Nucleotide monomer, and method for producing nucleic acid oligomer

Abstract

Provided is a novel nucleotide monomer that has a nucleobase with an unsubstituted amino group and yields a nucleic acid oligomer at a high yield. Also provided is a method for producing the nucleic acid oligomer at a high yield.　The nucleotide monomer according to the present invention is represented by general formula (1). In the formula: R1 represents a hydrogen atom or the like; R2 represents a hydrogen atom; R1 and R2 are bonded to each other to form a prescribed 5-member or higher ring; R3 is a hydrogen atom or a protecting group of a hydroxyl group; R4 is an aryl group having or not having a substituent; R5 is a hydrogen atom or the like and R6 represents an alkyl group of three or more carbons, the alkyl group having or not having a substituent, and having or nor having a heteroatom in the molecular chain, or R5 and R6 are bonded to each other to form a prescribed 5-member or higher ring having a branched chain; BNH2 represents a nucleobase that is adenine A, guanine G, cytosine C, 5-methylcytosine mC, thymine, or uracil; and the amino group in A, G, C, and mC is not substituted.

Description

Method for producing nucleotide monomers and nucleic acid oligomers

[0001] The present invention relates to a method for producing a nucleotide monomer and a nucleic acid oligomer.

[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] In order to efficiently inhibit the production of a target protein, antisense molecules (nucleic acid oligomers) are primarily required to have cell membrane permeability, nuclease resistance, chemical stability in the body (e.g., in an environment of pH 7.4), and the property of forming a stable double strand only with a specific base sequence. For example, nucleic acid oligomers obtained using boranophosphate compounds (hereinafter referred to as "boranophosphate-type nucleic acid oligomers") are known as antisense molecules.

[0004] A known method for chemically synthesizing nucleic acids is to use a monomer in which an acyl-based protecting group has been introduced into the amino group of the nucleic acid base moiety, and to obtain an oligomer by subjecting it to appropriate chemical modification via a phosphite triester intermediate as shown in the formula on the left below. In principle, this phosphite triester intermediate can be borated by reacting it with a boranolation agent to obtain a boranophosphate-type nucleic acid oligomer as shown in the formula on the right below. (In the above left and right formulae, DMTr represents a 4,4'-dimethoxytrityl group, a circle represents a solid phase support, and B ＰＲＯ and B ＰＲＯ＊ represents a nucleic acid base represented by the following formula: (In the above formula, Ph represents a phenyl group.)

[0005] However, during the above-mentioned boranation process, the acyl-based protecting groups that protect the amino groups of adenine, cytosine, and guanine are reduced by the boranation agent and cannot be deprotected, so this method can only be used when the nucleic acid base is thymine or uracil.

[0006] Thus, methods for obtaining nucleic acid oligomers, including boranophosphate-type nucleic acid oligomers, using nucleotide monomers having nucleic acid bases in which the amino group is not protected are known (Non-Patent Documents 1 and 2). Non-Patent Documents 1 and 2 use the nucleotide monomers represented by the following formulae on the left and right, respectively, as the nucleotide monomers. By using a monomer having a cyclic structure such as the one shown below, side reactions with the amino group are suppressed even under mildly acidic conditions, making it possible to obtain oligomers even when the base moiety is unprotected. (In the above formula, DMTr and Ph are as described above, and B ＮＨ２ represents a nucleic acid base selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the amino group in the adenine, guanine, cytosine, and 5-methylcytosine is unsubstituted.

[0007] Nucl. Nucl. Nucleic Acids, 2010, Vol. 29, pp. 144-154Bioorg. Med. Chem. Lett. , 2006, Vol. 16, pp. 3111-3114

[0008] However, according to the studies of the present inventors, when conventional nucleotide monomers such as those disclosed in Non-Patent Documents 1 and 2 are used, there is a limit to the chain length of the resulting nucleic acid oligomer. The reasons for this are as follows: The nucleotide monomers have high polarity due to the unprotected amino group, and therefore have extremely high affinity with silica gel during purification by silica gel column chromatography, making purification difficult. This tends to reduce the purity of the nucleotide monomer, reducing the efficiency of nucleic acid oligomer synthesis and, as a result, making it difficult to increase the chain length of the nucleic acid oligomer. Another reason is that the nucleotide monomers have extremely small steric hindrance around the phosphorus atom, making them highly reactive to hydrolysis and rapidly hydrolyzing under condensation conditions, which tends to reduce the effective concentration of the nucleotide monomer.

[0009] An objective of the present invention is to provide a novel nucleotide monomer that has a nucleic acid base in which the amino group is not substituted and that gives a nucleic acid oligomer in high yield, and a method for producing a nucleic acid oligomer in high yield.

[0010] Specific means for solving the above problems include the following embodiments: <1> A nucleotide monomer represented by the following general formula (1): (In general formula (1), R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy 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; R ３ represents a hydrogen atom or a protecting group for a hydroxyl group, R ４ represents an aryl group having or without a substituent, R ５ represents a hydrogen atom, a halogen group, or an alkyl group having or without a substituent, R ６represents an alkyl group having 3 or more carbon atoms, which may or may not have a substituent and which may or may not have a heteroatom in the molecular chain, or R ５ and R ６ are bonded to each other to form a 5-membered or greater ring having a branched chain, which may or may not have a substituent other than the branched chain, and which may or may not have a heteroatom; B ＮＨ２ represents a nucleic acid base selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the amino group in the adenine, guanine, cytosine, and 5-methylcytosine is unsubstituted.

[0011] <2> The R ６ The nucleotide monomer according to <1>, wherein the alkyl group represented by is branched.

[0012] <3> The R ６ The nucleotide monomer according to <1> or <2>, wherein the alkyl group represented by the formula (I) has 6 to 14 carbon atoms.

[0013] <4> R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R ２ represents a hydrogen atom, or R １ and R ２ <1> to <3>, wherein the nucleotide monomers are bonded to each other to form a divalent group represented by the following general formula (2): (In general formula (2), R ７ and R ８ each independently represents 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 that in general formula (1) 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.)

[0014] <5> The nucleotide monomer according to any one of <1> to <4>, wherein the stereochemistry of the phosphorus atom in the nucleotide monomer is controlled.

[0015] <6> A condensation step of condensing a nucleotide monomer represented by the following general formula (1a) to obtain a first precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (3a): a boronation step of boronating the first precursor nucleic acid oligomer with a boronating agent to obtain a second precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (4a): or an oxidation step of oxidizing the first precursor nucleic acid oligomer with an oxidizing agent to obtain a third precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (5a): ３ａ a chain elongation step of performing a combination of the following steps once or repeatedly two or more times: a deprotection step of removing a protecting group represented by the following general formula (6) from a sixth precursor nucleic acid oligomer comprising a nucleotide unit represented by the following general formula (4c) or a seventh precursor nucleic acid oligomer comprising a nucleotide unit represented by the following general formula (5c), with or without capping the 5'-hydroxyl group and the amino group in the fourth precursor nucleic acid oligomer or the fifth precursor nucleic acid oligomer with a protecting group, and further removing the protecting group if the capping has been performed, to obtain a nucleic acid oligomer comprising at least one type selected from the group consisting of a nucleotide unit represented by the following general formula (7) and a nucleotide unit represented by the following general formula (8). (In general formula (1a), R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group; R ２ represents a hydrogen atom, or R１ 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; R ３ａ represents a protecting group for a hydroxyl group, R ４ represents an aryl group having or without a substituent, R ５ represents a hydrogen atom, a halogen group, or an alkyl group having or without a substituent, R ６ represents an alkyl group having 3 or more carbon atoms, which may or may not have a substituent and which may or may not have a heteroatom in the molecular chain, or R ５ and R ６ are bonded to each other to form a 5-membered or greater ring having a branched chain, which may or may not have a substituent other than the branched chain, and which may or may not have a heteroatom; B ＮＨ２ represents a nucleic acid base selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the amino group in the adenine, guanine, cytosine, and 5-methylcytosine is unsubstituted. (In general formula (3a), R １ , R ２ , R ３ａ , R ４ ~R ６ , and B ＮＨ２ is as described above.) (In general formulas (4a), (5a), (4b), and (5b), R １ , R ２ , R ３ａ , R ４ ~R ６ , and B ＮＨ２ is as described above.) (In general formulas (4c) and (5c), R １ , R ２ , and R ４ ~R ６ is as described above, and when the capping is performed, R ９ represents a protecting group for a hydroxyl group, R １０represents a protecting group for an amino group, Bs represents a nucleic acid base selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the amino group in the adenine, guanine, cytosine, and 5-methylcytosine is substituted with a protecting group for an amino group, and when the capping is not performed, R ９ and R １０ represents a hydrogen atom, Bs represents B ＮＨ２ Represents.) (In general formula (6), R ４ ~R ６ and R １０ is as described above.) (In general formulas (7) and (8), R １ , R ２ , and B ＮＨ２ is as described above, and X ＋ represents a counter cation.)

[0016] <7> The R ６ The method according to <6>, wherein the alkyl group represented by is branched.

[0017] <8> The R ６ The method according to <6> or <7>, wherein the alkyl group represented by

[0018] <9> R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy 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 (2): (In general formula (2), R ７ and R ８ each independently represents 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 a bond that is present in R １is bonded to the carbon atom directly bonded to the R ２ is bonded to the carbon atom directly attached to it.)

[0019] <10> The method according to any one of <6> to <9>, wherein the stereochemistry of phosphorus atoms in the nucleotide monomer and the nucleotide unit is controlled.

[0020] According to the present invention, it is possible to provide a novel nucleotide monomer that has a nucleic acid base in which the amino group is not substituted and that gives a nucleic acid oligomer in high yield, and a method for producing a nucleic acid oligomer in high yield.

[0021] Figures 1(a) and 1(b) are reverse-phase HPLC charts obtained in Comparative Synthesis Example 1 and Entry 3 of Synthesis Example 1, respectively. Figures 2(a) to 2(c) are reverse-phase HPLC charts obtained in Synthesis Example 2, Entries 2 and 6, and Comparative Synthesis Example 2, respectively. Figures 3(a) and 3(b) are reverse-phase HPLC charts obtained in Comparative Synthesis Example 3 and Synthesis Example 3, respectively. Figures 4(a) and 4(b) are reverse-phase HPLC charts obtained in Comparative Synthesis Example 4 and Synthesis Example 4, respectively. Figures 5(a) to 5(c) are reverse-phase HPLC charts obtained in Comparative Synthesis Examples 5 and 6 and Synthesis Example 5, respectively.

[0022] 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.

[0023] <Nucleotide Monomer> The nucleotide monomer according to this embodiment is represented by the above general formula (1). In the nucleotide monomer represented by the general formula (1), R ４ represents an aryl group having or without a substituent, R ６ represents an alkyl group having 3 or more carbon atoms, which may or may not have a substituent and which may or may not have a heteroatom in the molecular chain, or R ６ is R ５ and bond to form a 5- or greater-membered ring having a branched chain, which may or may not have a substituent other than the branched chain, and which may or may not have a heteroatom. As a result, the nucleotide monomer represented by general formula (1) has a nucleic acid base in which the amino group is not substituted, but has low polarity, which makes it easy to purify by silica gel column chromatography and can be obtained with high purity, thereby providing a nucleic acid oligomer in high yield. Furthermore, by providing appropriate steric hindrance around the phosphorus atom of the nucleotide monomer, hydrolysis is suppressed, allowing the effective concentration to be maintained high during the condensation reaction, and improving the condensation efficiency.

[0024] 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.

[0025] 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.

[0026] R １Examples of the acyloxy group represented by the formula (I) include alkyl-carbonyloxy groups having 1 to 6 carbon atoms (e.g., methylcarbonyloxy group, ethylcarbonyloxy group, etc.), and aryl-carbonyloxy groups having 6 to 10 carbon atoms (e.g., benzoyloxy group).

[0027] R １ Examples of the trihydrocarbylsilyloxy group represented by the formula (I) include trialkylsilyloxy groups such as trimethylsilyloxy group, triethylsilyloxy group, triisopropylsilyloxy group and t-butyldimethylsilyloxy group, and alkyldiarylsilyloxy groups such as t-butyldiphenylsilyloxy group.

[0028] R １ Examples of the alkoxyalkoxy group represented by include a methoxymethoxy group, a methoxyethoxy group, an ethoxymethoxy group, an ethoxyethoxy group, and a (2-cyanoethoxy)methoxy group.

[0029] R １ Examples of the haloalkoxyalkoxy group represented by the formula (I) include a (2-fluoro)ethoxymethoxy group, a (2,2-difluoro)ethoxymethoxy group, a (2-chloro)methoxyethoxy group, and a (2,2-dichloro)ethoxymethoxy group.

[0030] 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.

[0031] 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.

[0032] R １ and R ２may 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, specifically 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 heteroatoms include an oxygen atom, a nitrogen atom, and a sulfur atom. The ring preferably has 5 to 10 members, more preferably 5 to 8 members, and even more preferably 5 to 7 members.

[0033] In terms of duplex formation ability, R １ and R ２ are preferably bonded to each other to form a divalent group represented by the following general formula (2):

[0034] In general formula (2), R ７ and R ８ each independently represents 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 that in general formula (1) as R １ is bonded to the carbon atom directly bonded to the R ２ is bonded to the carbon atom directly connected to it.

[0035] R ７ or R ８ 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. ７ and R ８ 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.

[0036] R１ and R ２ When the groups bond to each other to form a 5- or more-membered ring which may or may not have a substituent and which may or may not have a heteroatom, specific examples of the ring include rings represented by the following formula: (In the formula, B ＮＨ２ is as described above.)

[0037] R ３ Examples of the hydroxyl-protecting group represented by the formula (I) 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, a cyanoethoxymethyl group, etc. 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.

[0038] R ４represents an aryl group having or without a substituent. In terms of ease of synthesis, reactivity, ease of deprotection of the phosphate moiety during oligomer formation, and high stereoselectivity during condensation when controlling stereochemistry, R preferably represents an aryl group having 6 to 20 carbon atoms having or without a substituent, more preferably an aryl group having 6 to 14 carbon atoms having or without a substituent, and even more preferably an aryl group having 6 to 10 carbon atoms having or without a substituent. Examples of the substituent include an alkyl group having or without a substituent, a hydroxyl group, an amino group, an alkoxy group, a halogen group, etc. 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 a hydroxyl group, an amino group, an alkoxy group, an imino group, a halogen group, etc. Examples of the alkoxy group include a methoxy group and an ethoxy group. Examples of the halogen group include a fluoro group, a chloro group, a bromo group, etc. ４ Examples of the alkyl group include a phenyl group and a naphthyl group, with a phenyl group being preferred in terms of ease of synthesis, reactivity, and the like.

[0039] R ５ Examples of the halogenyl group represented by include a fluoro group, a chloro group, and a bromo group, with a fluoro group being preferred.

[0040] R ５ The alkyl group represented by R, which may or may not have a substituent, is preferably an alkyl group having 1 to 12 carbon atoms, which may or may not have a substituent, and more preferably an alkyl group having 1 to 6 carbon atoms, which may or may not have a substituent. Examples of the substituent include a hydroxyl group, an amino group, an alkoxy group, an imino group, a halogen group, etc. Examples of the alkoxy group include a methoxy group, an ethoxy group, etc. Examples of the halogen group include a fluoro group, a chloro group, a bromo group, etc. ５ Examples of the alkyl group represented by the formula (I) which may or may not have a substituent include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, and the like.

[0041] R ５ As the group, a hydrogen atom is preferred from the viewpoint of ease of synthesis.

[0042] R ６ represents an alkyl group having 3 or more carbon atoms, which may or may not have a substituent and which may or may not have a heteroatom in the molecular chain. ６ The alkyl group represented by preferably has 3 to 20 carbon atoms, more preferably 6 to 14 carbon atoms, and even more preferably 8 to 12 carbon atoms, in terms of the yield and purity of the monomer, and the ease of improving the yield of the resulting nucleic acid oligomer. Examples of the substituent include a hydroxyl group, an amino group, an alkoxy group, an imino group, and a halogenyl group. Examples of the alkoxy group include a methoxy group and an ethoxy group. Examples of the halogenyl group include a fluoro group, a chloro group, and a bromo group. Examples of the heteroatom include an oxygen atom, a nitrogen atom, and a sulfur atom. R ６ The alkyl group represented by R is preferably branched in terms of the ease of improving the yield and purity of the monomer and the yield of the resulting nucleic acid oligomer. ６ Examples of the alkyl group include an i-propyl group, a sec-butyl group, a tert-butyl group, a neopentyl group, an i-pentyl group, a sec-pentyl group, a 3-pentyl group, a tert-pentyl group, an i-hexyl group, a 2-ethylhexyl group, a 1-methylheptyl group, a 3-methyloctyl group, and a tetrahydrogeranyl group (i.e., a 3,7-dimethyloctyl group), with a tetrahydrogeranyl group being preferred.

[0043] R ５ and R ６may bond to each other to form a 5- or more-membered ring having a branched chain, which may or may not have a substituent other than the branched chain, and which may or may not have a heteroatom. The number of members in the ring is preferably 5 to 10, more preferably 5 to 8, and even more preferably 5 to 7. Examples of the substituent include an alkyl group, a hydroxyl group, an amino group, an alkoxy group, an imino group, a halogen group, 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 a hydroxyl group, an amino group, an alkoxy group, an imino group, a halogen group, and the like. Examples of the alkoxy group include a methoxy group and an ethoxy group. Examples of the halogen group include a fluoro group, a chloro group, a bromo group, and the like. Examples of the heteroatom include an oxygen atom, a nitrogen atom, a sulfur atom, and the like. Examples of the branched chain include an alkyl group, which may or may not have a substituent, and which may or may not have a heteroatom in the molecular chain. In terms of the yield of the resulting nucleic acid oligomer, ease of control of the stereochemistry of the phosphorus atom in the nucleotide unit in the resulting nucleic acid oligomer, and the like, the alkyl group preferably has from 1 to 15 carbon atoms, more preferably from 2 to 10 carbon atoms, and even more preferably from 3 to 8 carbon atoms. Examples of the substituent in the branched chain include a hydroxyl group, an amino group, an alkoxy group, an imino group, a halogenyl group, and the like. Examples of the alkoxy group include a methoxy group and an ethoxy group. Examples of the halogenyl group include a fluoro group, a chloro group, and a bromo group. Examples of the heteroatom in the branched chain include an oxygen atom, a nitrogen atom, and a sulfur atom. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, and the like.

[0044] From the viewpoint of ease of control of the stereochemistry of the phosphorus atom in the nucleotide unit of the obtained nucleic acid oligomer, it is preferable that the stereochemistry of the phosphorus atom is controlled in the nucleotide monomer. Examples of the nucleotide monomer in which the stereochemistry of the phosphorus atom is controlled include the Sp form represented by the formula on the left below and the Rp form represented by the formula on the right below. In the formulas on the left and right below, R １ ~R ６ , and B ＮＨ２ is as described above.

[0045] The nucleotide monomer according to this embodiment can be produced, for example, in the same manner as in the methods shown in the Examples.

[0046] <Method for Producing Nucleic Acid Oligomer> The method for producing a nucleic acid oligomer according to this embodiment includes a chain elongation step in which a combination of the condensation step, the boronation step or the oxidation step, and the deprotection step is carried out once or repeated two or more times, and the nucleic acid oligomer production step. By this production method, a nucleic acid oligomer can be obtained in high yield.

[0047] In the condensation step, the hydroxyl group bonded to the carbon atom at the 5' position of the ribose structure in the nucleoside structure supported on the solid support or in the nucleoside structure of the nucleic acid oligomer is condensed with the phosphorous acid moiety of the nucleotide monomer represented by the general formula (1a) above. ３ａ The protecting group for the hydroxyl group represented by R ３ The protecting group is the same as the protecting group for a hydroxyl group represented by

[0048] In the condensation step, an acidic activator can be used. Examples of the acidic activator include 1-phenylimidazolium triflate, N-cyanomethylpyrrolidium triflate, and N-cyanomethyldimethylammonium triflate. The acidic activators may be used alone or in combination of two or more. The condensation reaction in the condensation step can be carried out, for example, under ice-cooling (0°C) or higher and room temperature (25°C) or lower, preferably 20°C or higher and 25°C or lower, for example, from one minute to several hours or lower, preferably from two minutes to one hour or lower. Examples of the reaction solvent include an inert solvent. Examples of the inert solvent include acetonitrile and isobutyronitrile. The reaction solvent may be used alone or in combination of two or more.

[0049] In the boronation step, a boronating agent can be used. Examples of the boronating agent include borane compounds such as borane / tetrahydrofuran complex and dimethyl sulfide borane. The boronating agent may be used alone or in combination of two or more. The boronation reaction in the boronation step can be carried out, for example, under ice cooling (0°C) or higher, at room temperature (25°C) or lower, preferably 20°C or higher and 25°C or lower, for example, 30 seconds to several hours or lower, preferably 1 minute to 1 hour or lower. Examples of the reaction solvent include an inert solvent. Examples of the inert solvent include acetonitrile, tetrahydrofuran, toluene, etc. The reaction solvent may be used alone or in combination of two or more.

[0050] In the oxidation step, an oxidizing agent can be used. Examples of the oxidizing agent include peroxides such as t-butyl hydroperoxide, m-chloroperbenzoic acid, (2R,8aS)-(+)-(camphorylsulfonyl)oxaziridine, and (1S)-(+)-(8,8-dichlorocamphorylsulfonyl)oxaziridine. The oxidizing agent may be used alone or in combination of two or more. The oxidation reaction in the oxidation step can be carried out, for example, under ice-cooling (0°C) or higher and room temperature (25°C) or lower, preferably 20°C or higher and 25°C or lower, for example, from one minute to several hours, preferably from two minutes to one hour. Examples of the reaction solvent include an inert solvent. Examples of the inert solvent include acetonitrile, toluene, and decane. The reaction solvent may be used alone or in combination of two or more.

[0051] In the deprotection step, a deprotecting agent can be used. Examples of the deprotecting agent include carboxylic acids and halogenated alkyl carboxylic acids. Examples of the carboxylic acid include acetic acid. Examples of the halogenated alkyl carboxylic acids include trifluoroacetic acid, trichloroacetic acid, and dichloroacetic acid. The deprotecting agent may be used alone or in combination of two or more. In the precursor nucleic acid oligomer to be deprotected, the protecting group R ３ａ is a trityl-based protecting group such as a triphenylmethyl group, a 4,4'-dimethoxytrityl (DMTr) group, or a 4-methoxytrityl (MMTr) group, and the precursor nucleic acid oligomer further contains a borano group, 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 deprotection step. Examples of cation scavenger include triethylsilane. The cation scavenger may be used alone or in combination of two or more.

[0052] In the chain elongation step, a combination of the condensation step, the boranolation step or the oxidation step, and the deprotection step is performed once or repeatedly performed two or more times. This results in the formation of a precursor nucleic acid oligomer having a dimer or more. Specifically, performing the combination once can yield a dimeric precursor nucleic acid oligomer, and repeating the combination n times (n is an integer of 2 or more) can yield an (n+1)-mer precursor nucleic acid oligomer.

[0053] In the nucleic acid oligomer production step, the phosphate protecting group represented by general formula (7) can be removed using a phosphate protecting group removal agent. Examples of phosphate protecting group removal agents include bases such as diazabicycloundecene, 1,8-(dimethylamino)naphthalene, and aqueous ammonia. The phosphate protecting group removal agents may be used alone or in combination of two or more. The phosphate protecting group removal reaction can be carried out, for example, under ice-cooling (0°C) or higher and room temperature (25°C) or lower, preferably 20°C or higher and 25°C or lower, for example, 15 minutes to 24 hours or lower, preferably 30 minutes to 20 hours or lower. Examples of reaction solvents include inert solvents. Examples of inert solvents include acetonitrile and ethanol. The reaction solvents may be used alone or in combination of two or more.

[0054] When the 5'-hydroxyl group is capped with a protecting group, R ９Examples of the hydroxyl-protecting group represented by the formula (I) include an acetyl group, a phenylacetyl group, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a chloroacetyl group, a trifluoroacetyl group, a propionyl group, a butyryl group, an isobutyryl 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 9-fluorenylmethyloxycarbonyl group, a t-butoxycarbonyl group, a trimethylsilyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a cyanomethoxymethyl group, a 2-(cyanoethoxy)ethyl group, a cyanoethoxymethyl group, a (dimethylamino)methyl group, and the like.

[0055] When the amino group is capped with a protecting group, R １０ and the amino group protecting group in Bs representing adenine, guanine, cytosine, or 5-methylcytosine include, for example, an acetyl group, a phenylacetyl group, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a chloroacetyl group, a trifluoroacetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a 4- Examples include a methoxybenzoyl group, a triphenylmethyl group, a 4,4'-dimethoxytrityl (DMTr) group, a 4-methoxytrityl (MMTr) group, a 9-phenylxanthenyl group, a 9-fluorenylmethyloxycarbonyl group, a t-butoxycarbonyl group, a trimethylsilyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a cyanomethoxymethyl group, a 2-(cyanoethoxy)ethyl group, a cyanoethoxymethyl group, and a (dimethylamino)methyl group.

[0056] Capping of the 5'-hydroxyl group and amino group with a protecting group and elimination of the protecting group can be carried out by known methods.

[0057] X ＋Examples of the counter cation represented by the formula (I) include ammonium ions, cations derived from organic amine compounds, and metal cations. One type of counter cation may be used alone, or two or more types may be used in combination. Examples of the cation derived from organic amine compounds include, from the viewpoints 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, for example, the cation HNEt derived from triethylamine. ３ ＋ (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.

[0058] In the method for producing a nucleic acid oligomer according to this embodiment, it is preferable that at least the steps from the condensation step to the nucleic acid oligomer production step be carried out by a reaction using a solid phase support (solid phase method). 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.

[0059] The type of the solid phase 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 phase support can be used to link the solid phase support to the monomer. Examples of amino groups on the solid phase support include 3-aminopropyl groups and long-chain alkylamino groups (LCAA). A linker may be present between the solid phase support and the monomer. Examples of linkers include succinyl groups, sarkosyl groups, succinylsarkosyl groups, and oxalyl groups.

[0060] The nucleic acid oligomer obtained as described above can be appropriately cleaved from the solid phase support. Cleavage of the nucleic acid oligomer from the solid phase support can be carried out, for example, by treating the solid phase support to which the nucleic acid oligomer is bound with aqueous ammonia. Examples of the aqueous ammonia used in cleavage of the nucleic acid oligomer from the solid phase support include 25% aqueous ammonia by mass or a 25% aqueous ammonia-ethanol mixed solution (3:1, v / v). Cleavage of the nucleic acid oligomer from the solid phase support may be carried out, for example, simultaneously with the removal of the protecting group from the 5'-hydroxyl group or the removal of the phosphate protecting group described above.

[0061] 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.

[0062] In the method for producing a nucleic acid oligomer according to this embodiment, when the stereochemistry of the phosphorus atom of the nucleotide monomer is controlled, the stereochemistry of the phosphorus atom in the nucleotide unit is also controlled. This allows the method for producing a nucleic acid oligomer according to this embodiment to stereoselectively synthesize a nucleic acid oligomer, which can effectively improve, for example, the efficacy and safety of the antisense molecule. The stereochemical purity is preferably 90% or more, and more preferably 95% or more.

[0063] Nucleic Acid Oligomer The nucleic acid oligomer obtained by the method for producing a nucleic acid oligomer according to this embodiment contains at least one nucleotide unit selected from the group consisting of a nucleotide unit represented by the general formula (7) and a nucleotide unit represented by the general formula (8). The nucleotide unit represented by the general formula (7) may be used alone or in combination of two or more types. The nucleotide unit represented by the general formula (8) may be used alone or in combination of two or more types.

[0064] Nucleic acid oligomers containing nucleotide units represented by general formula (7) tend to have excellent affinity with target RNA and RNase H induction activity, and are particularly resistant to degradative enzymes, while being less toxic. Nucleic acid oligomers containing nucleotide units represented by general formula (8) tend to have excellent affinity with target RNA and RNase H induction activity, and are less toxic, but are likely to have low degradative enzyme resistance. When the nucleic acid oligomer contains a nucleotide unit represented by general formula (7) and a nucleotide unit represented by general formula (8), it is expected that the nucleic acid oligomer will maintain high affinity with target RNA, RNase H induction activity, and degradative enzyme resistance, while having low toxicity.

[0065] The length (base length) of the nucleic acid oligomer 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 pharmaceuticals, it is preferably 8 to 50 bases, more preferably 10 to 30 bases, and even more preferably 10 to 21 bases.

[0066] 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 a base sequence 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 unit represented by general formula (7) and the nucleotide unit represented by general formula (8) is appropriately selected so as to maintain high affinity with the target RNA, RNase H-inducing activity, resistance to degradative enzymes, and blood retention.

[0067] The nucleic acid oligomer according to this embodiment may be a nucleic acid oligomer having nucleotide units other than the nucleotide units represented by general formula (7) and the nucleotide units represented by general formula (8). In the nucleic acid oligomer, the total proportion of the nucleotide units represented by general formula (7) and the nucleotide units represented by general formula (8) is not particularly limited, and from the viewpoint of double-strand formation 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.

[0068] In the nucleic acid oligomer according to this embodiment, the 5'-end may be a hydroxyl group or a protecting group for a hydroxyl group, and the 3'-end may be a hydroxyl group or a protecting group for a hydroxyl group. Specific examples of the protecting group for a hydroxyl group are as described above.

[0069] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. The following types of analyzers were used. 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 (162 MHz) １ Tetramethylsilane (TMS) was used as an internal standard for H-NMR. ３１ P-NMR shows 85% H ３P.O. ４ was used as an external standard.

[0070] HRMS (ESI-TOF): Sciex X500R QTOF. The silica gel packed in the column chromatography was Silica Gel 60N from Kanto Chemical and CHROMATOREX NH-DM1020 from Fuji Silysia Chemical Ltd. Column used for reversed-phase HPLC (purification): Japan Analytical Industry JAIGEL-ODS, AP-SP-120-15. Column used for reversed-phase HPLC (analysis): Waters μ-BOUNDASPHERE, C18 5 μm, 100 Å, 3.9 mm × 159 mm.

[0071] 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.

[0072] <Synthesis of amino alcohol>

[0073] The amino alcohol derivative 1 (11.33 g, 83 mmol) shown on the left side of the scheme above was azeotropically dried with pyridine (10 mL x 3) and toluene (10 mL x 3) under an argon atmosphere and dissolved in tetrahydrofuran (500 mL). While stirring the resulting tetrahydrofuran solution, triethylamine (TEA, 17.5 mL, 124.5 mmol) and chlorotrimethylsilane (TMSCl, 15.7 mL, 124.5 mmol) were added, and the mixture was stirred for 2 hours. Then, methanol (5.5 mL) was added to terminate the reaction, and the solvent was evaporated under reduced pressure. The resulting colorless crystalline mixture was dissolved in dichloromethane (160 mL), and the solution was cooled to 0°C. Triethylamine (TEA, 29.0 mL, 166 mmol) was added, and 2-nitrobenzenesulfonyl chloride (NsCl, 19.31 g, 87.15 mmol) was added in 10 portions, followed by stirring for 3 hours. The reaction was stopped by adding methanol (5 mL), dichloromethane (150 mL) was added, and the mixture was washed with saturated aqueous sodium bicarbonate (300 mL x 3 times). The aqueous layer was back-extracted with dichloromethane (100 mL x 2 times). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a colorless crystalline mixture.

[0074] The resulting mixture was dissolved in THF (260 mL), and tetrahydrogeraniol (22.3 mL, 116 mmol) and triphenylphosphine (44.0 g, 166 mmol) were added. The mixture was then cooled to 0°C. A diisopropyl azodicarboxylate / tetrahydrofuran solution (DIAD, 33.0 mL, 166 mmol / 150 mL) was added, and the mixture was warmed to room temperature and stirred for 24 hours. Methanol (7 mL) was added to terminate the reaction, and the solvent was evaporated under reduced pressure. 350 mL of hexane-ethyl acetate (6:1, v / v) was added to the resulting mixture, and the mixture was cooled to 0°C. The precipitated crystals were filtered off, and the resulting solution was evaporated under reduced pressure. This procedure was repeated three times. Ethyl acetate (500 mL) was added to the resulting mixture, and the mixture was washed with saturated brine (200 mL x 3 times). The aqueous layer was back-extracted with ethyl acetate (200 mL x 2 times). The combined organic layers were dried over anhydrous sodium sulfate and filtered, and the solvent was evaporated under reduced pressure to give a pale yellow oily mixture.

[0075] Methanol (41.5 mL) and a 3% trifluoroacetic acid / dichloromethane solution (360 mL) were added sequentially to the resulting mixture, and the mixture was stirred overnight. The organic layer was then washed with saturated aqueous sodium bicarbonate (200 mL x 3), and the aqueous layer was back-extracted with dichloromethane (200 mL x 3). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (developing solvent: chloroform-hexane (1:1, v / v) → chloroform) to obtain target compound 2 (pale yellow oil). Yield: 32.4 g, 70 mmol, 84% yield.

[0076] １ H-NMR (400MHz, CDCl ３ ) δ8.05-8.02 (m, 1H), 7.72-7.60 (m, 3H), 7.36-7.26 (m, 5H), 4.90 (dt, J=8.5, 3.5Hz, 1H) , 3.56-3.29 (m, 4H), 2.76 (dd, J=8.7, 3.2Hz, 1H), 1.60-1.03 (m, 10H), 0.90-0.81 (m, 9H) HRMS (ESI-TOF): Calcd for [M+1H] １＋ ,463.2262;found,463.2262

[0077]

[0078] Aminoalcohol derivative 2 (32.4 g, 70.0 mmol) was dissolved in dimethylformamide (350 mL) under an argon atmosphere. 18-crown-6 (35.0 g, 140 mmol), potassium carbonate (37.0 g, 280 mmol), and 4-mercaptobenzoic acid (19.0 g, 140 mmol) were added sequentially, and the mixture was heated to 40°C and stirred for 12 hours. Approximately half of the solvent was then removed by distillation under reduced pressure. After dilution with ethyl acetate-hexane (1:4, v / v, 300 mL), the mixture was washed with aqueous sodium hydroxide (300 mL x 5 times) and back-extracted with ethyl acetate-hexane (1:4, v / v, 300 mL x 1 time). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (developing solvent: chloroform-methanol (100:0-97:3, v / v)) to obtain the target compound 3 (pale yellow oil). Yield: 12.99 g, 46.8 mmol, 67%.

[0079] １ H-NMR (400MHz, CDCl ３ ) δ7.39-7.25 (m, 5H), 4.75 (dd, J=9.1, 3.7Hz, 1H), 2.93 (dt, J=11.9, 3.2Hz, 1H), 2.78-2.60 (m, 3H), 2.08-2.58 (br, 2H), 1.58-1.07 (m, 10H), 0.87 (q, J = 3.2Hz, 9H) HRMS (ESI-TOF): Calcd for [M+1H] ＋ ,278.2479;found,278.2474

[0080]

[0081] The amino alcohol derivatives 3i, 3t, and 3r (3i: 0.67 g, 3.8 mmol; 3t: 13.9 g, 50 mmol; 3r: 1.4 g, 5.0 mmol) were azeotropically dried with toluene (5 mL x 3) under an argon atmosphere, and toluene (3i: 3.7 mL; 3t: 40.0 mL; 3r: 3.3 mL) and N-methylmorpholine (3i: 0.9 mL, 7.9 mmol; 3t: 11.5 mL, 105 mmol; 3r: 1.15 mL, 10.5 mmol) were added. The mixture was added over 10 min to a solution of phosphorus trichloride (3i: 0.3 mL, 3.9 mmol; 3t: 11.5 mL, 105 mmol; 3r: 0.5 mL, 5.3 mmol) in toluene (3i: 2.5 mL; 3t: 40 mL; 3r: 3.0 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours, and the resulting salt was filtered off at −78° C. under an argon atmosphere. The solvent was then evaporated under reduced pressure to give the target compounds 4i, 4t, and 4r (4i: pale yellow oil, 1.18 g; 4t: pale yellow oil, 18.2 g; 4r: pale yellow oil, 2.0 g). These compounds were used in the next reaction without further purification.

[0082] <Synthesis of Monomer>

[0083] In the above formula, B ＮＨ２ Ad ＮＨ２ , Cy ＮＨ２ , Gu ＮＨ２ , or Th, and Ad ＮＨ２ represents adenine with no amino group substitution, and Cy ＮＨ２ represents cytosine with no amino group substitution, and Gu ＮＨ２ represents guanine with no amino group substitution, and Th represents thymine.

[0084] (Synthesis of Oxazaphospholidine Monomers) Oxazaphospholidine monomers 6a, 6c, 6g, 6t, 6l, 6r, and 6i were synthesized with reference to a known method (J. Am. Chem. Soc., 2008, 130, 47, pp. 16031-16037).

[0085] Nucleoside derivative 5a, 5c, 5g, 5t, or 5l (A: 2.2 g, 4.0 mmol; C: 3.2 g, 6.0 mmol; G: 2.6 g, 4.5 mmol; T: 1.4 g, 2.5 mmol; LNAT: 1.7 g, 3.0 mmol) shown on the left side of the scheme above was azeotropically dried with pyridine, toluene, and THF three times (1 mL each) under an argon atmosphere, and THF (A: 10 mL; C: 15 mL; G: 10.3 mL; T: 10 mL; LNAT: 7.5 mL) and triethylamine (A: 3.9 mL, 28 mmol; C: 5.8 mL, 42 mmol; G: 4.4 mL, 31.5 mmol; T: 3.9 mL, 28 mmol; LNAT: 2.9 mL, 21 mmol) were added. While stirring the resulting THF solution at a predetermined temperature (A, C, T, LNAT: -78°C; G: -40°C), a 0.6 M solution of compound 4t in tetrahydrofuran (A: 3.4 g, 10 mmol; C: 5.1 g, 15 mmol; G: 3.8 g, 11.3 mmol; T: 2.1 g, 6.25 mmol; LNAT: 2.6 g, 7.7 mmol) was added over 5 minutes. The thymidine derivative was then warmed to room temperature, while the other derivatives were stirred for 2 hours at the predetermined temperature. The solution was diluted with 280 mL of chloroform and washed with saturated aqueous sodium bicarbonate (50 mL x 3). The aqueous layer was back-extracted with chloroform (50 mL x 3). The organic layer was collected and dehydrated with sodium sulfate. The solvent in the organic layer was then evaporated under reduced pressure. The resulting residue was azeotropically dried with toluene (1 mL x 3) under an argon atmosphere. The solution was purified by aminosilica gel column chromatography. Only for 6 g of the oxazaphospholidine monomer, a portion of the residue was then separated and purified by reversed-phase HPLC. The specific procedure for separation and purification is as follows.

[0086] A: 70% of the obtained residue was separated and purified by amino silica gel column chromatography [NH-silica gel, toluene-ethyl acetate (100:0-80:20, v / v), triethylamine 0.5%]. C: 51% of the obtained residue was separated and purified by amino silica gel column chromatography [NH-silica gel, toluene → ethyl acetate-tBuOH (99:1, v / v), triethylamine 0.5%]. G: The entire amount of the obtained residue was separated and purified by amino silica gel column chromatography [NH-silica gel, chloroform → chloroform-tBuOH (93:7, v / v), triethylamine 0.5%], and 51% of the crude product obtained by distilling off the solvent under reduced pressure was separated and purified by reversed-phase HPLC (mobile phase: acetonitrile-chloroform (85:15, v / v)). T: The entire residue was purified by amino silica gel column chromatography [NH-silica gel, toluene → toluene-ethyl acetate (80:20, v / v), triethylamine 0.5%]. LNAT: 95% of the residue was purified by amino silica gel column chromatography [NH-silica gel, toluene-ethyl acetate (100:0-80:20, v / v), triethylamine 0.5%].

[0087] The solvent was removed from the isolated and purified product by distillation under reduced pressure to obtain the target compound (oxazaphospholidine monomer 6a, 6c, 6g, 6t, or 6l). Yields: A: 0.82 g, 0.95 mmol, 34%; C: 0.80 g, 0.96 mmol, 39%; G: 0.61 g, 0.70 mmol, 30%; T: 1.02 g, 1.20 mmol, 55%; LNAT: 0.82 g, 0.93 mmol, 31%

[0088] Oxazaphospholidine Monomer 6a １ H-NMR (400MHz, CDCl ３) δ8.31 (d, J=2.3Hz, 1H), 7.99 (s, 1H), 7.41-7.16 (m, 14H), 6.80-6.74 (m, 4H), 6 .48-6.44 (m, 1H), 5.67 (br, 2H), 5.59-5.54 (m, 1H), 5.01 (qd, J=5.8, 2.9Hz, 1H) , 4.23 (t, J=3.4Hz, 1H), 3.77-3.74 (m, 6H), 3.58-3.34 (m, 3H), 3.09-2.97 (m, 2H ), 2.95-2.85 (m, 2H), 2.61-2.54 (m, 1H), 1.62-1.04 (m, 10H), 0.88-0.82 (m, 9H) ３１ P-NMR (162MHz, CDCl ３ ) δ142.7, 142.1, 142.0, 141.5.

[0089] Oxazaphospholidine Monomer 6c １ H-NMR (400MHz, CDCl ３ ) δ7.92 (td, J=4.8, 2.4Hz, 1H), 7.43-7.17 (m, 14H), 6.84-6.77 (m, 4H), 6 .33-6.28 (m, 1H), 5.51 (qd, J=7.3, 2.4Hz, 1H), 5.42-5.34 (m, 1H), 4.90-4 .81 (m, 1H), 3.77-3.71 (m, 6H), 3.50-3.36 (m, 3H), 3.09-2.84 (m, 3H), 2.6 0-2.53 (m, 1H), 2.33-2.21 (m, 1H), 1.58-1.02 (m, 10H), 0.88-0.80 (m, 9H) ３１ P-NMR (162MHz, CDCl ３ ) δ142.6, 144.0, 142.1, 141.6.

[0090] Oxazaphospholidine monomer 6g １ H-NMR (400MHz, CDCl ３) δ7.64 (s, 1H), 7.43-7.15 (m, 14H), 6.78 (dd, J=11.4, 8.7Hz, 4H), 6.27-6.20 (m , 3H), 5.55 (t, J = 7.1Hz, 1H), 4.97 (d, J = 2.7Hz, 1H), 4.20 (d, J = 2.7Hz, 1H), 3.74 -3.71 (m, 6H), 3.55-3.31 (m, 3H), 3.13-2.98 (m, 2H), 2.96-2.86 (m, 1H), 2.79 (t d, J=13.6, 7.9Hz, 1H), 2.50-2.46 (m, 1H), 1.68-1.05 (m, 10H), 0.88-0.81 (m, 9H) ３１ P-NMR (162MHz, CDCl ３ ) δ142.8, 142.3, 142.2, 141.9.

[0091] Oxazaphospholidine Monomer 6t １ H-NMR (400MHz, CDCl ３ ) δ8.48 (br, 1H), 7.62 (d, J = 1.4Hz, 1H), 7.42-7.19 (m, 14H), 6.85-6.79 (m, 4H) , 6.42 (dd, J = 7.5, 6.2 Hz, 1H), 5.51-5.45 (m, 1H), 4.92-4.86 (m, 1H), 4.09 (t, J = 2.5Hz, 1H), 3.76 (dt, J = 8.2, 3.4Hz, 6H), 3.53-3.33 (m, 3H), 3.07-2.95 (m, 2H) ), 2.92-2.83 (m, 1H), 2.47-2.29 (m, 2H), 1.60-1.01 (m, 13H), 0.88-0.80 (m, 9H) ３１ P-NMR (162MHz, CDCl ３ ) δ143.9, 143.4, 142.5, 142.1.

[0092] Oxazaphospholidine Monomer 6l １ H-NMR (400MHz, CDCl ３) δ8.99 (s, 1H), 7.73 (d, J=1.4Hz, 1H), 7.48-7.45 (m, 2H), 7.37-7.18 (m, 12H), 6. 86-6.81 (m, 2H), 6.78-6.73 (m, 2H), 5.66 (d, J = 9.6Hz, 1H), 5.47 (dt, J = 56.3, 7.3 Hz, 1H), 4.61-4.58 (m, 1H), 4.53-4.47 (m, 1H), 3.87-3.70 (m, 8H), 3.61-3.49 (m, 1H), 3.44-3.34 (m, 1H), 3.12-2.79 (m, 3H), 1.57-1.02 (m, 13H), 0.88-0.73 (m, 9H) ３１ P-NMR (162MHz, CDCl ３ ) δ145.3, 144.9, 142.3, 141.7.

[0093]

[0094] Nucleoside derivative 5c (1.1 g, 2.0 mmol) shown on the left side of the above scheme was azeotropically dried under an argon atmosphere with pyridine, toluene, and THF (1 mL x 3 times), followed by the addition of THF (5.0 mL) and triethylamine (1.9 mL, 14.0 mmol). While stirring the resulting THF solution at −78°C, a 0.6 M solution of compound 4r in tetrahydrofuran (1.7 g, 5.0 mmol) was added over 5 minutes. The mixture was then stirred for 2 hours while maintaining the temperature at −78°C. After dilution with 280 mL of chloroform, the mixture was washed with saturated aqueous sodium bicarbonate (50 mL x 3 times). The aqueous layer was back-extracted with chloroform (50 mL x 3 times). The organic layer was collected and dehydrated with sodium sulfate. The solvent in the organic layer was then evaporated under reduced pressure. The resulting residue was azeotropically dried with toluene (1 mL x 3 times) under an argon atmosphere. The product was then purified by aminosilica gel column chromatography. The specific procedure for the separation and purification is as follows.

[0095] All the residues obtained were separated and purified by aminosilica gel column chromatography [NH-silica gel, toluene → ethyl acetate-tBuOH (99:1, v / v), triethylamine 0.5%].

[0096] The solvent was removed from the separated and purified product by distillation under reduced pressure to obtain the target compound (oxazaphospholidine monomer 6r). Yield 6r: 0.86 g, 1.02 mmol, 51%

[0097] Oxazaphospholidine Monomer 6r １ H-NMR (400MHz, CDCl ３ ) δ7.96 (1H), 7.42-7.17 (14H), 6.82-6.73 (4H), 6.25-6.32 (1H), 5.56-5.48 (1H), 5.35 (1H), 4.91-4.84 (1H), 4.07-3.9 8 (1H), 3.75-3.70 (6H), 3.46-3.39 (3H), 3.09-2.81 (3H), 2.58 (1H), 2.40-2.28 (1H), 1.60-1.07 (10H), 0.89-0.84 (9H) ３１ P-NMR (162MHz, CDCl ３ ) δ144.7, 144.2

[0098]

[0099] Nucleoside derivative 5c (0.8 g, 1.5 mmol) shown on the left side of the scheme above was azeotropically dried under an argon atmosphere with pyridine, toluene, and THF (1 mL x 3 times), followed by the addition of THF (3.8 mL) and triethylamine (1.5 mL, 10.5 mmol). The resulting THF solution was stirred at −78°C, and a 0.6 M solution of compound 4i in tetrahydrofuran (0.9 g, 3.8 mmol) was added over 5 minutes. The mixture was then stirred for 2 hours while maintaining the temperature at −78°C. The mixture was diluted with 280 mL of chloroform and washed with saturated aqueous sodium bicarbonate (50 mL x 3 times). The aqueous layer was back-extracted with chloroform (50 mL x 3 times). The organic layer was collected and dehydrated with sodium sulfate. The organic solvent was then evaporated under reduced pressure, and the resulting residue was azeotropically dried with toluene (1 mL x 3 times) under an argon atmosphere. The product was then purified by aminosilica gel column chromatography. The specific procedure for the separation and purification is as follows.

[0100] All the residues obtained were separated and purified by aminosilica gel column chromatography [NH-silica gel, chloroform → chloroform-tBuOH (89:11, v / v), triethylamine 0.5%].

[0101] The solvent was removed from the separated and purified product by distillation under reduced pressure to obtain the target compound (oxazaphospholidine monomer 6i). Yield 6i: 0.30 g, 0.41 mmol, 27%

[0102] Oxazaphospholidine Monomer 6i １ H-NMR (400MHz, CDCl ３ ) δ7.98-8.07 (1H), 7.14-7.45 (14H), 6.72-6.85 (4H), 6.23-6.32 (1H), 5.42-5.53 (1H), 5.25-5.35 (1H), 4.80-4.97 (1H), 3.99- 4.11 (1H), 3.69-3.79 (6H), 3.26-3.56 (4H), 2.79-2.90 (1H), 2.52-2.68 (1H), 2.22-2.38 (1H), 1.17-1.24 (3H), 1.09-1.16 (3H) ３１ P-NMR (162MHz, CDCl ３ ) δ144.1, 141.8

[0103] <Synthesis of Nucleic Acid Oligomer by Solid Phase Method> Hereinafter, among internucleotide bonds, a boranophosphate bond will be represented by the subscript PB, and a phosphodiester bond will be represented by the subscript PO.

[0104] (dN ＰＢSolid-Phase Synthesis of 5'-O-Dimethoxytritylthymidine (0.5 μmol) supported on a solid support via a succinyl linker was detritylated by adding a 3% dichloroacetic acid / dichloromethane solution (5 times × 12 s, 1 mL / time). The solid support was then washed with acetonitrile and dichloromethane dried over molecular sieves. The solid support was dried for 5 minutes, and then [N-Thg,Ph]-type oxazaphospholidine monomer 6a, 6c, 6g, or 6t (A: 25.8 mg (60 equivalents, 30 μmol); C: 25.0 mg (60 equivalents, 30 μmol); G: 26.3 mg (60 equivalents, 30 μmol); T: 25.5 mg (60 equivalents, 30 μmol)) was added. After drying the solid support for 5 minutes, 150 μL of an acetonitrile-isobutyronitrile (7:3, v / v) solution containing 1.0 M 1-phenylimidazolium triflate as an acidic activator was added as a reaction solvent under an argon atmosphere, and condensation was carried out by stirring on a vortex mixer for 10 minutes. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile (1 mL x 3) and dichloromethane (1 mL x 3). After drying the solid support for 5 minutes, 950 μL of tetrahydrofuran dried over molecular sieves and 50 μL of a 1.0 M borane / tetrahydrofuran complex solution were added under an argon atmosphere, and boronation was carried out by stirring on a vortex mixer for 2 minutes. The solid support was then washed with tetrahydrofuran (1 mL x 3) and dichloromethane (1 mL x 3). The dimethoxytrityl group was deprotected using a 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution (5 times for 12 seconds, 1 mL each time). The solid support was then washed with dried dichloromethane (1 mL x 3 times) and acetonitrile (1 mL x 3 times) and then dried for 5 minutes. The free 5'-hydroxyl group was acetylated for 1 minute by adding 10 mg of 4-dimethylaminopyridine, 450 μL of 2,6-lutidine, and 50 μL of acetic anhydride. The solid support was washed with dried dichloromethane (1 mL x 3 times) and acetonitrile (1 mL x 3 times) and then dried for 5 minutes.A previously prepared 10% diazabicycloundecene / acetonitrile solution was added, and the protecting groups at the phosphate moieties were removed for 60 minutes, resulting in conversion to boranophosphodiester bonds. The solid support was washed with dried acetonitrile (1 mL x 3 times), and then reacted with a 25% aqueous ammonia / ethanol (3:1, v / v, 5 mL) solution under the conditions described below to deprotect the acetyl group at the nucleic acid base moiety, cleave the coordinate bond between the nitrogen atom at the nucleic acid base moiety and borane, and release the nucleic acid from the solid support (dC). ＰＢ T, dG ＰＢ T and T ＰＢ T: room temperature, 17 hours; dA ＰＢ (T: room temperature, 3 hours, 50°C, 17 hours). After the reaction, the reaction solution was filtered and washed with acetonitrile. The solvent was removed by distillation under reduced pressure, and the crude product was analyzed by reversed-phase HPLC. Reverse-phase HPLC was performed in 0.1 M TEAA buffer (pH 7) using a linear gradient of 0 to 20% acetonitrile for 60 minutes at 30°C and a flow rate of 0.5 mL / min. The results are shown in Table 1.

[0105] Comparative Synthesis Example 1: 7 g (30.0 mg, 80 equivalents, 40 μmol) of an [N-Me, Ph]-type oxazaphospholidine monomer was added to 0.5 μmol of 5'-hydroxythymidine supported on a solid support via a succinyl linker. After drying the solid support, 200 μL of an acetonitrile solution containing 1.0 M N-cyanomethylpyrrolidinium triflate as an acidic activator was added as a reaction solvent under an argon atmosphere, and condensation was carried out by stirring for 3 minutes. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile and dichloromethane. After drying the solid support, 200 μL of dichloromethane and 20 μL of dimethylsulfide borane were added under an argon atmosphere, and boronation was carried out by stirring for 5 minutes. The solid support was washed with dichloromethane. The dimethoxytrityl group was deprotected using a 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution. The solid support was then washed with dried dichloromethane and acetonitrile and dried. 300 μL of a 36% N-methylimidazole / tetrahydrofuran solution and 250 μL of a 22% acetic anhydride / tetrahydrofuran solution, which had been prepared in advance, were added sequentially to the free 5'-hydroxyl group, and the mixture was stirred for 30 seconds to perform acetylation. The solid support was then washed with dried acetonitrile and dried. The protecting group at the phosphate moiety was removed by reacting with a 4% diazabicycloundecene / acetonitrile solution for 1.5 hours. The product was then cleaved from the solid support by reacting with a 25% aqueous ammonia solution for 17 hours, yielding a mixture containing the target product. The mixture was analyzed by HPLC. The results are shown in Table 1.

[0106]

[0107] The percentages in Table 1 were calculated from the area ratio of the target product to all observed products in the HPLC analysis results.

[0108] Figures 1(a) and 1(b) show the results of reverse-phase HPLC analysis for Comparative Synthesis Example 1 and Entry 3 of Synthesis Example 1, respectively. In Comparative Synthesis Example, a large amount of unreacted thymidine was observed in the short elution time portion, and a large amount of by-products was observed in the long elution time portion. On the other hand, in Entry 3 of Synthesis Example 1, the amount of unreacted material and by-products was significantly lower. The percentage (%) of the target product relative to all products was 19% higher in Entry 3 of Synthesis Example 1. From these results, it is believed that the [N-Me,Ph]-type oxazaphospholidine monomer is prone to hydrolysis and oxidation of the monomer, making it susceptible to side reactions. However, the [N-Thg,Ph]-type oxazaphospholidine monomer has sufficient steric hindrance around the nitrogen atom, making the monomer stable and less likely to undergo side reactions, thereby allowing the desired condensation reaction to proceed efficiently. These findings confirm that the [N-Thg,Ph]-type is more suitable for synthesizing nucleic acid oligomers.

[0109] (HPLC elution conditions) Synthesis Example 1: 0-20% CH ３ CN in 0.1 M TEAA buffer for 60 min Comparative Synthesis Example 1: 0-8% CH ３ CN in 0.1M TEAA buffer for 20min, 8%CH ３ CN in 0.1M TEAA buffer for 40min

[0110] (dN ＰＯ Solid-phase synthesis of T

[0111] Synthesis Example 2 5′-O-dimethoxytrityl-thymidine (0.5 μmol) supported on a solid support via a succinyl linker was detritylated by adding a 3% dichloroacetic acid / dichloromethane solution (5 times × 12 s, 1 mL / time), and the solid support was then washed with acetonitrile and dichloromethane that had been dried using molecular sieves. The solid support was dried for 5 minutes, and then oxazaphospholidine monomer 6a, 6c, 6g, 6t, 6l, or 6i (A: 25.8 mg (60 equivalents, 30 μmol); C(Thg): 25.0 mg (60 equivalents, 30 μmol); G: 26.3 mg (60 equivalents, 30 μmol); T: 25.5 mg (60 equivalents, 30 μmol); LNAT: 26.3 mg (60 equivalents, 30 μmol); C(iPr): 22.1 mg (60 equivalents, 30 μmol)) was added. After drying the solid support for 5 minutes, 150 μL of an acetonitrile-isobutyronitrile (7:3, v / v) solution containing 1.0 M 1-phenylimidazolium triflate as an acidic activator was added as a reaction solvent under an argon atmosphere, and condensation was carried out by stirring on a vortex mixer for 10 minutes. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile (1 mL x 3 times) and dichloromethane (1 mL x 3 times). After drying the solid support for 5 minutes, 500 μL of a 1.0 M t-butyl hydroperoxide / toluene solution, previously prepared and dried over molecular sieves, was added under an argon atmosphere, and oxidation was carried out by stirring on a vortex mixer for 5 minutes. The solid support was washed with dichloromethane (1 mL x 6 times). The dimethoxytrityl group was deprotected using a 3% dichloroacetic acid / dichloromethane solution (5 times for 12 seconds, 1 mL per wash). The solid support was then washed with dried dichloromethane (1 mL x 3) and acetonitrile (1 mL x 3), and then reacted with 25% aqueous ammonia / ethanol (3:1, v / v, 5 mL) solution at room temperature for 3 hours to remove the protecting group of the phosphate moiety and cleave it from the solid support. After the reaction, the reaction solution was filtered and washed with acetonitrile. The solvent was evaporated under reduced pressure, and the crude product was analyzed by reverse-phase HPLC as described above. The results are shown in Table 2.

[0112] Comparative Synthesis Example 2 A dimer was synthesized and analyzed under the same conditions as in Synthesis Example 2, except that the [N-Me,Ph]-type oxazaphospholidine monomer 7c was used as the monomer instead of the [N-Thg,Ph]-type oxazaphospholidine monomer 6. The results are shown in Table 2.

[0113]

[0114] In Table 2, the HPLC yield is the peak area ratio dN in the reversed-phase HPLC results. ＰＯ T / (T+dN ＰＯ T).

[0115] 2(a) to 2(c) show the results of reversed-phase HPLC analysis of entries 2 and 6 of Synthesis Example 2 and Comparative Synthesis Example 2. While unreacted thymidine was observed in Comparative Synthesis Example 2, no unreacted thymidine was observed in entries 2 and 6 of Synthesis Example 2 compared to Comparative Synthesis Example 2, and the HPLC yield of the target product was 2% higher in each case. In Synthesis Example 2 above, a dimer was synthesized, but since the number of condensations increases during oligomer synthesis, it is desirable to use a monomer with high condensation efficiency. It was confirmed that a monomer having a branched alkyl group with 3 or more carbon atoms is suitable for synthesizing nucleic acid oligomers.

[0116] (HPLC elution conditions) 0-20% CH ３ CN in 0.1M TEAA buffer for 60min

[0117] ((Rp)-C ＰＢ Solid-phase synthesis of (Rp)-C with controlled stereochemistry at the phosphorus atom using a monomer with controlled stereochemistry at the phosphorus atom ＰＢ Solid phase synthesis of T was carried out. The following [N-Me, Ph]-type oxazaphospholidine monomer is a known compound, the synthesis of which is described in Non-Patent Document 2.

[0118] Comparative Synthesis Example 3: A stereocontrolled [N-Me,Ph]-type oxazaphospholidine monomer (Rp)-7c (30.0 mg, 80 equivalents, 40 μmol) was added to 5'-hydroxythymidine (0.5 μmol) supported on a solid support via a succinyl linker. After drying the solid support, an acetonitrile solution (200 μL) containing N-cyanomethylpyrrolidinium triflate (1.0 M) as an acidic activator was added as a reaction solvent under an argon atmosphere, and condensation was carried out by stirring for 3 minutes. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile and dichloromethane. After drying the solid support, 200 μL of dichloromethane and 20 μL of dimethylsulfide borane were added under an argon atmosphere, and boronation was carried out by stirring for 5 minutes. The solid support was washed with dichloromethane. The dimethoxytrityl group was deprotected using a 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution. The solid support was then washed with dried dichloromethane and acetonitrile and dried. The free 5'-hydroxyl group was acetylated by sequentially adding 300 μL of a 36% N-methylimidazole / tetrahydrofuran solution and 250 μL of a 22% acetic anhydride / tetrahydrofuran solution, which had been previously prepared, and stirring for 30 seconds. The solid support was then washed with dried acetonitrile and dried. The protecting group at the phosphate moiety was removed by reacting with a 4% diazabicycloundecene / acetonitrile solution for 1.5 hours. The solid support was then washed with dried acetonitrile, dried, and then reacted with 25% aqueous ammonia for 1 hour to cleave the product from the solid support, yielding a mixture containing the target product. The mixture was analyzed by HPLC. The results are shown in Table 3.

[0119] Synthesis Example 3: Stereoregulated [N-Thg,Ph]-type oxazaphospholidine monomer (Rp)-6c (18.8 mg, 45 equivalents, 23 μmol) was added to 5'-hydroxythymidine (0.5 μmol) supported on a solid support via a succinyl linker. After drying the solid support, an acetonitrile-isobutyronitrile (7:3, v / v, 160 μL) solution containing N-cyanomethylpyrrolidinium triflate (1.0 M) as an acidic activator was added as a reaction solvent under an argon atmosphere, and the mixture was stirred for 5 minutes to carry out condensation. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile and dichloromethane. After drying the solid support, boronation was carried out under an argon atmosphere using a 0.05 M borane / tetrahydrofuran complex solution in tetrahydrofuran for 2 minutes. The solid support was washed with dichloromethane. The dimethoxytrityl group was deprotected using a 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution. The solid support was then washed with dried dichloromethane and acetonitrile and dried. The free 5'-hydroxyl group was acetylated by adding 10 mg of 4-dimethylaminopyridine, 450 μL of 2,6-lutidine, and 50 μL of acetic anhydride and stirring for 60 seconds. The solid support was then washed with dried acetonitrile and dried. The protecting group at the phosphate moiety was removed by reacting with a 10% diazabicycloundecene / acetonitrile solution for 1 hour. The solid support was then washed with dried acetonitrile and dried, and then reacted with 25% aqueous ammonia-ethanol (3:1, v / v) for 17 hours to cleave the target product from the solid support, yielding a mixture containing the target product. The mixture was analyzed by HPLC. The results are shown in Table 3.

[0120]

[0121] In Table 3, the stereochemical purity is Sp isomer C as determined by HPLC. ＰＢ T and Rp body C ＰＢ The HPLC yield was calculated from the area ratio (Rp)-C ＰＢ T / (T+(Rp)-C ＰＢ T).

[0122] 3(a) and 3(b) show the results of reversed-phase HPLC analysis in Comparative Synthesis Example 3 and Synthesis Example 3, respectively. Compared to Comparative Synthesis Example 3, Synthesis Example 3 resulted in a higher yield and stereochemical purity of the target product. From these results, it is believed that epimerization is significant under the condensation reaction conditions in the [N-Me,Ph]-type, whereas the stereochemical purity is maintained in the [N-Thg,Ph]-type due to sufficient steric hindrance around the nitrogen atom. Therefore, it was confirmed that the [N-Thg,Ph]-type is more suitable for performing a synthesis with controlled stereochemistry.

[0123] (HPLC elution conditions) Comparative Synthesis Example 3: 0-15% CH ３ CN in 0.1M TEAA buffer for 45 min. Synthesis Example 3: 5-25% CH ３ CN in 0.1M TEAA buffer for 20min

[0124] (T ＰＢ T ＰＢ Solid-phase synthesis of T) T was synthesized using a thymidine monomer having a tetrahydrogeranyl group at the nitrogen atom of the oxazaphospholidine ring. ＰＢ T ＰＢ Solid phase synthesis of T was performed.

[0125] Comparative Synthesis Example 4 A chain elongation reaction was carried out on 5'-hydroxythymidine (0.5 μmol) supported on a solid support via a succinyl linker by repeating the following combination of steps (i), (ii), and (iii) twice.

[0126] Step (i): [N-Me, Ph]-type oxazaphospholidine monomer 7t (30.0 mg, 80 equivalents, 40 μmol) was added to the solid support. After drying the solid support, an acetonitrile solution (200 μL) containing N-cyanomethylpyrrolidinium triflate (1.0 M) as an acidic activator was added as a reaction solvent under an argon atmosphere, and the mixture was stirred for 3 minutes to carry out condensation. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile and dichloromethane.

[0127] Step (ii): After drying the solid support, 200 μL of dichloromethane and 20 μL of dimethylsulfide borane were added under an argon atmosphere and stirred for 5 minutes to carry out boronation. The solid support was then washed with dichloromethane and methanol.

[0128] Step (iii): After step (ii), the solid support was subjected to deprotection of the dimethoxytrityl group using a 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution.

[0129] The solid support was then washed with dried dichloromethane and acetonitrile and dried. 10 mg of 4-dimethylaminopyridine, 450 μL of 2,6-lutidine, and 50 μL of acetic anhydride were added to the free 5'-hydroxyl group, and acetylation was performed for 60 seconds. The solid support was then washed with dried dichloromethane and acetonitrile and dried. A previously prepared 4% diazabicycloundecene / acetonitrile solution was added and the protecting group at the phosphate moiety was removed for 17 hours. The solid support was then washed with dried acetonitrile and dried. The solid support was reacted with 25% aqueous ammonia for 1 hour to separate it from the solid phase, yielding a mixture containing the target product. The mixture was analyzed by HPLC. The results are shown in Table 4.

[0130] Synthesis Example 4 A chain elongation reaction was carried out on 5'-hydroxythymidine (0.5 µmol) supported on a solid support via a succinyl linker by repeating the following combination of steps (i), (ii), and (iii) twice.

[0131] Step (i): [N-Thg,Ph]-type oxazaphospholidine monomer 6t (25.5 mg, 60 equivalents, 30 μmol) was added to the solid support. After drying the solid support, acetonitrile-isobutyronitrile (7:3, v / v, 200 μL) containing N-phenylimidazolium triflate (1.0 M) as an acidic activator was added as a reaction solvent under an argon atmosphere, and the mixture was stirred for 10 minutes to carry out condensation. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile and dichloromethane.

[0132] Step (ii): After drying the solid support, boronation was carried out with a 1 M dimethylsulfide borane / toluene solution for 15 minutes under an argon atmosphere, and the solid support was washed with dichloromethane and ethanol.

[0133] Step (iii): After step (ii), the solid support was subjected to deprotection of the dimethoxytrityl group using a 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution.

[0134] The solid support was then washed with dried dichloromethane and acetonitrile and dried. The free 5'-hydroxyl group was acetylated for 60 seconds by adding 10 mg of 4-dimethylaminopyridine, 450 μL of 2,6-lutidine, and 50 μL of acetic anhydride. The solid support was then washed with dried dichloromethane and acetonitrile and dried. A previously prepared 10% diazabicycloundecene / acetonitrile solution was added and the protecting group at the phosphate moiety was removed for 1 hour. The solid support was then washed with dried acetonitrile and dried. The target product was cleaved from the solid support by reaction with 25% aqueous ammonia-ethanol (3:1, v / v) for 17 hours, yielding a mixture containing the target product. The mixture was analyzed by HPLC. The results are shown in Table 4.

[0135]

[0136] In Table 4, the HPLC yield is the area ratio T ＰB T ＰB T / (T+T ＰB T+T ＰB T ＰB T).

[0137] 4(a) and 4(b) show the results of reversed-phase HPLC analysis in Comparative Synthesis Example 4 and Synthesis Example 4, respectively. In Comparative Synthesis Example 4, a large amount of dimer was observed, whereas in Synthesis Example 4, the proportion of dimer was lower than in Comparative Synthesis Example 4, and the HPLC yield of the target product was 15% higher. In the above Synthesis Example, a trimer was synthesized, but in oligomer synthesis, the number of condensations increases, so it is more desirable to use a monomer with high condensation efficiency, and it was confirmed that the [N-Thg,Ph]-type is more suitable for synthesizing nucleic acid oligomers.

[0138] (HPLC elution conditions) Comparative Synthesis Example 4: 0-25% CH ３ CN in 0.1M TEAA buffer for 70 min. Synthesis Example 4: 0-30% CH ３ CN in 0.1M TEAA buffer for 60min

[0139] (Comparative study of conditions for removing the protecting group at the phosphate moiety in a phosphotriester trimer or tetramer intermediate) Using a monomer having a Ph group on the oxazaphospholidine ring, solid-phase synthesis of an oxidized trimer or tetramer was carried out. The following [N-Me,H]-type oxazaphospholidine monomer is a known compound, the synthesis of which is described in Non-Patent Document 1.

[0140] Comparative Synthesis Example 5 A chain elongation reaction was carried out on 5'-hydroxythymidine (0.5 µmol) supported on a solid support via a succinyl linker by repeating the following combination of steps (i), (ii), and (iii) twice.

[0141] Step (i): [N-Me,H]-type oxazaphospholidine monomer 8t (13.0 mg, 40 equivalents, 20 μmol) was added to the solid support. After drying the solid support, an acetonitrile solution (200 μL) containing 1H-tetrazole (0.45 M) as an acidic activator was added as a reaction solvent under an argon atmosphere, and the mixture was stirred for 3 minutes to carry out condensation. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile and dichloromethane.

[0142] Step (ii): After drying the solid support, 200 μL of 1 M t-butyl hydroperoxide was added under an argon atmosphere and the mixture was stirred for 3 minutes to carry out oxidation. The solid support was then washed with dichloromethane.

[0143] Step (iii): After step (ii), the solid phase support was subjected to deprotection of the dimethoxytrityl group with a 3% dichloroacetic acid / dichloromethane solution.

[0144] The solid support was then washed with dried dichloromethane and acetonitrile and dried. The solid support was then reacted with 25% aqueous ammonia at room temperature for 1 hour to remove the target compound T. ＰＯ T ＰＯ A mixture containing T was obtained. The mixture was analyzed by HPLC. The results are shown in Table 5.

[0145] Comparative Synthesis Example 6 Target compound T was obtained in the same manner as in Comparative Synthesis Example 4, except that the ammonia treatment was carried out at 55° C. for 17 hours instead of at room temperature for 1 hour. ＰＯ T ＰＯ The mixture was analyzed by HPLC. The results are shown in Table 5.

[0146] Synthesis Example 5 A chain elongation reaction was carried out on 5'-hydroxythymidine (0.5 µmol) supported on a solid support via a succinyl linker by repeating the following combination of steps (i), (ii), and (iii) three times.

[0147] Step (i): [N-Thg,Ph]-type oxazaphospholidine monomer 6a, 6c, or 6 g (60 equivalents, 30 μmol) was added to the solid support. After drying the solid support, an acetonitrile-isobutyronitrile (7:3, v / v, 200 μL) solution containing N-phenylimidazolium triflate (1.0 M) as an acidic activator was added as a reaction solvent under an argon atmosphere, and the mixture was stirred for 10 minutes to carry out condensation. After condensation of the oxazaphospholidine monomer, the solid support was washed with dried acetonitrile and dichloromethane.

[0148] Step (ii): After drying the solid support, 200 μL of 1 M t-butyl hydroperoxide was added under an argon atmosphere and the mixture was stirred for 5 minutes to carry out oxidation. The solid support was then washed with dichloromethane.

[0149] Step (iii): After step (ii), the solid phase support was subjected to deprotection of the dimethoxytrityl group with a 3% dichloroacetic acid / dichloromethane solution.

[0150] The solid support was then washed with dried dichloromethane and acetonitrile and dried. The solid support was then reacted with 25% aqueous ammonia-ethanol (3:1, v / v) at room temperature for 3 hours to remove the fragment from the solid support and deprotect the phosphoric acid moiety, yielding target compound A. ＰＯ G ＰＯ C ＰＯ A mixture containing T was obtained. The mixture was analyzed by HPLC. The results are shown in Table 5.

[0151]

[0152] The percentages in Table 5 indicate the progress rate of phosphodiester deprotection, and were calculated from the area ratio in the HPLC analysis results: phosphodiester derivative (completely deprotected after the deprotection reaction) / (phosphodiester derivative (incompletely deprotected before the deprotection reaction)+phosphodiester derivative (completely deprotected after the deprotection reaction)).

[0153] 5(a) to 5(c) show the results of reverse-phase HPLC analysis of Comparative Synthesis Examples 5 and 6 and Synthesis Example 5. In Comparative Synthesis Example 5, deprotection proceeded by only 4% after 1 hour of ammonia treatment, suggesting that the deprotection reaction was extremely slow. As shown in Comparative Synthesis Example 6, to achieve a 100% progress rate in deprotection of the phosphodiester, it was necessary to increase the reaction temperature to 55°C and extend the reaction time to 12 hours. On the other hand, in Synthesis Example 5, deprotection was completed in 3 hours at room temperature, indicating that the reaction proceeded rapidly under mild conditions. In the above Synthesis Examples, trimers and tetramers were synthesized. However, during oligomer synthesis, the number of protecting groups at the phosphate moieties increases, so it is desirable that the protecting groups at the phosphate moieties be easily deprotected. It was confirmed that the [N-Thg,Ph]-type monomer used in Synthesis Example 5 is more suitable for synthesizing nucleic acid oligomers.

[0154] (HPLC elution conditions) Comparative Synthesis Examples 5 and 6 and Synthesis Example 5: 0-20% CH ３ CN in 0.1M TEAA buffer for 60min

[0155] Example 1 Synthesis of PB Nucleic Acid Oligomer (Tetromer) and PB / PO Chimeric Nucleic Acid Oligomer (Tetromer) 5'-O-Dimethoxytritylthymidine (0.5 μmol) supported on a solid support via a succinyl linker was detritylated by adding a 3% dichloroacetic acid / dichloromethane solution (5 times x 12 s, 1 mL / time). The solid support was then washed with acetonitrile and dichloromethane that had been dried using molecular sieves, and vacuum dried. Next, a chain elongation reaction of the oligomer was carried out by repeating the following combination of steps (i), (ii), and (iii) three times.

[0156] Step (i): After adding the oxazaphospholidine monomer (60 equivalents, 30 μmol) selected according to the sequence to the solid support and drying it for 5 minutes, an acetonitrile-isobutyronitrile (7:3, v / v, 150 μL) solution containing 1-phenylimidazolium triflate (1.0 M) as an acidic activator was added as a reaction solvent under an argon atmosphere, and the mixture was stirred with a vortex mixer for 10 minutes to carry out condensation. After condensation, the solid support was washed with dried acetonitrile (1 mL x 3 times) and dichloromethane (1 mL x 3 times).

[0157] Step (ii): A 0.05 M borane / tetrahydrofuran complex solution in tetrahydrofuran or a 1.0 M t-butyltetrahydroperoxide / toluene solution, selected depending on the modification of the phosphoric acid site, was reacted for 2 or 5 minutes under an argon atmosphere. The reaction solution was then removed, and the solid support after the reaction was washed as follows, depending on the reaction performed. For boronation, the solid support was washed with dried tetrahydrofuran (1 mL x 3 times), dried ethanol (1 mL x 3 times), and dichloromethane (1 mL x 3 times). For oxidation, the solid support was washed with dichloromethane (1 mL x 6 times).

[0158] Step (iii): The solid support after step (ii) was reacted with 1 mL of a 3% dichloroacetic acid / dichloromethane-triethylsilane (1:1, v / v) solution for 12 seconds. After repeating the same procedure five times, the solid support was washed three times with 1 mL of dichloromethane and three times with acetonitrile, and then vacuum-dried.

[0159] Step (IV): The solid support was then washed with dried dichloromethane (1 mL x 3 times) and acetonitrile (1 mL x 3 times) and dried for 5 minutes. The free 5'-hydroxyl group was acetylated for 1 minute by adding 10 mg of 4-dimethylaminopyridine, 450 μL of 2,6-lutidine, and 50 μL of acetic anhydride. The solid support was washed with dried dichloromethane (1 mL x 3 times) and acetonitrile (1 mL x 3 times) and dried for 5 minutes. A previously prepared 10% diazabicycloundecene / acetonitrile solution was added and the protecting groups at the phosphate moieties were removed for 60 minutes, resulting in conversion to boranophosphodiester or phosphodiester bonds. The solid support was then washed with acetonitrile (1 mL x 3 times).

[0160] The washed solid phase carrier was treated with 25% aqueous ammonia-ethanol (3:1, v / v) for 3 hours at room temperature and then for 17 hours at 50°C to deprotect the nucleic acid base moiety, cleave the coordinate bond between the nitrogen atom of the nucleic acid base moiety and borane, and excise the nucleic acid oligomer from the solid phase carrier. The solid phase carrier was filtered, the filtrate was recovered, and the solvent was evaporated under reduced pressure.

[0161] After distilling off the solvent under reduced pressure, the crude product was analyzed by reversed-phase HPLC. Reverse-phase HPLC was carried out in 0.1 M TEAA buffer (pH 7) using a linear gradient of 0 to 60% acetonitrile for 60 minutes at 30°C and a flow rate of 0.5 mL / min. Thus, PB nucleic acid oligomer C ＰＢ A ＰＢ G ＰＢ T and PB / PO chimeric nucleic acid oligomer C ＰＢ A ＰＯ G ＰＢ I got a T.

[0162] PB nucleic acid oligomer C ＰＢ A ＰＢ G ＰＢ Yield: 28% HRMS (ESI-TOF): Calcd for [M-2H] ２－ , 582.6729; found, 582.6742 ・PB / PO chimeric nucleic acid C ＰＢ A ＰＯ G ＰＢYield: 33% HRMS (ESI-TOF): Calcd for [M-2H] ２－ ,583.6540;found,583.6559

[0163] [Example 2] Synthesis of PB / PO chimeric nucleic acid oligomer (12-mer) An oligomer was synthesized under the same conditions as in [Example 1], except that the combination of steps (i), (ii), and (iii) was repeated 11 times.

[0164] The resulting PB / PO chimeric nucleic acid oligomer (12-mer) was separated and purified by reversed-phase HPLC. Reverse-phase HPLC and separation and purification were carried out at 60°C for 20 minutes at a flow rate of 0.5 mL / min using a 0.1 M hexafluoroisopropanol-0.008 M triethylamine buffer solution containing 5 to 40% methanol as the mobile phase. In this way, PB / PO chimeric nucleic acid oligomer C ＰＢ A ＰＯ G ＰＢ T ＰＯ C ＰＢ A ＰＯ G ＰＢ T ＰＯ C ＰＢ A ＰＯ G ＰＢ T (SEQ ID NO: 1) was obtained. Yield: 3% HRMS (ESI-TOF): Calcd for [M-4H] ４－ ,907.2133;found,907.2129

[0165] [Example 3] Synthesis of PO nucleic acid oligomer (12-mer) An oligomer was synthesized under the same conditions as in [Example 1], except that the combination of steps (i), (ii), and (iii) was repeated 11 times, and step (IV) was not performed.

[0166] The resulting PO nucleic acid oligomer (12-mer) was separated and purified by reversed-phase HPLC. Reverse-phase HPLC and separation and purification were carried out at 50°C for 10 minutes at a flow rate of 0.5 mL / min using a 0.4 M hexafluoroisopropanol-0.016 M triethylamine buffer solution of 5 to 25% methanol as the mobile phase. In this way, PO nucleic acid oligomer C ＰＯ A ＰＯ G ＰＯ T ＰＯ CＰＯ A ＰＯ G ＰＯ T ＰＯ C ＰＯ A ＰＯ G ＰＯ T (SEQ ID NO: 2) was obtained. Yield: 14% HRMS (ESI-TOF): Calcd for [M-4H] ４－ ,910.1530;found,910.1565

Claims

1. A nucleotide monomer represented by the following general formula (1). 【Chemistry 1】 (In general formula (1), R １ represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy 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 having 5 or more members with or without a substituent and with or without a heteroatom, R ３ represents a hydrogen atom or a protecting group for a hydroxyl group, R ４ represents an aryl group with or without a substituent, R ５ represents a hydrogen atom, a halogenyl group, or an alkyl group with or without a substituent, R ６ represents an alkyl group having 6 to 14 carbon atoms with or without a substituent and with or without a heteroatom in the molecular chain, B ＮＨ２ represents a nucleobase selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the amino groups in the adenine, the guanine, the cytosine, and the 5-methylcytosine are unsubstituted.)

2. The aforementioned R ６ The alkyl group represented by is branched, as per claim 1, the nucleotide monomer.

3. R １ R represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group. ２ represents a hydrogen atom, or R １ and R ２ The nucleotide monomer according to claim 1, wherein the elements combine with each other to form a divalent group represented by the following general formula (2). 【Chemistry 2】 (In general formula (2), R ７ and R ８ Each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or they are bonded to each other to form a ring, and * and ** represent bonds, and the bond represented by * is R in general formula (1). １ The bond formed by ** which is directly connected to the carbon atom, is R in general formula (1). ２ It bonds to the carbon atom it is directly connected to.

4. The nucleotide monomer according to any one of claims 1 to 3, wherein the stereochemistry of the phosphorus atom in the nucleotide monomer is controlled.

5. A condensation step to obtain a first precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (3a) by condensing nucleotide monomers represented by the following general formula (1a), A boranoization step is to boranoize the first precursor nucleic acid oligomer with a boranoizing agent to obtain a second precursor nucleic acid oligomer containing nucleotide units represented by the following general formula (4a), or An oxidation step is performed to oxidize the first precursor nucleic acid oligomer with an oxidizing agent to obtain a third precursor nucleic acid oligomer containing nucleotide units represented by the following general formula (5a): From the second precursor nucleic acid oligomer or the third precursor nucleic acid oligomer, R ３ａ A deprotection step to remove the protecting group represented by the following general formula (4b) to obtain a fourth precursor nucleic acid oligomer containing a nucleotide unit represented by the following general formula (5b), A chain lengthening process in which the combination of the following is performed once or repeatedly two or more times, A nucleic acid oligomer production process to obtain a nucleic acid oligomer containing at least one selected from the group consisting of a nucleotide unit represented by general formula (7) and a nucleotide unit represented by general formula (8) by removing the phosphate protecting group represented by general formula (6) from a sixth precursor nucleic acid oligomer containing a nucleotide unit represented by general formula (4c) or a seventh precursor nucleic acid oligomer containing a nucleotide unit represented by general formula (5c) from the fourth precursor nucleic acid oligomer or the fifth precursor nucleic acid oligomer, with or without capping the 5'-hydroxyl group and amino group with a protecting group, and further removing the protecting group if capping is performed. A method for producing nucleic acid oligomers containing [specific ingredient / component]. 【Transformation 3】 (In general formula (1a), R １ R represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group. ２ represents a hydrogen atom, or R １ and R ２ These elements are bonded to each other to form a ring of five or more members, which may or may not have substituents and may or may not have heteroatoms, R ３ａ R represents a protecting group for hydroxyl groups. ４ R represents an aryl group with or without substituents, ５ R represents a hydrogen atom, a halogenyl group, or an alkyl group with or without a substituent. ６ B represents an alkyl group having or not having substituents, and having or not having heteroatoms in the molecular chain, with 6 to 14 carbon atoms. ＮＨ２ (wherein is a nucleic acid base selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the amino groups in adenine, guanine, cytosine, and 5-methylcytosine are not substituted.) 【Chemistry 4】 (In general formula (3a), R １ , R ２ , R ３ａ , R ４ ~R ６ , and B ＮＨ２ (As stated above.) 【Transformation 5】 (In general formulas (4a), (5a), (4b), and (5b), R １ , R ２ , R ３ａ , R ４ ~R ６ , and B ＮＨ２ (As stated above.) 【Transformation 6】 (In general formulas (4c) and (5c), R １ , R ２ , and R ４ ~R ６ As stated above, When the aforementioned capping is performed, R ９ R represents a protecting group for hydroxyl groups. １０ represents an amino group protecting group, Bs represents a nucleic acid base selected from the group consisting of adenine, guanine, cytosine, 5-methylcytosine, thymine, and uracil, and the amino groups in adenine, guanine, cytosine, and 5-methylcytosine are substituted with amino group protecting groups. If the aforementioned capping is not performed, R ９ and R １０ represents a hydrogen atom, and Bs is B ＮＨ２ (This represents...) 【Transformation 7】 (In general formula (6), R ４ ~R ６ and R １０ (As stated above.) 【Transformation 8】 (In general formulas (7) and (8), R １ , R ２ , and B ＮＨ２ As stated above, X ＋ (This represents a counter-cation.)

6. The aforementioned R ６ The manufacturing method according to claim 5, wherein the alkyl group represented is branched.

7. R １ R represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkenyloxy group, an acyloxy group, a trihydrocarbylsilyloxy group, an alkoxyalkoxy group, a haloalkoxyalkoxy group, or a halogenyl group. ２ represents a hydrogen atom, or R １ and R ２ The manufacturing method according to claim 5, wherein the two elements combine with each other to form a divalent group represented by the following general formula (2). 【Chemistry 9】 (In general formula (2), R ７ and R ８ Each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or they are bonded to each other to form a ring, and * and ** represent bonds, and the bond represented by * is R in general formula (1a). １ The bond formed by **, which is directly connected to the carbon atom, is R in general formula (1a). ２ It bonds to the carbon atom it is directly connected to.

8. The manufacturing method according to any one of claims 5 to 7, wherein the stereochemistry of the phosphorus atom in the nucleotide monomer and the nucleotide unit is controlled.

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