JP2023168030A - amidite monomer - Google Patents

Abstract

To provide a technique for incorporating a compound having a colchicine-like structure into a nucleic acid.SOLUTION: The invention provides a compound represented by a general formula (1) or a salt thereof, or their solvate. [In the formula: R1 are the same or different and each represent an alkoxy or phosphate group; R2 are the same or different and each represent an alkoxy or phosphate group; R3 represents -N= or -CH=; R4 represents =N- or =CH-; R5 represents a protecting group of a hydroxy group; R6 and R7 are the same or different and each represent an alkyl group; m represents an integer from 1 to 5; and n represents an integer from 1 to 4.]SELECTED DRAWING: None

Description

Application for application of Article 30, Paragraph 2 of the Patent Act (Part 1) Website publication date January 17, 2022 Website address https://confit. atlas. jp/guide/event/csj102nd/top https://confit. atlas. jp/guide/event/csj102nd/tables https://confit. atlas. jp/guide/event/csj102nd/table/20220324 https://confit. atlas. jp/guide/event/csj102nd/session/2G10109-24/tables? jhFhGgGRJj (Part 2) Website publication date March 9, 2022 Website address https://confit. atlas. jp/guide/event/csj102nd/top https://confit. atlas. jp/guide/event/csj102nd/proceedings/list https://confit. atlas. jp/guide/event-img/csj102nd/csj102nd_APA_all/proceedings (Part 3) Date March 24, 2022 (Duration: March 23, 2022 to March 26, 2022) Meeting name, venue Nippon Chemical 102nd Spring Annual Meeting (2022) (held online)

The present invention relates to amidite monomers and the like.

Various anticancer drugs and prodrugs thereof having structures similar to colchicine have been reported (for example, Non-Patent Document 1). Like colchicine, this anticancer drug suppresses cell division in cancer cells by specifically binding to tubulin and inhibiting the formation of microtubules. Therefore, the development of drug delivery technology, especially drug delivery technology that can specifically transport cancer cells, is desired.

In general, liposomes, polymerized nanoparticles, inorganic nanoparticles, dendrimers, micelles, nanoemulsions, polymer drug conjugates, and the like have been reported as drug delivery technologies. However, some of them lack homogeneity in shape, some have suboptimal biocompatibility, and some are often found to have insufficient selectivity for diseased cells. Therefore, the development of new drug delivery technology is desired.

Borowiak et al., 2015, Cell, 162, 403-411.

The present inventor has focused on nucleic acid structures such as DNA origami as a drug delivery technology. DNA origami is biocompatible (an inherent property of DNA), biodegradable (an inherent property of DNA), and has the ability to control the location of drug uptake.

An object of the present invention is to provide a technique for incorporating a compound having a structure similar to colchicine into a nucleic acid.

As a result of intensive research in view of the above problems, the present inventors have discovered that by using an amidite monomer represented by general formula (1), a nucleotide having a compound having a structure similar to colchicine is added and has anticancer activity. or polynucleotides, and furthermore, nucleic acid structures containing these, have been found to be obtainable. The present inventor has completed the present invention as a result of further research based on this knowledge. That is, the present invention includes the following aspects.

Item 1. General formula (1):

[In the formula: R ¹ is the same or different and represents an alkoxy group or a phosphoric acid group. R ² is the same or different and represents an alkoxy group or a phosphoric acid group. R ³ represents -N= or -CH=. R ⁴ represents =N- or =CH-. R ⁵ represents a hydroxy protecting group. R ⁶ and R ⁷ are the same or different and represent an alkyl group. m represents an integer from 1 to 5. n represents an integer from 1 to 4. ]
A compound represented by: or a salt thereof or a solvate thereof.

Item 2. The compound has general formula (1A):

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

Item 3. Item 3. The compound, salt thereof, or solvate thereof according to Item ¹ or 2, wherein R 1 and R ² are an alkoxy group.

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

Item 5. The terminal phosphate group has the general formula (2):

[In the formula: R ¹ is the same or different and represents an alkoxy group or a phosphoric acid group. R ² is the same or different and represents an alkoxy group or a phosphoric acid group. R ³ represents -N= or -CH=. R ⁴ represents =N- or =CH-. R ⁸ represents O or S. R ⁹ represents O ^- or S ^- . m represents an integer from 1 to 5. n represents an integer from 1 to 4. ]
A nucleotide or polynucleotide substituted with a group represented by

Item 6. General formula (2A):

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

Section 7. Item 7. A nucleic acid structure comprising the nucleotide or polynucleotide according to item 5 or 6.

Section 8. 8. The nucleic acid structure according to item 7, which is a DNA origami.

Item 9. A medicament 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.

Item 10. Item 9. The medicament according to item 9, which is an anticancer agent.

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.

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, each acid structure containing the nucleotide or polynucleotide, and the like.

^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of Compound 1 are shown below. ^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of compound 2 are shown below. ^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of compound 3 are shown below. ^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of Photostatin are shown below. The mass spectra of the PST-amidite monomer are shown. Mass spectra of PST-A, PST-G, PST-C, PST-T, and PST-T9 are shown. The HPLC purification results of PST-T and the mass spectra of the recovered peaks are shown. The results of HPLC purification of PST-T9 are shown. The mass spectrum of the recovered peak of PST-T9 is shown. The HPLC measurement evaluation results of cis-trans isomerization in Example 3 are shown. The results of UV measurement evaluation of cis-trans isomerization in Example 4 are shown. The results of UV measurement (time change) evaluation of cis-trans isomerization in Example 5 are shown. The Tm measurement results of Example 6 are shown. The thermal stability evaluation results of Example 7 are shown. The results of the anticancer activity test of Example 8 are shown. An overall diagram of the DNA origami dendrimer produced in Example 10 is shown. An enlarged view of the tip of a portion of the DNA origami dendrimer produced in Example 10 is shown. An enlarged view of a portion of the tip of the DNA origami dendrimer produced in Example 10 (left side) and an enlarged view of a portion of the tip of the PST-modified DNA origami dendrimer (right side) are shown.

In this specification, the expressions "contain" and "including" include the concepts of "containing", "comprising", "consisting essentially" and "consisting only".

1. In one embodiment of the present invention, the amidite monomer has the general formula (1):

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

R ¹ is the same or different and represents an alkoxy group or a phosphoric acid group. R ¹ is preferably an alkoxy group.

The alkoxy group represented by R ¹ includes either a linear or branched (preferably linear) group. 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, still more preferably 1 to 2, particularly preferably 1. Specific examples of the alkoxy group include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, and the like.

The phosphoric acid group represented by R ¹ is a group represented by H ₂ PO ₄ -.

m represents an integer from 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, particularly preferably 3.

The position of R ¹ is not particularly limited, and can be, for example, at the para position, meta position, or ortho position with respect to R ³ . The position of R ¹ is preferably the para or meta position. When a plurality of R ¹ exists, it is preferable to include R ¹ at para and/or meta positions, and more preferably to include R ¹ at para and meta positions.

R ² is the same or different and represents an alkoxy group or a phosphoric acid group. R ² is preferably an alkoxy group.

The definition of the alkoxy group represented by R ² is the same as the definition of the alkoxy group represented by R ¹ . The definition of the phosphoric acid group represented by R ² is the same as the definition of the phosphoric acid group represented by R ¹ .

n represents an integer from 1 to 4. n is preferably an integer of 1 to 3, more preferably an integer of 1 to 2, and even more preferably 1.

The position of R ² is not particularly limited, and can be, for example, at the para position, meta position, or ortho position with respect to R ⁴ . The position of R ¹ is preferably the para position, the meta position, and more preferably the para position.

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

Regarding the numbers (m, n) and positions of R ¹ and R ² , in one embodiment of the present invention, the compound represented by general formula (1) is particularly preferably represented by general formula (1A):

It can be a compound represented by

R ³ represents -N= or -CH=. R ³ is preferably −N=.

R ⁴ represents =N- or =CH-. R ⁴ is preferably =N-.

In a preferred embodiment of the invention, R ³ can be -N= and R ⁴ can be =N-, or R ³ can be -CH= and R ⁴ can be =CH-.

R ⁵ represents a hydroxy protecting group.

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

R ⁵ 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, an arylsulfonyl group, a trihalomethyl group, a trialkylamino group, and preferably a cyano group. R ⁵ is particularly preferably -(CH ₂ ) ₂ -CN.

R ⁶ and R ⁷ are the same or different and represent an alkyl group.

The alkyl group represented by R ⁶ and R ⁷ may be linear or branched, and is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms. Alkyl groups include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl. The alkyl group herein also includes alkyl moieties such as an alkoxy group. R ⁶ and R ⁷ may be bonded to each other to form a cyclic structure.

R ⁶ and R ⁷ are particularly preferably both isopropyl groups.

The compound represented by the general formula (1) includes both a cis form and a trans form with respect to the double bond of ^R3 = ^R4 . That is, the compound represented by general formula (1) has general formula (11):

A compound represented by or general formula (12):

It can be a compound represented by

The cis and trans isomers can be converted into the other isomer by irradiation with light. The conversion can be performed according to a known method (for example, according to the method described in Non-Patent Document 1).

Salts of the compound represented by formula (1) are not particularly limited, and include, for example, salts with inorganic bases such as sodium salts, magnesium salts, potassium salts, calcium salts, and aluminum salts; methylamine, ethylamine, and ethanol. Examples include salts with organic bases such as amines; salts with basic amino acids such as lysine, ornithine, and arginine, and ammonium salts. The salt may be an acid addition salt, and examples thereof include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; formic acid, acetic acid, Organic acids such as 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; aspartic acid, glutamic acid, etc. and acid addition salts with acidic amino acids.

Solvates of the compound represented by general formula (1) or a salt thereof are not particularly limited, and include, for example, solvates with solvents such as water, ethanol, glycerol, and acetic acid.

Amidite monomers of the present invention can be produced in a variety of ways.

The compound represented by general formula (1) can be produced, for example, according to or analogously to the following scheme.

X is a halogen atom. Examples of the halogen atom represented by X include fluorine atom, chlorine atom, bromine atom, and iodine atom. Among these, halogen atoms are preferred.

As the compound represented by general formula (101) and the compound represented by general formula (102), commercially available compounds can be used as they are, or if necessary, compounds synthesized according to known methods or analogously. You can also use

The amount of the compound represented by general formula (102) to be used is usually 1 to 3 mol per 1 mol of the compound represented by general formula (101) from the viewpoint of yield, ease of synthesis, etc. Preferably, 1.5 to 2.5 mol is more preferable.

This reaction is usually carried out in the presence of a reaction solvent. Examples of the reaction solvent include, but are not limited to, acetonitrile, pyridine, dimethylformamide, dichloromethane, tetrahydrofuran, acetone, toluene, ethanol, and the like, with acetonitrile being preferred. A single solvent may be used, or a plurality of solvents may be used in combination.

This reaction is preferably carried out in the presence of a base (preferably a non-nucleophilic base) such as N,N-diisopropylethylamine. The amount of the base to be used is usually preferably 3 to 15 mol, more preferably 4 to 10 mol, per 1 mol of the compound represented by general formula (101), from the viewpoint of yield, ease of synthesis, etc. .

In this reaction, in addition to the above-mentioned components, appropriate additives can also be used within the range that does not significantly impair the progress of the reaction.

The reaction temperature can be any of heating, room temperature, and cooling, and is usually preferably carried out at a temperature of 10 to 100°C. The reaction time is not particularly limited and can generally be from 30 minutes to 30 hours.

Progress of the reaction can be followed by conventional methods such as chromatography. After the reaction is completed, the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. Furthermore, the structure of the product can be identified by elemental analysis, MS (ESI-MS) analysis, IR analysis, ¹ H-NMR, ¹³ C-NMR, etc.

2. In one embodiment of the nucleotide or polynucleotide of the present invention, the terminal phosphate group has the general formula (2):

nucleotide or polynucleotide (herein sometimes referred to as "(poly)nucleotide of the present invention"), which is replaced with This will be explained below.

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

R ⁸ represents O or S. R ⁸ is preferably O.

R ⁹ represents O ^- or S ^- . R ⁹ is preferably O ^- .

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

The terminal phosphoric acid group may be either a 5'-terminal phosphoric acid group or a 3'-terminal phosphoric acid group, but is preferably a 5'-terminal phosphoric acid group.

Nucleotides and polynucleotides are not particularly limited, and for example, structural units of natural nucleic acids and various artificial nucleic acids including aTNA and SNA can be employed. Specifically, in addition to DNA, RNA, etc., the nucleic acids that can be employed may be those subjected to known chemical modifications, as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide are replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. I can do it. In addition, the hydroxyl group at the 2' position of the sugar (ribose) of each ribonucleotide is replaced by -OR (R is, for example, CH ₃ (2´-O-Me), CH ₂ CH ₂ OCH ₃ (2´-O-MOE) ), CH ₂ CH ₂ NHC(NH)NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, such as introducing a methyl group or cationic functional group to the 5-position of the pyrimidine base, or replacing the carbonyl group at the 2-position with thiocarbonyl. can be mentioned. Further examples include, but are not limited to, those in which the phosphoric acid moiety or hydroxyl moiety is modified with biotin, an amino group, a lower alkylamine group, an acetyl group, or the like. Furthermore, BNA (LNA), etc., in which the conformation of the sugar part of the nucleotide is fixed to N-type by cross-linking the 2' oxygen and 4' carbon of the sugar part of the nucleotide, can also be used.

More specifically, the (poly)nucleotide of the present invention preferably has the general formula (2A):

It is a compound represented by

R ^8A are the same or different and represent O or S. R ^{8 A} is preferably O.

R ^9A are the same or different and represent O ^- or S ^- . R ^{9 A} is preferably O ^- .

R ¹⁰ is the same or different and represents a divalent group obtained 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 nucleobase). One hydroxy group is the 5' hydroxy group of ribose, deoxyribose or the corresponding hydroxy group in other nucleic acid backbones, and the other hydroxy group is the 3' hydroxy group of ribose, deoxyribose or other sugar moieties. The corresponding hydroxy group is preferred.

R ^10A is the same or different and represents a divalent group 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 nucleobase). One hydroxy group is the 5' hydroxy group of ribose, deoxyribose or the corresponding hydroxy group in other nucleic acid backbones, and the other hydroxy group is the 3' hydroxy group of ribose, deoxyribose or other sugar moieties. The corresponding hydroxy group is preferred.

The sugar moiety of a nucleoside is not particularly limited as long as it can constitute a nucleic acid, but includes sugar moieties such as ribose, deoxyribose, and modified sugars (for example, sugar moieties constituting DNA, RNA, BNA, LNA, etc.). ).

As the nucleic acid base of the nucleoside, any base constituting a nucleic acid can be employed without particular limitation. The bases that make up nucleic acids include only typical bases found in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.). However, bases other than these, such as hypoxanthine (I) and modified bases, are also included. Examples of the modified base 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-carboxymethyl Aminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxy Aminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxy Examples include acetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, and the like.

R ¹¹ represents a hydroxy group, a phosphoric acid group, or a thiophosphoric acid group.

p represents an integer of 0 or 1 or more. p is preferably 1 to 100,000, more preferably 5 to 100,000, even more preferably 10 to 100,000, even more preferably 15 to 100,000, particularly preferably 20 to 100,000. The upper and/or lower limit of p can be, for example, 50, 100, 500, 1000, 5000, 10000, or 50000.

The (poly)nucleotides of the invention can be in salt form. Salts are not particularly limited, and include, for example, 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; lysine, Examples include salts with basic amino acids such as ornithine and arginine, and ammonium salts. The salt may be an acid addition salt, and examples thereof include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; formic acid, acetic acid, Organic acids such as 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; aspartic acid, glutamic acid, etc. and acid addition salts with acidic amino acids.

The (poly)nucleotides of the invention can be in the form of solvates. Solvates are not particularly limited, and include, for example, solvates with solvents such as water, ethanol, glycerol, and acetic acid.

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

The phosphoramidite method can be carried out according to a known method using, for example, a commercially available automatic nucleic acid synthesizer. Specifically, (A) a step of deprotecting the hydroxyl group at the 5' position (or a position corresponding to it), (B) a step of condensing the amidite monomer, and (C) a step of deprotecting the hydroxyl group at the 5' position (or a position corresponding to it) of the unreacted compound. (D) converting the phosphorous acid group into a phosphoric acid group or a thiophosphoric acid group; (E) cutting out the obtained compound from the solid support, and converting the phosphoric acid moiety and the nucleic acid group into It includes steps such as a step of deprotecting the base and a step of deprotecting the hydroxyl group at the (F) 5' position (or a position corresponding thereto). By repeating steps (A) to (D), a compound containing a polynucleotide skeleton of a desired chain length can be produced. In step (B), the (poly)nucleotide of the present invention can be produced by using the amidite monomer of the present invention.

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

In the deprotection in step (E), deprotection is performed using a base such as aqueous ammonia. Although it was expected that the bond between the phosphorus atom and the aromatic hydroxy group in the (poly)nucleotide of the present invention would be unstable to bases, unexpectedly, the bond between the phosphorus atom and the aromatic hydroxy group in the (poly)nucleotide of the present invention Cleavage of the above bond was inhibited during deprotection of the nucleotide.

After step (E) and before step (F), it is preferable to perform chromatography purification (for example, reversed phase chromatography purification) using the hydrophobicity of the protecting group (hydrophobic group) at the 5' position. Thereby, the purity of the single-stranded polynucleotide of interest can be further increased.

The obtained single-stranded polynucleotide may be isolated and purified as necessary. Generally, RNA can be isolated by precipitation, extraction, and purification methods. Specifically, RNA is precipitated by adding a solvent with low solubility to RNA, such as ethanol or isopropyl alcohol, to the reaction solution, or by adding a solution of phenol/chloroform/isoamyl alcohol to the reaction solution. A method is adopted in which RNA is extracted into the aqueous layer. Thereafter, it can be isolated and purified by known high performance liquid chromatography (HPLC) techniques such as reverse phase column chromatography, anion exchange column chromatography, and affinity column chromatography.

3. Nucleic acid construct In one aspect, the present invention relates to a nucleic acid construct (herein sometimes referred to as "nucleic acid construct of the present invention") containing the (poly)nucleotide of the present invention. This will be explained below.

The nucleic acid structure is not particularly limited as long as it mainly contains a nucleic acid. The content of nucleic acid in the nucleic acid structure is, for example, 70% by mass or more, preferably 80% by mass or more, more preferably 90% by mass or more, still more preferably 95% by mass or more, even more preferably 98% by mass or more, especially It is preferably 99% by mass or more, particularly preferably 100% by mass. Substances other than nucleic acids in the nucleic acid structure are not particularly limited, and include, for example, proteins, peptides, sugars, labeling substances, metal ions, and the like.

The nucleic acid structure can be a three-dimensional structure, a two-dimensional structure (planar structure), or a combination of these structures.

The nucleic acid structure is preferably a structure formed by folding a nucleic acid. From the viewpoint of being suitable for drug delivery, the nucleic acid structure is a structure (DNA origami) formed from a material containing a single-stranded circular nucleic acid and a stapled nucleic acid containing a complementary sequence to the single-stranded circular nucleic acid. is preferred.

The single-stranded circular nucleic acid that can constitute the nucleic acid structure is not particularly limited as long as it can be folded to form a DNA origami. No matter what base sequence a single-stranded circular nucleic acid is composed of, DNA origami can be formed by designing the staple DNA sequence accordingly. The number of bases in the single-stranded circular nucleic acid is not particularly limited, but is, for example, 500 to 50,000, preferably 1,000 to 25,000, more preferably 2,000 to 15,000, still more preferably 3,000 to 12,000, even more preferably 4,000 to 10,000. The base sequence of the single-stranded circular nucleic acid is not particularly limited, but includes, for example, a base sequence containing an M13 bacteriophage DNA sequence or a sequence derived from the DNA sequence.

Staple nucleic acids that can constitute a nucleic acid structure include a complementary sequence to a single-stranded circular nucleic acid, and can form DNA origami by folding the single-stranded circular nucleic acid through complementary base pairing with the single-stranded circular nucleic acid. There are no particular restrictions as far as possible. The number of bases in the staple nucleic acid is not particularly limited, but is, for example, 10 to 500, preferably 20 to 250, more preferably 30 to 100. In the staple nucleic acid, the number of bases in the complementary sequence to the single-stranded circular nucleic acid is not particularly limited, but is, for example, 10 to 500, preferably 20 to 250, more preferably 30 to 100. One stapled nucleic acid is usually complementary to each of multiple (e.g., 2 to 5, preferably 2 to 4, more preferably 2 to 3) strands that are present adjacent to each other when the single-stranded circular nucleic acid is folded. Contains arrays. Staple nucleic acids having such sequence characteristics can further stabilize single-stranded circular nucleic acids in a folded state through complementary base pairing with single-stranded circular nucleic acids. Various DNA origami have been reported so far, and the sequence of staple nucleic acids for forming a nucleic acid structure with a desired shape and structure can be designed according to known methods and information.

The size of the nucleic acid structure is not particularly limited and can be adjusted as appropriate depending on the purpose. The nucleic acid structure is preferably nanosized (nanostructure). For example, the longest diameter (major axis) of the nucleic acid structure is 1000 nm or less. In one embodiment of the present invention, the upper limit and/or lower limit of the long axis of the nucleic acid structure can be, for example, 5 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, or 800 nm. By making the nucleic acid structure 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.

The nucleic acid structure 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 structure. More specifically, the (poly)nucleotide of the present invention can be used, for example, as a nucleic acid that can hybridize to a portion of a staple nucleic acid (a portion that does not hybridize with a single-stranded circular nucleic acid), or as a staple nucleic acid. The nucleic acid structure of the present invention can be obtained by using it, or by using it as part or all of a single-stranded circular nucleic acid. Alternatively, the nucleic acid construct of the present invention can also be obtained by including the (poly)nucleotide of the present invention in the nucleic acid construct of the present invention.

In order to effectively exhibit the anticancer activity derived from the (poly)nucleotide of the present invention, the nucleic acid structure of the present invention may contain a plurality of nucleic acids (for example, 2 to 1000, 10 to 1000, 50 to 1000, or 10 to 100). 500), and each of the protruding structures preferably contains the (poly)nucleotide of the present invention.

Four. Applications The present invention, in one aspect, provides medicines, reagents, etc. (herein referred to as "the present invention") containing at least one selected from the group consisting of the (poly)nucleotide of the present invention and the nucleic acid structure of the present invention. More specifically, it can be used as an active ingredient of anti-cancer drugs and the like.

The drug 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. Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients. Other ingredients include ingredients that have pharmacological effects as well as additives. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, Examples include chelating agents.

The mode of use of the drug of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type thereof. Depending on the intended use, the drug of the present invention can be used, for example, in vitro (e.g., added to the culture medium of cultured cells) or in vivo (e.g., administered to animals). You can also do it.

The subject to which the drug of the present invention can be applied is not particularly limited, but examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Furthermore, examples of cells include animal cells. The type of cells is not particularly limited, and examples include blood cells, hematopoietic stem cells/progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, and muscle cells. , epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, etc.

When the drug of the present invention is used as an anticancer agent or for cancer cells, the target cancers are not particularly limited, and include, for example, hepatocellular carcinoma, pancreatic cancer, kidney cancer, leukemia, and esophagus. These include stomach cancer, colon cancer, lung cancer, prostate cancer, skin cancer, breast cancer, and cervical cancer. Among these, solid cancer is preferred, and hepatocellular carcinoma is more preferred.

The drug of the present invention can be in any dosage form, such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, Oral preparations such as hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies, and injectable preparations (e.g., intravenous drip preparations) ), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (e.g. ointments, poultices, lotions), suppository inhalants, eye preparations, eye ointments, nasal drops. Parenteral preparations such as tablets, ear drops, and liposomes can be used.

The route of administration of the drug of the present invention is not particularly limited as long as the desired effect is obtained, and includes oral administration, tube feeding, enteral administration such as enema administration; intravenous administration, transarterial administration, intramuscular administration; Examples include parenteral administration such as intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

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, etc., and is not limited to, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight. %.

The dosage when administering the drug of the present invention to animals is not particularly limited as long as it is an effective amount that exhibits the medicinal effect, and is usually 0.1 to 0.1 to 0.1 per day by weight of the active ingredient in the case of oral administration. 1000 mg/kg body weight, preferably 0.5 to 500 mg/kg body weight per day, and in the case of parenteral administration 0.01 to 100 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight per day. . The above dosage can be adjusted as appropriate depending on age, pathological condition, symptoms, etc.

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

Example 1. Synthesis of PST-amidite monomer
PST-amidite monomer was synthesized according to the following scheme.

<Example 1-1. Synthesis of compound 1>
Cathechol (5.2 g, 47.2 mmol) and K ₂ CO ₃ (6.3 g, 45.6 mmol) were weighed into a two-necked flask and dissolved in acetone (85 mL). Afterwards, a reflux device was installed and reflux started. Then, Allyl bromide (4.0 mL, 46.3 mmol) was slowly added dropwise using a syringe, and the mixture was refluxed at 75°C for 4 hours. The color of the solution changed from white to reddish-purple. Progress of the reaction was confirmed by TLC (hexane: ethyl acetate = 2: 1). After filtration, the solvent was completely removed using an evaporator, and the product was purified by flash column chromatography using hexane:ethyl acetate (0→20%) as the mobile phase. The desired product, a white solid (4.70 g), was obtained in a yield of 66.6%. ^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of Compound 1 are shown in Figure 1.

<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 using a syringe, and then 2.5 M NaNO ₂ (1.4 mL, 3.6 mmol) was added dropwise using a syringe as well. The solution turned from reddish-purple to black. After stirring for 45 minutes, Compound 1 (0.60 g, 4.0 mmol) was dissolved in 2 M NaOH (3.6 mL, 7.2 mmol) and added dropwise. Further, the mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding 3 M HCl (3.0 mL, 9.0 mmol). Progress of the reaction was confirmed by TLC (hexane: ethyl acetate = 2: 1). It was filtered and the solvent was removed using an evaporator. Thereafter, it was dissolved in ethyl acetate, extracted twice with saturated NaHCO ₃ aq, washed once with saturated NaCl aq, and the organic layer was collected. It was dried by adding Na ₂ SO ₄ and stirring for 30 minutes. Thereafter, it was filtered and the solvent was removed using an evaporator. Purification was performed by flash column chromatography using hexane:ethyl acetate (0→30%) as the mobile phase. The desired product, a yellow solid (0.71 g), was obtained in a yield of 57.7%. ^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of Compound 2 are shown in Figure 2.

<Example 1-3. Synthesis of compound 3>
Compound 2 (0.30 g, 0.87 mmol) was weighed into a two-necked flask and dissolved in DMF (50 mL). Then, K ₂ CO ₃ (0.26 g, 1.88 mmol) was added, and the atmosphere was replaced with nitrogen three times. Then, MeI (0.14 mL, 2.2 mmol) was added dropwise using a syringe, and the mixture was stirred at room temperature for 3 days. 2 M NaOH (30 mL) was added to quench the reaction. After confirming the progress of the reaction with TLC (hexane: ethyl acetate = 2: 1), it was filtered and the solvent was removed using an evaporator. Thereafter, it was dissolved in ethyl acetate, extracted twice with saturated NaHCO _{3 aq} , and washed once with saturated NaCl _aq , and the organic layer was collected. It was dried by adding Na ₂ SO ₄ and stirring for 30 minutes. Thereafter, it was filtered and the solvent was removed using an evaporator. It was dissolved in ethyl acetate and purified by flash column chromatography using hexane:ethyl acetate (0→30%) as the mobile phase. The desired product, a pale yellow solid (0.28 g), was obtained in a yield of 93.3%. ^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of Compound 3 are shown in Figure 3.

<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 mixed solution (40 mL). The atmosphere was replaced with nitrogen, and Pd (PPh ₃ ) ₄ (0.011 g, 0.0095 mmol) was added. After stirring at room temperature for 5 minutes, K ₂ CO ₃ (0.53 g, 3.8 mmol) was added, and the mixture was stirred at room temperature for 90 minutes. The color of the solution changed from yellow to reddish-orange. After 90 minutes, the progress of the reaction was confirmed by TLC (hexane: ethyl acetate = 2: 1), followed by filtration, and the solvent was removed using an evaporator. Thereafter, it was dissolved in ethyl acetate, extracted twice with saturated NaHCO _{3 aq} , and washed once with saturated NaCl _aq , and the organic layer was collected. It was dried by adding Na ₂ SO ₄ and stirring for 30 minutes. Thereafter, it was filtered and the solvent was removed using an evaporator. It was dissolved in ethyl acetate and purified by flash column chromatography using hexane:ethyl acetate (0→30%) as the mobile phase. The desired product, a red-orange oily solid (0.17 g), was obtained with a yield of 70.8%. ^{The 1} H NMR [CDCl ₃ ] spectrum and synthesis scheme of PST are shown in Figure 4.

<Example 1-5. Synthesis of PST-amidite monomer>
Photostatin (0.08 g, 0.25 mmol) was dissolved in acetonitrile (5 mL), transferred to a two-necked flask, and 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 slowly added dropwise with a pipette while cooling on ice. These series of steps were always performed under nitrogen. Stirred at room temperature for 90 minutes. The color of the solution changed from red-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 collected by extracting twice with saturated NH ₄ Claq and washing once with saturated NaCl aq. . It was dried by adding Na ₂ SO ₄ and stirring for 30 minutes. Thereafter, it was filtered and the solvent was removed using an evaporator. At this time, evaporation was performed without soaking in a hot water bath. It was dissolved in ethyl acetate and purified by flash column chromatography using hexane with triethylamine: ethyl acetate: (0→40%) as the mobile phase. The desired product, an orange oily solid (0.12 g), was obtained in a yield of 75.2%.

When the obtained substance was confirmed by TLC (developing solvent: hexane: ethyl acetate: triethylamine = 50:50:3), there was only a spot with an Rf value of 0.57 before the reaction, but a spot with an Rf value of 0.57 after the reaction. In addition, a spot with an Rf value of 0.65 was observed (these were the only two spots). Furthermore, the obtained substance was fractionated and purified by HPLC and confirmed by mass spectrometry. The mass spectrum of mass spectrometry is shown in FIG. It was confirmed that PST-amidite monomer was obtained.

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

The PST-amidite monomer was azeotropically dried three times with dry acetonitrile (2 mL). It was dissolved in dry acetonitrile, transferred to a brown bottle using a syringe, and introduced into an automatic DNA synthesizer. After DNA synthesis, transfer to a screw tube, add 500 μL of ammonia aqueous solution, and let stand at room temperature for 1 hour to obtain T and T9. By standing still, the protecting groups of the nucleobase portion and phosphate group of G were deprotected. Mass spectrometry was then performed using MALDI-TOF/MS (Figure 6). Furthermore, for PST-T9 and PST-T, mass spectrometry was performed after HPLC purification (Figures 7 to 9).

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

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 10× PBS. First, visible light was irradiated at 50% for 30 seconds and UV measurements were performed on the trans body. After that, UV measurements in the cis form were performed by irradiating the sample with ultra violet light at 50% for 30 seconds in a dark place. The results are shown in FIG. It was found that cis-trans isomerization occurs.

Example 5. Time change measurement To investigate the reversibility of time changes, we performed time change measurements. A similar sample was irradiated with ultra violet light at 50% for 30 seconds in a dark place to convert it to the cis form. The system was set to automatically perform measurements every 10 minutes, and the presence or absence of reversibility was investigated from the absorption wavelength and absorbance. The results are shown in FIG. It was found to exhibit cis and trans reversibility.

Example 6. Tm measurement
By mixing PST-T9 and a complementary single strand (A9), a double strand was assembled and the melting point was measured. PST-T9 and A9, each adjusted to 10 μM, were mixed and samples were prepared in 10× PBS to a final concentration of 5 μM. First, the internal temperature of the device was raised to 80°C. From there, the temperature was lowered to 10°C (-1°C/min). It was held in that state for 5 minutes. Next, the temperature was raised from 10°C to 80°C (1°C/min). Finally, it was converted to the cis form by irradiating it with ultra violet light at 50% for 30 seconds in the dark, and measurements were performed at 10°C → 60°C (1°C/min). Note that all of these steps were performed while flowing nitrogen gas to suppress the effects of dew condensation. The results are shown in FIG. When converted to cis form, Tm decreased slightly.

Example 7. Evaluation of thermal stability The structure of PST-T9 in the high temperature range was confirmed by HPLC. A sample of purified PSR-T9 was prepared with sterile water and 10x PBS to a final concentration of 5 μM. It was heated to 80°C using a thermal cycler and maintained at that temperature for 10 minutes. Next, the heated sample was injected into an HPLC injector using a syringe, and the elution peaks were examined. The results are shown in FIG. When the temperature was increased, only the peak due to the trans form appeared.

Example 8. Anticancer activity test
The anticancer activity against HeLa cells was investigated using PST-T. To prepare the sample, purified PST-T was prepared in sterilized water to a final concentration of 20 μM. HeLa cells were seeded in a 96-well plate and precultured for 2 days. After 2 days, the medium was removed. After sterilizing the PST-T solution by filtration, 50 μL each was dispensed into two Eppendorf tubes. After that, one side was irradiated with UV at 50% for 30 seconds to change it to the cis form, and the other side was irradiated with vis at 50% for 30 seconds to change it to the trans form. Thereafter, each was added to a 96-well plate along with 2×E-MEM (50 μL) so that the final concentration of the medium was 1×E-MEM and the final concentration of the sample was 10 μM, and incubated for 8 days. After 8 days, the solution was removed, Live/Dead reagent was added, and the mixture was incubated at 37°C. After 30 minutes, the state of the cells was observed using a confocal laser microscope. As a control, a well containing only 1×E-MEM was prepared. The results are shown in FIG. The cis form exhibited stronger anticancer activity.

Example 9. Synthesis of CA4-amidite monomer

CA4 (0.10 g, 0.31 mmol) was dissolved in acetonitrile (5 mL), transferred to a two-necked flask, and azeotropically dried three times with dry acetonitrile (2 mL) under nitrogen. 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. These series of steps were always performed under nitrogen. Stirred at room temperature for 90 minutes. The color of the solution changed from white to pale yellow. After 90 minutes, the progress of the reaction was confirmed by TLC (hexane : ethyl acetate triethylamine = 60 : 40 : 3), and the organic layer was collected by extracting twice with saturated NH ₄ Claq and washing once with saturated NaCl aq. . It was dried by adding Na ₂ SO ₄ and stirring for 30 minutes. Thereafter, it was filtered and the solvent was removed using an evaporator. At this time, evaporation was performed without soaking in a hot water bath. It was dissolved in ethyl acetate and purified by flash column chromatography using hexane with triethylamine: ethyl acetate: (0→30%) as the mobile phase. The desired product, a white solid (0.10 g), was obtained with a yield of 63.2%. When the obtained substance was confirmed by TLC (developing solvent: hexane: ethyl acetate: triethylamine = 50:50:3), there was only a spot with an Rf value of 0.34 before the reaction, but a spot with an Rf value of 0.34 after the reaction. In addition, a spot with an Rf value of 0.47 was observed (these were the only two spots). Furthermore, the obtained substance was fractionated and purified by HPLC, and it was confirmed that CA4-amidite monomer was obtained.

Example 10. Preparation of PST-modified DNA origami dendrimer
We created a PST-modified DNA origami dendrimer. Specifically, it was performed as follows.

Prepare a staple DNA mix to form a DNA origami dendrimer (overall view: Figure 16, enlarged view of the tip of part of the dendrimer: Figure 17), and add this, M13mp18 ssDNA, and 10× TAE/Mg ²⁺ buffer. Mixed. At this time, they were diluted with sterile water so that the staple DNA mix was 20 nM and the M13mp18 ssDNA was 4 nM. Thereafter, annealing was performed using a thermal cycler (90°C for 10 min, 90°C→25°C, -1°C/min) to form DNA origami dendrimers. Note that the staple DNA placed at the tip has a portion (free strand) that does not anneal to M13mp18 ssDNA, as shown on the left side of FIG. Staple DNA (PST-staple DNA 1 and PST-staple DNA 2), which is a DNA whose 5′ end is modified with PST and has a complementary sequence to the free strand, is annealed to the free strand (see the right side of Figure 18). ), a PST-modified DNA origami dendrimer can be obtained.

After forming a DNA origami dendrimer, 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 left overnight in the refrigerator. Thereafter, excess stapled DNA was removed by ultrafiltration (Amicon Ultra 50K). After purification, the solution was concentrated 50 times and UV absorption was measured using Nanodrop, confirming the presence of PST absorption (375 nm).

Claims (11)

General formula (1):

[In the formula: R ¹ is the same or different and represents an alkoxy group or a phosphoric acid group. R ² is the same or different and represents an alkoxy group or a phosphoric acid group. R ³ represents -N= or -CH=. R ⁴ represents =N- or =CH-. R ⁵ represents a hydroxy protecting group. R ⁶ and R ⁷ are the same or different and represent an alkyl group. m represents an integer from 1 to 5. n represents an integer from 1 to 4. ]
A compound represented by: or a salt thereof or a solvate thereof. The compound has general formula (1A):

[In the formula: R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are the same as above. ]
The compound according to claim 1, a salt thereof, or a solvate thereof, which is a compound represented by: The compound according to claim 1, a salt thereof, or a solvate thereof, wherein R ¹ and R ² are an alkoxy group. The compound or its salt or its solvent according to any one of claims 1 to 3, wherein R ⁵ is an alkyl group substituted with an electron-withdrawing group, and R ⁶ and R ⁷ are branched alkyl groups. Japanese item. The terminal phosphate group has the general formula (2):

[In the formula: R ¹ is the same or different and represents an alkoxy group or a phosphoric acid group. R ² is the same or different and represents an alkoxy group or a phosphoric acid group. R ³ represents -N= or -CH=. R ⁴ represents =N- or =CH-. R ⁸ represents O or S. R ⁹ represents O ^- or S ^- . m represents an integer from 1 to 5. n represents an integer from 1 to 4. ]
A nucleotide or polynucleotide substituted with a group represented by General formula (2A):

[In the formula: R ¹ , R ² , R ³ , R ⁴ , R ⁸ , R ⁹ , m, and n are the same as above. R ^8A are the same or different and represent O or S. R ^9A are the same or different and represent O ^- or S ^- . R ¹⁰ is the same or different and represents a divalent group formed by removing two hydroxy groups from the sugar moiety of a nucleoside. R ^10A is the same or different and represents a divalent group formed by removing two hydroxy groups from the sugar moiety of a nucleoside. R ¹¹ represents a hydroxy group, a phosphoric acid group, or a thiophosphoric acid group. p represents an integer of 0 or 1 or more. ]
The nucleotide or polynucleotide according to claim 5, which is a compound represented by: A nucleic acid construct comprising the nucleotide or polynucleotide according to claim 5. The nucleic acid structure according to claim 7, which is a DNA origami. A medicament 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. The medicament according to claim 9, which is an anticancer agent. 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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