Amidite monomer

Abstract

To provide a technique for incorporating a compound having a colchicine-like structure into a nucleic acid.SOLUTION: A compound represented by the general formula (1): wherein R1 represents an alkoxy group or a phosphoric acid group. R2 represents an alkoxy group or a phosphoric acid group; R3 represents -N = or -CH =. R4 represents = N - or = CH -. R5 represents a hydroxy-protecting group. R6 and R7 are identical or different and each represent an alkyl group. M represents an integer of 1 to 5. N represents an integer of 1 to 4. Or a salt thereof, or a solvate thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an amidite monomer and the like. [Background technology]

[0002] Various anticancer drugs and their prodrugs with colchicine-like structures have been reported (e.g., Non-Patent Document 1). Like colchicine, these anticancer drugs specifically bind to tubulin and inhibit the formation of microtubules, thereby suppressing cell division of cancer cells. Therefore, there is a need for the development of drug delivery technologies, particularly those that can transport drugs specifically to cancer cells.

[0003] Generally, drug delivery technologies reported include liposomes, polymeric nanoparticles, inorganic nanoparticles, dendrimers, micelles, nanoemulsions, and polymer drug conjugates. However, some of these technologies lack uniformity in shape, have suboptimal biocompatibility, and often lack selectivity for diseased cells. Therefore, the development of new drug delivery technologies is desired. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Borowiak et al., 2015, Cell, 162, 403-411. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have focused on nucleic acid structures such as DNA origami as a drug delivery technology. DNA origami has the inherent properties of biocompatibility, biodegradability, and the ability to control the site of drug uptake.

[0006] An objective of the present invention is to provide a technique for incorporating a compound having a colchicine-like structure into a nucleic acid. [Means for solving the problem]

[0007] In view of the above problems, the present inventors have conducted extensive research and have found that an amido group represented by general formula (1) The present inventors have found that by using a colchicine-like monomer, it is possible to obtain a nucleotide or polynucleotide having an anticancer activity and a nucleic acid construct containing the same. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Section 1. General formula (1):

[0009] [ka]

[0010] [In the formula: R 1 are the same or different and represent an alkoxy group or a phosphate group. 2 are the same or different R represents an alkoxy group or a phosphate group. 3 represents -N= or -CH=. 4 represents =N- or =CH-. 5 represents a protecting group for a hydroxy group. 6 and R 7 are the same or different and represent an alkyl group. m represents an integer of 1 to 5. n represents an integer of 1 to 4.] A compound represented by the formula (I), a salt thereof, or a solvate thereof.

[0011] Item 2. The compound represented by general formula (1A):

[0012] [ka]

[0013] [In the formula: R1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is the same as above.] Item 2. The compound according to Item 1, or a salt thereof, or a solvate thereof, which is a compound represented by the formula:

[0014] Section 3.R 1 and R 2 Item 3. The compound or salt thereof, or a solvate thereof according to Item 1 or 2, wherein is an alkoxy group.

[0015] Section 4.R 5 is an alkyl group substituted with an electron-withdrawing group, and R 6 and R 7 Item 4. The compound or salt thereof, or a solvate of the compound or salt according to any one of Items 1 to 3, wherein is a branched alkyl group.

[0016] Item 5. The terminal phosphate group is represented by the general formula (2):

[0017] [ka]

[0018] [In the formula: R 1 are the same or different and represent an alkoxy group or a phosphate group. 2 are the same or different and represent an alkoxy group or a phosphate group. 3 represents -N= or -CH=. 4 represents =N- or =CH-. 8 represents O or S. R 9 O - or S - m represents an integer of 1 to 5. n represents an integer of 1 to 4.] A nucleotide or polynucleotide in which the nucleotide or polynucleotide is replaced with a group represented by the following formula:

[0019] Section 6. General formula (2A):

[0020] [ka]

[0021] [In the formula: R 1 , R 2 , R 3 , R 4 , R 8 , R 9 , m, and n are the same as above. 8A are the same or different It indicates O or S. 9A are the same or different, O - or S - Indicates R 10 R may be the same or different and represent a divalent group formed by removing two hydroxy groups from the sugar moiety of a nucleoside. 10A R may be the same or different and represent a divalent group formed by removing two hydroxy groups from the sugar moiety of a nucleoside. 11 represents a hydroxy group, a phosphate group, or a thiophosphate group, and p represents an integer of 0 or 1 or more. ] Item 6. The nucleotide or polynucleotide according to Item 5, which is a compound represented by the formula:

[0022] Item 7. A nucleic acid construct comprising the nucleotide or polynucleotide according to Item 5 or 6.

[0023] Item 8. The nucleic acid structure according to Item 7, which is a DNA origami.

[0024] Item 9. A pharmaceutical comprising at least one selected from the group consisting of the nucleotide or polynucleotide according to Item 5 or 6 and the nucleic acid construct according to Item 7 or 8.

[0025] Item 10. The pharmaceutical agent according to Item 9, which is an anticancer agent.

[0026] Item 11. A reagent comprising at least one selected from the group consisting of the nucleotide or polynucleotide according to Item 5 or 6 and the nucleic acid structure according to Item 7 or 8. [Effects of the Invention]

[0027] According to the present invention, it is possible to provide an amidite monomer of a compound having a colchicine-like structure, a nucleotide or polynucleotide obtained using the amidite monomer, and various acid structures containing the nucleotide or polynucleotide. [Brief explanation of the drawings]

[0028] [Figure 1] The 1H NMR [CDCl3] spectrum and synthesis scheme of compound 1 are shown below. [Figure 2] The 1H NMR [CDCl3] spectrum and synthesis scheme of compound 2 are shown below. [Figure 3] The 1H NMR [CDCl3] spectrum and synthesis scheme of compound 3 are shown below. [Figure 4] The 1H NMR [CDCl3] spectrum and synthesis scheme of Photostatin are shown below. [Figure 5] 1 shows a mass spectrum of a PST-amidite monomer. [Figure 6] Mass spectra of PST-A, PST-G, PST-C, PST-T, and PST-T9 are shown. [Figure 7] The results of HPLC purification of PST-T and the mass spectrum of the collected peak are shown. [Figure 8] The results of HPLC purification of PST-T9 are shown. [Figure 9] The mass spectrum of the collected peak of PST-T9 is shown. [Figure 10] 1 shows the results of HPLC measurement evaluation of cis-trans isomerization in Example 3. [Figure 11] 1 shows the results of UV measurement evaluation of cis-trans isomerization in Example 4. [Figure 12] 1 shows the results of UV measurement (time course) evaluation of cis-trans isomerization in Example 5. [Figure 13] 1 shows the results of Tm measurement in Example 6. [Figure 14] 1 shows the results of thermal stability evaluation of Example 7. [Figure 15] 1 shows the results of the anticancer activity test in Example 8. [Figure 16] 1 shows an overall view of the DNA origami dendrimer prepared in Example 10. [Figure 17] 1 shows an enlarged view of a portion of the tip of the DNA origami dendrimer prepared in Example 10. [Figure 18] An enlarged view (left) of the tip of a portion of the DNA origami dendrimer prepared in Example 10 and an enlarged view (right) of the tip of a portion of the PST-modified DNA origami dendrimer are shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0030] 1. Amidite Monomers In one aspect, the present invention provides a compound represented by general formula (1):

[0031] [ka]

[0032] or a salt thereof, or a solvate thereof (these may be collectively referred to as the "amidite monomer of the present invention" in this specification). This will be explained below.

[0033] R 1 are the same or different and represent an alkoxy group or a phosphate group. 1 is preferably an alkoxy group.

[0034] R 1The alkoxy group represented by the formula (I) includes both straight-chain and branched-chain (preferably straight-chain) alkoxy groups. The number of carbon atoms in the alkoxy group is not particularly limited, but is, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 2, and particularly preferably 1. Specific examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, and an isopropyl group. Examples of the alkyl group include an epoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, and a tert-butoxy group.

[0035] R 1 The phosphate group represented by is a group represented by H2PO4-.

[0036] m represents an integer of 1 to 5. m preferably represents an integer of 1 to 4, more preferably an integer of 2 to 4, still more preferably an integer of 2 to 3 or an integer of 3 to 4, and particularly preferably 3.

[0037] R 1 The position of R 3 For example, it can be in the para position, meta position, or ortho position relative to R 1 The position of R is preferably the para position or the meta position. 1 When there are multiple Rs, Rs at the para and / or meta positions 1 and preferably includes R 1 It is more preferred that the composition contains:

[0038] R 2 are the same or different and represent an alkoxy group or a phosphate group. 2 is preferably an alkoxy group.

[0039] R 2 The definition of the alkoxy group represented by R 1 The definition of the alkoxy group is the same as that of the alkoxy group represented by R 2 The definition of the phosphate group is R 1 This is the same as the definition of the phosphate group shown below.

[0040] n represents an integer of 1 to 4. n is preferably an integer of 1 to 3, more preferably an integer of 1 or 2, and even more preferably 1.

[0041] R 2 The position of R 4 For example, it can be in the para position, meta position, or ortho position relative to R 1 The position of is preferably the para position or the meta position, more preferably the para position.

[0042] In one preferred embodiment of the present invention, R 1 and R 2 can be an alkoxy group.

[0043] R 1 and R 2 In one embodiment of the present invention, the number (m, n) and position of The compound to be treated is particularly preferably represented by the general formula (1A):

[0044] [ka]

[0045] The compound may be represented by the formula:

[0046] R 3 represents -N= or -CH=. 3 is preferably -N=.

[0047] R 4 represents =N- or =CH-. 4 is preferably =N-.

[0048] In one preferred embodiment of the present invention, R 3 is -N= and R 4 is =N-, or R 3 is -CH= and R 4 can be =CH-.

[0049] R 5indicates a protecting group for a hydroxy group.

[0050] R 5 As the protecting group for the hydroxy group represented by R, a wide range of known protecting groups used in amidite monomers can be used. 5 Examples of the alkyl group include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a haloalkyl group, an aryl group, a heteroaryl group, an arylalkyl group, a cycloalkenyl group, a cycloalkylalkyl group, a cyclylalkyl group, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a heterocyclylalkenyl group, a heterocyclylalkyl group, a heteroarylalkyl group, a silyl group, a silyloxyalkyl group, a mono-, di-, or trialkylsilyl group, and a mono-, di-, or trialkylsilyloxyalkyl group, which may be substituted with an electron-withdrawing group.

[0051] R 5 R is preferably an alkyl group substituted with an electron-withdrawing group. Examples of the electron-withdrawing group include a cyano group, a nitro group, an alkylsulfonyl group, a halogen atom, an arylsulfonyl group, a trihalomethyl group, and a trialkylamino group, and is preferably a cyano group. 5 is particularly preferably —(CH 2 ) 2 —CN.

[0052] R 6 and R 7 are the same or different and represent alkyl groups.

[0053] R 6 and R 7 The alkyl group represented by the formula (I) may be either a straight-chain or branched-chain alkyl group, and is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl. Alkyl groups also include alkyl moieties such as alkoxy groups. 6 and R7 may be bonded to each other to form a cyclic structure.

[0054] R 6 and R 7 and are particularly preferably both isopropyl groups.

[0055] The compound represented by general formula (1) is R 3 =R 4 The double bond of That is, the compound represented by general formula (1) is a compound represented by general formula (11):

[0056] [ka]

[0057] or a compound represented by general formula (12):

[0058] [ka]

[0059] The compound may be represented by the formula:

[0060] The cis- and trans-isomers can be converted to the other isomer by irradiation with light. The conversion can be carried out according to known methods (for example, according to the method described in Non-Patent Document 1).

[0061] The salt of the compound represented by general formula (1) is not particularly limited, and examples thereof include sodium salts. Examples of the salt include salts with inorganic bases such as methylamine, ethylamine, ethanolamine, etc.; salts with basic amino acids such as lysine, ornithine, arginine, etc., and ammonium salts. The salt may be an acid addition salt, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, etc.; and acidic amino acids such as aspartic acid, glutamic acid, etc.

[0062] The solvate of the compound represented by formula (1) or a salt thereof is not particularly limited, and examples thereof include Examples include solvates with solvents such as water, ethanol, glycerol, and acetic acid.

[0063] The amidite monomers of the present invention can be prepared in a variety of ways.

[0064] The compound represented by the general formula (1) can be produced, for example, according to the following scheme or a similar scheme. It is possible.

[0065] [ka]

[0066] X is a halogen atom. Examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Among these, a halogen atom is preferred.

[0067] As the compound represented by the general formula (101) and the compound represented by the general formula (102), commercially available compounds can be used as they are, or compounds synthesized according to or in accordance with known methods can be used as needed.

[0068] The amount of the compound represented by the general formula (102) to be used is usually determined from the viewpoint of yield, ease of synthesis, etc. The amount is preferably 1 to 3 moles, more preferably 1.5 to 2.5 moles, per mole of the compound represented by the general formula (101).

[0069] This reaction is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, but examples thereof include acetonitrile, pyridine, dimethylformamide, dichloromethane, tetrahydrofuran, acetone, toluene, ethanol, etc., and preferably acetonitrile, etc. The solvent may be used alone or in combination.

[0070] This reaction is preferably carried out in the presence of a base (preferably a non-nucleophilic base) such as N,N-diisopropylethylamine. From the viewpoints of yield, ease of synthesis, etc., the amount of the base used is usually preferably 3 to 15 moles, more preferably 4 to 10 moles, per mole of the compound represented by general formula (101).

[0071] In addition to the above components, additives may also be used in this reaction as appropriate, provided that they do not significantly impair the progress of the reaction.

[0072] The reaction can be carried out under heating, at room temperature, or under cooling. The reaction time is not particularly limited, and can usually be 30 minutes to 30 hours.

[0073] The progress of the reaction can be monitored by conventional methods such as chromatography. After the reaction is complete, the solvent is removed by distillation, and the product can be isolated and purified by conventional methods such as chromatography and recrystallization. The structure of the product can also be determined by elemental analysis, MS (ESI-MS), IR analysis, and the like. 1 H-NMR, 13 It can be identified by C-NMR or the like.

[0074] 2. Nucleotides or polynucleotides In one aspect, the present invention provides a polymer having a terminal phosphate group represented by general formula (2):

[0075] [ka]

[0076] This is described below.

[0077] R 1 , R 2 , R 3 , R 4 The definitions of m and n are as explained above in "1. Amidite Monomer." It is.

[0078] R 8 represents O or S. R 8 is preferably O.

[0079] R 9 O - or S - Indicates R 9 is preferably O - is.

[0080] The (poly)nucleotide of the present invention has a structure in which the terminal phosphate group of a nucleotide or polynucleotide is replaced with a group represented by general formula (2).

[0081] The terminal phosphate group may be either a 5'-terminal phosphate group or a 3'-terminal phosphate group, but is preferably a 5'-terminal phosphate group.

[0082] The nucleotides and polynucleotides are not particularly limited, and for example, natural nucleic acids and various artificial nucleic acid building blocks including aTNA and SNA can be used. Specifically, in addition to DNA, RNA, etc., known chemical modifications may also be applied, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphoric acid residue (phosphate) of each nucleotide can be substituted with a chemically modified phosphoric acid residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. In addition, the hydroxy group at the 2'-position of the sugar (ribose) of each ribonucleotide can be substituted with -OR (where R is, for example, CH3(2 The base moiety (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Furthermore, the phosphate moiety or hydroxyl moiety may be modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, or the like, but is not limited to these. Furthermore, by bridging the 2' oxygen and 4' carbon of the sugar moiety of a nucleotide, the conformation of the sugar moiety can be changed to N- Fixed-type BNA (LNA) and the like can also be used.

[0083] The (poly)nucleotide of the present invention more particularly preferably has the general formula (2A):

[0084] [ka]

[0085] It is a compound represented by the formula:

[0086] R 8A are the same or different and represent O or S. R 8 A is preferably O.

[0087] R 9A are the same or different, O - or S - Indicates R 9 Ais preferably O - is.

[0088] R 10 Nucleosides (sugar moieties such as ribose and deoxyribose) and nucleic acid salts may be the same or different. It refers to a divalent group formed by removing two hydroxy groups from the sugar moiety of a nucleoside (a nucleoside consisting of a nucleotide group). One hydroxy group is the 5' hydroxy group of ribose or deoxyribose or the corresponding hydroxy group in another nucleic acid backbone, and the other hydroxy group is the 5' hydroxy group of ribose or deoxyribose. The 3' hydroxyl group of ribose or the corresponding hydroxyl group of other sugar moieties preferable.

[0089] R 10A are the same or different divalent groups formed by removing two hydroxy groups from the sugar moiety of a nucleoside (a nucleoside consisting of a sugar moiety such as ribose or deoxyribose and a nucleic acid base). One hydroxy group is the 5' hydroxy group of ribose or deoxyribose or the corresponding hydroxy group in another nucleic acid backbone, and the other hydroxy group is the 5' hydroxy group of ribose or deoxyribose. The 3' hydroxyl group of ribose or the corresponding hydroxyl group of other sugar moieties preferable.

[0090] The sugar moiety of a nucleoside is not particularly limited as long as it can constitute a nucleic acid, and examples include sugar moieties such as ribose, deoxyribose, and modified sugars (e.g., sugar moieties that constitute DNA, RNA, BNA, LNA, etc.).

[0091] As the nucleic acid base of the nucleoside, any base constituting a nucleic acid can be used without any particular limitation. The base constituting a nucleic acid includes not only typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also the bases constituting a nucleic acid. Other bases, such as hypoxanthine (I) and modified bases, are also included. Examples of uracils include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl Uracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methyl cytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine Anine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5- Methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil , 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine , purine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, etc.

[0092] R 11 represents a hydroxy group, a phosphate group, or a thiophosphate group.

[0093] p represents 0 or an integer of 1 or more. p is preferably 1 to 100,000, more preferably 5 to 100,000, even more preferably 10 to 100,000, still more preferably 15 to 100,000, and particularly preferably 20 to 100,000. The upper and / or lower limits of p can be, for example, 50, 100, 500, 1,000, 5,000, 10,000, or 50,000.

[0094] The (poly)nucleotide of the present invention may be in the form of a salt. The salt is not particularly limited. Examples of the salt include salts with inorganic bases such as sodium salts, magnesium salts, potassium salts, calcium salts, and aluminum salts; salts with organic bases such as methylamine, ethylamine, and ethanolamine; salts with basic amino acids such as lysine, ornithine, and arginine; and ammonium salts. The salt may be an acid addition salt, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0095] The (poly)nucleotide of the present invention may be in the form of a solvate. The solvate is not particularly limited, and examples thereof include solvates with solvents such as water, ethanol, glycerol, and acetic acid.

[0096] The (poly)nucleotide of the present invention can be produced by a method for producing a single-stranded polynucleotide by the phosphoramidite method, using the amidite monomer of the present invention.

[0097] The phosphoramidite method can be carried out according to a known method using, for example, a commercially available automated nucleic acid synthesizer, etc. Specifically, the method comprises the steps of: (A) deprotecting the hydroxyl group at the 5'-position (or a position corresponding thereto); (B) condensing the amidite monomer; (C) capping the hydroxyl group at the 5'-position (or a position corresponding thereto) of the unreacted compound; (D) converting the phosphite group to a phosphate group or a thiophosphate group; (E) cleaving the resulting compound from the solid support and deprotecting the phosphate moiety and nucleic acid base; and (F) deprotecting the hydroxyl group at the 5'-position (or a position corresponding thereto). By repeating steps (A) to (D), a compound containing a polynucleotide backbone of a desired chain length can be produced. By using the amidite monomer of the present invention in step (B), The (poly)nucleotides of the present invention can be produced.

[0098] In step (D), it is preferable to use a solution containing iodine / water as the oxidizing agent.

[0099] In the deprotection step (E), deprotection is carried out using a base such as aqueous ammonia. Although the bond between the phosphorus atom and the aromatic hydroxy group in the (poly)nucleotide of the present invention was expected to be unstable to bases, unexpectedly, cleavage of the above bond was suppressed during deprotection in the (poly)nucleotide of the present invention.

[0100] After step (E), before step (F), chloroform is formed by utilizing the hydrophobicity of the 5'-position protecting group (hydrophobic group). It is preferable to perform chromatography purification (for example, reverse phase chromatography purification), which can further increase the purity of the single-stranded polynucleotide of interest.

[0101] The resulting single-stranded polynucleotide may be isolated and purified as necessary. The RNA can be isolated by precipitation, extraction, and purification. Specifically, the reaction solution is added to a solvent with low solubility for RNA, such as ethanol or isopropyl alcohol. RNA can be precipitated by adding phenol / chloroform / isoamyl alcohol. A solution of ethanol is added to the reaction solution, and the RNA is extracted into the aqueous layer. The compound can be isolated and purified by known high performance liquid chromatography (HPLC) techniques such as column chromatography, anion exchange column chromatography, and affinity column chromatography.

[0102] 3.Nucleic acid construct In one aspect, the present invention provides a nucleic acid construct ( In this specification, this may be referred to as the "nucleic acid construct of the present invention."), which will be explained below.

[0103] The nucleic acid construct is not particularly limited as long as it is a construct mainly containing nucleic acids. The content of nucleic acids in the nucleic acid construct is, for example, 70% by mass or more, preferably 80% by mass or more, more preferably 90% by mass or more, even more preferably 95% by mass or more, still more preferably 98% by mass or more, particularly preferably 99% by mass or more, and particularly preferably 100% by mass. The substance is not particularly limited, but examples thereof include proteins, peptides, sugars, labeling substances, metal ions, and the like.

[0104] The nucleic acid structure can be a three-dimensional structure, a two-dimensional structure (planar structure), or a structure that is a combination of these.

[0105] The nucleic acid construct is preferably a structure formed by folding nucleic acid. From the viewpoint of suitability for drug delivery, the nucleic acid construct is a structure (DNA origami) formed from a material containing a single-stranded circular nucleic acid and a staple nucleic acid containing a complementary sequence to the single-stranded circular nucleic acid. It is preferable that:

[0106] The single-stranded circular nucleic acid that can constitute the nucleic acid construct is not particularly limited as long as it can be folded to form a DNA origami. Even if the number of bases in the single-stranded circular nucleic acid is not particularly limited, it can be formed into a DNA origami by designing the staple DNA sequence accordingly. The number of bases in the single-stranded circular nucleic acid is, for example, 500 to 50,000, preferably 1,000 to 25,000, more preferably 2,000 to 15,000, even more preferably 3,000 to 12,000, and still more preferably 4,000 to 10,000. The base sequence of the single-stranded circular nucleic acid is, for example, not particularly limited, and may include, for example, an M13 bacteriophage DNA sequence or a sequence derived from the DNA sequence. Examples include base sequences.

[0107] The staple nucleic acid that can constitute the nucleic acid structure is a nucleic acid having a complementary sequence to the single-stranded circular nucleic acid. The single-stranded circular nucleic acid is folded by complementary base pairing with the single-stranded circular nucleic acid to form a DNA origami. The number of bases in the staple nucleic acid is not particularly limited, but is, for example, 10 to 500, preferably 20 to 250, and more preferably 30 to 100. In stapled nucleic acids, the number of bases in the complementary sequence to the single-stranded circular nucleic acid is not particularly limited. The number of staple nucleic acids is not limited to 10 to 500, but is, for example, 10 to 500, preferably 20 to 250, and more preferably 30 to 100. One staple nucleic acid is usually composed of multiple ( For example, the staple nucleic acid contains complementary sequences to each of the strands (2 to 5, preferably 2 to 4, more preferably 2 to 3). The staple nucleic acid having such sequence characteristics can form complementary base pairs with the single-stranded circular nucleic acid. By this synthesis, the single-stranded circular nucleic acid can be more stabilized in the folded state. Various DNA origami methods have already been reported, and they can be used to form nucleic acid structures with desired shapes and structures. The sequence of the staple nucleic acid for this purpose can be designed according to known methods and information.

[0108] The size of the nucleic acid construct is not particularly limited and can be adjusted appropriately depending on the purpose. The nucleic acid construct is preferably nano-sized (nanostructure). For example, the longest diameter (major axis) of the nucleic acid construct is 1000 nm or less. In one embodiment of the present invention, the upper and / or lower limits of the major axis of the nucleic acid construct are, for example, 5 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, or 800 nm. By making the nucleic acid construct of the present invention larger than a certain size, it can be selectively delivered to cancer tissues based on the EPR (enhanced permeability and retention) effect.

[0109] The nucleic acid construct of the present invention can be obtained by using the (poly)nucleotide of the present invention as a part of the nucleic acid constituting the nucleic acid construct. The nucleotide is, for example, a part of the staple nucleic acid (hybridized with the single-stranded circular nucleic acid). The nucleic acid constructs of the present invention can be produced by using them as nucleic acids that can hybridize to the nucleic acid (parts that are not included in the nucleic acid), as staple nucleic acids, or as part or all of single-stranded circular nucleic acids. Alternatively, the nucleic acid construct of the present invention can be obtained by encapsulating the (poly)nucleotide of the present invention in the nucleic acid construct of the present invention.

[0110] The nucleic acid construct of the present invention contains a plurality of (for example, 2 to 1000, 10 to 1000, 50 to 1000, or 100 to 500) (poly)nucleotides in order to effectively exert the anticancer activity derived from the (poly)nucleotide of the present invention. It is preferred that the nucleic acid has overhanging structures, and each overhanging structure comprises a (poly)nucleotide of the present invention.

[0111] 4.Applications In one aspect, the present invention provides a pharmaceutical, a reagent, etc. (referred to herein as "pharmaceuticals," "reagents," etc.) comprising at least one selected from the group consisting of the (poly)nucleotide of the present invention and the nucleic acid construct of the present invention. (sometimes referred to as "the agent of the present invention"). More specifically, it can be used as an active ingredient of an anticancer agent or the like.

[0112] The pharmaceutical preparation of the present invention is not particularly limited as long as it contains the active ingredient of the present invention, and may further contain other ingredients as necessary. The other ingredients are not particularly limited as long as they are pharmaceutically acceptable. The other ingredients include not only ingredients with pharmacological effects but also additives. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0113] The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type of agent. Depending on the application, the agent of the present invention can be used in vitro (for example, by adding it to the medium of cultured cells) or in vivo (for example, by administering it to animals). Administered.)

[0114] The agents of the present invention may be applied to, but are not limited to, mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, and examples include blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0115] When the agent of the present invention is used as an anticancer agent or when used on cancer cells, the target cancer is not particularly limited and includes, for example, hepatocellular carcinoma, pancreatic cancer, kidney cancer, leukemia, esophageal cancer, stomach cancer, colon cancer, lung cancer, prostate cancer, skin cancer, breast cancer, cervical cancer, etc. Among these, solid cancers are preferred, and hepatocellular carcinoma is more preferred.

[0116] The agents of the present invention may take any dosage form, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; and parenteral preparations such as injectable preparations (for example, drip injections (e.g., intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (for example, ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

[0117] The route of administration of the drug of the present invention is not particularly limited as long as the desired effect can be obtained, and oral administration is one example. enteral administration such as oral administration, tube feeding, enema administration, etc.; parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration, etc.

[0118] The content of the active ingredient in the drug of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0119] The dosage of the agent of the present invention when administered to animals is not particularly limited as long as it is an effective amount that exhibits a medicinal effect. Generally, the dosage is 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in terms of the weight of the active ingredient in the case of oral administration, and 0.1 to 1000 mg / kg body weight per day in the case of parenteral administration. The dosage is 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day. The dosage can be increased or decreased as appropriate depending on age, pathological condition, symptoms, etc. [Example]

[0120] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0121] Example 1. Synthesis of PST-amidite monomers The PST-amidite monomer was synthesized according to the following scheme.

[0122] [ka]

[0123] Example 1-1. Synthesis of Compound 1 Cathechol (5.2 g, 47.2 mmol) and K2CO3 (6.3 g, 45.6 mmol) were weighed into a two-necked flask and dissolved in acetone (85 mL). A reflux apparatus was then attached and reflux was initiated. Allyl bromide (4.0 mL, 46.3 mmol) was then slowly added dropwise via syringe, and the mixture was refluxed at 75°C for 4 hours. The color of the solution changed from white to reddish purple. The progress of the reaction was confirmed. After filtration, the solvent was completely removed using an evaporator, and the residue was purified by flash column chromatography using hexane:ethyl acetate (0→20%) as the mobile phase. The target product was obtained as a white solid (4.70 g) in a yield of 66.6%. Compound 1 of 1 The H NMR [CDCl3] spectrum and synthesis scheme are shown in Figure 1.

[0124] Example 1-2: Synthesis of Compound 2 3,4,5-trimethoxyaniline (0.68 g, 3.7 mmol) was dissolved in ethanol (20 mL) and cooled on ice. Then, 3 M HCl (3.0 mL, 9.0 mmol) was slowly added via syringe, followed by dropwise addition of 2.5 M NaNO2 (1.4 mL, 3.6 mmol) via syringe. The solution changed color from reddish purple to black. After stirring for 45 min, compound 1 (0.60 g, 4.0 mmol) was dissolved in 2 M NaOH (3.6 mL, 7.2 mmol). The mixture was stirred at room temperature for 2 hours. 3 M HCl (3.0 mL, 9.0 mmol) was added to the mixture to quench the reaction. The progress of the reaction was confirmed by TLC (hexane: ethyl acetate = 2:1). The solvent was removed using an evaporator. The residue was then dissolved in ethyl acetate, extracted twice with saturated aqueous NaHCO3, and washed once with saturated aqueous NaCl, and the organic layer was recovered. Na2SO4 was added and the mixture was stirred for 30 minutes. The residue was dried with hexane. After filtration, the solvent was removed using an evaporator. The residue was purified by flash column chromatography using hexane:ethyl acetate (0% to 30%) as the mobile phase. The target product was obtained as a yellow solid (0.71 g) in a yield of 57.7%. 1 The H NMR [CDCl3] spectrum and synthesis scheme are shown in Figure 2.

[0125] <Example 1-3. Synthesis of Compound 3> Compound 2 (0.30 g, 0.87 mmol) was weighed into a two-neck flask and dissolved in DMF (50 mL). K2CO3 (0.26 g, 1.88 mmol) was then added, and the mixture was purged with nitrogen three times. Mel (0.14 mL, 2.2 mmol) was then added dropwise via syringe, and the mixture was stirred at room temperature for 3 days. The reaction was quenched by adding 2 M NaOH (30 mL). The progress of the reaction was confirmed by TLC (hexane: ethyl acetate = 2:1), and the mixture was filtered. The solvent was removed using an evaporator. 3aq Extracted twice with saturated NaCl aq The organic layer was collected by washing once with Na2SO4. The mixture was dried by stirring for 30 minutes after adding Na2SO4. After that, the mixture was filtered and the solvent was removed using an evaporator. The eluate was dissolved in hexane and ethyl acetate (0% → 30%) in the mobile phase and subjected to flash column chromatography. The target product was a pale yellow solid (0.28 g) in a yield of 93.3%. 1 The H NMR [CDCl3] spectrum and synthesis scheme are shown in Figure 3.

[0126] Example 1-4: Synthesis of PST (Photostatin) Compound 3 (0.28 g, 0.45 mmol) was weighed into a two-necked flask and dissolved in a 5:3 MeOH:CH2Cl2 mixture (40 mL). The atmosphere was replaced with nitrogen, and Pd(PPh3)4 (0.011 g, 0.0095 mmol) was added. After stirring at room temperature for 5 minutes, K2CO3 (0.53 g, 3.8 mmol) was added and the mixture was stirred at room temperature for 90 minutes. The color changed from yellow to reddish orange. After 90 minutes, TLC (hexane: ethyl acetate = 2:1) showed After confirming the progress of the reaction, the reaction mixture was filtered and the solvent was removed using an evaporator. 3aq Extracted twice with saturated NaCl aq The organic layer was collected after washing once with Na2SO4. The mixture was dried by stirring for 30 minutes after adding Na2SO4. After that, the mixture was filtered and the solvent was removed using an evaporator. The residue was dissolved in ethyl acetate and purified by flash column chromatography using hexane:ethyl acetate (0→30%) as the mobile phase. The target product was a reddish-orange oily solid (0.17 g) in a yield of 70.8%. 1 The H NMR [CDCl3] spectrum and synthesis scheme are shown in Figure 4.

[0127] <Example 1-5. Synthesis of PST-amidite monomer> Photostatin (0.08 g, 0.25 mmol) was dissolved in acetonitrile (5 mL) and transferred to a two-neck flask. The solution was azeotropically dried three times with dry acetonitrile (2 mL) under nitrogen. Then, acetonitrile (5 mL) was added dropwise. Next, DIPEA (348 μL, 2.0 mmol, 8 eq) and 2-cyanoethyldiisopropylchlorophosphoramidite (111 μL, 0.50 mmol, 2 eq) were added with a pipette while cooling on ice. The reaction mixture was slowly added dropwise. This series of steps was always carried out under nitrogen. The mixture was stirred at room temperature for 90 minutes. The color of the solution changed from reddish-orange to bright orange. After 90 minutes, the progress of the reaction was confirmed by TLC (hexane: ethyl acetate: triethylamine = 60:40:3), and the organic layer was extracted twice with saturated aqueous NH4Cl and washed once with saturated aqueous NaCl to recover the organic layer. Na2SO4 was added and the mixture was stirred for 30 minutes to dry. The mixture was then filtered and the solvent was removed using an evaporator. The evaporation was carried out without immersion in a water bath. The product was dissolved in ethyl acetate and purified by flash column chromatography using hexane with triethylamine: ethyl acetate (0 → 40%) as the mobile phase. Purpose The product was obtained as an orange oily solid (0.12 g) in 75.2% yield.

[0128] The obtained substance was confirmed by TLC (developing solvent: hexane:ethyl acetate:triethylamine = 50:50:3). Before the reaction, only a spot with an Rf value of 0.57 was observed, but after the reaction, a spot with an Rf value of 0.65 was also observed in addition to the spot with an Rf value of 0.57 (these were the only two spots). The obtained substance was further purified by HPLC and confirmed by mass spectrometry. The mass spectrum of the mass spectrometry is shown in Figure 5. It was confirmed that the PST-amidite monomer was obtained.

[0129] Example 2. Synthesis of PST-modified nucleic acids Photostatin was modified with five types of DNA: T9, A, T, G, and C. The structural formula of the target product is shown below. vinegar.

[0130] [ka]

[0131] The PST-amidite monomer was azeotropically dried three times with dry acetonitrile (2 mL). It was then dissolved in dry acetonitrile, transferred to a brown bottle with a syringe, and introduced into the automated DNA synthesizer. The tubes were transferred to screw tubes, and 500 μL of aqueous ammonia was added. The protecting groups of the nucleic acid base and phosphate groups were deprotected by leaving them at room temperature for 1 hour (T and T9), at 37°C for 8 hours (A and C), and at 65°C for 8 hours (G). Mass spectrometry was then performed using MALDI-TOF / MS (Figure 6). Furthermore, PST-T9 and PST-T were purified by HPLC before mass spectrometry. was carried out (Figures 7 to 9).

[0132] Example 3. Evaluation of cis-trans isomerization The purified PST-T9 was added to sterile water to a final concentration of 5 μM. The samples were prepared by irradiating visible light at 50% for 30 seconds and ultra violet light. The system was irradiated with 50% UV for 30 seconds, and 10 μL of each was injected into an HPLC injector using a syringe, and the change in peaks was examined. The results are shown in Figure 10. The proportion of peaks derived from cis isomers increased with UV irradiation. It was found that the structure of PST-modified nucleic acids can also be controlled by UV irradiation. It was.

[0133] Example 4. UV measurement We investigated how the absorption wavelength changes between the cis and trans forms of Photostatin. Purified PST-T9 was adjusted to a final concentration of 5 μM with sterile water and 10x PBS. First, visible light was irradiated at 50% for 30 seconds, and UV measurement was performed on the trans form. Then, in a dark place, ultraviolet light was irradiated at 50% for 30 seconds, and UV measurement was performed on the cis form. The results are shown in Figure 11. Cis-trans isomerization I found out that.

[0134] Example 5. Time-varying measurements To examine the reversibility of the time-dependent change, time-dependent measurements were performed. The same sample was irradiated with 50% ultra violet light for 30 seconds in the dark to convert it to the cis form. Measurements were taken every 10 minutes. The system was set to operate automatically, and the presence or absence of reversibility was examined based on the absorption wavelength and absorbance. The results are shown in Figure 12. It was found that cis-trans reversibility was observed.

[0135] Example 6. Tm measurement PST-T9 was mixed with a complementary single strand (A9) to form a double strand, and its melting point was measured. PST-T9 and A9, each adjusted to 10 μM, were mixed and the sample was prepared in 10xPBS to a final concentration of 5 μM. First, the internal temperature of the device was raised to 80°C, then lowered to 10°C (-1°C / min), and held at that temperature for 5 minutes. Next, the temperature was raised from 10°C to 80°C (1°C / min). Finally, the cis isomer was detected by irradiating it with 50% ultra violet light in a dark place for 30 seconds. The temperature was changed from 10°C to 60°C (1°C / min), and measurements were taken. The experiment was carried out under a nitrogen gas flow to suppress the influence of the cis isomer. This resulted in a slight decrease in Tm.

[0136] Example 7. Evaluation of thermal stability The structure of PST-T9 at high temperatures was confirmed by HPLC. Purified PST-T9 was prepared as a sample with sterilized water and 10xPBS to a final concentration of 5 μM. The sample was heated to 80°C in a thermal cycler and held at that temperature for 10 minutes. The heated sample was then injected into an HPLC injector using a syringe, and the elution peak was examined. The results are shown in Figure 14. As the temperature increased, the trans isomer was formed. The only peak was due to

[0137] Example 8. Anticancer activity test The anti-cancer activity of PST-T against HeLa cells was examined. For sample preparation, purified PST-T was diluted with sterile water to a final concentration of 20 μM. HeLa cells were placed in a 96-well plate. The seeds were seeded and pre-cultured for two days. After two days, the medium was removed. After filtration sterilization of the PST-T solution, 50 μL of the solution was dispensed into two Eppendorf tubes. One of the tubes was then irradiated with UV light at 50% for 30 seconds. One was converted to cis isomer by irradiation with 50% vis for 30 seconds, and the other was converted to trans isomer by irradiation with 50% vis for 30 seconds. Then, each sample was added to a 96-well plate together with 2x E-MEM (50 μL) so that the final concentration of the medium was 1x E-MEM and the final concentration of the sample was 10 μM, and the mixture was incubated for 8 days. After that, the solution was removed, Live / Dead reagent was added, and the cells were incubated at 37°C. After 30 minutes, the cells were analyzed by confocal imaging. The state of the cells was observed using a laser microscope. As a control, a well containing only 1x E-MEM was prepared. The results are shown in Figure 15. By using the cis isomer, stronger anti-cancer activity was exhibited. Ta.

[0138] Example 9. Synthesis of CA4-amidite monomer

[0139] [ka]

[0140] CA4 (0.10 g, 0.31 mmol) was dissolved in acetonitrile (5 mL) and transferred to a two-neck flask. Under nitrogen, the solution was azeotropically dried three times with dry acetonitrile (2 mL). Then, acetonitrile (5 mL) was added dropwise. Next, DIPEA (431 μL, 2.48 mmol, 8 eq) and 2-cyanoethyldiisopropylchlorophosphoramidite (137 μL, 0.62 mmol, 2 eq) were slowly added dropwise with a pipette while cooling on ice. This series of steps was always carried out under nitrogen. The mixture was stirred at room temperature for 90 minutes. The color of the solution changed from white to pale yellow. After 90 minutes, TLC (hexane: ethyl acetate triethylamine = After checking the progress of the reaction in a 60:40:3 mixture, the organic layer was extracted twice with saturated NH4Cl aq. and washed once with saturated NaCl aq. The organic layer was recovered. Na2SO4 was added and the mixture was stirred for 30 minutes to dry. After that, the mixture was filtered and the solvent was removed using an evaporator. The evaporation was performed without immersing the mixture in a hot water bath. The mixture was dissolved in ethyl acetate and the mobile phase was hexane with triethylamine:ethyl acetate: (0→30% The product was purified by flash column chromatography using hexane. The target product was obtained as a white solid (0.10 g) in a yield of 63.2%. The obtained substance was analyzed by TLC (eluent: hexane). When checked with a solvent (a mixture of ethanol, ethyl acetate, and triethylamine = 50:50:3), only a spot with an Rf value of 0.34 was observed before the reaction, but after the reaction, in addition to the spot with an Rf value of 0.34, a spot with an Rf value of 0.47 was also observed (these were the only two spots). Furthermore, the obtained substance was fractionated by HPLC. After purification, it was confirmed that a CA4-amidite monomer was obtained.

[0141] Example 10. Preparation of PST-modified DNA origami dendrimers PST-modified DNA origami dendrimers were prepared as follows.

[0142] DNA origami dendrimer (overall view: Figure 16, enlarged view of a part of the tip of the dendrimer: Figure 17) was prepared by adding the staple DNA mix to M13mp18 ssDNA and 10× TAE / Mg 2+ The mixture was mixed with the buffer. The staple DNA mix was adjusted to 20 nM and the M13mp18 ssDNA to 4 nM with sterile water. The mixture was then annealed (90°C for 10 min, 90°C → 25°C, -1°C / min) using a thermal cycler to form a DNA origami dendrimer. The staple DNA placed at the tip is M13mp18 ssDNA as shown on the left side of Figure 18. It has a portion (free strand) that does not anneal to the DNA. Staple DNAs (PST-staple DNA 1 and PST-staple DNA 2) modified with PST at the 5' end can be annealed to the free strand (right side of Figure 18) to obtain a PST-modified DNA origami dendrimer.

[0143] [Table 1]

[0144] After the DNA origami dendrimer was formed, 1.1 μL and 1.3 μL of 67.3 μM PST-staple DNA 1 and 49.6 μM PST-staple DNA 2 were added, respectively, and the mixture was left to stand overnight in a refrigerator. Excess staple DNA was removed by ultrafiltration (Amicon Ultra 50K). After purification, the solution was concentrated 50 times and UV absorption was measured using Nanodrop to confirm the presence of PST absorption (375 nm). did.

Claims

1. General formula (1): 【Chemistry 1】 [In the formula: R 1 are the same or different and represent an alkoxy group or a phosphate group. 2 are the same or different and represent an alkoxy group or a phosphate group. 3 indicates -N= or -CH=. 4 indicates =N- or =CH-. 5 represents a protecting group for a hydroxy group. 6 and R 7 are the same or different and represent an alkyl group; m represents an integer of 1 to 5; and n represents an integer of 1 to 4. A compound represented by the formula (I), a salt thereof, or a solvate thereof.

2. The compound has the general formula (1A): 【Chemistry 2】 [In the formula: R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 is the same as above.] The compound according to claim 1, or a salt thereof, or a solvate thereof, which is a compound represented by the formula:

3. R 1 and R 2 The compound according to claim 1, or a salt thereof, or a solvate thereof, wherein is an alkoxy group.

4. R 5 is an alkyl group substituted with an electron-withdrawing group, and R 6 and R 7 The compound or salt thereof, or a solvate of the compound or salt according to any one of claims 1 to 3, wherein is a branched alkyl group.

5. The terminal phosphate group has the general formula (2): 【Transformation 3】 [In the formula: R 1 are the same or different and represent an alkoxy group or a phosphate group. 2 are the same or different and represent an alkoxy group or a phosphate group. 3 indicates -N= or -CH=. 4 indicates =N- or =CH-. 8 represents O or S. R 9 O - or S - m represents an integer of 1 to 5, and n represents an integer of 1 to 4. A nucleotide or polynucleotide in which the nucleotide or polynucleotide is replaced with a group represented by the following formula:

6. General formula (2A): 【Chemistry 4】 [In the formula: R 1 , R 2 , R 3 , R 4 , R 8 , R 9 , m, and n are the same as above. 8A are the same or different It indicates O or S. 9A are the same or different, O - or S - Indicates R 10 R may be the same or different and represent a divalent group formed by removing two hydroxy groups from the sugar moiety of a nucleoside. 10A R may be the same or different and represent a divalent group formed by removing two hydroxy groups from the sugar moiety of a nucleoside. 11 represents a hydroxy group, a phosphate group, or a thiophosphate group, and p represents an integer of 0 or 1 or more. ］ The nucleotide or polynucleotide according to claim 5, which is a compound represented by the formula:

7. A nucleic acid construct comprising the nucleotide or polynucleotide of claim 5.

8. The nucleic acid structure of claim 7, which is a DNA origami.

9. A pharmaceutical comprising at least one selected from the group consisting of the nucleotide or polynucleotide according to claim 5 or 6 and the nucleic acid structure according to claim 7 or 8.

10. The pharmaceutical composition according to claim 9, which is an anticancer agent.

11. A reagent comprising at least one selected from the group consisting of the nucleotide or polynucleotide according to claim 5 or 6 and the nucleic acid structure according to claim 7 or 8.

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