Production method of oligonucleotide
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Filing Date
- 2023-07-21
- Publication Date
- 2026-06-24
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Figure 2024019137000001 
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Abstract
Description
Methods for producing oligonucleotides
[0001] This patent application claims priority under the Paris Convention to and the benefit of Japanese Patent Application No. 2022-117422 (filed July 22, 2022), the entire contents of which are incorporated herein by reference. The present invention relates to a method for producing oligonucleotides using the phosphoramidite method.
[0002] Oligonucleotides, which are nucleic acid oligomers, are useful materials used in a variety of fields, such as nucleic acid probes for gene detection, DNA probes used in PCR, antisense nucleic acids as pharmaceuticals, siRNA, and aptamers.
[0003] Oligonucleotides can be produced by solid-phase synthesis using the phosphoramidite method. In the phosphoramidite method, phosphoramidites (hereinafter referred to as "amidites") of nucleoside monomers are sequentially linked on a glass or resin support to produce oligonucleotides with any desired sequence. The specific production process includes a deprotection step in which the protecting group of the hydroxyl group at the 5' end of the nucleotide is removed using an acidic solution; a condensation step in which the hydroxyl group at the 5' end of the deprotected nucleotide reacts with the amidite group of the amidite in the presence of an activator; an oxidation step in which the newly formed internucleoside bond is oxidized from trivalent phosphorus to pentavalent phosphorus; and a capping step in which the unreacted hydroxyl group at the 5' end is acylated. By sequentially repeating these steps, the amidites can be linked in any order to produce oligonucleotides with the desired sequence.
[0004] As disclosed in Patent Document 1, etc., the amidite used in the condensation step is dissolved in acetonitrile and used as an amidite solution. However, in the case of amidites with low solubility in acetonitrile, this method is difficult to use.
[0005] 2'-OMe-U amidite is one of the amidites with low solubility in acetonitrile. To improve the solubility of 2'-OMe-U amidite, it has been reported that dichloromethane (Patent Document 2, Non-Patent Document 1) is added to acetonitrile to prepare an amidite solution. The addition of these solvents improves the solubility of 2'-OMe-U amidite.
[0006] Although methods for producing such oligonucleotides have been developed, the purity of the synthesized oligonucleotides has not always been satisfactory. One of the reasons for this is the presence of n-1 mer impurities. n-1 mer impurities are impurities that arise during oligonucleotide production and have a chain length that is one step shorter than the target chain length. It is known that these n-1 mer impurities are difficult to separate from oligonucleotides of the target chain length (Non-Patent Document 2). Therefore, reducing the n-1 mer impurities that arise during production is important for efficient oligonucleotide production.
[0007] International Publication No. 2019 / 170731 Chinese Patent Application Publication No. 112007040
[0008] Bioorganic & Medicinal Chemistry Letters 28 (2018) 3774-3779Mass Spectrometry Reviews 40 (2021) 75-109
[0009] An object of the present invention is to provide a method for producing a highly pure oligonucleotide having one or more 2'-OMe-Us.
[0010] As a result of extensive research conducted by the present inventors to achieve the above object, the present inventors have provided a method for producing nucleic acids, which is characterized by using acetonitrile and an aromatic hydrocarbon as the solvent for the 2'-OMe-U amidite solution.
[0011] The present invention includes, but is not limited to, the following embodiments: 1. Formula (2): [In the formula, G ２ represents a protecting group for a hydroxyl group, B ａrepresents a nucleic acid base which may be protected with a protecting group, R represents a protected hydroxyl group, hydrogen atom, fluorine atom, methoxy group, 2-methoxyethyl group, or OQ' group, Q' represents an alkylene group bonded to the 4'-position carbon atom of ribose, X represents an oxygen atom or a sulfur atom, and the bond marked with * represents a bond to the 3'-terminal side of a nucleic acid.] with a compound represented by formula (4): [In the formula, G １ represents a protecting group for a hydroxyl group. ] in the presence of an activator, and the 2'-OMe-U amidite represented by the formula (3): [In the formula, Y １ ~Y ６are each independently the same or different and represent a hydrogen atom, a methyl group, an ethyl group, or a halogen atom.] 2. A method for producing an oligonucleotide according to [1], wherein the aromatic hydrocarbon represented by formula (3) is benzene having one or two substituents selected from the group consisting of a methyl group, an ethyl group, and a halogen atom, and when the benzene has two substituents, the substituents may be the same or different. 3. A method for producing an oligonucleotide according to [1], wherein the aromatic hydrocarbon represented by formula (3) is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and a mixture of two or more thereof. 4. 4. The method for producing an oligonucleotide according to any one of [1] to [3], wherein the volume ratio of acetonitrile to the aromatic hydrocarbon represented by formula (3) contained in the mixture in which the 2'-OMe-U amidite represented by formula (4) is dissolved is 99:1 to 1:99 (acetonitrile:aromatic hydrocarbon represented by formula (3)). 5. The method for producing an oligonucleotide according to any one of [1] to [3], wherein the volume ratio of acetonitrile to the aromatic hydrocarbon represented by formula (3) contained in the mixture in which the 2'-OMe-U amidite represented by formula (4) is 90:10 to 10:90 (acetonitrile:aromatic hydrocarbon represented by formula (3).
[0023] 6. The method for producing an oligonucleotide according to any one of [1] to [3], wherein the volume ratio of acetonitrile to aromatic hydrocarbon represented by formula (3) contained in a mixture solution in which the 2'-OMe-U amidite represented by formula (4) is dissolved is 90:10 to 40:60 (acetonitrile:aromatic hydrocarbon represented by formula (3). 7. The method for producing an oligonucleotide according to any one of [1] to [6], wherein the concentration of the 2'-OMe-U amidite represented by formula (4) in the solution is 0.01 to 0.4 M. 8. The method for producing an oligonucleotide according to any one of [1] to [6], wherein the concentration of the 2'-OMe-U amidite represented by formula (4) in the solution is 0.05 to 0.2 M.9. The method for producing an oligonucleotide according to any one of [1] to [8], wherein the activator is 5-benzylthio-1H-tetrazole. 10. When R in the compound represented by formula (2) is a protected hydroxyl group, the protecting group for the hydroxyl group is a compound represented by formula (10): [wherein q represents an integer of 0 to 5; R ａ and R ｂ are the same or different and represent a methyl group, an ethyl group, or a hydrogen atom, the bond marked with an asterisk is bonded to the oxygen of the 2'-hydroxyl group, and Ew represents an electron-withdrawing group. 11. The method for producing an oligonucleotide according to any one of [1] to [9], wherein in the 2'-OMe-U amidite represented by formula (4), G １ is a 4,4'-dimethoxytrityl group. 12. The method for producing the oligonucleotide according to any one of [1] to
[11] , wherein the oligonucleotide is produced by solid-phase synthesis. 13. Formula (4): [In the formula, G １ represents a protecting group for a hydroxyl group.], acetonitrile, and a compound represented by formula (3): [In the formula, Y １ ~Y ６ are each independently the same or different and represent a hydrogen atom, a methyl group, an ethyl group, or a halogen atom.] 14. A method for producing an oligonucleotide using a solution containing 2'-OMe-U amidite represented by formula (4), acetonitrile, and an aromatic hydrocarbon represented by formula (3) described in
[13] .
[0012] The present invention provides a method for producing highly pure oligonucleotides containing one or more 2'-OMe-U amidites.
[0013] 1 is a diagram showing a scheme (Scheme A) of steps (1) to (6) of the production method of the present invention.
[0014] The method for producing the oligonucleotide of the present invention comprises the step of: [In the formula, G ２represents a protecting group for a hydroxyl group, B ａ represents a nucleic acid base which may be protected with a protecting group, R represents a protected hydroxyl group, hydrogen atom, fluorine atom, methoxy group, 2-methoxyethyl group, or OQ' group, Q' represents an alkylene group bonded to the 4'-position carbon atom of ribose, X represents an oxygen atom or a sulfur atom, and the bond marked with * represents a bond to the 3'-terminal side of a nucleic acid.] with a compound represented by formula (4): [In the formula, G １ represents a protecting group for a hydroxyl group. ] in the presence of an activator, and the 2'-OMe-U amidite represented by the formula (3): [In the formula, Y １ ~Y ６ and each independently represent a hydrogen atom, a methyl group, an ethyl group, or a halogen atom, which may be the same or different.
[0015] The method for producing an oligonucleotide may include a step of reacting a 2'-OMe-U amidite solution, in which 2'-OMe-U amidite represented by formula (4) is dissolved, with a mixed solution of acetonitrile and an aromatic hydrocarbon represented by formula (3), and an activator. The method for producing an oligonucleotide will be described below.
[0016] In the present invention, the n-1 mer contained in the produced oligonucleotide can be reduced by using a mixed solution containing acetonitrile and the aromatic hydrocarbon represented by formula (3) as the solvent for the 2'-OMe-U amidite solution. The mixing ratio of the mixed solution containing acetonitrile and the aromatic hydrocarbon represented by formula (3) is not particularly limited, but the volume ratio of acetonitrile to aromatic hydrocarbon is, for example, 99:1 to 1:99, preferably 90:10 to 10:90, and more preferably 90:10 to 40:60.
[0017] As the aromatic hydrocarbon, usable is benzene having a substituent selected from the group consisting of a methyl group, an ethyl group, or a halogen atom, preferably benzene having one or two substituents selected from the group consisting of a methyl group, an ethyl group, or a halogen atom, more preferably toluene, o-xylene, m-xylene, p-xylene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, or a mixture of two or more thereof.
[0018] The 2'-OMe-U amidite solution may be prepared by mixing a solvent other than acetonitrile and aromatic hydrocarbons.
[0019] The concentration of 2'-OMe-U amidite in the 2'-OMe-U amidite solution is adjusted to preferably 0.01 M to 0.4 M, more preferably 0.05 M to 0.2 M.
[0020] It is also possible to dissolve 2'-OMe-U amidite in the above-mentioned mixed solvent, and then reduce the moisture content with a drying agent such as Zeolum before use.
[0021] The 2'-OMe-U amidite solution can be stored in a glass, plastic, or metal container. Plastic containers include polyethylene or polypropylene containers, and metal containers include stainless steel containers and Hastelloy containers.
[0022] The reaction precursor with 2'-OMe-U amidite represented by formula (4) is specifically represented by the following formula (12): (In the formula, G ２ represents a protecting group for a hydroxyl group, B ａeach independently represent a nucleobase which may be protected by a protecting group; R represents a protected hydroxyl group, a hydrogen atom, a fluorine atom, a methoxy group, a 2-methoxyethyl group, or an OQ' group; Q' represents an alkylene group bonded to the carbon atom at the 4'-position of ribose; each X is independently the same or different and represents an oxygen atom or a sulfur atom; Z represents a solid-phase support and a group consisting of a linking moiety connecting the solid-phase support and the oxygen atom of the hydroxyl group at the 3'-position of ribose at the 3'-end of the oligonucleotide; and 1 represents an integer of 0 to 300. (In the formula, G １ represents a protecting group for a hydroxyl group, ２ , B ａ , R, X, Z, and 1 are as defined above.
[0023] In formulas (2), (12), and (13), when R represents an OQ′ group and Q′ represents an alkylene group bonded to the 4′-position carbon atom of ribose, specific examples of the structure include the following LNA-1 to LNA-4. (In the formula, B ａ represents an optionally protected nucleobase.
[0024] Nucleosides (ribose and deoxyribose) contained in the oligonucleotides used in the present invention include, but are not limited to, DNA, RNA, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-Me, 2'-F RNA, and the above-mentioned LNA.
[0025] More specifically, the group represented by Z, which is composed of a solid phase carrier and a linking moiety connecting the solid phase carrier and the oxygen atom of the hydroxyl group at the 2'- or 3'-position of ribose at the 3'-end of the nucleic acid oligomer, includes a structure represented by the following formula (18): In formula (18), Sp represents a spacer. Examples of the spacer (Sp) include those having the structural formula shown in formula (19) below.
[0026]
[0027] The linker may have, for example, a structure shown in the following formula (20), or a structure in which the structure of formula (20) does not have a hexamethyleneamino group portion and an aminopropyl group is bonded to Si. Alternatively, the linker may have a structure shown in the following formula (21). (In the formula, A may be any of a hydroxyl group, an alkoxy group, or an alkyl group. Examples of alkoxy groups include a methoxy group and an ethoxy group. Examples of alkyl groups include a methyl group, an ethyl group, an isopropyl group, and an n-propyl group. Si indicates that it is bonded to the oxygen of a hydroxyl group on the surface of the support.) Examples of solid supports include inorganic porous supports and organic resin supports. Examples of inorganic porous supports include controlled pore glass (CPG) and zeolite. Examples of organic resin supports include supports made of polystyrene.
[0028] The method for synthesizing an oligonucleotide typically includes the following steps: (1) a step of deprotecting the 5'-hydroxyl group of a nucleoside whose hydroxyl group is protected and bound to a solid-phase support via a linker; (2) a step of condensing the 5'-hydroxyl group produced in the above step with an amidite compound to obtain a phosphite triester compound; (3) a step of oxidizing the phosphite triester produced in the above step to convert it into a phosphate triester bond to produce an extended nucleic acid molecule, or an optional step of converting it into a thiophosphate triester; (4) a step of synthesizing a nucleic acid molecule on a solid-phase support by repeating a series of reaction cycles consisting of steps (1) to (3), i.e., a step of deprotecting the 5'-hydroxyl group of the produced nucleic acid molecule, a step of condensing the 5'-hydroxyl group with an amidite compound, and a step of oxidizing the produced phosphite triester, any number of times; (5) a step of subjecting the nucleic acid molecule on the solid-phase support produced in step (4) to a step of excising and deprotecting it, and releasing it from the solid-phase support to produce an oligonucleotide from which the protecting groups have been removed; (6) A step of deprotecting the protecting group of the 2' hydroxyl group of ribose constituting the nucleic acid molecule.
[0029] However, the method for synthesizing the oligonucleotide may include, following step (2) or (3), a step of capping the hydroxyl group at the 5'-position that has not undergone the condensation reaction with the amidite compound, or a capping step may be added between any of the steps in the series of reaction cycles that constitute step (4).
[0030] More specifically, the step (5) involves carrying out the following steps (5-1) and (5-2) of reactions on the nucleic acid molecule on the solid support produced in step (4), in this order. The reaction in step (5-1) may be carried out arbitrarily, and the reaction in step (5-2) may be carried out using the method described in Japanese Patent No. 4705716. As a result, an oligonucleotide in which the protecting group has been removed from the nucleic acid molecule released from the solid support, or an oligonucleotide in which the hydroxyl group at the 5'-end is protected, can be produced. (5-1) A reaction to deprotect the protecting group on the hydroxyl group at the 5'-end of the nucleic acid molecule, and (5-2) A reaction to cleave and release the nucleic acid molecule from the solid support.
[0031] The scheme of steps (1) to (6) is shown in Scheme A of Figure 1. Among the substituents in the chemical formula in Scheme A, G １ , G ２ , B ａ The definitions of R and G are as defined above. ３ , G ４ , G ５ , B ｃ The definitions of R and R' are as described below. In addition, in the chemical formula of Scheme A, each Y is independently the same or different and represents an oxygen atom or a sulfur atom, X represents an R group or an OZ group, where Z is as defined above, W represents an OZ group when X represents an R group, where Z is as defined above, or W represents an OV group when X represents an OZ group, where V represents a protecting group for a hydroxyl group, W １ is a W group or a group derived from a W group (e.g., a residue cleaved from a solid support, a deprotected group, etc.), and W １０ Is W １ group, or W １a group derived from a group (e.g., a residue cleaved from a solid support, a deprotected group, etc.), １ is an X group or a group derived from an X group (e.g., a residue cleaved from a solid support, a deprotected group, etc.), and X １０ is X １ group, or X １ group (for example, a residue cleaved from a solid support, a deprotected group, etc.), n represents an integer of 1 to 300, and m represents an integer of 1 to 300.
[0032] The nucleic acid compound of formula (A5) can be further extended to any desired chain length using a nucleotide or non-nucleotide linker by the amidite method and used to produce a nucleic acid compound of formula (A5').The nucleic acid compound alone can be cut out from the nucleic acid compound of formula (A5') bound to a solid support to obtain an oligonucleotide of formula (A6), which can then be further deprotected to obtain an oligonucleotide of formula (A7).
[0033] The substituents in each formula will be explained in more detail below.
[0034] G １ There are no particular limitations on the protecting group G as long as it can function as a protecting group, and a wide range of known protecting groups used in amidite compounds can be used. １ is preferably a protecting group represented by the following formula (14): (In the formula, R １ , R ２ and R ３ are each independently the same or different and represent hydrogen or an alkoxy group.
[0035] R １ , R ２ and R ３ Preferably, one of G is hydrogen and the remaining two are the same or different (preferably the same) alkoxy groups, and a methoxy group is particularly preferred as the alkoxy group. １Specifically, 4,4'-dimethoxytrityl group (DMTr group), 4-monomethoxytrityl group, and 4,4',4"-trimethoxytrityl group are preferred, with 4,4'-dimethoxytrityl group being particularly preferred.
[0036] G ２ There are no particular limitations on the protecting group G as long as it can function as a protecting group, and a wide range of known protecting groups used in amidite compounds can be used. ２ Examples of the alkyl group include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a haloalkyl group, an aryl group, a heteroaryl group, an arylalkyl group, a cycloalkenyl group, a cycloalkylalkyl group, a cyclylalkyl group, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a heterocyclylalkenyl group, a heterocyclylalkyl group, a heteroarylalkyl group, a silyl group, a silyloxyalkyl group, a mono-, di-, or trialkylsilyl group, and a mono-, di-, or trialkylsilyloxyalkyl group, which may be substituted with one or more electron-withdrawing groups.
[0037] G ２ is preferably an alkyl group substituted with an electron-withdrawing group. Examples of the electron-withdrawing group include a cyano group, a nitro group, an alkylsulfonyl group, a halogen atom, an arylsulfonyl group, a trihalomethyl group, and a trialkylamino group, and a cyano group is preferred. ２ Particularly preferred as the alkyl group is a 2-cyanoethyl group (a group represented by the following formula).
[0038] G ３ is an alkyl group, and two G ３ may be bonded to each other to form a cyclic structure, and preferably both are isopropyl groups.
[0039] The R １ , R ２ , R ３ and G ２ , G ３The alkyl group in the definition may be either linear or branched, and is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl. The alkyl group moiety constituting the alkoxy group in the definition of the substituent has the same definition as the alkyl group herein.
[0040] G ４ represents a hydrogen atom, an alkali metal ion, an ammonium ion, an alkylammonium ion, or a hydroxyalkylammonium ion. Examples of alkali metal ions include sodium ions and lithium ions. Specific examples of alkyl groups for alkylammonium ions include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl, with more specific examples including diethylammonium ion, triethylammonium ion, tetrabutylammonium ion, hexylammonium ion, and dibutylammonium ion. Specific examples of hydroxyalkyl moieties for hydroxyalkylammonium ions include hydroxymethyl, hydroxyethyl, hydroxy-n-propyl, hydroxyisopropyl, hydroxy-n-butyl, and trishydroxymethyl, with more specific examples of hydroxyalkylammonium ions including trishydroxymethylammonium ions.
[0041] G ５ represents a hydrogen atom or a protecting group, and when representing a protecting group, １ represents the same protecting group as ５ is a hydrogen atom when deprotected, and the nucleotide compound in this state is also subjected to a series of steps in a nucleic acid extension reaction.
[0042] In the method of the present invention, the amidite compound can be used in its free state or in its salt state. Examples of salts of the amidite compound include, but are not limited to, base addition salts and acid addition salts. Specific examples of base addition salts include salts with inorganic bases such as sodium salts, magnesium salts, potassium salts, calcium salts, and aluminum salts; salts with organic bases such as methylamine, ethylamine, and ethanolamine; salts with basic amino acids such as lysine, ornithine, and arginine; and ammonium salts. Specific examples of acid addition salts include salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. The amidite compounds also include salts, hydrates, solvates, crystalline polymorphs, and other forms.
[0043] As used herein, the term "nucleobase" refers to a group having a natural or non-natural nucleobase backbone. The nucleobase also includes modified forms of the natural or non-natural nucleobase backbone.
[0044] B ａThe nucleobase that may be protected with a protecting group represented by the formula (I) is not particularly limited. Examples of the nucleobase include adenine, cytosine, guanine, uracil, thymine, 5-methylcytosine, pseudouracil, and 1-methylpseudouracil. The nucleobase may also be substituted with a substituent. Examples of such substituents include halogen atoms such as fluoro, chloro, bromo, and iodo groups, acyl groups such as acetyl groups, alkyl groups such as methyl and ethyl groups, arylalkyl groups such as benzyl groups, alkoxy groups such as methoxy groups, alkoxyalkyl groups such as methoxyethyl groups, cyanoalkyl groups such as cyanoethyl groups, hydroxy groups, hydroxyalkyl groups, acyloxymethyl groups, amino groups, monoalkylamino groups, dialkylamino groups, carboxy groups, cyano groups, and nitro groups, as well as combinations of two or more of these substituents.
[0045] B ａ More specifically, examples of the nucleic acid base represented by the formula (I) include the following structures: (In the above formula, R ４ represents a hydrogen atom, a methyl group, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group, or a benzoyl group; R ５ represents a hydrogen atom, an acetyl group, an isobutyryl group, or a benzoyl group; R ６ represents a hydrogen atom, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group, or an isobutyryl group; R ７ represents a 2-cyanoethyl group, R ８ represents a hydrogen atom, a methyl group, a benzoyl group, a 4-methoxybenzoyl group, or a 4-methylbenzoyl group, and R ９ represents a dimethylaminomethylene group.
[0046] When a nucleic acid base has an amino group outside the ring, the protecting group for the amino group is not particularly limited, and any protecting group known in nucleic acid chemistry can be used. Examples of such protecting groups include a benzoyl group, a 4-methoxybenzoyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a phenylacetyl group, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, and a (dimethylamino)methylene group, as well as combinations of two or more of these protecting groups.
[0047] B ｃ indicates an unprotected nucleic acid base, but the type thereof is not particularly limited. Examples of such nucleic acid bases include adenine, cytosine, guanine, uracil, thymine, 5-methylcytosine, pseudouracil, and 1-methylpseudouracil. The nucleic acid base may also be substituted with a substituent. Examples of such substituents include halogen atoms such as fluoro, chloro, bromo, and iodo groups, acyl groups such as acetyl groups, alkyl groups such as methyl and ethyl groups, arylalkyl groups such as benzyl groups, alkoxy groups such as methoxy groups, alkoxyalkyl groups such as methoxyethyl groups, cyanoalkyl groups such as cyanoethyl groups, hydroxy groups, hydroxyalkyl groups, acyloxymethyl groups, amino groups, monoalkylamino groups, dialkylamino groups, carboxy groups, cyano groups, and nitro groups, as well as combinations of two or more of these substituents.
[0048] When R represents a protected hydroxyl group, the protecting group may be any that can be used in the amidite method, for example, 2'-tert-butyldimethylsilyl (TBDMS) group, 2'-bis(2-acetoxy)methyl (ACE) group, 2'-(triisopropylsilyloxy)methyl (TOM) group, 2'-(2-cyanoethoxy)ethyl (CEE) group, 2'-(2-cyanoethoxy)methyl (CEM) group (WO 2006 / 022323), 2'-para-tolylsulfonylethoxymethyl (TEM) group, 2'-EMM group (WO 2013 / 027843), as well as those described in WO 2019 / 208571 can be used. Of these ribonucleoside (RNA) 2'-protecting groups, the protecting group represented by formula (10) is exemplified as a preferred protecting group. More preferably, E Ｗ An example of such a protecting group is a protecting group represented by formula (15) having a cyano group as the electron-withdrawing group. (wherein q represents an integer of 0 to 5; R ａ and R ｂ are the same or different and represent a methyl group, an ethyl group, or a hydrogen atom, the bond marked with an * is bonded to the oxygen of the 2' hydroxyl group, and Ew represents an electron-withdrawing group.
[0049] The protecting group represented by formula (15) can be synthesized, for example, according to the description in WO 2013 / 027843 and WO 2019 / 208571, and an amidite compound having such a protecting group can be used in the production of an oligonucleotide.
[0050] Nucleotides and amidites in which the R group is a substituent other than a hydroxyl group can be produced from nucleosides synthesized by known methods described in Japanese Patent No. 3745226, WO 2001 / 053528, JP 2014-221817 A, and known methods cited therein. Furthermore, they can be produced using commercially available products in accordance with the methods described in the Examples below or by methods with appropriate modifications to these methods.
[0051] R' represents a hydroxyl group, a hydrogen atom, a fluorine atom, a methoxy group, a 2-methoxyethyl group, or an OQ' group, and Q' represents a methylene group bonded to the 4'-position carbon atom of ribose, an ethylene group bonded to the 4'-position carbon atom, or an ethylidene group bonded to the 4'-position carbon atom.
[0052] The synthesis of the oligonucleotide can be carried out by repeating the deprotection step, condensation step, and other steps according to a generally known method (e.g., the method described in the aforementioned Japanese Patent No. 5,157,168 or Japanese Patent No. 5,554,881), except for the condensation step using 2'-OMe-U amidite according to the present invention. Each step is described below.
[0053] (Nucleic acid extension reaction) In this specification, the term "nucleic acid extension reaction" refers to a reaction in which a nucleic acid molecule is extended by sequentially linking nucleotides via phosphodiester bonds. The nucleic acid extension reaction can be carried out according to the procedure of the general amidite method (phosphoramidite method). The nucleic acid extension reaction may be carried out using an automatic nucleic acid synthesizer that employs the amidite method, or may be carried out by liquid phase synthesis.
[0054] The chain length of the oligonucleotide may be, for example, 20 mer or more, 40 mer or more, 50 mer or more, 60 mer or more, 80 mer or more, 100 mer or more, 200 mer or more, 2 to 300 mer, 2 to 250 mer, 2 to 200 mer, 10 to 300 mer, 10 to 250 mer, 10 to 200 mer, 10 to 150 mer, 15 to 300 mer, 15 to 250 mer, 15 to 200 mer, 15 to 150 mer, or 15 to 110 mer.
[0055] The deprotection step of step (1) is a step of deprotecting the protecting group of the 5' hydroxyl group at the end of the RNA strand supported on the solid phase carrier (see Scheme A in Figure 1). １Examples of common protecting groups represented by the formula (I) include a 4,4'-dimethoxytrityl group (DMTr group), a 4-monomethoxytrityl group, and a 4,4',4"-trimethoxytrityl group. Deprotection can be carried out using an acid. Examples of acids for deprotection include trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid, methanesulfonic acid, hydrochloric acid, acetic acid, and p-toluenesulfonic acid.
[0056] The condensation step of step (2) is a reaction in which a nucleoside phosphoramidite represented by the following formula (A3) shown in Scheme A of Figure 1 is bonded to the 5' hydroxyl group at the end of the oligonucleotide chain deprotected in the deprotection step. Examples of phosphoramidites represented by the following formula (A3) used for nucleic acid elongation include those in which R is a protected hydroxyl group, 2'-OMe, 2'-F, 2'-O-tert-butyldimethylsilyl group, 2'-O-methoxyethyl group, 2'-H, 2'-fluoro-2'-deoxy-β-D-arabinofuranosyl, etc. The nucleoside phosphoramidite used has its 5' hydroxyl group protected with a protecting group (e.g., a DMTr group). The condensation step can be carried out using an activator that activates the nucleoside phosphoramidite. Examples of the activator include 5-benzylthio-1H-tetrazole (BTT), 1H-tetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), N-methylbenzimidazolium triflate (N-MeBIT), benzimidazolium triflate (BIT), N-phenylimidazolium triflate (N-PhIMT), imidazolium triflate (IMT), 5-nitrobenzimidazolium triflate (NBT), 1-hydroxybenzotriazole (HOBT), and 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, with 5-benzylthio-1H-tetrazole (BTT) being preferred.
[0057] The nucleoside phosphoramidite (hereinafter referred to as amidite) represented by formula (A3) in Scheme A of Figure 1 is as follows: Formula (A3): (In the formula, G１ , G ２ , G ３ , B ａ and R is as defined above.
[0058] In the present invention, 2'-OMe-U amidite (a compound represented by formula (4)) is used as the amidite of formula (A3) in at least one condensation step. In the condensation step, a solution in which 2'-OMe-U amidite is dissolved in a mixed liquid containing acetonitrile and the aromatic hydrocarbon represented by formula (3) can be used. An activator can be added to the mixed liquid. The activator may be dissolved in the mixed liquid.
[0059] After the condensation step, any unreacted 5' hydroxyl groups may be capped, if desired, using a known capping solution such as an acetic anhydride-tetrahydrofuran solution or a phenoxyacetic anhydride / N-methylimidazole solution.
[0060] The oxidation step (3) is a step of converting the phosphite group formed in the condensation step into a phosphate group or a thiophosphate group. This step is a reaction of converting trivalent phosphorus to pentavalent phosphorus using an oxidizing agent, and can be carried out by allowing the oxidizing agent to act on an oligonucleic acid derivative supported on a solid phase carrier.
[0061] When converting a phosphorous group to a phosphate group, for example, iodine can be used as an "oxidizing agent." The oxidizing agent can be prepared to a concentration of 0.005 to 2 M. Water can be used as the oxygen source for oxidation, and pyridine, N-methylimidazole (NMI), N-methylmorpholine, or triethylamine can be used as the base to promote the reaction. Furthermore, the solvent is not particularly limited as long as it is not involved in the reaction, and acetonitrile, tetrahydrofuran (THF), or a mixture of these in any ratio can also be used. For example, iodine / water / pyridine / acetonitrile, iodine / water / pyridine, iodine / water / pyridine / NMI, or iodine / water / pyridine / THF can be used. The reaction temperature is preferably 5°C to 50°C. The reaction time is usually 1 to 30 minutes. The amount of reagent used is preferably 1 to 100 mol, more preferably 1 to 10 mol, per 1 mol of compound supported on the solid phase support.
[0062] When converting a phosphite triester group into a thiophosphate group, examples of the "oxidizing agent" that can be used include sulfur, 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3-amino-1,2,4-dithiazole-5-thione (ADTT), 5-phenyl-3H-1,2,4-dithiazol-3-one (POS), [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione (DDTT), and phenylacetyl disulfide (PADS). The oxidizing agent can be diluted with an appropriate solvent to a concentration of 0.001 to 2 M before use. The solvent used in the reaction is not particularly limited as long as it is inert to the reaction, and examples include dichloromethane, acetonitrile, pyridine, and any mixtures thereof. The oxidation step may be carried out after the capping step, or conversely, the capping step may be carried out after the oxidation step, and this order is not limited.
[0063] In step (5), after the synthesis of a nucleic acid having a desired sequence is completed, the phosphate protecting group is deprotected by the action of an amine compound to deprotect the protecting group of the phosphate moiety. Examples of the amine compound include diethylamine, which is described in Japanese Patent No. 4705716.
[0064] The protecting group of the 5' hydroxyl group of the nucleoside introduced at the end of elongation may be used for column purification using the 5' protecting group as a tag after cleavage from the solid phase support and deprotection of the protecting group as described below, or the protecting group of the 5' hydroxyl group may be deprotected after column purification.
[0065] In step (5), the oligonucleotide elongated to a desired chain length on the solid support is cleaved from the solid support usually using concentrated aqueous ammonia as a cleavage agent.
[0066] Furthermore, the oligonucleotide chain is cleaved from the solid support and recovered using ammonia or an amine compound, etc. Examples of the amine compound include methylamine, ethylamine, isopropylamine, ethylenediamine, and diethylamine.
[0067] In step (6), the protecting group of the 2' hydroxyl group of the ribose of the nucleic acid compound (A6) cleaved from the solid phase support in step (5) can be removed according to the method described in WO 2006 / 022323, WO 2013 / 027843, or WO 2019 / 208571 to obtain a deprotected oligonucleotide (A7).
[0068] Oligonucleotides that can be produced using the production method of the present invention include, but are not limited to, oligonucleotides in which the nucleosides contained therein are RNA, DNA, RNA having 2'-O-MOE, 2'-O-Me, or 2'-F, and LNA. For example, examples of various nucleosides include those described in Xiulong, Shen et al., Nucleic Acids Research, 2018, Vol. 46, No. 46, 1584-1600, and Daniel O'Reilly et al., Nucleic Acids Research, 2019, Vol. 47, No. 2, 546-558.
[0069] Typical examples of oligonucleotides that can be used in the production method of the present invention are shown below in addition to the examples described in the Examples, but are not limited to these.
[0070] In the following explanation of the sequences, U represents uridine (ST.25 format), C represents cytidine, A represents adenosine, and G represents guanosine. Furthermore, T, according to the ST.26 format, represents uridine.
[0071] Examples include oligonucleotides having the following sequences (A) and (B) described in WO 2019 / 060442. Sequence (A): 5'-AUGGAAUmACUCUUGGUUmACdTdT-3' (based on the ST.25 format) (5'-ATGGAATmACTCTTGGTTmACdTdT-3' (based on the ST.26 format)) (Antisense) (SEQ ID NO: 1) 21 mer Sequence (B): 5'-GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT-3' (based on the ST.25 format) (5'-GTmAACmCmAAGAGTmATmTmCmCmATmdTdT-3' (based on the ST.26 format)) (Sense) (SEQ ID NO: 2) 21 mer In sequences (A) and (B), Um represents 2'-O-methyluridine (ST.25 format), Tm represents 2'-O-methyluridine (ST.26 format), Cm represents 2'-O-methylcytidine, and dT represents thymidine. Unless otherwise specified, the abbreviations in the sequences herein apply to both the ST.25 format and the ST.26 format.
[0072] Examples include the oligonucleotides described in JP-A-2017-537626. A typical example is an oligonucleotide having the following sequence (C): Sequence (C): 5'-AmsGmsUmsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUmsUmsU-3' (based on the ST.25 format) (5'-AmsGmsTmsCCTCATCTCCCTCAAGCGTTTAAGAGCTATGCTGGTAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTmsTmsTmsT-3' (based on the ST.26 format)) (SEQ ID NO: 3) 113mer In sequence (C), Um represents 2'-O-methyluridine (ST.25 format), Tm represents 2'-O-methyluridine (ST.26 format), Am represents 2'-O-methyladenosine, Gm represents 2'-O-methylguanosine, and s represents a phosphorothioate modification.
[0073] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.
[0074] Measurement Methods First, the various measurement methods used in the following tests are shown below.
[0075] (Measurement Method 1: Measurement of n-1 mer impurities and FLP ratio in oligonucleotides) Oligonucleotide purity was measured using HPLC. FLP stands for full length product. The HPLC measurement conditions are shown in Table 1 below.
[0076] (Measurement Method 2: Measurement of Oligonucleotide Yield) OD of the crude product ２６０ The OD was measured. ２６０ represents the UV 260 nm absorbance per 10 mm path length in 1 mL of solution (pH = 7.5). It is generally known that 1 OD = 40 μg for RNA. ２６０ The yield was calculated based on the measured values.
[0077] Solid-phase synthesis of oligonucleotides Sequence (I): 5'-UmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUm-3' (based on ST.25 format) (5'-TmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTmTm-3' (based on ST.26 format)) (SEQ ID NO: 4) 25mer In sequence (I), Um represents 2'-O-methyluridine (ST.25 format), and Tm represents 2'-O-methyluridine (ST.26 format).
[0078] The sequence (I) has the following structure (16):
[0079] The oligonucleotide consisting of the above sequence (I) was synthesized from the 3' to 5' end by phosphoramidite solid-phase synthesis using Controlled Pore Glass (CPG) as the solid support and an AKTA Oligopilot Plus 100 (GE Healthcare) as the nucleic acid synthesizer. The synthesis was carried out on an approximately 53 μmol scale. The synthesis used a 2'-OMe-U amidite solution prepared by dissolving the 2'-OMe-U amidite represented by formula (4) in a suitable solvent. A high-purity dichloroacetic acid toluene solution was used as the deblocking solution. A 5-benzylthio-1H-tetrazole solution was used as the condensing agent. An iodine solution was used as the oxidizing agent. A phenoxyacetic anhydride solution and an N-methylimidazole solution were used as the capping solution.
[0080] Next, specific examples of oligonucleotides produced by the method of the present invention will be described. In the following examples, the oligonucleotides produced by the method of the present invention are oligonucleotides having the sequence (I) shown in SEQ ID NO:4.
[0081] Furthermore, the 2'-OMe-U derivative CPG described in the following examples and comparative examples refers to the compound represented by the following formula (17), where the circle shown in formula (17) is a schematic representation of CPG.
[0082] Example 1: Using 53.7 μmol of 2'-OMe-U derivative CPG and a 2'-OMe-U amidite solution adjusted to 75 mM in a 9:1 acetonitrile:toluene solvent, the oligonucleotide shown in sequence (I) was synthesized automatically from the 3' to the 5' end using an AKTA Oligopilot Plus 100 (GE Healthcare). The automated synthesis procedure consisted of first loading a 3% dichloroacetic acid toluene solution into the CPG to deprotect the trityl protecting group at the 5' position. Subsequently, the 2'-OMe-U amidite solution and a 5-benzylmercapto-1H-tetrazole acetonitrile solution as a condensation agent were loaded into the CPG, allowing the condensation reaction to proceed with the hydroxyl group at the 5' position. Subsequently, a 50 mM iodine solution was loaded to convert the phosphite group to a phosphate group. Next, 0.1 M phenoxyacetic anhydride acetonitrile solution and 10 wt% N-methylimidazole / 10 wt% 2,6-lutidine acetonitrile solution were used as capping solutions to cap reaction sites where the condensation reaction had not progressed. These steps were repeated a total of 24 times to synthesize the nucleic acid oligonucleotide shown in Sequence (I) on the CPG support. The trityl protecting group at the 5' position was then deprotected with a 3% dichloroacetic acid toluene solution. The oligonucleotide was then released from the solid support using 4.09 g of aqueous ammonia and 1.21 g of ethanol for 8.0 μmol of oligonucleotide-loaded CPG support. The solid support was then removed by filtration, and the aqueous ammonia and ethanol were removed by drying under reduced pressure, yielding the desired oligonucleotide as a dry solid. Measurement using Measurement Method 2 yielded 57.3 mg. Measurement using Measurement Method 1 yielded an FLP ratio of 76.04% and an n-1 mer ratio of 7.17%.
[0083] Example 2: The oligonucleotide of sequence (I) was obtained in the same manner as in Example 1, except that 55.6 μmol of 2'-OMe-U derivative CPG was used and the solvent for the 2'-OMe-U amidite solution was a 4:1 mixture of acetonitrile and toluene. The yield was 58.3 mg, with an FLP ratio of 78.01% and an n-1 mer ratio of 5.24%.
[0084] Example 3: The oligonucleotide of sequence (I) was obtained in the same manner as in Example 1, except that 53.5 μmol of 2′-OMe-U derivative CPG was used and the solvent for the 2′-OMe-U amidite solution was a 4:6 mixture of acetonitrile and toluene. The yield was 54.3 mg, with an FLP ratio of 74.24% and an n-1 mer ratio of 4.14%.
[0085] Example 4: The oligonucleotide of sequence (I) was obtained in the same manner as in Example 1, except that 53.1 μmol of 2'-OMe-U derivative CPG was used and the solvent for the 2'-OMe-U amidite solution was a 4:1 mixture of acetonitrile and o-xylene. The yield was 58.6 mg, with an FLP ratio of 77.84% and an n-1 mer ratio of 5.22%.
[0086] Example 5: The oligonucleotide of sequence (I) was obtained in the same manner as in Example 1, except that 53.1 μmol of 2'-OMe-U derivative CPG was used and the solvent for the 2'-OMe-U amidite solution was a 4:1 mixture of acetonitrile and chlorobenzene. The yield was 57.3 mg, with an FLP ratio of 77.82% and an n-1 mer ratio of 5.78%.
[0087] Example 6: The oligonucleotide of sequence (I) was obtained in the same manner as in Example 1, except that 52.4 μmol of 2'-OMe-U derivative CPG was used and the solvent for the 2'-OMe-U amidite solution was a 4:1 mixture of acetonitrile and o-dichlorobenzene. The yield was 56.4 mg, with an FLP ratio of 76.62% and an n-1 mer ratio of 5.50%.
[0088] Comparative Example 1: The oligonucleotide of sequence (I) was obtained in the same manner as in Example 1, except that 52.8 μmol of 2′-OMe-U derivative CPG was used and the solvent for the 2′-OMe-U amidite solution was a 4:1 mixture of acetonitrile and dichloromethane. The yield was 59.4 mg, with an FLP ratio of 76.87% and an n-1 mer ratio of 7.68%.
[0089] The results of Examples 1 to 6 and Comparative Example 1 regarding the FLP ratio and n-1 mer ratio are shown in Table 2.
[0090] The results in Table 2 above show that when acetonitrile and aromatic hydrocarbons are used as solvents for the 2'-OMe-U amidite solution, the n-1 mer impurity can be relatively reduced compared to when the solution of Comparative Example 1 is used.
[0091] SEQ ID NOs: 1 to 4 in the sequence listing represent the base sequences of the oligonucleotides produced according to the production method of the present invention.
Claims
1. Formula (2): 【Chemistry 1】 [During the ceremony, G ２ This indicates a protecting group for hydroxyl groups. B ａ This indicates a nucleic acid base that may be protected by a protecting group. R represents a protected hydroxyl group, hydrogen atom, fluorine atom, methoxy group, 2-methoxyethyl group, or OQ' group. Q' represents the alkylene group bonded to the carbon atom at the 4' position of ribose. X represents an oxygen atom or a sulfur atom, and, *Bonds marked with an asterisk indicate binding to the 3' end of the nucleic acid. The compound shown by and formula (4): 【Chemistry 2】 [During the ceremony, G １ This indicates a protecting group for hydroxyl groups. The process includes condensing the 2'-OMe-U amidite shown in the formula with an activator, The 2'-OMe-U amidite is acetonitrile and formula (3): 【Transformation 3】 [In the formula, Y １ ~Y ６ Each of these independently represents a hydrogen atom, a methyl group, an ethyl group, or a halogen atom, either identical or distinct. It is dissolved in a mixture containing aromatic hydrocarbons, Method for producing oligonucleotides.
2. The method for producing an oligonucleotide according to claim 1, wherein the aromatic hydrocarbon represented by formula (3) is a benzene having one or two substituents selected from the group consisting of a methyl group, an ethyl group, and a halogen atom, and if the benzene has two substituents, the substituents may be the same or different.
3. The method for producing an oligonucleotide according to claim 1, wherein the aromatic hydrocarbon represented by formula (3) is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and mixtures of two or more thereof.
4. A method for producing oligonucleotides according to any one of claims 1 to 3, wherein the volume ratio of acetonitrile and the aromatic hydrocarbon represented by formula (3) in a mixture in which the 2'-OMe-U amidite represented by formula (4) is dissolved is 99:1 to 1:99 in terms of acetonitrile:aromatic hydrocarbon represented by formula (3).
5. A method for producing oligonucleotides according to any one of claims 1 to 3, wherein the volume ratio of acetonitrile and aromatic hydrocarbon represented by formula (3) in a mixed solution in which 2'-OMe-U amidite represented by formula (4) is dissolved is 90:10 to 10:90 in terms of acetonitrile:aromatic hydrocarbon represented by formula (3).
6. A method for producing oligonucleotides according to any one of claims 1 to 3, wherein the volume ratio of acetonitrile and aromatic hydrocarbon represented by formula (3) in a mixed solution in which 2'-OMe-U amidite represented by formula (4) is dissolved is 90:10 to 40:60 in terms of acetonitrile:aromatic hydrocarbon represented by formula (3).
7. A method for producing oligonucleotides according to any one of claims 1 to 3, wherein the concentration of 2'-OMe-U amidite in the 2'-OMe-U amidite solution represented by formula (4) is 0.01 to 0.4 M.
8. A method for producing oligonucleotides according to any one of claims 1 to 3, wherein the concentration of 2'-OMe-U amidite in the 2'-OMe-U amidite solution represented by formula (4) is 0.05 to 0.2 M.
9. A method for producing an oligonucleotide according to any one of claims 1 to 3, wherein the activator is 5-benzylthio-1H-tetrazole.
10. If R in the compound represented by formula (2) is a protected hydroxyl group, then the protecting group of that hydroxyl group is formula (10): 【Chemistry 4】 [During the ceremony, q represents an integer from 0 to 5. R ａ and R ｂ These are identical or distinct, representing a methyl group, an ethyl group, or a hydrogen atom. Bonds marked with an asterisk are bonded to the oxygen of the 2'-hydroxyl group. Ew represents an electron-withdrawing group. A method for producing an oligonucleotide according to any one of claims 1 to 3, wherein the protecting group is represented by .
11. In the 2'-OMe-U amidite represented by equation (4), G １ A method for producing an oligonucleotide according to any one of claims 1 to 3, wherein the group is a 4,4'-dimethoxytrityl group.
12. A method for producing an oligonucleotide according to any one of claims 1 to 3, carried out by a solid-phase synthesis method.
13. Formula (4): 【Transformation 5】 [During the ceremony, G １ represents a protecting group for a hydroxyl group. The 2'-OMe-U amidite, acetonitrile, and formula (3) shown: 【Transformation 6】 [In the formula, Y １ ~Y ６ Each of these independently represents a hydrogen atom, a methyl group, an ethyl group, or a halogen atom, either identical or distinct. A solution containing aromatic hydrocarbons represented by [the symbol].
14. A method for producing oligonucleotides using a solution containing a 2'-OMe-U amidite represented by formula (4) as described in claim 13, acetonitrile, and an aromatic hydrocarbon represented by formula (3).