (aI) and the aforementioned compound (bI) with Pg of **LYZ, or a combination of the aforementioned compound (a-I') and the aforementioned compound (b-I') with Pg of **LYZ.

[0184] Step (1) is carried out in a solution containing a nonpolar solvent. Examples of nonpolar solvents include: halogen 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, cyclopentylmethyl ether, and tert-butylmethyl ether. Only one nonpolar solvent may be used, or two or more may be used in combination.

[0185] In one embodiment of the invention, the nonpolar solvent is preferably selected from at least one of halogen solvents, aromatic solvents, ester solvents, and aliphatic solvents, more preferably from at least one of halogen solvents and toluene, further preferably from at least one of chloroform, dichloromethane, and toluene, and particularly preferably dichloromethane and / or toluene. In another embodiment of the invention, the nonpolar solvent is preferably a halogen solvent, more preferably from at least one of dichloromethane and chloroform, and further preferably dichloromethane. Steps following step (1) are also carried out in a solution containing a nonpolar solvent.

[0186] Solutions containing nonpolar solvents may further contain aprotic polar solvents. Examples of aprotic polar solvents include nitrile solvents such as acetonitrile and propionitrile; and polar ether solvents such as tetrahydrofuran, with nitrile solvents being preferred, and acetonitrile being more preferred. The molar ratio of the aprotic polar solvent relative to 100 mL of nonpolar solvent is preferably 10 to 100 mL, and more preferably 10 to 50 mL.

[0187] The molar ratio of the aforementioned phosphoramidized nucleoside, nucleotide, or oligonucleotide (b) relative to 1 mol of the aforementioned 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.

[0188] The reaction temperature of step (1) is not particularly limited as long as condensation is carried out; for example, it can be 0 to 100°C, but preferably 20 to 50°C. The reaction time varies depending on the type of raw materials used, the reaction temperature, etc., and can range from 5 minutes to 24 hours.

[0189] To promote condensation, known activators can be used. Examples of activators include pyridine trifluoroacetate, tetrazolium, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole, and 4,5-dicyanimidazolium. Only one activator may be used, or two or more may be used in combination. When using an activator, its amount relative to 1 mol of the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b) is preferably 0.5 to 10 mol, more preferably 1 to 5 mol.

[0190] (Step (2) (Quenching of phosphoramidized nucleosides, nucleotides or oligonucleotides (b))) In step (2), the solution after step (1) is mixed with the quencher (i) of the phosphoramidated nucleoside, nucleotide or oligonucleotide (b) to quench the phosphoramidated nucleoside, nucleotide or oligonucleotide (b).

[0191] Quenching agent (i) may be used in combination with only one type or in combination with two or more types. Examples of quenching agents (i) include water, alcohols, phenols and amines.

[0192] Examples of alcohols that can be used as quenchers (i) include, for example, monohydric alcohols that can be halogenated, such as methanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol, tetrahydrofurfuryl alcohol, furfuryl alcohol, 2,3-O-isopropylidene-D-ribofuranoside, and 3'-O-triisopropylsilyl-thymidine; and polyhydric alcohols that can be halogenated, such as ethylene glycol and diethylene glycol.

[0193] Examples of phenols that can be used as quenchers (i) include 4-nitrophenol and pentafluorophenol. Examples of amines that can be used as quenchers (i) include morpholine.

[0194] The amount of quencher (i) relative to 1 mol of phosphoramidized nucleoside, nucleotide or oligonucleotide (b) used in step (1) is preferably 1 to 20 mol, more preferably 1 to 10 mol, and even more preferably 1 to 5 mol.

[0195] The temperature of the solution after mixing with quencher (i) is not particularly limited, as long as it can quench the phosphoramidized nucleosides, nucleotides, or oligonucleotides (b), but is preferably 5–40°C, and more preferably 15–30°C. The stirring time of the solution after mixing with quencher (i) varies depending on the type of quencher (i) used, the temperature, etc., and is, for example, 10 minutes to 3 hours.

[0196] (Process (3) (Oxidation or Sulfidation)) In step (3), the solution from 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 the hydroxyl group being protected by a temporary protecting group that can be removed under acidic conditions. Only one oxidizing agent or one or more sulfurizing agents may be used.

[0197] There are no particular limitations on the oxidizing agent; any known oxidizing agent may be used. Examples of oxidizing agents include iodine, (1S)-(+)-(10-camphenylsulfonyl)oxazolidinyl propane, tert-butyl hydroperoxide (TBHP), 2-butanone peroxide, 1,1-dihydroperoxycyclododecane, bis(trimethylsilyl)peroxide, and m-chloroperoxybenzoic acid. Furthermore, the oxidizing agents described in International Publication No. 2022 / 172994 may also be used.

[0198] There are no particular limitations on the vulcanizing agent, and known vulcanizing agents can be used. Examples of vulcanizing agents include 5-phenyl-3H-1,2,4-dithiazol-3-one (POS), 5-[(N,N-dimethylaminomethylene)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-dithiazol-5-thione (ADTT).

[0199] The ratio of oxidant or sulfidant to 1 mole of the above oligonucleotide precursor (c) is, for example, 1 to 50 moles, preferably 1 to 5 moles.

[0200] The reaction temperature is not particularly limited as long as the reaction proceeds, but it is preferably 0℃ to 100℃, and more preferably 20℃ to 50℃. The reaction time varies depending on the type of oligonucleotide precursor (c), the type of oxidizing or sulfiding agent used, the reaction temperature, etc., and may range from 1 minute to 3 hours.

[0201] (Use of aromatic amines) It is preferable to mix the solution with an aromatic amine before, during, or after oxidation or sulfidation. That is, it is preferable to carry out oxidation or sulfidation in the presence of an aromatic amine, and / or to mix the oxidized or sulfidated solution with a cation scavenger. Only one aromatic amine may be used, or two or more may be used in combination. By using aromatic amines, the formation of byproducts can be suppressed.

[0202] 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-dimethylaniline).

[0203] From the viewpoint of suppressing byproducts, the amount of aromatic amine relative to 1 mol of oxidant or sulfiding 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.

[0204] (Step (4) (Quenching of oxidant)) If an oxidant is used in step (3), step (4) is performed as needed. In step (4), the solution after step (3) is mixed with an oxidant quencher (ii) to quench the oxidant. Only one quencher (ii) may be used, or two or more may be used together.

[0205] Quenching agent (ii) is preferably an organophosphorus compound. Only one organophosphorus compound may be used, or two or more may be used in combination.

[0206] The organophosphorus compound is preferably selected from at least one of phosphine derivatives, triphosphite, hypophosphite esters, phosphonate diesters, and hypophosphite esters, and more preferably phosphine derivatives. Only one type of phosphine derivative may be used, or two or more may be used in combination.

[0207] Phosphines are compounds represented by the formula: P(R)3 (where the three Rs independently represent a hydrogen atom, an alkyl group, or an aryl group). Examples of phosphines include triphenylphosphine and methyldiphenylphosphine.

[0208] Triphosphite is a compound represented by the formula: P(OR')3 (where the three R's independently represent alkyl or aryl groups). Examples of triphosphite include triethyl phosphite.

[0209] Hypophosphonates are compounds represented by the formula: P(OR')(R)2 (where the two Rs independently represent a hydrogen atom, an alkyl group, or an aryl group, and R' represents an alkyl group or an aryl group). Examples of hypophosphonates include ethoxydiphenylphosphine.

[0210] Phosphite diesters are compounds represented by the formula: P(R)(OR')2 (where R represents a hydrogen atom, alkyl or aryl group, and the two R's independently represent an alkyl or aryl group). Examples of phosphite diesters include, for instance, diethoxyphenylphosphine.

[0211] Phosphinates are compounds represented by the formula: P(=O)(R)2(OR') (where the two Rs independently represent a hydrogen atom, an alkyl group, or an aryl group, and R' represents an alkyl group or an aryl group, and one R and R' can form a heterocycle together with the phosphorus atom and oxygen atom they are bonded to). Examples of phosphinates include 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide, represented by the following formula.

[0212] [Chemical Formula 21] .

[0213] The amount of quencher (ii) should be sufficient to quench the remaining portion of the oxidant used in step (3), and is preferably 1 to 10 mol, more preferably 1 to 5 mol, relative to the remaining amount of 1 mol of oxidant (i.e., “amount of oxidant used in step (3)” - “amount of phosphoramidized nucleosides, nucleotides or oligonucleotides (b) used in step (1)”).

[0214] The temperature of the solution after mixing quencher (ii) is only required to quench the oxidant and is not particularly limited, but preferably 0℃~50℃, more preferably 10℃~40℃. The stirring time of the solution after mixing quencher (ii) varies depending on the type of quencher (ii) used, the temperature, etc., but preferably 5 minutes to 5 hours, more preferably 5 minutes to 2 hours.

[0215] (Step (5) (Removal of temporary protective substrate)) In step (5), the solution after step (3), or the solution after step (4) in the case of step (4), is mixed with an acid to remove the temporary protecting group of the oligonucleotide (d) that can be removed under acidic conditions, forming an oligonucleotide (e) with unprotected hydroxyl groups and hydrophobic protecting groups. Only one acid may be used, or two or more acids may be used together.

[0216] The acid is only required to effectively remove the temporary protecting group; there are no particular limitations. Examples include trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid, methanesulfonic acid, hydrochloric acid, acetic acid, and p-toluenesulfonic acid. From the viewpoint of effectively removing the temporary protecting group, trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, and trichloroacetic acid are preferred; further preferred are trifluoroacetic acid, dichloroacetic acid, and trifluoromethanesulfonic acid; even further preferred are trifluoroacetic acid and trifluoromethanesulfonic acid; and most preferably are trifluoroacetic acid.

[0217] The acid content relative to 1 mol oligonucleotide (d) is, for example, 1 to 100 mol, preferably 1 to 40 mol.

[0218] The reaction temperature in step (5) can be set as long as the reaction proceeds, without any particular limitation. For example, it can be -10℃ to 50℃, and preferably 0℃ to 40℃. The reaction time varies depending on the oligonucleotide (d) used, the type of acid and the type of nonpolar solvent, the reaction temperature, etc., and can be, for example, 5 minutes to 5 hours.

[0219] (Use of cation scavengers) It is preferable to mix the cation scavenger with the solution before, during, or after the removal of the temporary protecting group of the oligonucleotide (d). That is, it is preferable to remove the temporary protecting group in the presence of the cation scavenger, or to mix the solution after the temporary protecting group has been removed with the cation scavenger. Only one cation scavenger may be used, or two or more may be used in combination.

[0220] As a cation scavenger, there are no particular limitations as long as reprotection from the removed temporary protecting group or side reactions to the deprotected functional groups do not occur. In one embodiment of the invention, the cation scavenger is at least one selected from pyrrole, pyrrole derivatives, indole, indole derivatives, furan derivatives, and mercapto-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 mentholfuran. Examples of thiol-containing compounds include thiomalic acid, 3-mercaptopropionic acid, cysteine, cysteylglutamic acid, 1-octylthiol, 1-dodecylthiol, cyclohexylthiol, and 2,2'-(1,2-ethylenedioxy)diethylthiol. The molar ratio of the cation scavenger relative to 1 mol of the aforementioned oligonucleotide (d) is preferably 1–50 mol, more preferably 5–20 mol.

[0221] (Process (6) (Neutralization)) To neutralize the acid used in step (5), step (6) is performed as needed. In step (6), the solution from step (5) is mixed with alkali. It should be noted that the acid used in step (5) can be removed from the oligonucleotide (e) by performing solid-liquid separation and washing, therefore step (6) (neutralization) is not necessary.

[0222] A base may be used in isolation or in combination of two or more. Organic bases are preferred. Examples of organic bases 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 them, 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 even more preferred; pyridine, 2,4,6-trimethylpyridine, benzimidazole, and 1,2,4-triazole are even more preferred; and pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, and benzimidazole are particularly preferred.

[0223] The amount of alkali in step (6) is preferably 1 to 10 mol relative to 1 mol of acid used in step (5), and more preferably 1 to 3 mol.

[0224] (Process (7) (Precipitation)) In step (7), the oligonucleotide (e) is precipitated from a solution containing the oligonucleotide (e) and a nonpolar solvent (i.e., the solution after step (5) or, if necessary, the solution after step (6)) by the precipitation method of the present invention. The precipitation method of the present invention is described above.

[0225] Deprotection After the precipitation method of the present invention, the protecting groups (e.g., phosphate protecting groups, hydrophobic protecting groups) of the obtained oligonucleotide can be removed by known methods. For example, 2-cyanoethyl and the aforementioned protecting groups (Pg-5), which are phosphate protecting groups, can be removed by treatment with ammonia, an ammonia / ethanol solution, or a mixture of ammonia and methylamine aqueous solution. Example

[0226] The present invention will be described in more detail below with examples and embodiments. The present invention is not limited to the following examples and embodiments, and may be implemented by appropriate changes within the scope of the spirit of the context, all of which are included within the technical scope of the present invention.

[0227] In the following examples, "room temperature" refers to "20°C to 30°C". Furthermore, the meanings of abbreviations used in the following examples are as follows.

[0228] Monomer (1m): Thymidine-3'-yl-[3,4,5-tris(octadecoxy)benzyl]succinate 5'-DMT-dG(iBu)-3'-phosphoramide: 5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyryl-deoxyguanosine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide 5'-DMT-dA(dma)-3'-phosphoramide: 5'-O-(4,4'-dimethoxytriphenylmethyl)-N6-[1-(dimethylamino)ethylene]-deoxyadenosine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide 5'-DMT-dmeC(Bz)-3'-phosphoramide: 5'-O-(4,4'-dimethoxytriphenylmethyl)-N4-benzoyl-2'-deoxy-5-methylcytidine-3'-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide DMTr: 4,4'-Dimethoxytriphenylmethyl POS: 5-Phenyl-3H-1,2,4-Dithiazolyl-3-one.

[0229] Example 1: Synthesis and solid-liquid separation of dimers [Chemical Formula 22] .

[0230] 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 sieve 3A (6.1 g) was added to this mixture, and the mixture was stirred at room temperature for 30 min. Then, 5'-DMT-dG(iBu)-3'-phosphoramide (3.4 g, 4.0 mmol) was added, and the mixture was stirred at room temperature for 15 min. 5-Ethylthio-1H-tetrazole (0.53 g, 4.0 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 h. 2,2,2-trifluoroethanol (1.5 mL, 20.2 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 min. Then, 2,6-dimethylaniline (1.9 mL, 15.2 mmol) and POS (0.99 g, 5.1 mmol) were added, and the mixture was stirred at room temperature for 19 h. After adding 2,3-dimethylfuran (2.1 mL, 20.2 mmol), a mixed solution of trifluoroacetic acid (5.2 mL, 67.5 mmol) and 2,6-dimethylaniline (0.083 mL, 0.67 mmol) dissolved in anhydrous chloroform (10.4 mL) was added dropwise, and the mixture was stirred at room temperature for 1.5 hours. Then, 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. Filter the reaction mixture, and wash the funnel used for filtration with chloroform (10 mL) to obtain a target solution as a mixture of chloroform used for washing and filtrate (volume of target solution: 190 mL, volume of nonpolar solvent (chloroform): 120.5 mL, volume of secondary polar solvent (acetonitrile, methanol and 2,2,2-trifluoroethanol): 32.46 mL, volume of secondary polar solvent / volume of nonpolar solvent: 0.27).

[0231] Acetonitrile, acting as the first polar solvent, was added dropwise to the obtained target solution to adjust the volume of the mixture of the target solution and the first polar solvent 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). Simultaneously, the solvent was removed by distillation at a temperature of 14.8–23.4 °C and a pressure of 28–254 mmHg (total volume of the first polar solvent added: 285 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.37). The precipitated solid was filtered and dried to obtain a dimer (2m) (3.2 g, yield: 92.6%) as a white solid.

[0232] m / z (TOF-MS): Theoretical value 1705.08, measured value 1706.0773 [M+H] + .

[0233] Example 2: Synthesis and solid-liquid separation of trimers [Chemical Formula 23] .

[0234] Under an argon atmosphere, the dimer (2m) (3.0 g, 1.8 mmol) and triphenylphosphine (0.23 g, 0.90 mmol) obtained in Example 1 were dissolved in a mixed solvent of acetonitrile (26.6 mL) and dichloromethane (88.5 mL). Molecular sieve 3A (5.3 g) was added to the mixture, and after stirring at room temperature for 30 minutes, 5'-DMT-dA(dma)-3'-phosphoramide (2.9 g, 3.6 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Then, 5-ethylthio-1H-tetrazole (0.46 g, 3.6 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 hours. 2,2,2-Trifluoroethanol (1.3 mL, 1.8 mmol) was added to the reaction mixture, and the mixture was 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 the mixture was stirred at room temperature for 19 hours. 2,3-Dimethylfuran (1.9 mL, 17.7 mmol) was added, followed by the addition dropwise 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 used for filtration was washed with dichloromethane (12 mL) to obtain a target solution as a mixture of dichloromethane used for washing and filtrate (volume of target solution: 167.3 mL, volume of nonpolar solvent (dichloromethane): 109.5 mL, volume of secondary polar solvent (acetonitrile, methanol and 2,2,2-trifluoroethanol): 28.48 mL, volume of secondary polar solvent / volume of nonpolar solvent: 0.26).

[0235] Acetonitrile, acting as the first polar solvent, was added dropwise to the obtained target solution to adjust the volume of the mixture of the target solution and the first polar solvent 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). Simultaneously, the solvent was removed by distillation at a temperature of 17.3–24.0 °C and a pressure of 74–360 mmHg (total volume of the first polar solvent added: 251 mL; total volume of the first polar solvent / volume of the non-polar solvent: 2.29). The precipitated solid was filtered and dried to obtain a trimer (3m) (3.7 g, yield: 97.8%) as a white solid.

[0236] m / z (TOF-MS): Theoretical value 2156.20, measured value 2157.20 [M+H]+ .

[0237] Example 3: Synthesis and solid-liquid separation of tetramers [Chemical Formula 24] .

[0238] Under an argon atmosphere, the trimer (3m) (3.4 g, 1.6 mmol) and triphenylphosphine (0.21 g, 0.81 mmol) obtained in Example 2 were dissolved in a mixed solvent of acetonitrile (24.1 mL) and dichloromethane (80.4 mL). Molecular sieve 3A (4.8 g) was added to the mixture, and after stirring at room temperature for 30 minutes, 5'-DMT-dA(dma)-3'-phosphoramide (2.6 g, 3.2 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Then, 5-ethylthio-1H-tetrazole (0.42 g, 3.2 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 hours. Add 1.2 mL (1.6 mmol) of 2,2,2-trifluoroethanol to the reaction mixture, stir for 30 minutes at room temperature, then add 1.5 mL (12.2 mmol) of 2,6-dimethylaniline and POS (0.78 g, 4.0 mmol), and stir for 19 hours at room temperature. Add 1.7 mL (16.1 mmol) of 2,3-dimethylfuran, then add dropwise a mixture of 4.1 mL (53.9 mmol) of trifluoroacetic acid and 0.067 mL (0.54 mmol) dissolved in anhydrous dichloromethane (8.3 mL), and stir for 2 hours at room temperature. Add 15.7 mL (134.6 mmol) of 2,6-dimethylpyridine and 0.52 mL of methanol to the reaction mixture, and stir for 45 minutes at room temperature. Filter the reaction mixture, and wash the funnel used for filtration with dichloromethane (10 mL) to obtain a target solution as a mixture of dichloromethane used for washing and filtrate (volume of target solution: 153 mL, volume of nonpolar solvent (dichloromethane): 98.7 mL, volume of secondary polar solvent (acetonitrile, methanol and 2,2,2-trifluoroethanol): 25.82 mL, volume of secondary polar solvent / volume of nonpolar solvent: 0.26).

[0239] Acetonitrile, acting as the first polar solvent, was added dropwise to the obtained target solution to adjust the volume of the mixture of the target solution and the first polar solvent 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). Simultaneously, the solvent was removed by distillation at a temperature of 13.7–21.2 °C and a pressure of 36–400 mmHg (total volume of the first polar solvent added: 230 mL; total volume of the first polar solvent / volume of the non-polar solvent: 2.33). The precipitated solid was filtered and dried to obtain a tetramer (4m) (4.1 g, yield: 97.9%) as a white solid.

[0240] m / z (TOF-MS): Theoretical value 2608.32, measured value 2608.32 [M+H] + .

[0241] Example 4: Synthesis and solid-liquid separation of pentamers [Chemical Formula 25] .

[0242] Under an argon atmosphere, the tetramer (4m) (3.8 g, 1.5 mmol) and triphenylphosphine (0.19 g, 0.74 mmol) obtained in Example 3 were dissolved in a mixed solvent of acetonitrile (22.1 mL) and dichloromethane (73.7 mL). Molecular sieve 3A (4.4 g) was added to the mixture, and after stirring at room temperature for 30 minutes, 5'-DMT-dG(iBu)-3'-phosphoramide (2.5 g, 3.0 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Then, 5-ethylthio-1H-tetrazole (0.38 g, 2.9 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 hours. Add 1.1 mL (1.5 mmol) of 2,2,2-trifluoroethanol to the reaction mixture, stir for 30 minutes at room temperature, then add 1.4 mL (11.1 mmol) of 2,6-dimethylaniline and POS (0.72 g, 4.0 mmol), and stir for 17 hours at room temperature. Add 1.5 mL (14.7 mmol) of 2,3-dimethylfuran, then add dropwise a mixture of 3.8 mL (49.2 mmol) of trifluoroacetic acid and 0.061 mL (0.49 mmol) dissolved in anhydrous dichloromethane (7.5 mL), and stir for 2 hours at room temperature. Add 14.3 mL (122.8 mmol) of 2,6-dimethylpyridine and 0.48 mL of methanol to the reaction mixture, and stir for 45 minutes at room temperature. Filter the reaction mixture, and wash the funnel used for filtration with dichloromethane (9 mL) to obtain a target solution as a mixture of dichloromethane used for washing and filtrate (volume of target solution: 140 mL, volume of nonpolar solvent (dichloromethane): 90.2 mL, volume of secondary polar solvent (acetonitrile, methanol and 2,2,2-trifluoroethanol): 23.68 mL, volume of secondary polar solvent / volume of nonpolar solvent: 0.26).

[0243] Acetonitrile, acting as the first polar solvent, was added dropwise to the obtained target solution to adjust the volume of the mixture of the target solution and the first polar solvent 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). Simultaneously, the solvent was removed by distillation under conditions of 10.3–23.9 °C and 43–400 mmHg (total volume of the first polar solvent added: 211 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.34). The precipitated solid was filtered and dried to obtain a pentamer (5m) (4.5 g, yield: 100.0%) as a white solid.

[0244] m / z (TOF-MS): Theoretical value 3075.42, measured value 1538.71 [M+2H]+ .

[0245] Example 5: Synthesis and solid-liquid separation of hexamer [Chemical Formula 26] .

[0246] Under an argon atmosphere, the pentamer (5m) (4.3 g, 1.4 mmol) and triphenylphosphine (0.18 g, 0.71 mmol) obtained in Example 4 were dissolved in a mixed solvent of acetonitrile (20.9 mL) and dichloromethane (69.8 mL). Molecular sieve 3A (4.2 g) was added to the mixture, and after stirring at room temperature for 30 minutes, 5'-DMT-dmeC(Bz)-3'-phosphoramide (2.4 g, 2.8 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Then, 5-ethylthio-1H-tetrazole (0.36 g, 2.8 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 hours. Add 1.0 mL (1.4 mmol) of 2,2,2-trifluoroethanol to the reaction mixture, stir for 30 minutes at room temperature, then add 1.3 mL (10.5 mmol) of 2,6-dimethylaniline and POS (0.68 g, 3.5 mmol), and stir for 18 hours at room temperature. Add 1.5 mL (13.9 mmol) of 2,3-dimethylfuran, then add dropwise a mixture of 3.4 mL (43.8 mmol) of trifluoroacetic acid and 0.054 mL (0.44 mmol) dissolved in anhydrous dichloromethane (6.7 mL), and stir for 2 hours at room temperature. Add 13.6 mL (116.4 mmol) of 2,6-dimethylpyridine and 0.45 mL of methanol to the reaction mixture, and stir for 45 minutes at room temperature. Filter the reaction mixture, and wash the funnel used for filtration with dichloromethane (9 mL) to obtain a target solution as a mixture of dichloromethane used for washing and filtrate (volume of target solution: 134 mL, volume of nonpolar solvent (dichloromethane): 85.5 mL, volume of secondary polar solvent (acetonitrile, methanol and 2,2,2-trifluoroethanol): 22.35 mL, volume of secondary polar solvent / volume of nonpolar solvent: 0.26).

[0247] Acetonitrile, acting as the first polar solvent, was added dropwise to the obtained target solution to adjust the volume of the mixture of the target solution and the first polar solvent 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). Simultaneously, the solvent was removed by distillation under conditions of 10.5–21.2 °C and 48–400 mmHg (total volume of the first polar solvent added: 201 mL, total volume of the first polar solvent / volume of the non-polar solvent: 2.35). The precipitated solid was filtered and dried to obtain hexamer (6m) (4.8 g, yield: 98.6%) as a white solid.

[0248] m / z (TOF-MS): Theoretical value 3551.51, measured value 1776.77 [M+2H] + .

[0249] <Deprotection reaction> The hexamer (6m) (5 mg) obtained in Example 5 was mixed with 28% (w / w) ammonia (5 mL), and the mixture was heated at 65°C for 4 hours and then cooled to room temperature. After removing unwanted substances from the reaction mixture through a syringe filter, the mixture was concentrated under reduced pressure using a centrifugal evaporator to obtain the deprotected form of the hexamer.

[0250] Comparative Example 1-1: Synthesis and Solid-Liquid Separation of Dimers (Precipitation with Added Polar Solvents 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 sieve 3A (3.7 g) was added to this mixture, and the mixture was stirred at room temperature for 30 min. Then, 5'-DMT-dG(iBu)-3'-phosphoramide (2.1 g, 2.5 mmol) was added, and the mixture was stirred at room temperature for 15 min. 5-Ethylthio-1H-tetrazole (0.31 g, 2.4 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 h. 2,2,2-trifluoroethanol (0.91 mL, 12.5 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 min. Then, 2,6-dimethylaniline (1.2 mL, 9.4 mmol) and POS (0.61 g, 3.1 mmol) were added, and the mixture was stirred at room temperature for 1 h. After adding 2,3-dimethylfuran (1.3 mL, 12.4 mmol), a mixed solution of trifluoroacetic acid (3.0 mL, 39.1 mmol) and 2,6-dimethylaniline (0.048 mL, 0.42 mmol) dissolved in anhydrous chloroform (6.4 mL) was added dropwise, and the mixture was stirred at room temperature for 1.5 hours. Then, 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. After filtering the reaction mixture, acetonitrile (525 mL) was added to the filtrate, and the precipitated solid was collected and dried to obtain a white solid as a dimer (2 m) (2.0 g, yield: 94.3%). m / z (TOF-MS): Theoretical value 1705.08, measured value 1706.0773 [M+H] + .

[0251] Comparative Examples 1-2: Synthesis and Solid-Liquid Separation of Trimers Under an argon atmosphere, the dimer (2m) (1.8 g, 1.1 mmol) and triphenylphosphine (0.14 g, 0.53 mmol) obtained in Comparative Example 1-1 were dissolved in a mixed solvent of acetonitrile (15.8 mL) and dichloromethane (52.7 mL). Molecular sieve 3A (3.2 g) was added to the mixture, and after stirring at room temperature for 30 minutes, 5'-DMT-dA(dma)-3'-phosphoramide (1.7 g, 2.1 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Then, 5-ethylthio-1H-tetrazole (0.26 g, 2.0 mmol) was added to the mixture, and the mixture was stirred at room temperature for 4 hours. Add 2,2,2-trifluoroethanol (0.77 mL, 10.5 mmol) to the reaction mixture, stir for 30 minutes at room temperature, then add 2,6-dimethylaniline (0.98 mL, 7.9 mmol) and POS (0.51 g, 2.6 mmol), and stir for 1 hour at room temperature. Add 2,3-dimethylfuran (1.1 mL, 10.5 mmol), then add dropwise a mixture of trifluoroacetic acid (2.5 mL, 33.0 mmol) and 2,6-dimethylaniline (0.048 mL, 0.35 mmol) dissolved in anhydrous chloroform (5.4 mL), and stir for 1.5 hours at room temperature. Add 2,6-dimethylpyridine (10.2 mL, 87.7 mmol) and methanol (0.34 mL) to the reaction mixture, and stir for 45 minutes at room temperature. After filtering the reaction mixture, acetonitrile (443 mL) was added to the filtrate, and the precipitated solid was filtered off and dried to obtain trimer (3 m) (2.2 g, yield: 98.4%) as a white solid. m / z (TOF-MS): Theoretical value 2156.20, measured value 2157.20 [M+H] + .

[0252] Comparative Examples 1-3: Synthesis and Solid-Liquid Separation of Tetramers Except for the trimer (3m) (2.0 g, 0.94 mmol) and 5'-DMT-dA(dma)-3'-phosphoramide (1.5 g, 1.8 mmol) obtained in Comparative Examples 1-2, the procedure was performed in the same manner as in Comparative Examples 1-2, and a tetramer (4m) (2.3 g, yield: 97.1%) was obtained as a white solid. m / z (TOF-MS): Theoretical value 2608.32, Measured value 2608.32 [M+H] + .

[0253] Comparative Examples 1-4: Synthesis and Solid-Liquid Separation of Pentamers Except for the tetramer (4m) (1.9 g, 0.72 mmol) and 5'-DMT-dG(iBu)-3'-phosphoramide (1.2 g, 1.4 mmol) obtained in Comparative Examples 1-3, the procedure was performed in the same manner as in Comparative Examples 1-2, and pentamer (5m) (2.1 g, yield: 96.7%) was obtained as a white solid. m / z (TOF-MS): Theoretical value 3075.42, measured value 1538.71 [M+2H] + .

[0254] Comparative Examples 1-5: Synthesis and Solid-Liquid Separation of Hexamers Except for the use of the pentamer (5m) (1.7 g, 0.55 mmol) and 5'-DMT-dmeC(Bz)-3'-phosphoramide (0.94 g, 1.1 mmol) obtained in Comparative Examples 1-4, the same procedure was performed as in Comparative Examples 1-2 to obtain the hexamer (6m) (1.6 g, yield: 83.9%) as a white solid. m / z (TOF-MS): Theoretical value 3551.51, measured value 1776.76 [M+2H] + .

[0255] <Deprotection reaction> The hexamer (6m) (5 mg) obtained in Comparative Examples 1-5 was mixed with 28% ammonia (5 mL), and the mixture was heated at 65°C for 4 hours and then cooled to room temperature. After removing unwanted substances from the reaction mixture through a syringe filter, the mixture was concentrated under reduced pressure using a centrifugal evaporator to obtain the deprotected form of the hexamer.

[0256] Comparative Example 2-1: Synthesis and solid-liquid separation of dimers (using concentration and precipitation with the addition of polar solvents) 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 sieve 3A (6.1 g) was added to this mixture, and the mixture was stirred at room temperature for 30 min. Then, 5'-DMT-dG(iBu)-3'-phosphoramide (3.4 g, 4.0 mmol) was added, and the mixture was stirred at room temperature for 15 min. 5-Ethylthio-1H-tetrazole (0.53 g, 4.0 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 h. 2,2,2-trifluoroethanol (1.5 mL, 20.2 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 min. Then, 2,6-dimethylaniline (1.9 mL, 15.2 mmol) and POS (0.99 g, 5.1 mmol) were added, and the mixture was stirred at room temperature for 1 h. After adding 2,3-dimethylfuran (2.1 mL, 20.2 mmol), a mixed solution of trifluoroacetic acid (5.2 mL, 67.5 mmol) and 2,6-dimethylaniline (0.083 mL, 0.68 mmol) dissolved in anhydrous chloroform (10.4 mL) was added dropwise, and the mixture was stirred at room temperature for 1.5 hours. Then, 2,6-dimethylpyridine (19.7 mL, 169.1 mmol) and methanol (0.66 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 45 minutes. After filtering the reaction mixture, the filtrate was concentrated to 63 mL. Acetonitrile (444 mL) was added to the concentrate, and the precipitated solid was filtered off and dried to obtain a white solid as a dimer (2 m) (3.1 g, yield: 92.2%). m / z (TOF-MS): Theoretical value 1705.08, measured value 1706.0773 [M+H] + .

[0257] Comparative Example 2-2: Synthesis and Solid-Liquid Separation of Trimers Under an argon atmosphere, the dimer (2m) (3.0 g, 2.0 mmol) and triphenylphosphine (0.23 g, 0.89 mmol) obtained in Comparative Example 2-1 were dissolved in a mixed solvent of acetonitrile (26.4 mL) and dichloromethane (87.8 mL). Molecular sieve 3A (5.3 g) was added to the mixture, and after stirring at room temperature for 30 minutes, 5'-DMT-dA(dma)-3'-phosphoramide (2.9 g, 3.5 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Then, 5-ethylthio-1H-tetrazole (0.46 g, 3.5 mmol) was added to the mixture, and the mixture was stirred at room temperature for 3 hours. Add 1.3 mL (17.6 mmol) of 2,2,2-trifluoroethanol to the reaction mixture and stir for 30 minutes at room temperature. Then add 1.6 mL (13.3 mmol) of 2,6-dimethylaniline and POS (0.86 g, 4.4 mmol) and stir for 1 hour at room temperature. Add 1.8 mL (17.6 mmol) of 2,3-dimethylfuran and add dropwise a mixture of 4.2 mL (54.9 mmol) of trifluoroacetic acid and 0.068 mL (0.59 mmol) dissolved in anhydrous chloroform (8.5 mL) and stir for 1.5 hours at room temperature. Add 17.1 mL (146.7 mmol) of 2,6-dimethylpyridine and 0.57 mL of methanol to the reaction mixture and stir for 45 minutes at room temperature. Filter the reaction mixture and concentrate the filtrate to 55 mL. Add acetonitrile (387 mL) to the concentrate, filter out the precipitated solid, dry it, and obtain trimer (3m) (3.6 g, yield: 96.9%) as a white solid; m / z (TOF-MS): Theoretical value 2156.20, measured value 2157.20 [M+H] + .

[0258] Comparative Examples 2-3: Synthesis of Tetramers and Solid-Liquid Separation Except for the trimer (3m) (3.4 g, 1.5 mmol) and 5'-DMT-dA(dma)-3'-phosphoramide (2.6 g, 3.1 mmol) obtained in Comparative Example 2-2, the procedure was performed in the same manner as in Comparative Example 2-2, and a tetramer (4m) (4.0 g, yield: 99.6%) was obtained as a white solid. m / z (TOF-MS): Theoretical value 2608.32, Measured value 2608.32 [M+H] + .

[0259] Comparative Examples 2-4: Synthesis and Solid-Liquid Separation of Pentamers Except for the tetramer (4m) (3.7 g, 1.4 mmol) and 5'-DMT-dG(iBu)-3'-phosphoramide (2.4 g, 2.9 mmol) obtained in Comparative Example 2-2, the same procedure was performed to obtain the pentamer (5m) (4.2 g, yield: 97.7%) as a white solid. m / z (TOF-MS): Theoretical value 3075.42, measured value 1538.71 [M+2H] + .

[0260] Comparative Examples 2-5: Synthesis and Solid-Liquid Separation of Hexamers Except for the use of the pentamer (5m) (4.1 g, 1.3 mmol) and 5'-DMT-dmeC(Bz)-3'-phosphoramide (2.3 g, 2.7 mmol) obtained in Comparative Examples 2-4, the procedure was performed in the same manner as in Comparative Example 2-2 to obtain the hexamer (6m) (4.5 g, yield: 97.5%) as a white solid. m / z (TOF-MS): Theoretical value 3551.51, measured value 1776.77 [M+2H] + .

[0261] <Deprotection reaction> The hexamer (6m) (5 mg) obtained in Comparative Examples 2-5 was mixed with 28% (w / w) ammonia (5 mL), and the mixture was heated at 65°C for 4 hours and then cooled to room temperature. After removing unwanted substances from the reaction mixture through a syringe filter, the mixture was concentrated under reduced pressure using a centrifugal evaporator to obtain the deprotected form of the hexamer.

[0262] <Evaluation of the purity of the deprotected body> The purity (area %) of the deprotected hexamer obtained as described above was calculated based on the peak area of ​​the chromatogram obtained by high performance liquid chromatography (HPLC). The results are shown in Table 1 below.

[0263] [Table 1] .

[0264] As shown in Table 1, the purity difference between Example 5 and Comparative Examples 1-5 was 2.31% by area, and the purity difference between Example 5 and Comparative Examples 2-5 was 3.27% by area. As described above, in the liquid-phase synthesis of oligonucleotides using hydrophobic protecting groups, the influence of impurities in the next synthesis cycle is greater compared to the solid-phase synthesis of oligonucleotides using solid-phase supports. Therefore, the aforementioned purity difference can be considered significant in the liquid-phase synthesis of oligonucleotides using hydrophobic protecting groups. Thus, the precipitation method of the present invention achieves excellent results (high purity).

[0265] Industrial applications The oligonucleotides obtained by the precipitation method of the present invention can be used in various applications, such as pharmaceuticals (RNA, DNA, oligonucleotide drugs, etc.) for human or animal use, functional foods, foods for specific health care, food products, chemical products, and polymer materials for biological or industrial use.

[0266] This application is based on Japanese Patent Application No. 2024-056447, the contents of which are fully contained in this application specification.

Claims

1. A method for precipitating an oligonucleotide from a solution containing an oligonucleotide having a hydrophobic protecting group and a nonpolar solvent, wherein, The method includes: Mix the solution with a first polar solvent; and While maintaining the volume of the mixture of the solution and the first polar solvent at more than 1.00 times the volume of the solution, the solvent is removed from the mixture by distillation.

2. The method according to claim 1, wherein, The solvent is removed by distillation from the mixture under conditions of 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, Nonpolar solvents include halogen solvents.

4. The method according to claim 1 or 2, wherein, The first polar solvent includes at least one selected from 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 according to claim 1 or 2, wherein, The solution contains a second polar solvent.

7. The method according to claim 6, wherein, The second polar solvent includes at least one selected from 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 that of the nonpolar solvent.

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

10. The method according to claim 1 or 2, wherein, The hydrophobic protecting group is represented by the following formula (Pg-5). **LYZ (Pg-5) In the formula, **Indicates the binding site with the protected group; L represents a single bond, or a group represented by the formula (Pg-i-1) or (Pg-i-2). In the formula, * indicates the position where it is associated with Y. **Same as the meaning above, R 1p and R 2p Represent C independently. 1-22 hydrocarbon group, L 1 Indicates divalent C with optional substitution 1-22 Hydrocarbon group, the divalent C 1-22 The -CH2- in the hydrocarbon group may be optionally replaced by a linking group. L 2 Represents a single bond, or represents a bond with ***C(=O)N(R) 3p )-R 4p -N(R 5p **** represents the group, where *** represents the group with L. 1 The binding position, **** indicates the binding position with C=O, R 4p C represents 1-22 Alkylene, R 3p and R 5p Each can be used independently to represent a hydrogen atom or a carbon atom. 1-22 Alkyl, or R 3p and R 5p They can be arbitrarily combined to form a ring; Y represents a single bond, oxygen atom, or NR; R represents a hydrogen atom, alkyl group, or aralkyl group; and Z represents any one of the groups represented by formulas (Pg-ii-1) to (Pg-ii-5). In the formula, * indicates the binding position. Ring A' represents the benzene ring. Ring B' represents the cyclohexane ring. Ring D' represents a naphthalene ring or a bicyclic fused aromatic heterocycle. R 6p For hydrogen atoms, or when R b In the case of a group represented by the following formula (Pg-iii), R of ring A' or ring B' 6p Optional with R 8p Together they represent a single bond or -O-, forming a fused ring together with ring A' or ring B' and ring C'. k represents an integer from 1 to 4. Each of the k Q's independently represents -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, or -NR'C(=O)-, where R' independently represents a hydrogen atom or a C atom. 1-6 Alkyl groups, k Qs preferably represent -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, or -NHC(=O)-, respectively. Each of the k Q's independently represents a single bond, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR'-, -C(=O)NR'-, or -NR'C(=O)-, where R' independently represents a hydrogen atom or a C atom. 1-6 alkyl, k R 7p Each can be independently represented as a hydrocarbon group with a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms bonded together via a single bond or a linking group. Rings A' and B' are independent, except that they have k QRs. 7p In addition, it may optionally have substituents selected from halogen atoms, C atoms optionally substituted with halogen atoms. 1-6 Alkyl groups, and C atoms optionally substituted with halogen atoms 1-6 Alkoxy R a Represents hydrogen atoms, and R b Represents a hydrogen atom, or a group represented by formula (Pg-iii), In the formula, * indicates the binding position. The ring C' represents the benzene ring. j represents an integer from 0 to 4. Each of the j Q's independently has the same meaning as described above. j R 9p Each can be independently represented as a hydrocarbon group with a straight-chain aliphatic hydrocarbon group having 10 or more carbon atoms bonded together via a single bond or a linking group. R 8p Represents a hydrogen atom, or optionally a R atom of ring A' or ring B'. 6p Together they represent a single bond or -O-, forming a fused ring together with ring A' or ring B' and ring C'. In addition to having j QRs 9p In addition, it may optionally have substituents selected from halogen atoms, C atoms optionally substituted with halogen atoms. 1-6 Alkyl groups, and C atoms optionally substituted with halogen atoms 1-6 alkoxy, or R a and R b Together they form an oxygen group. u represents 1 or 2, and R 10p C represents 2-21 Perfluoroalkyl.

11. A method for producing oligonucleotides via a one-pot synthesis, wherein, The method includes: Step (1): In a solution containing a nonpolar solvent, 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 by a temporary protecting group that can be removed under acidic conditions, and which is 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 that can be removed under acidic conditions; Step (2) involves mixing the solution from step (1) with the quencher (i) of the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b) to quench the phosphoramidized nucleoside, nucleotide, or oligonucleotide (b). Step (3) involves mixing the solution from step (2) with an oxidizing agent or a sulfiding agent to oxidize or sulfidate 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 the hydroxyl group being protected by a temporary protecting group that can be removed under acidic conditions. In step (4), if an oxidant is used in step (3), the solution after step (3) is mixed with an oxidant quencher (ii) as needed to quench the oxidant; In step (5), the solution after step (3) or the solution after step (4) in the case of step (4) is mixed with acid to remove the temporary protecting group of the oligonucleotide (d) that can be removed under acidic conditions, forming an oligonucleotide (e) with unprotected hydroxyl groups and hydrophobic protecting groups. Step (6): As needed, mix the solution from step (5) with alkali; and Step (7) involves precipitating the oligonucleotide (e) from a solution containing the oligonucleotide (e) and a nonpolar solvent by the method described in claim 1 or 2.

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

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

Images