Chimeric nucleic acid oligomer containing phosphorothioate and boranophosphate, and method for producing the same

Abstract

Provided are a novel nucleic acid oligomer and a method for producing the same.　The nucleic acid oligomer according to the present invention includes a nucleotide unit represented by general formula (1) and a nucleotide unit represented by general formula (2), and may or may not include a nucleotide unit represented by general formula (3). In general formulas (1), (2), and (3): R1 represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group, and R2 represents a hydrogen atom, or R1 and R2 bond together to form a five- or more-membered ring optionally having a substituent and optionally having a hetero atom; Bs represents a nucleobase selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, the nucleobase optionally having a nucleobase protecting group; and X+ represents a counter cation.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid oligomer and a method for producing the same. [Background technology]

[0002] Antisense molecules, which have a base sequence complementary to that of a target nucleic acid, form a complementary duplex with the target nucleic acid and can inhibit protein production from the target nucleic acid. When a disease-related gene is selected as the target nucleic acid, antisense molecules act directly on the disease-related gene, and are therefore attracting attention as effective medicines for gene therapy.

[0003] Antisense molecules (nucleic acid oligomers) efficiently inhibit the production of targeted proteins From the viewpoint of the use of nucleotides, the main factors are cell membrane permeability, nuclease resistance, and bioavailability (e.g., pH 7.4). It has chemical stability under various environmental conditions and the property of forming a stable double strand only with a specific base sequence. As an antisense molecule, for example, a phosphorothioate compound The nucleic acid oligomer obtained using the method (hereinafter referred to as "phosphorothioate type nucleic acid oligomer") Nucleic acid oligomers obtained using boranophosphate compounds (hereinafter referred to as "boranophosphate-type nucleic acid oligomers") (Non-Patent Document 1) and nucleic acid oligomers obtained using boranophosphate compounds (hereinafter referred to as "boranophosphate-type nucleic acid oligomers") (Non-Patent Document 2) are known. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nucleic Acids Research, 2010, Vol.38, No.20, pp.7100-7111 [Non-patent document 2] Chem. Rev., 2007, Vol.107, pp.4746-4796 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a novel nucleic acid oligomer and a method for producing the same. [Means for solving the problem]

[0006] Specific means for solving the above problems include the following embodiments. <1> A nucleic acid oligomer comprising a nucleotide unit represented by the following general formula (1) and a nucleotide unit represented by the following general formula (2), and which may or may not contain a nucleotide unit represented by the following general formula (3): [ka] (In general formulas (1), (2), and (3), R 1 represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R 2 represents a hydrogen atom, or R 1 and R 2 are bonded to each other to form a ring of 5 or more members which may or may not have a substituent and which may or may not have a heteroatom, Bs represents a nucleobase selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the nucleobase may have a protecting group for the nucleobase, X + represents the counter cation.)

[0007] <2> R 1 represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R 2 represents a hydrogen atom, or R 1 and R 2 are bonded to each other to form a divalent group represented by the following general formula (4): <1> The nucleic acid oligomer according to claim 1. [ka] (In general formula (4), R 3 and R 4 are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or are bonded to each other to form a ring; * and ** represent a bond; the bond represented by * is the same as R 1 is bonded to the carbon atom to which R is directly bonded, and the bond represented by ** is 2 is bonded to the carbon atom directly connected to it.)

[0008] <3> <1> or <2> A method for producing a nucleic acid oligomer according to claim 1, a condensation step of sequentially condensing nucleotide monomers selected from the group consisting of compounds represented by the following general formula (5) and compounds represented by the following general formula (6), or nucleotide monomers selected from the group consisting of compounds represented by the following general formula (5), compounds represented by the following general formula (6), and compounds represented by the following general formula (7), to obtain a precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (8) and a nucleotide unit represented by the following general formula (9), and which may or may not contain a nucleotide unit represented by the following general formula (10); oxidizing the precursor nucleic acid oligomer with an oxidizing agent; <1> an oxidation step to obtain the nucleic acid oligomer according to A manufacturing method comprising: [ka] (In general formulas (5), (6), and (7), R 1 , R 2 , Bs, and X + is as described above, and R 5 represents a protecting group for a hydrogen atom or a hydroxyl group. [ka] (In general formulas (8), (9), and (10), R 1 , R 2 , and Bs are as described above.)

[0009] <4> At least the condensation step is carried out by a reaction using a solid phase support. <3> The manufacturing method described in [Effects of the Invention]

[0010] According to the present invention, a novel nucleic acid oligomer and a method for producing the same can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] 1 shows charts of reversed-phase HPLC performed in Comparative Synthesis Examples or Synthesis Examples: (a) Comparative Synthesis Example 1, (b) Comparative Synthesis Example 2, (c) Synthesis Example 2, (d) Synthesis Example 3, (e) Synthesis Example 6, and (f) Synthesis Example 7. [Figure 2] 1 shows reverse-phase HPLC charts of the nucleic acid oligomer of Example 1. (a) Before separation and purification, (b) After separation and purification. [Figure 3] 1 shows reverse-phase HPLC charts of the nucleic acid oligomer of Example 2. (a) Before separation and purification, (b) After separation and purification. [Figure 4] 1 shows reverse-phase HPLC charts of the nucleic acid oligomer of Example 3. (a) Before separation and purification, (b) After separation and purification [Figure 5] 1 shows reverse-phase HPLC charts of the nucleic acid oligomer of Example 4. (a) Before separation and purification, (b) After separation and purification. [Figure 6] FIG. 1 shows a chart of reversed-phase HPLC performed in Synthesis Example 12. [Figure 7] FIG. 1 shows a chart of reverse-phase UHPLC performed on the nucleic acid oligomer of Example 5. [Figure 8] 1 shows reverse-phase HPLC charts of the nucleic acid oligomer of Example 6. (a) Before separation and purification, (b) After separation and purification DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments. In this specification, the term "process" includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved. In addition, in this specification, "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively. Furthermore, in this specification, when multiple substances corresponding to each component are present in the composition, the amount of each component in the composition means the total amount of the multiple substances present in the composition, unless otherwise specified.

[0013] <Nucleic acid oligomer> The nucleic acid oligomer according to this embodiment contains a nucleotide unit represented by the following general formula (1) and a nucleotide unit represented by the following general formula (2), and may or may not contain a nucleotide unit represented by the following general formula (3). [ka]

[0014] In general formulas (1), (2), and (3), R 1 represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R 2 represents a hydrogen atom, or R 1 and R 2 are bonded to each other to form a ring of 5 or more members which may or may not have a substituent and which may or may not have a heteroatom, Bs represents a nucleobase selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the nucleobase may have a protecting group for the nucleobase, X + represents a counter cation.

[0015] Nucleic acid oligomers containing nucleotide units represented by general formula (1) tend to have excellent affinity for target RNA, RNase H induction activity, and resistance to degradative enzymes, and are particularly long-lasting in blood, but are prone to high toxicity. Nucleic acid oligomers containing nucleotide units represented by general formula (2) tend to have excellent affinity for target RNA, RNase H induction activity, and are particularly long-lasting in blood, but are unlikely to be highly toxic, and have not been shown to have a tendency toward excellent blood retention. Nucleic acid oligomers containing nucleotide units represented by general formula (3) tend to have excellent affinity for target RNA and RNase H induction activity, and are unlikely to be highly toxic, but are likely to have low resistance to degradative enzymes and low blood retention. The nucleic acid oligomer of this embodiment contains a nucleotide unit represented by general formula (1) and a nucleotide unit represented by general formula (2), or contains a nucleotide unit represented by general formula (1), a nucleotide unit represented by general formula (2), and a nucleotide unit represented by general formula (3), and is therefore expected to have low toxicity while maintaining high affinity for the target RNA, RNase H induction activity, resistance to degradative enzymes, and blood retention.

[0016] The length (base length) of the nucleic acid oligomer according to this embodiment is not particularly limited as long as the nucleic acid oligomer has two or more bases, and a desired length can be appropriately selected depending on the type, length, etc. of the target nucleic acid. The length of the nucleic acid oligomer is not particularly limited, and from the viewpoint of application to antisense medicines, it is preferably 8 to 50 bases, more preferably 10 to 30 bases, and even more preferably 10 to 21 bases.

[0017] The base sequence of the nucleic acid oligomer is set based on the base sequence of the target nucleic acid. From the viewpoint of double-strand formation ability, the base sequence of the nucleic acid oligomer is appropriately selected so as to be complementary to the base sequence of the target nucleic acid, but in some cases, the base sequence may contain one or more different bases. Furthermore, in the nucleic acid oligomer, the ratio of the nucleotide units represented by general formula (1), the nucleotide units represented by general formula (2), and the nucleotide units represented by general formula (3) is appropriately selected so as to maintain high affinity for the target RNA, RNase H induction activity, resistance to degradative enzymes, and blood retention while reducing toxicity.

[0018] R 1 The alkoxy group represented by is preferably an alkoxy group having 1 to 12 carbon atoms, more preferably an alkoxy group having 1 to 6 carbon atoms, and examples thereof include a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxy group, a sec-butoxy group, a tert-butoxy group, and an n-pentyloxy group.

[0019] R 1 Examples of the alkenyloxy group represented by include a vinyloxy group, an allyloxy group, a 1-propenyloxy group, an isopropenyloxy group, a 2-methyl-1-propenyloxy group, a 2-methylallyloxy group, and a 2-butenyloxy group.

[0020] R 1 Examples of the acyloxy group represented by the formula (I) include alkyl-carbonyloxy groups having 1 to 6 carbon atoms (such as a methylcarbonyloxy group, an ethylcarbonyloxy group, etc.), and aryl-carbonyloxy groups having 6 to 10 carbon atoms (such as a benzoyloxy group).

[0021] R 1 Examples of the trialkylsilyloxy group represented by include a trimethylsilyloxy group and a triethylsilyloxy group.

[0022] R 1Examples of the alkoxyalkoxy group represented by include a methoxymethoxy group, a methoxyethoxy group, an ethoxymethoxy group, an ethoxyethoxy group, and the like.

[0023] R 1 Examples of the haloalkoxyalkoxy group represented by include a (2-fluoro)ethoxymethoxy group, a (2,2-difluoro)ethoxymethoxy group, a (2-chloro)methoxyethoxy group, and a (2,2-dichloro)ethoxymethoxy group.

[0024] R 1 Examples of the halogenyl group represented by include a fluoro group, a chloro group, and a bromo group, with a fluoro group being preferred.

[0025] R 1 Among the above, a hydrogen atom, a hydroxyl group, an alkoxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, and a halogenyl group are preferred in terms of double-strand formation ability.

[0026] R 1 and R 2 may be bonded to each other to form a 5-membered or greater ring, which may or may not have a substituent and which may or may not have a heteroatom. Examples of the substituent include substituents bonded to atoms forming the ring, and specific examples include alkyl groups, oxo groups (=O), and the like, which may or may not have a substituent. Examples of the alkyl group include a methyl group and an ethyl group. When the alkyl group has a substituent, examples of the substituent include halogenyl groups such as a fluoro group, a chloro group, and a bromo group, amino groups, and imino groups. Examples of the heteroatom include an oxygen atom, a nitrogen atom, and a sulfur atom. The number of members in the ring is preferably 5 to 10, more preferably 5 to 8, and even more preferably 5 to 7.

[0027] In terms of duplex formation ability, R 1 and R 2are preferably bonded to each other to form a divalent group represented by the following general formula (4). [ka]

[0028] In general formula (4), R 3 and R 4 are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or are bonded to each other to form a ring; * and ** represent a bond; the bond represented by * is the same as R 1 is bonded to the carbon atom to which R is directly bonded, and the bond represented by ** is 2 is bonded to the carbon atom directly connected to it.

[0029] R 3 or R 4 Examples of the alkyl group represented by R include linear or branched alkyl groups having 1 to 10 carbon atoms, and specific examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-hexyl, and n-octyl groups. 3 and R 4 When they are bonded to each other to form a ring, specific examples of the ring include a cyclopropane ring, a cyclobutane ring, and a cyclohexane ring.

[0030] R 1 and R 2 When these are bonded to each other to form a ring of 5 or more members which may or may not have a substituent and which may or may not have a hetero atom, specific examples of the ring include rings represented by the following formula: [ka] (In the formula, Bs is as defined above.)

[0031] Bs represents a nucleobase selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the nucleobase may have a protecting group. When the nucleobase has a protecting group, the type of protecting group is not particularly limited. Examples of the protecting group include a benzyl group, 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 chloroacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, and a (dimethylamino)methylene group.

[0032] X + represents a counter cation. Examples of the counter cation include ammonium ions, cations derived from organic amine compounds, and metal cations. Examples of the cation derived from organic amine compounds include, in terms of solubility in organic solvents and volatility of the counter cation, cations derived from tertiary alkylamine compounds and cations derived from quaternary ammonium compounds. Examples of the cation derived from tertiary alkylamine compounds include the cation HNEt3 derived from triethylamine. + (Et represents an ethyl group), and cations derived from 1,8-diazabicyclo[5.4.0]undecene. Examples of cations derived from quaternary ammonium compounds include, for example, tetrabutylammonium ions. Examples of metal cations include Na + , Li + , K. + etc.

[0033] The nucleic acid oligomer according to this embodiment may be a nucleic acid oligomer having nucleotide units other than nucleotide units represented by general formula (1), nucleotide units represented by general formula (2), and nucleotide units represented by general formula (3). In the nucleic acid oligomer, the total proportion of nucleotide units represented by general formula (1), nucleotide units represented by general formula (2), and nucleotide units represented by general formula (3) is not particularly limited, and from the viewpoint of double-strand forming ability, it is preferably 40 to 100 mol %, more preferably 50 to 100 mol %, even more preferably 60 to 100 mol %, and particularly preferably 70 to 100 mol %, relative to the total nucleotide units in the nucleic acid oligomer.

[0034] In the nucleic acid oligomer according to this embodiment, the 5'-end may be a hydroxyl group or a hydroxyl-protecting group, and the 3'-end may be a hydroxyl group or a hydroxyl-protecting group. Examples of hydroxyl-protecting groups include acetyl, phenylacetyl, phenoxyacetyl, chloroacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, 4-methoxybenzoyl, triphenylmethyl, 4,4'-dimethoxytrityl (DMTr), 4-methoxytrityl (MMTr), 9-phenylxanthenyl, t-butoxycarbonyl, trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, and cyanoethoxymethyl groups.

[0035] <Method of manufacturing nucleic acid oligomer> The method for producing a nucleic acid oligomer according to this embodiment includes the steps of: a condensation step of sequentially condensing nucleotide monomers selected from the group consisting of compounds represented by the following general formula (5) and compounds represented by the following general formula (6), or nucleotide monomers selected from the group consisting of compounds represented by the following general formula (5), compounds represented by the following general formula (6), and compounds represented by the following general formula (7), to obtain a precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (8) and a nucleotide unit represented by the following general formula (9), and which may or may not contain a nucleotide unit represented by the following general formula (10); an oxidation step of oxidizing the precursor nucleic acid oligomer with an oxidizing agent to obtain the nucleic acid oligomer according to this embodiment; Includes: [ka]

[0036] In general formulas (5), (6), and (7), R 1 , R 2 , Bs, and X + is as described above, and R 5 represents a protecting group for a hydrogen atom or a hydroxyl group. Examples of the protecting group for a hydroxyl group include an acetyl group, a phenylacetyl group, a phenoxyacetyl group, a chloroacetyl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a 4-methoxybenzoyl group, a triphenylmethyl group, a 4,4'-dimethoxytrityl (DMTr) group, a 4-methoxytrityl (MMTr) group, a 9-phenylxanthenyl group, a t-butoxycarbonyl group, a trimethylsilyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a cyanomethoxymethyl group, a 2-(cyanoethoxy)ethyl group, and a cyanoethoxymethyl group. Among these, the 4,4'-dimethoxytrityl group is preferred because it can be easily removed under acidic conditions and is suitable for chain elongation in solid-phase synthesis.

[0037] [ka]

[0038] In general formulas (8), (9), and (10), R 1 , R 2 , and Bs are as defined above.

[0039] In the condensation step, nucleotide monomers selected from the group consisting of compounds represented by general formula (5) and compounds represented by general formula (6), or nucleotide monomers selected from the group consisting of compounds represented by general formula (5), compounds represented by general formula (6), and compounds represented by general formula (7), are sequentially condensed to obtain a precursor nucleic acid oligomer containing a nucleotide unit represented by general formula (8) and a nucleotide unit represented by general formula (9), and which may or may not contain a nucleotide unit represented by general formula (10). During this process, the hydroxyl group bonded to the 5' carbon atom of the ribose structure in the nucleoside structure of the nucleic acid oligomer condenses with the phosphate moiety of the monomer.

[0040] A condensing agent can be used in the condensation step. Examples of condensing agents include N,N-bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride, 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP), and 1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphosphoridium hexafluorophosphate (MNTP). These condensing agents are less likely to undergo side reactions with hydroxyl groups on the solid support, making them suitable for synthesizing long-chain nucleic acid oligomers. The compound represented by general formula (5) contains nucleophilic oxygen and sulfur atoms within the molecule. Reaction of the oxygen atom with the condensing agent promotes the desired reaction, whereas reaction of the sulfur atom with the condensing agent results in a side reaction. From the viewpoint of suppressing side reactions, it is preferable to use PyNTP as a condensing agent when condensing a compound represented by general formula (5). From the viewpoint of quickly completing the reaction, it is preferable to use MNTP as a condensing agent when condensing a compound represented by general formula (6) or a compound represented by general formula (7).

[0041] The condensation step can be further carried out in the presence of a basic compound. Examples of basic compounds include 1,8-bis(dimethylamino)naphthalene (DMAN), 2,2,6,6-tetramethylpiperidine (TMP), diisopropylethylamine (DIPEA), 2,6-lutidine, 2,4,6-trimethylpyridine, pyridine, and quinoline. Among these, quinoline is preferred when condensing a compound represented by general formula (5) in which Bs is adenine, cytosine, or 5-methylcytosine, from the viewpoint of suppressing side reactions. 2,6-lutidine is preferred when condensing a compound represented by general formula (6) or a compound represented by general formula (7). Furthermore, from the viewpoint of suppressing side reactions, the condensation step is preferably carried out in the absence of a basic compound when condensing a compound represented by general formula (5) in which Bs is guanine, thymine, or uracil.

[0042] The condensation reaction in the condensation step can be carried out, for example, under ice-cooling (0°C) to room temperature (25°C), preferably in the range of 20°C to 25°C, for example, 1 minute to several hours, preferably 3 minutes to 1 hour.

[0043] Examples of the reaction solvent include an inert solvent, such as acetonitrile.

[0044] In the method for producing a nucleic acid oligomer according to this embodiment, it is preferable that at least the condensation step is carried out by a reaction using a solid phase support (solid phase method). Specifically, both the condensation step and the oxidation step may be carried out by a reaction using a solid phase support, or the condensation step may be carried out by a reaction using a solid phase support and the oxidation step may be carried out by another method, for example, a reaction in liquid phase. When producing a nucleic acid oligomer by a solid phase reaction, a monomer containing a base at the 3' end of the nucleic acid oligomer can be bound to the solid phase support, for example, via an oxygen atom bound to the 3' carbon atom of the ribose structure. In this case, a linker may be present between the monomer and the solid phase support, if necessary.

[0045] The type of the solid support is not particularly limited, and examples thereof include pore glass, oxalated pore glass, TentaGel support-aminopolyethylene glycol-derivatized support, highly cross-linked aminomethyl polystyrene, and Pros-polystyrene / divinylbenzene copolymer. Amino groups on the solid support can be used to link the solid support to the monomer. Examples of amino groups on the solid support include 3-aminopropyl groups and long-chain alkylamino groups (LCAAs). A linker may be present between the solid support and the monomer. Examples of the linker include succinyl groups and oxalyl groups.

[0046] In the oxidation step, the precursor nucleic acid oligomer obtained in the condensation step is oxidized with an oxidizing agent to obtain the nucleic acid oligomer according to this embodiment. Examples of oxidizing agents include (+)-camphorylsulfonyloxaziridine (CSO), (+)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine (DCSO), t-butyl hydroperoxide, methyl ethyl ketone peroxide, and a combination of a positive halogen reagent (carbon tetrachloride, carbon tetrabromide, iodine, N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, etc.) and water. The oxidation step may be performed in the presence of a basic compound as the oxidizing agent. Examples of basic compounds include triethylamine and diisopropylethylamine.

[0047] The condensation step in the method for producing a nucleic acid oligomer according to this embodiment involves condensation of the hydroxyl group attached to the 5' carbon atom of the ribose structure in the nucleotide structure of the nucleic acid oligomer with the phosphate moiety of the monomer. Therefore, if the hydroxyl group attached to the 5' carbon atom has a protecting group, the method includes, for example, a step of reacting the protecting group with a deprotecting reagent to remove the protecting group (first protecting group elimination step). The first protecting group elimination step may be performed at any time as long as the condensation reaction in the production of the nucleic acid oligomer proceeds sufficiently, and is preferably performed before the condensation reaction. A deprotecting agent can be used in the first protecting group elimination step. Examples of deprotecting agents that can be used in the first protecting group elimination step include halogenated alkyl carboxylic acids. Examples of halogenated alkyl carboxylic acids include trifluoroacetic acid, trichloroacetic acid, and dichloroacetic acid. When a trityl-based protecting group such as triphenylmethyl, 4,4'-dimethoxytrityl (DMTr), or 4-methoxytrityl (MMTr) is used as the protecting group for the hydroxyl group bonded to the 5' carbon atom, it is desirable to add a cation scavenger to avoid a side reaction between the trityl cation and the borano group that occurs in the first protecting group removal step. Examples of cation scavenger include triethylsilane. It is preferable that the nucleic acid oligomer having the protecting group reacts with 2 to 100 equivalents of the deprotecting agent.

[0048] Furthermore, when the base in the monomer used in the method for producing a nucleic acid oligomer has a protecting group, the method may further include a step of removing the protecting group from the base (second protecting group elimination step). The stage at which the second protecting group elimination step is performed is not particularly limited, as long as the base in the finally obtained nucleic acid oligomer does not have a protecting group. In the second protecting group elimination step, for example, the monomer or nucleic acid oligomer is treated with aqueous ammonia. Examples of the aqueous ammonia used in the second protecting group elimination step include 25% by mass aqueous ammonia or a 25% by mass aqueous ammonia-ethanol mixed solution (3:1, v / v).

[0049] The resulting nucleic acid oligomer can be purified by known purification methods such as reversed-phase high performance liquid chromatography (reverse-phase HPLC), ion-exchange HPLC, column chromatography, and recrystallization. [Example]

[0050] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples. The various analyzers used were the following models: 1 H-nuclear magnetic resonance spectrum ( 1 H-NMR):JEOL JNM-ECZ400S(400MHz) 31 P-nuclear magnetic resonance spectrum ( 31 P-NMR):JEOL JNM-ECZ400S(162MHz) Tetramethylsilane (TMS) was used as the internal standard for 1H-NMR. 31 For P-NMR, 85% H3PO4 was used as an external standard.

[0051] HRMS(ESI-TOF):Sciex X500R QTOF Ion-exchange HPLC: GE Healthcare AKTA purifier 10 S Silica Gel 60N from Kanto Chemical was used as the silica gel packed in the column chromatography. Column used for reversed-phase HPLC (analytical): Waters μ-BOUNDASPHERE, C18 5 μm, 100 Å, 3.9 mm × 159 mm Column used for ion-exchange HPLC (analytical / preparative): GE Healthcare MiniQTM 4.6 / 50PE, 3 μm, 4.6 mm x 50 mm Reversed-phase UHPLC:waters ACQUITY UPLC H-Class Bio PDA:waters ACQUITY UPLC PDA eλ Detector LRMS(ESI):waters ACQUITY QDa Detector Column used for reversed-phase UHPLC (analysis): Waters ACQUITY UPLC Oligonucleotide BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 100 mm

[0052] Unless otherwise specified, "%" is based on mass. In the examples, DMTr represents 4,4'-dimethoxytrityl, and other abbreviations have the same meanings as in the above explanation.

[0053] <Monomer synthesis 1> [ka]

[0054] (Synthesis of H-phosphonate and H-boranophosphonate monomers) H-phosphonate monomers 3a, 3c, 3g, and 3t and H-boranophosphonate monomers 4a, 4c, 4g, and 4t were synthesized by known methods (J. Org. Chem., 2019, Vol. 84, pp. 15032-15041; J. Org. Chem., 2014, Vol. 79, pp. 3465-3472).

[0055] (Synthesis of H-phosphonothioate monomers) With reference to a known method (J. Org. Chem., 1990, Vol. 55, pp. 3503-3506), an H-phosphonothioate monomer was synthesized according to the following scheme. [ka]

[0056] Nucleoside derivative 1a, 1c, 1g, or 1t (A: 0.90 g, 1.5 mmol; C: 0.96 g, 1.5 mmol; G: 0.97 g, 1.5 mmol; or T: 0.96 g, 1.5 mmol) shown in the leftmost part of the scheme above and 0.19 mL (1.0 mmol) of triethylammonium phosphinic acid were azeotropically dried with pyridine (3 mL x 3) under an argon atmosphere and dissolved in pyridine (5 mL). The resulting pyridine solution was stirred in an ice bath (0 °C) while adding 0.17 mL (1.5 mmol) of pivaloyl chloride and stirred for 15 minutes. The reaction temperature was then lowered to room temperature (25 °C), and 48 mg (1.5 mmol) of sulfur was added and stirred for an additional hour. This solution was diluted with 30 mL of chloroform and washed with 1 M TEAB buffer solution (pH 7.0, 30 mL x 3). The aqueous layer was back-extracted with chloroform (30 mL x 3 times), and the water in the organic layer was removed with anhydrous sodium sulfate. The organic layer was then evaporated, and the residue was purified by silica gel column chromatography (eluent: A: ethyl acetate-methanol-triethylamine (100:0:1-100:4:1, v / v / v), followed by chloroform-methanol-triethylamine (100:2:1, v / v / v); C: ethyl acetate-methanol-triethylamine (100:0:1-100:4:1, v / v / v), followed by chloroform-methanol-triethylamine (100:4:1, v / v / v); G: ethyl acetate-methanol-triethylamine (100:2:1, v / v / v), followed by chloroform-methanol-triethylamine (100:3:1-100:5:1, v / v / v); T: ethyl acetate-methanol-triethylamine (100:0:1-100:1:1, v / v / v), followed by chloroform-methanol-triethylamine). The resulting mixture was purified by elution with 0.2 M DBU bicarbonate (100:0:1-100:3:1, v / v / v), salt exchange was performed, the organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give the target compounds (H-phosphonothioate monomers 5a, 5c, 5g, or 5t). Yields: A: 0.88 g, 0.95 mmol, 95%; C: 0.89 g, 0.99 mmol, 99%; G: 0.58 g, 0.64 mmol, 64%; T: 0.64 g, 0.65 mmol, 65%.

[0057] H-phosphonothioate monomer 5a 1 H-NMR(400MHz,CDCl3)δ11.9-11.7(br,1H),9.2-8.9(br,1H),8.71(s,1H),8.20(s,1H),8.17(d,J=574Hz,0.5 H),8.13(d,J=574Hz,0.5H),8.01(d,J=7.3Hz,2H),7.62-7.57(m,1H),7.54-7.48(m,2H),7.46-7.38(m,3H),7 .34-7.10(m,6H),6.79(d,J=8.7Hz,4H),6.65-6.57(m,1H),5.43-5.35(m,1H),4.57-4.54(m,0.5H),4.48-4.4 4(m,0.5H),3.76(s,6H),3.52-3.36(m,8H),2.98-2.84(m,4H),1.99(quintet,J=5.8Hz,2H),1.80-1.60(m,6H) 31 P-NMR(162MHz,CDCl3)δ53.0,52.9.

[0058] H-phosphonothioate monomer 5c 1H-NMR(400MHz,CDCl3)δ11.7-11.4(br,1H),8.8-8.5(br,1H),8.19(d,J=5.0Hz,0.5H),8.17(d,J=5.0Hz,0.5H),8.13(d,J=574Hz,0.5H),8. 10(d,J=581Hz,0.5H),7.46-7.39(m,2H),7.13(d,J=6.0Hz,0.5H),7.11(d,J=6.0Hz,0.5H),6.90-6.81(m,4H),6.34-6.24(m,1H),6.30(t,J= 6.0Hz, 0.5H), 6.29(t, J=6.0Hz, 0.5H) 5.37-5.29(m, 0.5H), 5.25-5.17(m, 0.5H), 4.48(q, J=3.0Hz, 0.5H), 4.36(q, J=3.3Hz, 0.5H), 3.80(s, 6H), 3.54-3.29(m, 8H), 2.92-2.82(m, 3H), 2.69-2.61(m, 1H), 2.38-2.26(m, 1H), 2.00(q, J=5.8Hz, 2H), 1.82-1.60(m, 7H), 1.25-1.17(m, 6H) 31 P-NMR(162MHz,CDCl3)δ53.6,52.6.

[0059] H-ホスホノチオエートモノマー5g 1 H-NMR(400MHz,CDCl3)δ8.12(d,J=581Hz,0.5H),8.05(d,J=574Hz,0.5H),7.82(s,0.5H),7.80(s,0.5H),7.44-7.31 (m,3H),7.31-7.22(m,4H),7.22-7.13(m,2H),6.79-6.67(m,4H),6.18(t,J=6.0Hz,1H),6.18(t,J=6.4Hz,1H),5.75- 5.62(m,0.5H), 5.62-5.51(m,0.5H), 4.37(q,J=3.7Hz,0.5H), 4.26(q,J=3.7Hz,0.5H), 3.75(s,6H), 3.50-3.25(m,7H), 3.02-2.83(m,1H), 2.83-2.74(m,2H), 2.74-2.58(m,2H), 2.03-1.87(m,2H), 1.83-1.53(m,6H), 1.20-1.07(m,6H) 31 P-NMR(162MHz,CDCl3)δ52.8,52.5.

[0060] H-phosphonothioate monomer 5t 1 H-NMR(400MHz,CDCl3)δ11.6-11.5(br,1H),8.13(d,J=572Hz,0.5H),8.08(d,J=578Hz,0.5H),7.92(d,J=8.2Hz,2H),7.78(s,0.5H),7.77(s,0.5H), 7.64(t,J=7.3Hz,1H),7.53-7.39(m,5H),7.41(d,J=572Hz,0.5H),7.36(d ,J=578Hz,0.5H),7.38-7.27(m,5H),7.26-7.18(m,1H),6.91-6.82(m,4H) ,6.44(t,J=8.7Hz,0.5H),6.43(t,J=8.9Hz,0.5H),5.51-5.41(m,0.5H),5 .41-5.32(m,0.5H),4.41(d,J=1.4Hz,0.5H),4.30(d,J=2.3Hz,0.5H),3.7 9(s,6H),3.63-3.46(m,1H),3.45-3.31(m,1H),2.87-2.62(m,3H),2.54-2 .38(m,1H),1.93(q,J=5.8Hz,2H),1.78-1.54(m,6H),1.37(d,J=6.9Hz,3H) 31 P-NMR(162MHz,CDCl3)δ54.5,53.4.

[0061] (Synthesis of 2'-OMe-modified H-boranophosphonate monomers) 2'-OMe-modified H-boranophosphonate monomers were synthesized according to the following scheme. [ka]

[0062] Nucleoside derivatives 2a, 2c, 2g, or 2t (A: 0.69 g, 1.0 mmol; C: 0.68 g, 1.0 mmol; G: 0.70 g, 1.0 mmol; or U: 0.67 g, 1 mmol) shown in the leftmost part of the scheme above and pyridinium H-boranophosphonate (0.28 g, 2.0 mmol) were azeotropically dried with pyridine (3 mL x 3) under an argon atmosphere and dissolved in 25 mL of pyridine. BopCl (0.51 g, 2.0 mmol) was added to the resulting pyridine solution while stirring in an ice bath. The reaction temperature was then brought to room temperature and stirred for 1 hour. This solution was diluted with 30 mL of chloroform, and the organic layer was washed with 1.0 M TEAB buffer solution (pH 7.0) (30 mL x 3). The aqueous layer was back-extracted with chloroform (30 mL x 3). The organic layer was collected and water was removed with anhydrous sodium sulfate, and then the solvent in the organic layer was distilled off under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (developing solvent A: ethyl acetate-methanol-triethylamine (100:0:1-100:4:1, v / v / v), followed by chloroform-methanol-triethylamine (100:4:1, v / v / v); C: ethyl acetate-methanol-triethylamine (100:0:1-100:4:1, v / v / v), followed by chloroform-methanol-triethylamine (100:4:1, v / v / v); G: ethyl acetate-methanol-triethylamine (100:2:1, v / v / v), followed by chloroform-methanol-triethylamine (100:3:1-100:5:1, v / v / v); U: ethyl acetate-methanol-triethylamine (100:0:1, v / v / v), followed by chloroform-methanol-triethylamine (100:0:1-100:1:1, v / v / v), 1.0 M The mixture was extracted with TEAB buffer solution (pH 7.0) and chloroform (30 mL x 3 times). The water in the organic layer was removed with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give the target product (2'-OMe-modified H-boranophosphonate monomer 6a, 6c, 6g, or 6u). Yields: A: 0.81 g, 0.95 mmol, 95%; C: 0.75 g, 0.90 mmol, 90%; G: 0.78 g, 0.92 mmol, 92%; U: 0.62 g, 0.74 mmol, 74%

[0063] 2'-OMe modified H-ボラノホスホネートモノマー6a 1 H-NMR(400MHz, CDCl3)δ9.09(s,1H),8.70(s,0.5H),8.69(s,0.5H),8.24(s,0.5H),8.20(s,0.5H),8.02(d,J=7.8Hz,2H) ,7.9-7.8(br,0.5H),7.60(t,J=7.3Hz,1H),7.51(t,J=7.8Hz,2H),7.48-7.42(m,2H),7.36-7.31(m,4H),7.30-7.23(m,2H ),7.23-7.17(m,1H),6.84-6.78(m,4H),6.27(t,J=5.0Hz,0.5H),6.27(t,J=5.0Hz,0.5H),5.09-5.02(m,1H),4.74-4.68 (m,1H),4.55-4.50(m,1H),3.77(s,6H),3.55-3.48(m,5H),2.99(q,J=7.3Hz,6H),1.27(t,J=7.3Hz,9H),0.9-0.1(br,3H) 31 P-NMR(162MHz, CDCl3)δ109-104(br).

[0064] 2'-OMe modified H-ボラノホスホネートモノマー6c 1H-NMR(400MHz, CDCl3)δ8.65(d,J=7.8Hz,0.5H),8.59(d,J=7.3Hz,0.5H),7.95-7.86(m,2H),7.9-7.8(br,0.5H),7.60 (t,J=6.9Hz,1H),7.51(t,J=7.8Hz,2H),7.44(dd,J=7.1,5.7Hz,2H),7.39-7.24(m,7H),7.2-7.0(br,0.5H),6.92-6.85 (m,4H),6.05(s,0.5H),6.02(s,0.5H),5.05-4.92(m,0.5H),4.82-4.75(m,0.5H),4.40(dd,J=2.1,8.9Hz,1H),4.12-4 .03(m,1H),3.83(s,6H),3.67(s,3H),3.63-3.53(m,2H),3.01(q,J=7.2Hz,6H),1.27(t,J=7.3Hz,9H),0.9-0.1(br,3H) 31 P-NMR(162MHz,CDCl3)δ112-102(br)

[0065] 2'-OMe modified H-ボラノホスホネートモノマー6g 1H-NMR (400MHz, CDCl3) δ12.5-11.6(br,1H),10.5-9.7(br,1H),7.88(s,0.5H),7.87(s,0.5H),7.8-7.7(br,0.5H),7.53-7.36(m,2H),7.34-7.28(m, 4H),7.26-7.13(m,3H),6.93-6.83(br,0.5H),6.83-6.66(m,4H),5.93(d, J=4.6Hz,0.5H),5.90(d,J=4.6Hz,0.5H),5.37-5.29(m,0.5H),5.29-5.20( m,0.5H),4.58(t,J=4.8Hz,0.5H),4.56(t,J=4.8Hz,0.5H),4.44-4.36(m, 0.5H),4.36-4.28(m,0.5H),3.75,3.75,3.74(s,s,s,6H),3.54-3.37(m,4 H),3.37-3.22(m,1H),3.00(q,J=7.2Hz,6H),2.62-2.40(m,1H),1.25(t,J =7.3Hz,9H),1.11(d,J=6.9Hz,3H),1.04(d,J=5.5Hz,3H),1.0-0.1(br,3H) 31 P-NMR(162MHz,CDCl3)δ108-103(br)

[0066] 2'-OMe modified H-ボラノホスホネートモノマー6u 1H-NMR(400MHz,CDCl3)δ8.23(d,J=8.2Hz,0.5H),8.17(d,J=8.2Hz,0.5H),8.01-7.90(m,2H),7.65(t,J=7.3Hz,1H),7.51 (t,J=7.8Hz,2H),7.47-7.38(m,2H),7.38-7.21(m,7H),6.87(m,4H),5.97(d,J=2.3Hz,0.5H)5.97(d,J=2.1Hz,0.5H),5.3 0(d,J=2.7Hz,0.5H),5.27(d,J=2.7Hz,0.5H),5.10-5.03(m,0.5H),4.95-4.86(m,0.5H),4.38-4.33(m,1H),4.08-4.05(m ,1H),3.80(s,6H),3.67-3.58(m,1H),3.55(d,J=4.6Hz,4H),3.10(q,J=7.2Hz,6H),1.28(t,J=7.2Hz,9H),1.0-0.2(m,3H) 31 P-NMR(162MHz,CDCl3)δ113-103(br)

[0067] <Solid-phase synthesis of nucleic acid oligomers 1> Hereinafter, among internucleotide bonds, phosphorothioate bonds are represented by the subscript PS, boranophosphate bonds by the subscript PB, and phosphodiester bonds by the subscript PO.

[0068] (T PS Solid-phase synthesis of T [Comparative Synthesis Example 1] 5'-Dimethoxytrityl-N-methylpropional supported on a solid support via a succinyl linker 30.5 μmol of benzoylthymidine was detritylated with 3% dichloroacetic acid / dichloromethane solution (5 times for 12 seconds, 1 mL each time). The solid support was then washed with molecular sieve-dried acetonitrile and dichloromethane. After drying the solid support for 10 minutes, 19.7 mg (40 equivalents, 20 μmol) of H-phosphonothioate monomer 5t and 16.2 mg (100 equivalents, 50 μmol) of bis(2,6-dimethylphenyl)phosphorochloridate (hereinafter referred to as "(DMP)2CP") were added as a condensation agent according to Tetrahedron Lett. 2004, 45, 5803-5806. The reaction mixture was then mixed with a 1:4 (v / v) acetonitrile solution containing pyridine as a base. The mixture was then stirred slowly by hand for 3 minutes under argon. After condensation of the H-phosphonothioate monomer, the solid support was washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4), followed by deprotection of the dimethoxytrityl group with 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution (4 x 15 s, 1 mL each time). The solid support was then washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4) and dried for 10 min. The resulting H-phosphonothioate bond was converted to a phosphorothioate bond by oxidation with 0.1 M triethylamine / carbon tetrachloride-2,6-lutidine-water (5:12.5:1, v / v / v) for 90 min. The solid support was washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4), and then reacted with 25% aqueous ammonia / ethanol (3:1, v / v, 5 mL) at room temperature for 3 hours to deprotect the nucleic acid bases and cleave them from the solid support. After the reaction, the reaction solution was filtered and washed with ethanol. The solvent was removed under reduced pressure, and the crude product was analyzed by reverse-phase HPLC. Reverse-phase HPLC was performed in 0.1 M TEAA buffer (pH 7) using a linear gradient of 0–30% acetonitrile for 60 minutes at 30°C and a flow rate of 0.5 mL / min. The results are shown in Table 1 and Figure 1(a).

[0069] [Comparative Synthesis Example 2] Control experiment of Comparative Synthesis Example 1 The same experimental procedure as in Comparative Synthesis Example 1 was carried out, except that (DMP)2CP was added instead of H-phosphonothioate monomer 5t. The results are shown in Figure 1(b).

[0070] [Synthesis Example 1] The same procedure as in Comparative Synthesis Example 1 was carried out, except that BOMP (23.3 mg, 100 equivalents, 50 μmol) was used instead of (DMP)CP as the condensing agent and an acetonitrile solution of 2,6-lutidine (base, 0.6 M, 120 μmol) was used as the reaction solvent. The results are shown in Table 1.

[0071] [Synthesis Example 2] The same procedure as in Synthesis Example 1 was carried out, except that MNTP (22.3 mg, 100 equivalents, 50 μmol) was used as the condensing agent instead of BOMP. The results are shown in Table 1 and Figure 1(c).

[0072] [Synthesis Example 3] The same procedure as in Synthesis Example 1 was carried out, except that PyNTP (25.0 mg, 100 equivalents, 50 μmol) was used as the condensing agent instead of BOMP. The results are shown in Table 1 and Figure 1(d).

[0073] [Synthesis Example 4] The same operations as in Synthesis Example 3 were carried out, except that pyridine (0.6 M, 120 μmol) was used instead of 2,6-lutidine as the base added during the condensation reaction. The results are shown in Table 1.

[0074] [Synthesis Example 5] The same operations as in Synthesis Example 3 were carried out, except that quinoline (0.6 M, 120 μmol) was used instead of 2,6-lutidine as the base added during the condensation reaction. The results are shown in Table 1.

[0075] [Synthesis Example 6] The same procedure as in Synthesis Example 3 was carried out, except that quinoline (1.8 M, 355 μmol) was used instead of 2,6-lutidine as the base added during the condensation reaction. The results are shown in Table 1 and Figure 1(e).

[0076] [Synthesis Example 7] The same operations as in Synthesis Example 3 were carried out, except that acetonitrile alone, without adding a base, was used as the reaction solvent during the condensation reaction. The results are shown in Table 1 and Figure 1(f).

[0077] [Table 1]

[0078] In Table 1, the chemoselectivity is determined by the HPLC results, where the by-product T PO T and the target T PS The HPLC yield was calculated from the area ratio T PS T / (T+T PO T+T PS T).

[0079] [ka]

[0080] FIG. 1 shows the results of reverse-phase HPLC analysis of Comparative Synthesis Examples 1 and 2 and Synthesis Examples 2, 3, 6, and 7. Compared to Comparative Synthesis Example 1, Synthesis Examples 6 and 7 resulted in higher yields of the target product. An unidentified peak was observed around 28 minutes into the elution time in Comparative Synthesis Example 1 only. Therefore, a control experiment was conducted in Comparative Synthesis Example 2 in which no monomer was added during the condensation reaction, and a peak was observed at 28 minutes into the elution time in reverse-phase HPLC. Therefore, the peak observed around 28 minutes into the elution time in Comparative Synthesis Example 1 is thought to be a by-product resulting from the reaction of a hydroxyl group with a condensing agent. In the above Synthesis Examples and Comparative Synthesis Examples, a dimer was synthesized. However, if such a side reaction occurs during oligomer synthesis, the reaction efficiency may be significantly reduced. Therefore, it was confirmed that the method of the Synthesis Examples is suitable for synthesizing oligomers.

[0081] (N PS Solid-phase synthesis of T [Synthesis Example 8] 5'-Dimethoxytrityl-N-methylpropional supported on a solid support via a succinyl linker 3 0.5 μmol of benzoylthymidine was detritylated with 3% dichloroacetic acid / dichloromethane (4 times for 15 s, 1 mL each time), followed by washing with molecular sieve-dried acetonitrile and dichloromethane. After drying for 10 min, the solid support was mixed with H-phosphonothioate monomer 5a, 5c, or 5g (A: 19.0 mg (40 equiv., 20 μmol); C: 17.8 mg (40 equiv., 20 μmol); or G: 18.1 mg (40 equiv., 20 μmol)) and PyNTP (100 equiv., 50 μmol). The reaction mixture was then mixed with 1.8 M quinoline (1.8 M) in acetonitrile as a base under argon and stirred slowly by hand for 3 min. After condensation of the H-phosphonothioate monomer, the solid support was washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4), followed by deprotection of the dimethoxytrityl group with 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution (4 x 15 s, 1 mL each time). The solid support was then washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4) and dried for 10 min. The formed H-phosphonothioate bond was converted to a phosphorothioate bond by oxidation with 0.1 M triethylamine / carbon tetrachloride-2,6-lutidine-water (5:12.5:1, v / v / v) for 90 min. The solid support was washed with dried acetonitrile (1 mL x 4 times) and dichloromethane (1 mL x 4 times), and then reacted with a 25% aqueous ammonia / ethanol (3:1, v / v, 5 mL) solution under the following conditions to deprotect the nucleic acid base moiety and cleave it from the solid support (A PB T and C PB T: Room temperature condition, 20h; G PB After the reaction, the reaction solution was filtered and washed with ethanol. The solvent was removed under reduced pressure, and the crude product was analyzed by reverse-phase HPLC in the same manner as described above. The results are shown in Table 2.

[0082] [Synthesis Example 9] The same procedure as in Synthesis Example 8 was carried out, except that acetonitrile alone without adding a base was used as the reaction solvent. The results are shown in Table 2.

[0083] [Synthesis Example 10] Synthesis Example 10 was carried out using only 5 g of H-phosphonothioate monomer. The same procedure as in Synthesis Example 9 was carried out, except that the amount of the monomer was changed to 36.2 mg (80 equivalents, 40 μmol) and the amount of PyNTP was changed to 50.0 mg (200 equivalents, 100 μmol). The results are shown in Table 2.

[0084] [Table 2]

[0085] In Table 2, the chemoselectivity is determined by the HPLC results, which show that the by-product dN PO T and the target dN PS The HPLC yield was calculated from the area ratio dN PS T / (T+dN PO T+dN PS T). In entries 1 to 6, the equivalents of the monomer were 40 and the equivalents of PyNTP were 100, whereas in entry 7, the equivalents of the monomer were 80 and the equivalents of PyNTP were 200. Entries 1, 3, and 5 show the results of Synthesis Example 8, entries 2, 4, and 6 show the results of Synthesis Example 9, and entry 7 shows the results of Synthesis Example 10.

[0086] (N * PB Solid-phase synthesis of T [Synthesis Example 11] Below, N * indicates a 2'-OMe modified nucleotide unit. 5'-Dimethoxytrityl-N-methylpropional supported on a solid support via a succinyl linker 30.5 μmol of 1-benzoylthymidine was detritylated with 3% dichloroacetic acid / dichloromethane (4 times for 15 s, 1 mL each time), followed by washing with molecular sieve-dried acetonitrile and dichloromethane. After drying for 10 min, the solid support was added to the reaction system with 2'-OMe-modified H-boranophosphonate monomers 6a, 6c, 6g, or 6u (A: 17.1 mg (40 equiv., 20 μmol); C: 16.7 mg (40 equiv., 20 μmol); G: 16.9 mg (40 equiv., 20 μmol); U: 16.7 mg (40 equiv., 20 μmol)) and MNTPs (22.3 mg, 100 equiv., 50 μmol). The reaction mixture was then added with acetonitrile containing 2,6-lutidin as a base under argon and stirred slowly by hand for 3 min for condensation. After condensation of the 2'-OMe-modified H-boranophosphonate monomer, the solid support was washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4), followed by deprotection of the dimethoxytrityl group with 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution (4 x 15 s, 1 mL each time). The solid support was then washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4) and dried for 10 min. The formed H-boranophosphonate bond was converted to a boranophosphate bond by oxidation with 0.1 M triethylamine / carbon tetrachloride-2,6-lutidine-water (5:12.5:1, v / v / v) for 90 min. The solid support was washed with dried acetonitrile (1 mL x 4 times) and dichloromethane (1 mL x 4 times), and then reacted with a 25% aqueous ammonia / ethanol (3:1, v / v, 5 mL) solution under the following conditions to deprotect the nucleic acid base moiety and cleave it from the solid support (U * PB T: Room temperature condition, 3h;A * PB T and C * PB T: Room temperature condition, 20h; G * PBAfter the reaction, the reaction solution was filtered and washed with ethanol. The solvent was removed under reduced pressure, and the crude product was analyzed by reverse-phase HPLC in the same manner as described above. The results are shown in Table 3.

[0087] [Table 3]

[0088] In Table 3, the HPLC yield is the area ratio N * PB T / (T+N * PB T).

[0089] [Example 1] Synthesis of PB / PS / PO chimeric nucleic acid oligomer (12-mer) 5'-Dimethoxytrityl-N-methylpropional supported on a solid support via a succinyl linker 3 1 mL of 3% dichloroacetic acid / dichloromethane solution was reacted with 0.5 μmol of benzoylthymidine for 15 seconds to remove the protecting group, and the reaction solution was then removed. This procedure was repeated four times, followed by washing four times with 1 mL of dichloromethane and four times with acetonitrile (1 mL), and the solid support was vacuum dried. Next, the oligomer chain elongation reaction was carried out by repeating the following combination of steps (i) and (ii) 11 times.

[0090] Step (i): A monomer selected according to the sequence, MNTP or PyNTP, and optionally 2,6-lutidine or quinoline were mixed in acetonitrile (0.2 mL) and reacted for 3 minutes. After 3 minutes, the reaction solution was removed, and the solid support after the reaction was washed four times with acetonitrile and four times with dichloromethane. The condensation conditions using various monomers are shown below.

[0091] [ka]

[0092] [Table 4]

[0093] Step (ii): The solid support after step (i) was reacted with 1 mL of 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution for 15 seconds. After repeating the same procedure four times, the solid support was washed four times with 1 mL of dichloromethane and four times with acetonitrile, and then vacuum-dried.

[0094] After chain elongation, 0.5 mL of 0.1 M triethylamine / carbon tetrachloride-2,6-lutidine-HO (5:12.5:1, v / v / v) solution was added to the solid support and reacted for 90 minutes. After the reaction, the solid support was washed four times with acetonitrile (1 mL) and four times with dichloromethane (1 mL).

[0095] The washed solid support was then reacted with 25% aqueous ammonia-ethanol (3:1, v / v) for 20 hours at 50°C to deprotect the nucleic acid base moiety and cleave the nucleic acid oligomer from the solid support. The solid support was filtered, the filtrate was collected, and the solvent was evaporated under reduced pressure.

[0096] The resulting solution containing the PB / PS / PO chimeric nucleic acid oligomer (12-mer) was treated with a 25% NH3-ethanol aqueous solution (3:1, v / v, 50°C, 20 h) and washed with ethanol (2 ml x 4 times). The solvent was removed from the aqueous solution under reduced pressure. The resulting solution was then purified by reverse-phase HPLC. Reverse-phase HPLC and separation / purification were carried out at 60°C for 20 minutes at a flow rate of 0.5 mL / min using a 5-40% methanol-0.1 M hexafluoroisopropanol-0.008 M triethylamine buffer solution as the mobile phase. Figures 2(a) and 2(b) show the reverse-phase HPLC charts before and after separation / purification in Example 1, respectively. Yield 6%. HRMS(ESI-TOF):Calcd for [M-6H] 6- ,615.7786;found,615.7767

[0097] The resulting PB / PS / PO chimeric nucleic acid oligomer (12-mer) was d(C PS A PS G PS T PS C PB A PB G PB T PB C PO A PO G PO It is a nucleic acid oligomer having the base sequence of (SEQ ID NO: 1).

[0098] [Example 2] Synthesis of PB / PS / PO chimeric nucleic acid oligomer (apoB sequence-containing 12-mer) A portion of the mRNA encoding apoB (apoprotein B-100; Nature, 2006, Vol. 441, pp. 111-114) (3'-rCGU AAC CAU AAG-5': complementary strand RNA, SEQ ID NO: 2) was selected as the target nucleic acid. A PB / PS / PO chimeric nucleic acid oligomer with a base sequence complementary to the RNA base sequence was synthesized.

[0099] 5'-O-DMTr-N attached to a solid support via a succinyl linker 4 The PB / PS / PO chimeric nucleic acid oligomer was synthesized using 0.5 μmol of benzoylcytidine by the same procedure as in Example 1. The resulting nucleic acid oligomer was separated and purified by ion-exchange HPLC. Separation and purification was carried out at room temperature using a 10 mM Tris-HCl buffer solution (pH 7.5) containing 0-0.5 M NaCl and 30% acetonitrile at a flow rate of 0.4 mL / min for 40 minutes. Reverse-phase HPLC charts obtained in Example 2 before and after separation and purification are shown in Figure 3(a) and Figure 3(b), respectively. Yield 19%. HRMS(ESI-TOF):Calcd for [M-6H] 6- ,614.1138;found,614.1136

[0100] The resulting PB / PS / PO chimeric nucleic acid oligomer (12-mer) was PB C PS A PB TPO T PO G PO G PO T PS A PB T PS T PB C) An antisense molecule having the base sequence of (SEQ ID NO: 3).

[0101] [Example 3] Synthesis of PB / PS / PO-gapmer (12-mer) In the same manner as in Example 2, in addition to the monomer used in Example 2, N 4 -benzoylcytosine, N 6 -benzoyladenine, N 2 -isobutyrylguanine, or N 3 Using a 2'-OMe-modified boranophosphonate compound having -benzoylthymine as a base, the deprotection reaction of the hydroxyl group having a protecting group and the condensation reaction were repeated until a 12-mer was obtained, followed by an oxidation reaction, deprotection reaction of the nucleic acid base portion, and cleavage from the solid support to obtain a PB / PS / PO-gapmer (12-mer).

[0102] The resulting solution containing the PB / PS / PO-gapmer (12-mer) was treated with a 25% NH3-ethanol aqueous solution (3:1, v / v, 50°C, 20 h) and washed with ethanol (2 ml x 4 times). The solvent was removed from the aqueous solution under reduced pressure. The product was then purified by reverse-phase HPLC. Reverse-phase HPLC and separation / purification were performed at 60°C for 20 minutes at a flow rate of 0.5 mL / min using a 5-40% methanol-0.1 M hexafluoroisopropanol-0.008 M triethylamine buffer solution as the mobile phase. Figures 4(a) and 4(b) show the reverse-phase HPLC charts before and after separation / purification in Example 3, respectively. Yield 13%. HRMS(ESI-TOF) Calcd for [M-6H] 6- ,635.9695;found,635.9670

[0103] The resulting PB / PS / PO-gapmer (12-mer) was *PB C * PB A * PB d(T PS T PO G PO G PO T PS A PB )U * PB U * PB C * It is an antisense molecule having the base sequence of (SEQ ID NO: 4). * indicates a 2'-OMe modified nucleotide unit.)

[0104] [Example 4] Synthesis of PB / PS chimeric nucleic acid oligomer (12-mer) As in Example 2, deprotection reactions of the hydroxyl groups having protecting groups and condensation reactions were repeated until a 12-mer was obtained, so that the sequence was complementary to the base sequence of the target nucleic acid (apoB protein), followed by oxidation reactions, deprotection reactions of the nucleic acid bases, and cleavage from the solid support to obtain a PB / PS chimeric nucleic acid oligomer (12-mer).

[0105] The resulting solution containing the PB / PS chimeric nucleic acid oligomer (12-mer) was treated with a 25% NH3-ethanol aqueous solution (3:1, v / v, 50°C, 20 h) and washed with ethanol (2 ml x 4 times). The solvent was removed from the aqueous solution under reduced pressure. The resulting solution was then purified by reverse-phase HPLC. Reverse-phase HPLC and separation / purification were carried out at 60°C for 20 minutes at a flow rate of 0.5 mL / min using a 5-40% methanol-0.1 M hexafluoroisopropanol-0.008 M triethylamine buffer solution as the mobile phase. Figures 5(a) and 5(b) show the reverse-phase HPLC charts before and after separation / purification in Example 4, respectively. Yield 5%. HRMS(ESI-TOF) Calcd for [M-6H] 6- ,618.6194;found,618.6175

[0106] The resulting PB / PS chimeric nucleic acid oligomer (12-mer) was PB CPS A PB T PS T PS G PB G PB T PS A PB T PS T PB C) An antisense molecule having the base sequence of (SEQ ID NO: 5).

[0107] <Monomer synthesis 2> (Synthesis of 2'-O-4'-C-Locked-H-Boranophosphonate Monomer 7a or 7c) Hereinafter, the 2'-O-4'-C-Locked-H-boranophosphonate monomer is also referred to as a Locked Nucleic Acid (hereinafter referred to as "LNA")-modified H-boranophosphonate monomer. [ka]

[0108] The nucleoside derivative shown on the left side of the above scheme (A: 0.69 g, 1.0 mmol; or m C: 0.77 g, 1.1 mmol; m C represents 5-methylcytosine) and pyridinium H-boranophosphonate (A: 0.41 g, 2.0 mmol; or m C: 0.47 g, 2.3 mmol) was dried azeotropically with pyridine (3 mL x 3) under an argon atmosphere to obtain pyridine (A: 10 mL; or m The resulting pyridine solution was stirred in an ice bath while adding BopCl (A: 0.51 g, 2.0 mmol; or mC (0.58 g, 2.3 mmol) was added, and then the reaction temperature was brought to room temperature and stirred for 1 hour. After diluting this solution with 60 mL of chloroform, the organic layer was washed with 1.0 M TEAB buffer solution (pH 7.0) (80 mL x 3 times). The aqueous layer was back-extracted with chloroform (40 mL x 3 times). The organic layer was collected and the water was removed with anhydrous sodium sulfate. The organic solvent was then evaporated under reduced pressure, and the resulting residue was separated and purified by silica gel column chromatography (developing solvent A: ethyl acetate-methanol-triethylamine (99:0.5:0.5, v / v / v), followed by chloroform-methanol-triethylamine (98:2:0.5-96:4:0.5, v / v / v); m C: Ethyl acetate-methanol-triethylamine (99.5:0.5:0.5, v / v / v), followed by chloroform-methanol-triethylamine (99:1:0.5, v / v / v) and distilling off the solvent under reduced pressure to give LNA-modified H-boranophosphonate monomer 7a or 7c. 7a: Yield 0.79 g, 0.92 mmol, 92% yield; 7c: Yield 0.60 g, 0.67 mmol, 59% yield.

[0109] LNA-modified H-boranophosphonate monomer 7a 1 H-NMR(400MHz,CDCl3)δ9.50-9.10(br,1H),8.78(s,0.5H),8.77(s,0.5H),8.40(s,0.5H),8.40(s,0.5H) ,8.06-7.96(m,2H),7.84-7.68(br,0.5H),7.64-7.42(m,5H),7.38-7.19(m,7H),6.89-6.83(m,4H),6.79 -6.73(br,0.5H),6.17(s,0.5H),6.14(s,0.5H),4.95-4.89(m,1H),4.73(d,J=7.8Hz,1H),4.12-3.95(m, 2H),3.83-3.73(m,6H),3.65-3.49(m,2H),2.91(q,J=7.5Hz,6H),1.19(t,J=7.5Hz,9H),1.0-0.0(br,3H) 31 P-NMR(162MHz,CDCl3)δ110-103(br)

[0110] LNA modified H-ボラノホスホネートモノマー7c 1 H-NMR (400MHz, CDCl3) δ8.35-8.28(m,2H),7.90-7.86(m,1H),7.55-7.42(m,5H),7.41-7.30(m,6H),7 .27-7.22(m,1H),6.89-6.81(m,4H),6.84-6.78(br,0.5H)5.69(s,0.5H),5.65(s,0.5H),4.89(d,J=6 .4Hz,0.5H),4.70(s,0.5H),4.67(s,0.5H),4.58(d,J=8.0Hz,0.5H),3.98-3.96(m,1H),3.82-3.78(m ,6H),3.56-3.44(m,2H),2.84(q,J=7.2Hz,6H),1.87-1.82(m,3H),1.18(t,J=7.3Hz,9H),1.0-0.1(3H) 31 P-NMR(162MHz,CDCl3)δ111-104(br)

[0111] (Synthesis of 2'-O-4'-C-Locked-H-ボラノホスホネートモノマー7g)

change

[0112] 2'-O-4'-C-Locked-guanosine (0.443 g, 1.5 mmol) was dissolved in 5 mL of pyridine under an argon atmosphere, and 4-dimethylaminopyridine (0.189 g, 1.5 mmol) and isobutyric anhydride (1.25 mL, 7.5 mmol) were added sequentially. The mixture was reacted at 110 °C for 9 hours. Chloroform (20 mL) was added to the mixture, which was then washed with 1 M hydrochloric acid (20 mL). The aqueous layer was back-extracted with chloroform (20 mL). The combined organic layer was washed twice with saturated aqueous sodium bicarbonate (40 mL), and the aqueous layer was back-extracted with chloroform (40 mL). The combined organic layer was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting mixture was used in the next reaction without further purification. The mixture was dissolved in pyridine (4 mL) under an argon atmosphere, and diisopropylethylamine (0.4 mL, 2.25 mmol) and diphenylcarbamoyl chloride (0.69 g, 3.0 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour. Methanol (5 mL) was added to quench the reaction, and chloroform (20 mL) was added. The mixture was washed three times with saturated aqueous sodium bicarbonate (20 mL). The combined aqueous layer was back-extracted with chloroform (20 mL). The combined organic layer was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate-hexane (40:60-50:50, v / v)) to give 2'-O-4'-C-Locked-3',5'-O-diisobutyryl-2-N-isobutyryl-6-O-diphenylcarbamoyl-guanosine (0.859 g, 1.23 mmol, 82%).

[0113] The entire amount of the obtained 2'-O-4'-C-Locked-3',5'-O-diisobutyryl-2-N-isobutyryl-6-O-diphenylcarbamoyl-guanosine was dissolved in a pyridine-methanol mixed solvent (1:1, v / v, 21 mL), and sodium methoxide (26.4 mg, 0.488 mmol) was added stepwise in four portions at 0°C. The mixture was stirred for 14 hours. The temperature was then raised to room temperature, and after stirring for 4.5 hours, the reaction was stopped by adding cation exchange resin. The cation exchange resin was removed by filtration, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: chloroform-methanol (100:0-100:4, v / v)) to give 2'-O-4'-C-Locked-2-N-isobutyryl-6-O-diphenylcarbamoyl-guanosine (0.401 g, 0.715 mmol, 58%).

[0114] The entire amount of the obtained 2'-O-4'-C-Locked-2-N-isobutyryl-6-O-diphenylcarbamoyl-guanosine was dissolved in pyridine (7 mL) under an argon atmosphere, and 4,4'-dimethoxytrityl chloride (0.266 g, 0.79 mmol) was added. After stirring at room temperature for 1.5 hours, 4,4'-dimethoxytrityl chloride (61 mg, 0.18 mmol) was added and stirred for 40 minutes. The reaction was quenched by adding methanol (5 mL), and chloroform (20 mL) was added. The mixture was washed three times with saturated aqueous sodium bicarbonate (20 mL). The combined aqueous layer was back-extracted with chloroform (20 mL). The combined organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform-methanol-pyridine (100:0:0.5-100:1.5:0.5, v / v / v)) to obtain 2'-O-4'-C-Locked-5'-O-(4,4'-dimethoxytrityl)-2-N-isobutyryl-6-O-diphenylcarbamoyl-guanosine (0.617 g, 0.451 mmol, 63%).

[0115] [ka]

[0116] The LNA-modified guanosine derivative (0.39 g, 0.45 mmol) shown on the left side of the scheme above and pyridinium H-boranophosphonate (0.19 g, 0.90 mmol) were azeotropically dried with pyridine (3 mL x 3) under an argon atmosphere and dissolved in pyridine (5 mL). BopCl (0.23 g, 0.90 mmol) was added to the resulting pyridine solution while stirring in an ice bath, and the reaction temperature was then increased to room temperature and stirred for 1 hour. After diluting the solution with chloroform (30 mL), the organic layer was washed with 1.0 M TEAB buffer solution (pH 7.0) (30 mL x 3). The aqueous layer was back-extracted with chloroform (30 mL x 3). The organic layer was collected and the water was removed using anhydrous magnesium sulfate. The solvent in the organic layer was then evaporated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography (developing solvent: ethyl acetate-triethylamine (100:0.5, v / v), followed by chloroform-methanol-triethylamine (98:2:0.5, v / v / v)). The solvent was evaporated under reduced pressure to obtain 7 g (0.35 g, 0.92 mmol, 92%) of LNA-modified H-boranophosphonate monomer.

[0117] LNA-modified H-boranophosphonate monomer 7g 1H-NMR(400MHz,CDCl3)δ8.74(s,0.5H),8.68(s,0.5H),8.23(s,0.5H),8.20(s,0.5H),7.84-7.64(m,0.5H),7.44-7.41(m ,5H),7.38-7.28(m,10H),7.26-7.18(m,4H),6.91-6.75(m,4H),6.76-6.70(br,0.5H),6.00(s,0.5H),6.00(s,0.5H),5. 17(d,J=6.4Hz,0.5H),4.96(s,0.5H),4.89(s,0.5H),4.82(d,J=8.5Hz,0.5H),4.14-4.07(m,1.5H),3.99-3.95(m,0.5H) ,3.80-3.73(m,6H),3.59-3.44(m,2H),3.30-3.21(m,1H),2.86(q,J=7.3Hz,6H),1.27-1.19(m,6H),1.12(t,J=7.3Hz,9H) 31 P-NMR(162MHz,CDCl3)δ110-101(br)

[0118] (Synthesis of 2'-O-4'-C-Locked-H-boranophosphonate Monomer 7t) [ka]

[0119] 2'-O-4'-C-Locked-H-boranophosphonate monomer 7t was synthesized by the synthetic method described in the literature (The Journal of Organic Chemistry, 2014, Vol. 79, pp. 3465-3472).

[0120] (Synthesis of 2'-O-MOE-modified H-boranophosphonate monomers) 2'-O-MOE modified H-boranophosphonate monomers were synthesized according to the following scheme. [ka]

[0121] 8g of the guanosine derivative (0.722g, 1.0mmol) shown on the left side of the scheme above was dried azeotropically with pyridine (3mL x 3) under an argon atmosphere and dissolved in 25mL of pyridine together with pyridinium H-boranophosphonate (0.30g, 2.0mmol). BopCl (0.51g, 2.0mmol) was added to the resulting pyridine solution while stirring in an ice bath. The reaction temperature was then brought to room temperature and stirred for 6 hours. After diluting the solution with 30mL of chloroform, the organic layer was washed with 1.0M TEAB buffer solution (pH 7.0) (30mL x 3). The aqueous layer was back-extracted with chloroform (30mL x 3). The organic layer was collected and dehydrated with anhydrous sodium sulfate. The solvent was then evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate-methanol-triethylamine (100:0:1, v / v / v), followed by chloroform-methanol-triethylamine (100:10:1, v / v / v)). The solvent was evaporated under reduced pressure to give 9 g of 2'-O-MOE-modified H-boranophosphonate monomer. Yield: 0.57 g, 0.60 mmol, 60% yield.

[0122] 9g of 2'-O-MOE modified H-boranophosphonate monomer 1H-NMR(400MHz,CDCl3)δ11.4-10.5(br,1H),7.90(s,0.5H),7.89(s,0.5H),7.80-7.68(br,0.5H)7.42(d,J=7.8Hz,2H),7.30(d,J= 8.2Hz,4H),7.24-7.14(m,3H),6.93-6.87(br,0.5H),6.78-6.74(m,4H),5.97(d,J=4.1Hz,0.5H),5.93(d,J=4.6Hz,0.5H),5.40-5 .27(m,1H),4.82(t,J=4.3Hz,0.5H),4.80(t,J=4.3Hz,0.5H),4.43-4.34(m,1H)3.93-3.85(m,1H),3.76-3.71(m,6H),3.51-3.41( m,2H),3.23(d,J=7.3Hz,3H),2.91(q,J=7.3Hz,6H),2.63-2.49(m,1H),1.19(t,J=7.3Hz,9H),1.14-1.03(m,6H),0.9-0.2(br,3H) 31 P-NMR(162MHz,CDCl3)δ108-103(br)

[0123] <Solid-phase synthesis of nucleic acid oligomers 2> (N # PB Solid-phase synthesis of T [Synthesis Example 12] Below, N # indicates a 2'-O-MOE modified nucleotide unit. 5'-Dimethoxytrityl-N-methylpropional supported on a solid support via a succinyl linker 30.5 μmol of benzoylthymidine was detritylated with 3% dichloroacetic acid / dichloromethane (4 times for 15 s, 1 mL each time), followed by washing with acetonitrile and dichloromethane dried over molecular sieves. After drying for 10 min, 9 g of 2'-O-MOE-modified H-boranophosphonate monomer (18.9 mg, 40 equiv., 20 μmol) and MNTP (22.3 mg, 100 equiv., 50 μmol) were added to the reaction system. 0.2 mL of acetonitrile solution containing 2,6-lutidin as a base was added as a reaction solvent, and the reaction was carried out by slow manual stirring for 3 min under argon. After condensation of the 2'-O-MOE-modified H-boranophosphonate monomer, the solid support was washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4), followed by deprotection of the dimethoxytrityl group with 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution (4 x 15 s, 1 mL each time). The solid support was then washed with dried acetonitrile (1 mL x 4) and dichloromethane (1 mL x 4) and dried for 10 min. The formed H-boranophosphonate bond was converted to a boranophosphate bond by oxidation with 0.1 M triethylamine / carbon tetrachloride-2,6-lutidine-water (5:12.5:1, v / v / v) for 90 min. The solid support was washed with dried acetonitrile (1 mL x 4 times) and dichloromethane (1 mL x 4 times), and then reacted with a 25% aqueous ammonia / ethanol (3:1, v / v, 5 mL) solution under the following conditions to deprotect the nucleic acid base moiety and cleave it from the solid support (G # PB (T: 50°C, 20 h). After the reaction, the reaction solution was filtered and washed with ethanol. The solvent was removed under reduced pressure, and the crude product was analyzed by reverse-phase HPLC in the same manner as described above. The results are shown in Table 5 and Figure 6.

[0124] [Table 5]

[0125] In Table 5, the HPLC yield is the area ratio G # PB T / (T+G # PB T).

[0126] [Example 5] Synthesis of PB / PS-LNA-gapmer (12-mer) A nucleic acid oligomer having a sequence complementary to the base sequence of the target nucleic acid (apo B protein) was synthesized in the same manner as in Example 2. A unilinker having a 5'-dimethoxytrityl group (Universal UniLinker Support 1000 Å (ChemGenes), 0.5 μmol) was used as the solid support. Furthermore, in addition to the monomer used in Example 2, N 4 -benzoyl-5-methylcytosine, N 6 -benzoyladenine, N 2 -Isobutyryl-O 6 -diphenylcarbamoylguanine, or N 3 The LNA-modified H-boranophosphonate compound containing α-benzoylthymine as the base was used. Only the first base was introduced by the phosphoramidite method using an automated synthesizer (M-4 (Nihon Techno Service)) as shown in step (A) below.

[0127] Step (A): A 3% trichloroacetic acid / dichloromethane solution was passed through a solid-phase synthesis column packed with the solid support four times, and the reaction was allowed to proceed for 40 seconds (10 seconds per run) to perform detritylation. After the reaction was completed, the solid support was washed with acetonitrile and then dried by purging with argon gas. Next, N 4 -benzoyl-5-methylcytosine, 5'-dimethoxytrityl-3'-cyanoethyl phosphoramidite unit (N 4An acetonitrile solution containing 0.1 mol / L of 2-benzoyl-5-methyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-locked-cytidine-3'-cyanoethyl phosphoramidite (hereafter referred to as the "amidite solution") was passed through the column four times for 24 minutes (6 minutes per pass) to introduce the LNA-modified 5-methylcytidine derivative onto the solid support. A 0.25 mol / L solution of 5-benzylthio-1-H-tetrazole in acetonitrile was also passed through the column as an activator. The ratio of the amidite solution to the activator was 40:60 (v / v). After the reaction was complete, the solid support was washed with acetonitrile and then dried under argon gas. Next, the oxidizing agent (tetrahydrofuran-water-pyridine-iodine (66:12:22:0.6, v / v / v / w)) was passed through the solid support four times for 36 seconds (9 seconds per pass) to oxidize the phosphoric acid moiety. After the reaction was complete, the solid support was washed with acetonitrile and then dried under argon gas. Next, a 1:1 (v / v) mixture of Cap A solution (tetrahydrofuran-acetic anhydride-pyridine (8:1:1, v / v / v)) and Cap B solution (16% N-methylimidazole / tetrahydrofuran) was passed through the solid support four times for 80 seconds (20 seconds per pass) to cap the unreacted hydroxyl groups of the unilinker. Finally, the solid support was washed with acetonitrile and then dried under argon gas to obtain the desired solid support.

[0128] Step (B): After that, the deprotection reaction of the hydroxyl group having a protecting group and the condensation reaction were repeated for the second and subsequent bases in the same manner as in Example 2 until a 12-mer was obtained. However, in this sequence, MNTP was used as the condensation agent for the condensation using the LNA-modified H-boranophosphonate monomer, and PyNTP was used as the condensation agent for the condensation using the 2'-deoxyribonucleic acid-type H-boranophosphonate monomer (condensation conditions for various monomers are shown in Table 6). Finally, an oxidation reaction was performed to obtain a solid support containing a PB / PS-LNA gapmer (12-mer).

[0129] [Table 6]

[0130] The resulting PB / PS-LNA gapper (12-mer) was then cleaved from the solid support by reacting it with 28% NH3 water-ethanol (3:1, v / v) at 25°C (room temperature) for 6.5 hours. The solid support was filtered through a 0.45 μm filter, and the filtrate was further reacted at 55°C for 8 hours to deprotect the nucleobase moiety. The solvent was removed from the resulting solution under reduced pressure, followed by analysis by reversed-phase UHPLC-MS. Reverse-phase UHPLC analysis was performed at 60°C for 20 minutes using a 0.1 M hexafluoroisopropanol-0.008 M triethylamine buffer solution of 5-30% methanol as the mobile phase at a flow rate of 0.2 mL / min. The reversed-phase UHPLC chart used in Example 5 is shown in Figure 7. Purity 18.7% (elution time: 11.3 minutes). LRMS(ESI-MS) Calcd for [M-4H]4-,977.71;found,977.89.[M-5H]5-,781.97;found,781.87

[0131] The resulting PB / PS-LNA gapmer (12-mer) was † PB mC † PB A † PB d(T PS T PS G PS G PS T PS A PB )T † PB T † PB mC † It is an antisense molecule having the base sequence of (SEQ ID NO: 6). † indicates an LNA-modified nucleotide unit.)

[0132] [Example 6] Synthesis of PB / PS / PO-mixmer (12-mer) In the same manner as in Example 2, in addition to the monomer used in Example 2, N 2 Using 9 g of a 2'-O-MOE-modified H-boranophosphonate compound containing 1-isobutyrylguanine as the base, deprotection of the hydroxyl groups and condensation (condensation conditions for various monomers are shown in Table 7) were repeated until the 12-mer was obtained, followed by oxidation. The nucleobase moiety was then deprotected and cleaved from the solid support by treatment with 25% NH3 water-ethanol (3:1, v / v, 50°C, 20 h). The mixture was then washed with ethanol (2 ml x 3 times), and the solvent was evaporated under reduced pressure to obtain the PB / PS / PO mixmer (12-mer).

[0133] The product was then separated and purified by reversed-phase HPLC. Analysis and separation by reversed-phase HPLC were carried out at 60°C for 20 minutes at a flow rate of 0.5 mL / min using a 10-45% methanol 0.4 M hexafluoroisopropanol-0.008 M triethylamine buffer solution as the mobile phase. The charts of the reversed-phase HPLC performed in Example 6 before and after separation and purification are shown in Figure 8(a) and Figure 8(b), respectively. Yield 20%. HRMS(ESI-TOF) Calcd for [M-4H] 4- ,975.9717;found,975.9670

[0134] The resulting PB / PS / PO-mixmer (12-mer) was # PB d(C PO A PS T PB T PO )G # PB G # PB d(T PB A PS T PS T PB C) An antisense molecule having the base sequence of (SEQ ID NO: 7).

[0135] [Table 7]

Claims

1. A nucleic acid oligomer comprising a nucleotide unit represented by the following general formula (1), a nucleotide unit represented by the following general formula (2), and a nucleotide unit represented by the following general formula (3): 【Chemistry 1】 (In general formulas (1), (2), and (3), R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R ２ represents a hydrogen atom, or R １ and R ２ are bonded to each other to form a ring of 5 or more members which may or may not have a substituent and which may or may not have a heteroatom; Bs represents a nucleobase selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil; the nucleobase may have a nucleobase-protecting group which is selected from the group consisting of benzyl, benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, chloroacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, and (dimethylamino)methylene; X ＋ represents a counter cation.)

2. R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R ２ represents a hydrogen atom, or R １ and R ２ The nucleic acid oligomer according to claim 1, wherein: are bonded to each other to form a divalent group represented by the following general formula (4): 【Chemistry 2】 (In general formula (4), R ３ and R ４ each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or combines with each other to form a ring; * and ** represent a bond; the bond represented by * is the same as R １ is bonded to the carbon atom directly bonded to the R ２ is bonded to the carbon atom directly attached to it.)

3. A method for producing a nucleic acid oligomer comprising a nucleotide unit represented by the following general formula (1) and a nucleotide unit represented by the following general formula (2), and which may or may not contain a nucleotide unit represented by the following general formula (3), a condensation step of sequentially condensing nucleotide monomers selected from the group consisting of compounds represented by the following general formula (5) and compounds represented by the following general formula (6), or nucleotide monomers selected from the group consisting of compounds represented by the following general formula (5), compounds represented by the following general formula (6), and compounds represented by the following general formula (7), to obtain a precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (8) and a nucleotide unit represented by the following general formula (9), and which may or may not contain a nucleotide unit represented by the following general formula (10); an oxidation step of oxidizing the precursor nucleic acid oligomer with an oxidizing agent to obtain a nucleic acid oligomer containing a nucleotide unit represented by the following general formula (1) and a nucleotide unit represented by the following general formula (2), and which may or may not contain a nucleotide unit represented by the following general formula (3); A manufacturing method comprising: 【Transformation 3】 (In general formulas (1), (2), and (3), R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trialkylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R ２ represents a hydrogen atom, or R １ and R ２ are bonded to each other to form a ring of 5 or more members which may or may not have a substituent and which may or may not have a heteroatom; Bs represents a nucleobase selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil; the nucleobase may have a nucleobase-protecting group which is selected from the group consisting of benzyl, benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, chloroacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, and (dimethylamino)methylene; X ＋ represents a counter cation.) 【Chemistry 4】 (In general formulas (5), (6), and (7), R １ , R ２ , Bs, and X ＋ is as described above, and R ５ represents a protecting group for a hydrogen atom or a hydroxyl group. 【Transformation 5】 (In general formulas (8), (9), and (10), R １ , R ２ , and Bs are as described above.)

4. The method according to claim 3, wherein at least the condensation step is carried out by a reaction using a solid phase support.

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