WO2021177123A1 - Artificial nucleic acid - Google Patents

Abstract

Provided are, inter alia, novel compounds. Also provided are a nucleic acid detection substance and a base pair formed by this polymer, and a nucleic acid drug containing the polymer or base pair.　The compounds are represented by formula (A). [In formula (A), X is an aromatic ring group, R¹ is -OR^1a (In the formula, R^1a represents a hydrogen atom or a substituent.) or a phosphoramidite group, R² is a hydrogen atom or a substituent, R³ is a halogen atom or -OR^3a (In the formula, R^3a represents a hydrogen atom or a substituent.).]

Description

Artificial nucleic acid

The present invention relates to novel compounds and the like.

Development of artificially synthesized DNA (artificial DNA) in which a deoxyribose skeleton and a nucleobase skeleton are bound via a carbon-carbon triple bond has been developed (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 2). See Document 3 and Non-Patent Document 4).

An object of the present invention is to provide a novel compound or the like.

The artificial DNA has physicochemical properties such as _{a basic skeleton and double chain melting temperature (Tm} value) as compared with natural nucleic acids such as naturally occurring DNA (natural DNA) or RNA (natural RNA). It is very similar in terms of molecular structure such as hydrogen bond and double helix structure, and it is possible not only to form a double helix structure between artificial DNAs but also to form a double helix structure with natural DNA.

However, the above-mentioned artificial DNA has almost the same affinity for nucleic acid as that of natural nucleic acid, or has lower affinity than natural nucleic acid. Therefore, when natural nucleic acids form a double helix structure, even if heat treatment is performed, the artificial DNA replaces the natural nucleic acid having a sequence corresponding to the artificial DNA in one of the double strands. In addition, it was difficult to form a double helix structure with a natural nucleic acid having a sequence complementary to artificial DNA, which is the other of the double strands.

Under these circumstances, as a result of diligent studies to solve the above problems, the present inventor can obtain a novel compound in which the sugar skeleton is bonded to a specific structure via a carbon-carbon triple bond. Further, when a polymer was formed using such a novel compound, it was surprisingly found that it could have a high affinity with other nucleic acids such as natural DNA, and the present invention was completed. I arrived.

That is, the present invention relates to the following inventions and the like.
[1] The following formula (A)

[In the formula (A), X is an aromatic ring group,
R ¹ is -OR ^1a (in the formula, R ^1a represents a hydrogen atom or a substituent) or a phosphoramidite group.
R ² is a hydrogen atom or a substituent and is
R ³ is a halogen atom or −OR ^3a (in the formula, R ^3a represents a hydrogen atom or a substituent). ]
The compound represented by.
[2] X is a heteroaromatic ring group {or a group having an aromatic heterocyclic skeleton [for example, a group having only an aromatic heterocyclic skeleton (aromatic heterocyclic skeleton, an aromatic heterocycle which may have a substituent). The compound according to the above [1], which is a ring skeleton)]}.
[3] X is a pyridine ring group [for example, a pyridine skeleton (a pyridine skeleton that may have a substituent)], a pyrimidine ring group [for example, a pyrimidine skeleton (a pyrimidine skeleton that may have a substituent)]. Alternatively, the compound according to the above [1] or [2], which is a purine ring group [for example, a purine skeleton (a purine skeleton that may have a substituent)].
[4] The compound according to any one of the above [1] to [3], wherein ^{R 3} is −OR ^3a.
[5] The compound according to any one of the above [1] to [4], wherein ^{R 1} and R ³ are hydroxy groups and R ^{2 is a hydrogen atom.}
[6] The compound according to any one of the above [1] to [4], wherein ^{R 1} is a hydroxy group or a phosphoramidite group, R ³ is −OR ^3a , and R ² and R ^{3a are protecting groups.} ..
[7] A polymer of the compound according to any one of the above [1] to [6].
[8] The following formula (B)

[In formula (B), X is an aromatic ring group,
R ^a is −OR ^b (in the formula, R ^b represents a hydrogen atom or a substituent).
R ³ is a halogen atom or −OR ^3a (in the formula, R ^3a represents a hydrogen atom or a substituent).
n is 2 or more.
A plurality of X, R ^a and R ³ may be be the same or different. ]
The polymer according to the above [7], which has a structural unit represented by.
[9] The polymer according to the above [7] or [8], which can form a base pair with a nucleic acid.
[10] When a base pair is formed between nucleic acids having the same length, the double-chain melting temperature of the base pair with the natural DNA is higher than that between the natural DNAs [7] to [9]. ] The polymer according to any one of.
[11] The polymer according to any one of the above [8] to [10], wherein n is 10 or more.
[12] A base pair containing the polymer according to any one of the above [7] to [11].
[13] The base pair according to the above [12], which forms a double helix structure.
[14] A nucleic acid detection material containing the polymer according to any one of the above [7] to [11].
[15] A nucleic acid drug containing the compound, polymer or base pair according to any one of the above [1] to [13].

In the present invention, a novel compound can be obtained. Such a novel compound can be suitably used as a raw material for a polymer, for example.

Further, in the present invention, a polymer containing a novel compound as a polymerization component can be obtained. Such polymers have an affinity for, for example, nucleic acids (such as natural nucleic acids) and are often capable of forming base pairs.

In particular, such a polymer can efficiently form base pairs with high affinity for nucleic acids (and thus excellent thermal stability).

Such a polymer can be suitably used as a nucleic acid detection material because, for example, nucleic acid can be easily and easily detected. Moreover, the above-mentioned polymer or base pair can be suitably used as a nucleic acid medicine.

FIG. 1A is a diagram showing UV-vis spectrum measurement results using compounds 5 and 7 obtained in Examples. FIG. 1B is a diagram showing the results of CD spectrum measurement using compounds 5 and 7. FIG. 1C is a diagram showing the measurement results of the double chain melting temperature using compounds 5 and 7. FIG. 2A is a diagram showing UV-vis spectrum measurement results using the compounds 5 and 13 obtained in the examples. FIG. 2B is a diagram showing the results of CD spectrum measurement using compounds 5 and 13. FIG. 2C is a diagram showing the measurement results of the double chain melting temperature using the compounds 5 and 13. FIG. 3A is a diagram showing the results of UV-vis spectrum measurement using the compounds 5 and 6 obtained in the examples. FIG. 3B is a diagram showing the results of CD spectrum measurement using compounds 5 and 6. FIG. 3C is a diagram showing the measurement results of the double chain melting temperature using the compounds 5 and 6.

<Compound>
The compound according to one aspect of the present invention is represented by the following formula (A).

[In the formula (A), X is an aromatic ring group,
R ¹ is -OR ^1a (in the formula, R ^1a represents a hydrogen atom or a substituent) or a phosphoramidite group.
R ² is a hydrogen atom or a substituent and is
R ³ is a halogen atom or −OR ^3a (in the formula, R ^3a represents a hydrogen atom or a substituent). ]

The aromatic ring group represented by X in the above formula (A) may be a group having an aromatic ring skeleton whose ring structure exhibits aromaticity, and may have an aromatic ring skeleton (even if it has a substituent). It may be one having only a good aromatic ring skeleton (aromatic ring skeleton (aromatic ring skeleton which may have a substituent)].

The aromatic ring skeleton may be a single ring or a ring containing a plurality of rings (for example, a condensed ring).

Examples of the aromatic ring skeleton include an aromatic hydrocarbon ring skeleton and an aromatic heterocyclic ring skeleton. The number of ring members of these aromatic ring skeletons may be, for example, 5 to 20, preferably 5 to 15, and more preferably 6 to 14.

Examples of the aromatic hydrocarbon ring skeleton include benzene, naphthalene, anthracene, and tetracene.

The aromatic heterocyclic skeleton may have an aromatic heterocyclic skeleton containing at least one heteroatom other than a carbon atom on the ring structure, and the heteroatom may be, for example, an oxygen atom. Examples include a sulfur atom and a nitrogen atom. A nitrogen atom is preferable because it facilitates the formation of hydrogen bonds.
The aromatic heterocyclic skeleton may have one or more heteroatoms. When having two or more heteroatoms, the heteroatoms may be the same or different.
When the aromatic heterocyclic skeleton contains a plurality of rings, the hetero atom may have at least one ring, and all the rings may have a hetero atom.

Examples of the aromatic heterocyclic skeleton include an aromatic heterocyclic skeleton containing an oxygen atom (for example, furan, benzofuran, isobenzofuran, etc.) and an aromatic heterocyclic skeleton containing a sulfur atom (for example, thiophene, benzothiophene). Aromatic heterocyclic skeleton containing nitrogen atoms {eg, aromatic heterocyclic skeletons containing only nitrogen atoms (eg, pyrrole, indol, isoindole, pyridine, quinoline, isoquinolin, imidazole, benzoimidazole, pyrazole, etc.) Indazole, pyrazine, pyrimidine, pyridazine, quinoxalin, quinazoline, cinnoline, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, purine, etc.), nitrogen atoms and other heteroatoms. Aromatic heterocyclic skeletons containing [eg, aromatic heterocyclic skeletons containing oxygen and nitrogen atoms (eg, oxazole, benzoxazole, etc.), aromatic heterocyclic skeletons containing sulfur and nitrogen atoms (eg, thiazole). , Benzothiazole, etc.), etc.], etc.} and the like. Among these, a pyridine skeleton, a pyrimidine skeleton, a purine skeleton, a pyrazine skeleton or a pyridazine skeleton is preferable, and a pyridine skeleton, a pyrimidine skeleton or a purine skeleton is more preferable.

The aromatic ring skeleton may have a substituent. Examples of the substituent include a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), a hydroxy group, an amino group, a nitro group, a cyano group, a phosphoric acid group (-H ₂ PO ₃ ), and phosphorus. Examples thereof include an acid ester group, an oxo group, a mercapto group (thiol group), a thioether group, a thiocarbonyl group, a sulfo group, and an organic group (for example, an organic group having 1 to 30 carbon atoms). Incidentally, the phosphate group, which at least partially is ionized (e.g., -HPO ^{₃ -,} _{-PO 3} ^2-, etc.).
The organic group may be a monovalent organic group or a divalent organic group.
The substituent [for example, hydroxy group, amino group, oxo group, organic group (for example, alkyl group), etc.] is a nucleic acid base as the group X (for example, adenin, guanine, amino group of cytosine, guanine, uracil, etc. It may constitute a skeleton (such as cytosine, an oxo group of timine, a methyl group of timine, etc.).
The compound having an oxo group as a substituent may be an isomer (keto-enol isomer) of a compound having a hydroxy group as a substituent depending on the substitution position and the like. Therefore, in the present specification, the compound having an oxo group (or hydroxy group) as a substituent is assumed to contain the isomer.
When the aromatic ring skeleton has a substituent, the aromatic ring skeleton may have the above substituents alone or in combination of two or more.

The organic group is not particularly limited as long as it has at least one carbon atom, and may have a bond containing a hetero atom. Examples of such a hetero atom include an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a silicon atom and the like.

The bond containing the hetero atom, for example, -O -, - CO -, - COO -, - S -, - NR 4 - (R 4 is a hydrogen atom or a monovalent organic group), - PR ⁵ - ^{(R 5} is a hydrogen atom or a monovalent organic ^{group), - SiR 6 R 7 -} (R 6 and ^{R 7} are hydrogen atom or a monovalent organic group). The bond containing the heteroatom may be present alone or in combination of two or more. The bond containing the heteroatom may be included in the monovalent organic group, and may be present at the bond end of the monovalent organic group, for example, and the bonds containing a plurality of heteroatoms are bonded to each other. May be adjacent to each other.

Examples of the ^{monovalent organic group represented by R 4} to R ⁷ include those similar to the monovalent organic group that the aromatic ring group represented by X may have. Be done. The ^{monovalent organic group represented by R 4} to R ⁷ may be, for example, a monovalent organic group having 1 to 30 carbon atoms, or a monovalent organic group having 1 to 20 carbon atoms. May be good.

Specific organic groups include, for example, a hydrocarbon group [for example, an alkyl group (for example, a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, an n-butyl group, etc.), an alkenyl group (for example, vinyl). Group, allyl group, etc.), alkynyl group (eg, propargyl group, etc.), cycloalkyl group (eg, cyclohexyl group, etc.), cycloalkenyl group (eg, cyclobutenyl group, cyclopentenyl group, etc.), aryl group (eg, phenyl group) , Etc.), aralkyl group (eg, benzyl group, etc.)], substituted hydroxy group [eg, alkoxy group (eg, methoxy group, ethoxy group, phenoxy group, etc.), silyloxy group (eg, tert-butyldimethylsilyloxy group, etc.), etc. Trialkylsilyloxy group, etc.), alkoxysilyl group (eg, methoxysilyl group, ethoxysilyl group, triisopropylsiloxymethyl (TOM) group, etc.)], acyl group (eg, acetyl group, benzoyl group, phenoxyacetyl group, etc.) ), Formyl group, carboxy group, ester group (eg, alkoxycarbonyl group such as methoxycarbonyl group, ethoxycarbonyl group), substituted mercapto group [eg, thioalkoxy group (eg, methylthio group)], thioacyl group (eg, eg, methylthio group). Thioacetyl group), thioester group (eg, methylthiocarbonyl group), substituted amino group [eg, alkylamino group (eg, methylamino group, dimethylamino group, etc.), aralkylamino group (eg, benzylamino, etc.), acylamino group (eg, benzylamino group) For example, an acetylamino group, a 2-phenoxyacetylamino group), etc.], a substituted phosphate group [for example, a phosphate ester group (for example, a group in which a hydrogen atom constituting the phosphate group is replaced with an alkyl group, etc.)], Examples thereof include a silyl group (for example, a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl (TBS) group, etc.) and the like.

The substituent is a protected (protected by a protecting group (leaving group)) functional group (for example, a hydroxy group, an amino group, a thio group, a phosphate group (-H ₂ PO ₃ ), a carboxyl group, etc.). May be good. The protecting group may usually be a leaving group (a group that can be removed).

Examples of the desorbing group include a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.) and an imidate group [for example, 2,2,2-trichloroacetimideyloxy group (group-OC (=)). NH) -CCl ₃ ), (N-phenyl) trifluoroacetimideyloxy group (group-OC (= NPh) -CF ₃ ), etc.], carbonate group (or carbonate ester group, for example 2,2) , 2-Trichloroethoxydicarbonyloxy group, etc.) and the like.

Typical substituents include, for example, halogen atoms (eg, fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), nitro group, cyano group, oxo group, and optionally substituted hydroxy group [eg, hydroxy group. Group, substituted hydroxy group (eg, alkoxy group, silyloxy group, etc.), etc.], optionally substituted mercapto group [eg, mercapto group (thiol group), substituted mercapto group (eg, thioalkoxy group, etc.), etc.], Substituentally substituted amino groups [eg, amino groups, substituted amino groups (eg, alkylamino groups, acylamino groups, etc.), etc.], optionally substituted phosphate groups, phosphate ester groups, hydrocarbon groups (eg, alkylamino groups, acylamino groups, etc.) For example, an alkyl group), an acyl group, an ester group and the like can be mentioned.

The substitution site (substitution position with respect to the aromatic ring skeleton) of the substituent is not particularly limited and may be a carbon atom on the aromatic ring skeleton. When the aromatic ring skeleton is an aromatic heterocyclic skeleton, the aromatic heterocycle may be used. It may be a heteroatom on the ring skeleton (eg, a nitrogen atom, etc.) or both.

Examples of such an aromatic ring skeleton (or aromatic ring group) include structures (skeletons, groups) represented by the following formulas (X-1) to (X-5).

(In the formula, R ^c is a substituent independently of each other.
R ^d is a hydrogen atom or a substituent and is
m1 is an integer from 0 to 5 and
m2 is an integer from 0 to 4 and
m3 is an integer of 0 to 3. )

Examples of the ^{substituents represented by R c} and R ^d include the same groups as those exemplified as the substituents that the aromatic ring group (aromatic ring skeleton) represented by X may have. ..

The R ^d is preferably an alkyl group having 1 to 5 carbon atoms, an amino group, or a protecting group for an amino group.

In the above formulas (X-1) to (X-5), the bonding position of the aromatic ring group is not particularly limited, and for example, the position excluding one hydrogen atom constituting the aromatic ring group (aromatic ring group or aromatic). Any carbon atom or the like constituting the ring skeleton may be used.

In particular, as the aromatic ring group, the pyridine skeleton, the pyrimidine skeleton and the purine skeleton have the following formulas (X-1-1) to (X-1-2), (X-2-1) to (X-2-4). ), (X-3-1) to (X-3-2) may be the structure (skeleton, group).

(In the formula, R ^c and R ^d are synonymous with the above formulas (X-1) to (X-3).
^Re and R ^f are hydrogen atoms or substituents and are
m4 is an integer from 0 to 4 and
m5 is an integer from 0 to 3 and
m6 and m7 are independently integers of 0 to 2. )

The ^{substituents represented by Re} and R ^f are the same as the monovalent groups among the groups exemplified as the substituents that the aromatic ring group (aromatic ring skeleton) represented by X may have. Can be mentioned.

In the above formula (A), the bond position (substitution or bond position with respect to the acetylene group, -C≡C-) of the aromatic ring group (aromatic ring skeleton) is not particularly limited, and for example, constitutes a ring of the aromatic ring skeleton. It may be either a carbon atom, a hetero atom constituting the ring of the aromatic heterocyclic skeleton among the aromatic ring skeletons, or a substituent having the aromatic ring skeleton, but typically constitutes the ring of the aromatic ring skeleton. Of the carbon atoms or the aromatic ring skeleton, it may be a hetero atom constituting the ring of the aromatic heterocyclic skeleton, particularly a carbon atom constituting the ring of the aromatic ring skeleton.
To give a specific example, when the aromatic ring skeleton is a pyridine skeleton, it is preferably bonded at the carbon atom at the 3- or 5-position of the skeleton, and is bonded at the carbon atom at the 5-position of the skeleton. Is more preferable. When the aromatic ring skeleton is a pyrimidine skeleton, it is preferably bonded at the carbon atom at the 5-position of the skeleton.
Examples of the aromatic ring group represented by X include an aromatic heterocyclic skeleton containing a nitrogen atom (an aromatic heterocyclic skeleton in which the nitrogen atom is a heteroatom constituting the ring of the aromatic ring skeleton). Preferably, a pyridine skeleton (pyridine skeleton which may have a substituent), pyrimidine skeleton (pyrimidine skeleton which may have a substituent), purine skeleton (purine skeleton which may have a substituent). , Pyrazine skeleton (pyrazine skeleton which may have a substituent) or pyridazine skeleton (pyridazine skeleton which may have a substituent), and pyridine skeleton, pyrimidine skeleton or purine skeleton is preferable.

Examples of the ^{substituent represented by R 1a} include the same groups as those exemplified as the substituents that the aromatic ring group represented by X may have. As the ^{substituent represented by R 1a} , a group having at least one phosphoric acid group (for example, monophosphoric acid, diphosphoric acid, triphosphate, etc.) is preferable.

The R ¹ is preferably a hydroxy group or a phosphoramidite group.

Examples of the ^{halogen atom represented by R 3} include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

Examples of the ^{substituents represented by R 2} and R ^3a include the same groups as those exemplified as the substituents that the aromatic ring group represented by X may have. The ^{substituents represented by R 2} and R ^3a may be the same or different. Further, the ^{substituent represented by R 2} and R ^3a is preferably a protecting group of a hydroxy group bonded to the 2-position and / or 5-position of the sugar skeleton, and the protecting group of the hydroxy group is unnecessary. When it becomes, it is preferable that it is a group that can be deprotected. Examples of the hydroxy group protective group include an acyl group (for example, an acetyl group, a benzoyl group, etc.), an aralkyl group (for example, a benzyl group, etc.), a trialkylsilyl group (for example, a trimethylsilyl group, a triethylsilyl group, a TBS group, etc.). ), A group having a trityl group and the like.

Examples of the ^{substituent (protective group) represented by R 2} and R ^3a include a trityl group and a silyl group (for example, a trimethylsilyl group, a triethylsilyl group and a tert-butyldimethylsilyl) which may have a substituent. (TBS) group, etc.), acyl group (eg, acetyl group, benzoyl group, phenoxyacetyl group, etc.), alkoxysilyl group (eg, methoxysilyl group, ethoxysilyl group, triisopropylsiloxymethyl (TOM) group, etc.), etc. Can be mentioned.

As the ^{substituent (protecting group) represented by R 2} , a trityl group which may have a substituent is preferable.

As the substituent contained in the trityl group, an alkoxy group having 1 to 5 carbon atoms is preferable, and a methoxy group is more preferable.

As the ^{substituent (protecting group) represented by R 3a} , a silyl group and an alkoxysilyl group are preferable, and a TBS group and a TOM group are more preferable.

The phosphoramidite group has a structure represented by the following formula (I).

In formula (I), R ^1b , R ^1c and R ^1d are independently hydrogen atoms or substituents.
^* Indicates the binding site with the sugar skeleton in the above formula (A). )

Examples of the ^{substituent represented by R 1b} , R ^1c and R ^1d include the same groups as those exemplified as the substituent that X of the above formula (A) may have.

The R ^1b is preferably a protecting group for the hydroxy group of phosphoric acid, and the protecting group is preferably a group that can be deprotected when it is no longer needed.

As the ^{substituent (protecting group) represented by R 1b} , a hydrocarbon group having 1 to 5 carbon atoms substituted with a cyano group is preferable.

As the ^{substituents represented by R 1c} and R ^1d , a hydrocarbon group having 1 to 10 carbon atoms which may be substituted is preferable, and a hydrocarbon group having 1 to 5 carbon atoms which may be substituted is more preferable. Preferably, an alkyl group having 1 to 5 carbon atoms which may be substituted is more preferable, and a 2-propyl group is particularly preferable.

The compound represented by the above formula (A) can be prepared, for example, using the compound represented by the following formula (II) as a raw material. As the compound represented by the following formula (II), a commercially available product may be used as long as it is available, or a synthesized (manufactured) compound may be used.

Wherein (II), R ^{1 'is} -OR ^1a' (wherein, ^{R 1a 'is} a hydrogen atom or a substituent.) Or a phosphoramidite group,
R ^2a is a hydrogen atom or a substituent and is
R ^{3 'is} a halogen atom or -OR ^3a' (wherein, ^{R 3a 'is} a hydrogen atom or a substituent.) It is. ]

Examples of the substituent represented by R ^{1a ',} those similar to the substituents represented by R ^1a in the formula (A).

Examples of the substituent represented by R ^2a and R ^{3a'include the same substituents represented by R 2} and R ^3a of the above formula (A), respectively.

As a method for preparing the compound represented by the above formula (A) from the compound represented by the above formula (II), a method known in the art may be referred to, and for example, as follows. It may be carried out in various steps.

The compound represented by the above formula (II) is reacted with the compound represented by the following formula (III) (coupling reaction) to prepare the compound represented by the following formula (IV).

In the coupling reaction, the ratio of the compound represented by the following formula (III) to be used is, for example, 0.3 mol or more (for example, 0.4 to 0.4 to 1 mol) of the compound represented by the above formula (II). It may be 10 mol) or more, preferably 0.5 mol or more (for example, 0.6 to 5 mol), and more preferably 0.6 mol or more (for example, 0.7 to 3 mol).

The coupling reaction may be carried out in the presence of a catalyst. Examples of the catalyst include palladium (for example, palladium chloride (PdCl ₂ ), dichlorobis (triphenylphosphine) palladium (II) (PdCl ₂ (PPh ₃ ) ₂ ), palladium acetate (Pd (OAc) ₂ ), tetrakis (triphenyl). Phosphine) palladium (0) (Pd (PPh ₃ ) ₄ ), tris (dibenzilidenacetone) palladium (0) -chloroadium adduct (Pd ₂ (dba) ₃ · CHCl ₃ ), palladium black, etc.), copper (eg, phosphine) palladium (0) (Pd (PPh 3) 4), copper (eg Copper iodide (I) (CuI), copper (I) oxide (Cu ₂ O), copper bromide (I) (CuBr), copper trifluoromethanesulfonate (I) benzene complex ((CuOTf) _2- C ₆ H ₆ ), copper (I) chloride (CuCl), etc.), platinum and the like. The catalyst may be used alone or in combination of two or more. As the catalyst, palladium, copper and a combination thereof are preferable, and a combination of palladium and copper is more preferable.

The coupling reaction may be carried out in the presence of a base. Examples of the base include amines (eg, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, etc.), carbonates (eg, alkali metal carbonates such as sodium carbonate, potassium carbonate, cesium carbonate, etc.), silver oxide, and the like. Examples thereof include pyridines (pyridine, picolin, etc.). The above bases may be used alone or in combination of two or more. As the base, amine is preferable, and diethylamine and triethylamine are more preferable from the viewpoint that they can also be used as a solvent.

The coupling reaction may be carried out in a solvent. The solvent is not particularly limited as long as it does not inhibit the coupling reaction, and is, for example, hydrocarbons [for example, aliphatic hydrocarbons (for example, hexane, heptane, cyclohexane, etc.), aromatic hydrocarbons (for example, for example). , Benzene, toluene, xylene, etc.], halogenated hydrocarbons (eg, methylene chloride, dichloromethane, chloroform, etc.), ethers [eg, chain ethers (eg, diethyl ether, etc.), cyclic ethers (eg, eg, diethyl ether, etc.) , Tetrahydrofuran, dioxane, etc.], esters (eg, ethyl acetate, etc.), amides [eg, N-substituted amides (N-alkylalkaneamides, such as N, N-dimethylformamide), etc.], alcohols (eg, N-alkylalkaneamides, etc.) , Alkanels such as methanol, ethanol and isopropanol), aprotonic polar solvents (eg, acetonitrile, acetone and the like) and the like.
These solvents may be used alone or in combination of two or more.

The reaction may be carried out at room temperature (or room temperature), cooling, or heating. Further, the reaction may be carried out in air or in an inert atmosphere (noble gas such as nitrogen or argon).

The reaction time is not particularly limited, but is, for example, 1 minute or more (for example, 2 minutes to 48 hours), preferably 3 minutes or more (for example, 4 minutes or more to 24 hours), and more preferably 5 minutes or more (for example, 10 minutes). It may be about minutes to 12 hours). The progress of the reaction may be confirmed by using a conventional method such as thin layer chromatography (TLC).

(In formula (III), X'is an aromatic ring group,
A is a leaving group. )

As the aromatic ring group represented by X', the same group as the aromatic ring group represented by X in the above formula (A) can be mentioned.

The desorbing group represented by A is particularly any group capable of forming a glycoside bond by reacting with the hydrogen atom (hydrogen atom of the terminal alkin) of the compound represented by the above formula (II). Not limited, for example, halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), imidate group [eg, 2,2,2-trichloroacetimideyloxy group (group-OC (= NH)). -CCl ₃ ), (N-phenyl) trifluoroacetimideyloxy group (group-OC (= NPh) -CF ₃ ), etc.], carbonate group (or carbonate ester group, for example 2,2,2) -Trichloroethoxydicarbonyloxy group, etc.) and the like.

Of these, a halogen atom is preferable because it is easy to adjust the binding site in X.

The compound represented by the above formula (III) is not particularly limited as long as the above A is a group that can be eliminated during the coupling reaction, and the convenience of preparing the compound represented by the above formula (III) or It can be appropriately set from the viewpoint of detachability. The bond site is preferably bonded to a carbon atom constituting the aromatic ring skeleton in the aromatic ring group represented by X', and in particular, when the aromatic ring group is a pyridine ring group or a pyrimidine ring group. , It is preferable that it is bonded to the carbon atom at the 5-position of these aromatic ring skeletons.

(In formula (IV), X'is synonymous with formula (III) above.
R ^{1 ', R 2a} and R ^3' is as defined in the above formula (II). )

As described above, among the compounds represented by the above formula (A), the compound represented by the above formula (IV) can be obtained. The reaction mixture (mixture containing the compound represented by the formula (IV)) may be subjected to the reaction described later as it is without separation (or recovery), or may be separated (or recovered).

Using the compound represented by the above formula (IV) obtained as described above as an intermediate or a precursor, the above formula (A) is carried out by yet another reaction (for example, deprotection reaction, phosphoramidite conversion reaction, etc.). The compound represented by may be prepared.

The deprotection reaction is not particularly limited as long as it can deprotect the substituents at the 2-position and / or the 6-position of the sugar skeleton of the compound represented by the above formula (IV). When both the 2- and 6-positions of the sugar skeleton have substituents, the 2- and 6-position substituents of the sugar skeleton may be deprotected in a single reaction, and one of the substituents may be removed. After protection, the remaining substituents may be deprotected. Further, in the deprotection reaction, when the aromatic ring group represented by X in the above formula (A) has a substituent (protecting group of amino group), the above substituent (protecting group of amino group) is further deprotected. It may have a reaction to the effect.

In the deprotection reaction, the compound represented by the following formula (V) is prepared by deprotecting the substituents at the 2- and / or 6-positions of the sugar skeleton of the compound represented by the above formula (IV). .. When the aromatic ring group represented by X has a substituent (protecting group), the compound represented by the following formula (V) can be obtained by further deprotecting the substituent (protecting group). It may be prepared.

(In the formula (V), X'is synonymous with the above formula (III).
R ^3b is a halogen atom or a hydroxy group. )

The deprotection reaction can be carried out using acid and / or fluoride ions (or by reacting with acid and / or fluoride ions).

Acids include organic acids [eg, carboxylic acids (eg acetic acid, citric acid, oxalic acid, tartaric acid, trichloroacetic acid, trifluoroacetic acid, etc.)], inorganic acids [eg, hydrogen halide (eg, hydrochloric acid, hydrogen fluoride). Acids, etc.), sulfuric acid, nitric acid, phosphoric acid, chromium acid, boric acid, sulfonic acid, etc.] and the like. The acid may be used alone or in combination of two or more.
In particular, as the acid, a carboxylic acid is preferable, and trichloroacetic acid or trifluoroacetic acid is more preferable.

Examples of the fluoride ion include tetra-n-butylammonium fluoride (TBAF), hydrofluoric acid, cesium fluoride and the like. Fluoride ions may be used alone or in combination of two or more.
In particular, TBAF is preferable as the fluoride ion.

Further, when deprotecting the substituent (protecting group of amino group) of the aromatic ring group represented by X', the deprotection reaction is carried out using (or reacting with) a base. Can be done.

Examples of the base include a base having a small degree of ionization (weak base) such as ammonia, a base having a large degree of ionization (strong base) such as sodium hydroxide, calcium hydroxide and barium hydroxide. The bases may be used alone or in combination of two or more.
In particular, as the base, a base having a small degree of ionization (weak base) is preferable, and ammonia (ammonia water) is more preferable.

The deprotection reaction may be carried out in a solvent. The solvent is not particularly limited as long as it does not inhibit the deprotection reaction, and examples thereof include the same solvents as those exemplified in the coupling reaction.

The reaction may be carried out at room temperature (or room temperature), cooling, or heating. Further, the reaction may be carried out in air or in an inert atmosphere (noble gas such as nitrogen or argon).

The reaction time is not particularly limited, but is, for example, 1 minute or more (for example, 2 minutes to 48 hours), preferably 3 minutes or more (for example, 4 minutes or more to 24 hours), and more preferably 5 minutes or more (for example, 10 minutes). It may be about minutes to 12 hours). The progress of the reaction may be confirmed by using a conventional method such as thin layer chromatography (TLC).

The reaction mixture (mixture containing the compound represented by the formula (IV)) may be subjected to another deprotection reaction as it is without separation (or recovery).
Conventional methods (eg, filtration, extraction, concentration, washing, adsorption, membrane separation, chromatography, etc.) can be used for separation (or purification) from the reaction mixture.

The phosphoramidite-forming reaction is not particularly limited as long as it can phosphoramidite the hydroxy group at the 3-position of the sugar skeleton of the compound represented by the above formula (IV).

In the phosphoramidite conversion reaction, a compound represented by the following formula (VI) is prepared by reacting a compound represented by the above formula (IV) with a phosphoramidite agent.

(In the formula ^{(VI), X ', R} 2a and R ^3' are as defined in the above formula (IV).
R ^1e is a phosphoramidite group. )

The phosphoramidite agent is not particularly limited as long as it can change the hydroxy group at the 2-position of the sugar skeleton of the compound represented by the above formula (IV) to a phosphoramidite group. Examples of the phosphoramidite agent include compounds in which a cyanoethoxy group and a dialkylamino group are bonded (for example, i-Pr ₂ NP (Cl) O (CH ₂ ) ₂ CN, etc.).

In the phosphoramidite conversion reaction, the ratio of the phosphoramidite agent used is preferably 0.8 mol or more (for example, 1 to 10 mol) with respect to 1 mol of the compound represented by the above formula (IV). May be 1 mol or more (for example, 1.2 to 7 mol), more preferably 1.5 mol or more (for example, 1.6 to 5 mol).

The phosphoramidite formation reaction may be carried out in a solvent. The solvent is not particularly limited as long as it does not inhibit the phosphoramidite formation reaction, and examples thereof include the same solvents as those exemplified in the coupling reaction.

The reaction may be carried out at room temperature (or room temperature), cooling, or heating. Further, the reaction may be carried out in air or in an inert atmosphere (noble gas such as nitrogen or argon).

The reaction time is not particularly limited, but is, for example, 1 minute or more (for example, 2 minutes to 48 hours), preferably 3 minutes or more (for example, 4 minutes or more to 24 hours), and more preferably 5 minutes or more (for example, 10 minutes). It may be about minutes to 12 hours). The progress of the reaction may be confirmed by using a conventional method such as thin layer chromatography (TLC).

The reaction mixture (mixture containing the compound represented by the formula (IV)) may be subjected to another deprotection reaction as it is without separation (or recovery).
Conventional methods (eg, filtration, extraction, concentration, washing, adsorption, membrane separation, chromatography, etc.) can be used for separation (or purification) from the reaction mixture.

The compound represented by the above formula (VI) obtained by the phosphoramidite reaction may be a mixture containing optical isomers (for example, diastereomers, enantiomers, etc.). Even in such a mixture, the asymmetric atom is eliminated when a polymer is formed via a phosphodiester bond.

<Polymer>
Another aspect of the present invention includes a polymer of the above compounds.

The polymer may be a polymer containing a plurality of compounds represented by the above formula (A) as a polymerization component, and examples of the bonds between the polymerization components include phosphodiester bonds and phosphorothioate bonds. .. Further, the polymer may be one capable of forming a base pair with nucleic acid. Here, the "nucleic acid" may be a naturally occurring DNA or RNA in a living body or the like, or may be an artificially synthesized natural nucleic acid such as a naturally occurring DNA or RNA. As long as it is a nucleic acid-like substance having a structure capable of forming a base pair with a naturally occurring nucleic acid, the above-mentioned nucleic acid-like substance may be artificially synthesized.

The reason why the polymer has a high affinity for natural nucleic acids is not limited to the following speculation, but the sugar skeleton in the above formula (A) and the specific structure represented by X are carbon. -By being linked via a carbon triple bond, the conformation of the acetylene group (-C≡C-) and the sugar skeleton is similar to the conformation of sugar in natural nucleic acids (particularly natural DNA). Therefore, it is presumed that the affinity (the force of interaction) with the natural nucleic acid (particularly, the natural DNA) is high.

Examples of such a polymer include those having a structural unit represented by the following formula (B).

[In formula (B), X is an aromatic ring group,
A is an oxygen atom or a sulfur atom,
R ^a is -OR ^b (R ^b indicates a hydrogen atom or a substituent).
R ³ is a halogen atom or −OR ^3a (in the formula, R ^3a represents a hydrogen atom or a substituent).
n is 2 or more.
A plurality of X, R ^a and R ³ may be be the same or different. ]

Examples of the aromatic ring group include those similar to X in the above formula (A).

The above A is preferably an oxygen atom from the viewpoint of structural unity. Further, the above-mentioned A is preferably a sulfur atom from the viewpoint of excellent resistance to RNA nuclease.

The -OR ^b is what is ionized ^- may be a (-O).

Examples of the ^{substituent represented by R b} include the groups exemplified as the substituent represented by ^{R 1a} in the above formula (A).

The above n is preferably 10 or more, more preferably 15 or more, and even more preferably 20 or more from the viewpoint of forming a double helix structure. The upper limit of n is not particularly limited, but may be 100, for example.

The substituent represented by R ^3a, those similar to the substituents represented by R ^3a in the above formula (A).

The method for forming the polymer from the compound represented by the above formula (A) is not particularly limited as long as the compound represented by the above formula (A) can form a crosslinked structure, and is a nucleic acid synthesis method. A known method (for example, a phosphoramidite method, a synthetic method via phosphorochloridate, a phosphite triester method, etc.) may be used. From the viewpoint of enhancing reactivity, those using the phosphoramidite method are preferable.

The phosphoramidite method may be a solid phase synthesis or a liquid phase synthesis. From the viewpoint of enhancing reactivity, it is preferable to carry out solid phase synthesis.

For solid-phase synthesis, it is preferable to use a solid-phase carrier from the viewpoint of simplification of washing operations and the like.

Solid-phase synthesis by the phosphoramidite method can be performed by, for example, the following steps. The solid-phase synthesis by the phosphoramidite method may be performed manually or by using an apparatus for automatic synthesis under computer control.

(1) A step of introducing a compound into a solid phase carrier, (2) a step of deprotecting a substituent (protecting group) at the 5'position of the sugar skeleton, (3) a cup at the hydroxy group at the 5'position and the 3'position. It includes a ringing step, (4) a step of capping the hydroxy group at the 5'position of the unreacted sugar skeleton, (5) a step of oxidizing or sulfurizing, and (6) a step of deprotecting and cutting out from the solid phase carrier.

[(1) Step of introducing the compound into the solid phase carrier]
(1) In the step of introducing the compound into the solid phase carrier, the compound represented by the formula (A) located on the 3'side is introduced into the solid phase carrier.

The solid-phase carrier can be appropriately changed depending on the arrangement of the polymer to be prepared, the degree of polymerization, the concentration of the compound represented by the above formula (A) to be used, and the like.

[(2) Step of deprotecting the 5'-position substituent (protecting group) of the sugar skeleton]
(2) In the step of deprotecting the 5'-position substituent (protecting group) of the sugar skeleton, only the 5'-position hydroxy group substituent (protecting group) of the sugar skeleton can be selectively deprotected. , Anything that does not eliminate the substituent (protecting group) of the hydroxy group at the 2-position of the sugar skeleton may be used.

The above step (2) can be performed in the same manner as the above deprotection reaction.

[(3) Step of coupling at the hydroxy group at the 5'position and the 3'position]
(3) The step of coupling at the hydroxy group at the 5'position and the coupling at the ^3'position is represented by the above formula (I) among the phosphoramidite groups represented by R 1e in the compound represented by the above formula (VI). ^{The site represented by -NR 1c} R ^1d in the structure is desorbed and reacts with the 5'-position hydroxy group of another compound represented by the above formula (VI) bonded to the solid phase carrier to form the formula. A polymer having a structural unit represented by (B) may be prepared.

The above step (3) may be carried out in the presence of an activator. Examples of the activator include compounds having a tetrazole ring (for example, 1H-tetrazole, 2-ethylthiotetrazole, 2-benzylthiotetrazole, etc.).

The ratio of the compound to be bonded to 1 mol of the compound (or polymer) bonded to the solid phase carrier is not particularly limited as long as the reaction proceeds sufficiently, and is, for example, 1.5. It may be up to 100 mol, preferably 2 to 80 mol, more preferably 3 to 50 mol.

The ratio of the activator to 1 mol of the compound (or polymer) bonded to the solid phase carrier is not particularly limited as long as the reaction proceeds sufficiently, and is, for example, 0.5 to 0.5. It may be 30 mol, preferably 0.8 to 25 mol, more preferably 1 to 20 mol.

[(4) Step of capping the hydroxy group at the 5'position of the unreacted sugar skeleton]
(4) The step of capping the hydroxy group at the 5'position of the unreacted sugar skeleton is the sugar skeleton of the unreacted compound (or polymer) among the compounds (or polymers) bonded to the solid phase carrier. Anything can be used as long as it can cap the hydroxy group at the 5'position of the above so that the polymer does not extend any more.

In the above step (4), for example, the reaction may be carried out in the presence of an acylating agent. The acylating agent may be any as long as it can acylate the hydroxy group at the 5'position of the sugar skeleton in the unreacted compound (or polymer), and for example, acyl halide (for example, acetyl chloride), etc. Examples thereof include carboxylic acid anhydrides (for example, acetic anhydride, propionic anhydride, etc.).

[(5) Step of oxidizing or sulfurizing]
(5) The step of oxidizing or sulphurizing is a step of converting the phosphite ester bond formed in the above step (3) into a phosphate ester by oxidation or a thiophosphate ester bond by sulphurizing.

In the above step (5), for example, the reaction may be carried out in the presence of an oxidizing agent or a sulfurizing agent.

The oxidizing agent is not particularly limited as long as it can convert a phosphite bond into a phosphate ester bond, and is, for example, halogen (for example, chlorine (Cl ₂ ), fluorine (F ₂ ), iodine). (I ₂ ), etc.) and the like.

The sulfide agent is not particularly limited as long as it can convert a phosphite ester bond into a thiophosphate ester bond, and is, for example, ((dimethylaminomethylidene) amino) -3H-1,2,4. - dithiazoline-3-thione (DTTT), 5-phenyl-3H-1,2,4-dithiazole-3-one, molecular sulfur _(S 8), phenylacetyl disulfide, Beaucage reagent and the like.

The above steps (2) to (5) can be repeated until a polymer having a desired degree of polymerization is obtained.
When sulfurization is carried out in the above step (5), the above steps (4) and the above (5) may be interchanged to carry out the reaction.

[(6) Steps of deprotection and cutting out from solid-phase carrier]
(6) In the steps of deprotection and cutting out from the solid-phase carrier, after obtaining a polymer having a desired degree of polymerization, the protecting groups present in the polymer are deprotected and the solid-phase carrier is cut out.

The above step (6) can be performed in the same manner as the above deprotection reaction, for example.

In the polymer, all the protecting groups may be deprotected in the step (6), or some protecting groups may remain.

<Base pair>
Yet another aspect of the present invention includes base pairs containing the above polymers.

The base pair may be any one as long as at least one of the double strands constituting the base pair is the polymer in the present invention, and the other of the double strands is a natural nucleic acid such as DNA or RNA that may exist in nature. , It may be a nucleic acid such as an artificially synthesized nucleic acid-like substance, or it may be a polymer in the present invention.

The above base pair may have a double chain formed over the entire length, or a double chain may be formed in a part of the entire length.

The polymer of at least one of the double chains in the base pair may have, for example, 50% or more, preferably 60% or more, and 70% or more, as complementarity to the other of the double chains. It is more preferable to have, more preferably 80% or more, and particularly preferably 90% or more. The upper limit of the complementarity is, for example, 100%.

Complementarity means that one polymer of a double chain and the other of the double chains have at least two hydrogen bond modes (relationship between a hydrogen bond donor (D) and an acceptor (A)) per base. Those having a complementary relationship. It may have two hydrogen bonds per base, or it may have three hydrogen bonds.

For example, the hydrogen bonding modes of the commonly known nucleobases adenine, guanine, cytosine, thymine, and uracil are DA, DDA, AAD, AD, and AD, respectively, and the other of the double chains is a natural nucleic acid. If this is the case, it suffices to have complementarity to these hydrogen bond modes so that two or more hydrogen bonds can be formed per base. Further, when the other of the double chains is an artificially synthesized nucleic acid-like substance and has a hydrogen bonding mode (for example, DAD, ADA, etc.) that does not exist in nature, the weight of one of the double chains is heavy. The coalescence may have a complementary hydrogen bond mode.

It is preferable that the polymer of at least one of the double chains in the above base pair has a hydrophobic property that can form a hydrophobic interaction with the other of the double chains.

When one polymer of the double chain has a palindromic sequence over the entire length or a part of the entire length, it may be a single polymer forming a base pair.

When the other of the double chains is a natural nucleic acid, the base pair preferably has a higher _{double chain melting temperature (Tm value) than the base pair formed by the natural nucleic acids.} .. _{For example, when a base pair is formed between nucleic acids having the same length, the Tm} value of the base pair formed by the natural DNA and the polymer is higher than that of the base pair formed by the natural DNAs. Is preferable.
When the other of the double chains is a natural nucleic acid, the base pair preferably has a higher _{Tm value than the base pair formed by the polymers.} For example, when a base pair is formed between nucleic acids having the same length, the base pair formed by natural DNA and the polymer is compared with the base pair formed by the polymers because it is easy to handle. It is preferable that the T _m value of is high.

The T _m value can be measured by, for example, a conventional method.
For example, the measurement can be performed under the following measurement conditions.
Measurement conditions: A 1: 1 buffer solution of artificial or natural nucleic acid oligomer (2 μM duplexes, 10 mM HEPES (pH 7.0), 10 mM MgCl ₂ , 100 mM NaCl) was prepared, and a double chain melting experiment was performed while monitoring the absorbance at 270 nm. (Temperature range: 10 to 80 ° C., temperature change: 1 ° C./1 minute).
Equipment used: V-560 UV / vis spectrophotometer (manufactured by JASCO Corporation)

The T _m value may be appropriately calculated according to conditions such as the length of the nucleic acid, the base sequence, and the measurement solution.

T _m values base pairs having the above natural DNA and polymer, and T _m values having base pairs of the native DNA with each other, the measurement conditions (the length of the nucleic acid, the hydrogen bonding pattern of the base sequence, the sample solution and the like) when performing a measurement in the same, for example, preferably T _m values having base pairs of the native DNA and the polymer is higher, the difference between the two in T _m values, even 10 ° C. or higher It is often preferably 15 ° C. or higher, and more preferably 20 ° C. or higher. _{The upper limit of the difference between the T m} values of the above two base pairs is not particularly limited, but may be, for example, 50 ° C. or lower, preferably 45 ° C. or lower.

The above base pair may form a double helix structure.

The fact that the base pairs form a double helix structure can be confirmed by a conventional method such as circular dichroism (CD) spectrum measurement.
For example, the measurement can be performed under the following measurement conditions.
Measurement conditions: Prepare a 1: 1 buffer solution of artificial or natural nucleic acid oligomer (2 μM duplexes, 10 mM HEPES (pH 7.0), 10 mM MgCl ₂ , 100 mM NaCl), and prepare 10, 20, 30, 40, 50, 60, 70. , 80 ° C. for circular dichroism (CD) spectrum measurements.
Equipment used: J-720WI spectroscopy (manufactured by JASCO Corporation)

<Nucleic acid detection material>
Yet another aspect according to the present invention includes a nucleic acid detection material containing the above polymer.

<Nucleic acid medicine>
Yet another aspect of the present invention includes a nucleic acid drug containing the polymer or base pair.

[Manufacturing Example 1]
Compound 8 (570 mg, 1.86 mmol) represented by the following formula, 5-iodo-1-methyluracil (300 mg, 1.19 mmol), tris (dibenzylideneacetone) dipalladium (0) -chloroform adduct (Pd _2). ( _{Dba) 3.} CHCl ₃ ) (50 mg, 0.048 mmol), triphenylphosphine (PPh ₃ ) (70 mg, 0.19 mmol), and copper (I) (CuI) iodide (5 mg, 0.026 mmol). Under an argon atmosphere, it was dissolved in N, N-dimethylformamide (DMF) (10 mL) and triethylamine (10 mL), and the mixture was stirred at 70 ° C. for 4.5 hours. After cooling to room temperature, the solvent was evaporated under reduced pressure, ethyl acetate was added to the residue, and the organic layer was washed with saturated brine. Then, the organic layer was dried over magnesium sulfate, and then the solvent was distilled off under reduced pressure. Purification by silica gel column chromatography (ethyl acetate: hexane = 1: 3 to 1: 1) gave compound 9 (450 mg, yield 64%) represented by the following formula as a yellow foamy substance.

(Compound 9)
Mp97-100 ° C; IR (KBr) 3483,3194,306,2952,2930,2856,2360,2232,2045,1693,1509,1252cm ^-1 ; ^{1 1} H NMR (500MHz, CDCl ₃ ) δ7.63 (s, 1H), 7.53 (d, J = 7.8Hz, 2H), 7.40 (dd, J = 12.8, 8.7Hz, 4H), 7.30-7.20 (m, 3H), 6.97 (s, 1H), 6.84-6.80 (m, 4H), 4.70 (d, J = 6.0Hz, 1H), 4.65 (t, J = 5.3Hz, 1H) ), 4.18-4.16 (m, 1H), 4.10-4.06 (m, 1H), 3.79 (s, 6H), 3.46 (dd, J = 10.3, 3) 0.0Hz, 1H), 3.41 (s, 1H), 3.05 (d, J = 6.0Hz, 3H), 2.70 (d, J = 4.1Hz, 1H), 0.94 (s) ^{, 9H), 0.23-0.19 (m,} 6H) ppm; 13 C {1 H} NMR (101MHz, CDCl 3) δ158.53.149.60,148.46,136.37,135.99 , 130.29, 128.37, 127.90, 126.82, 113.21, 113.17, 113.11, 99.05, 91.94, 86.27, 84.24, 77.98, 76 .41, 73.59, 73.41, 64.33, 55.32, 36.15, 25.84, 25.77, 25.74, 25.68, 18.14, 0.09, -4. 24, -4.87 ppm; HRMS calcd for MH ⁺ , C ₃₉ H ₄₆ N _{2 NaO 8} Si: 721.2916; found 721.2930.

(In the formula, DMTr represents a dimethoxytrityl group.
TBS represents a tert-butyldimethylsilyl group. )

(In the formula, DMTr and TBS are synonymous with compound 8.
Me represents a methyl group. )

[Manufacturing Example 2]
Similarly, compound 9 was resynthesized and the combined compound 9 (850 mg, 1.2 mmol) was dissolved in tetrahydrofuran (THF) (50 mL) in a 1 M tetrabutylammonium fluoride (TBAF) / THF solution (1). .5 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The solvent was evaporated under reduced pressure, ethyl acetate was added to the residue, and the organic layer was washed with saturated brine. The organic layer was dried over sodium sulfate, and the residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1). Dichloromethane (10 mL) was added to the obtained crude product 1 to prepare a solution, trichloroacetic acid (300 mg, 1.8 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered, and the solid was washed with dichloromethane to obtain a crude product 2. This was purified by reverse phase HPLC (methanol) for preparative use to obtain Compound 1 (130 mg, yield 38%) represented by the following formula as a pale yellow solid.

(Compound 1)
Mp113-118 ° C; IR (KBr) 3410,3058,2943,2359,2233,1684,1332 cm ^-1 ; ^{1 1} H NMR (500 MHz, DMSO-d ₆ ) δ8.06 (s, 1H), 5.76 (s) , 1H), 5.21 (s, 1H), 4.97 (s, 1H), 4.75 (s, 1H), 4.38 (d, J = 5.7Hz, 1H), 3.87 ( dt, J = 24.6, 4.9Hz, 2H), 3.68 (d, J = 4.6Hz, 1H), 3.54-3.41 (m, 2H), 3.24 (s, 3H) ) ^{Ppm; 13} C { ¹ H} NMR (126 MHz, DMSO-d ₆ ) δ150.86,150.71,96.97,85.29,78.59,76.63,72.81,71.86, 62.64, 55.44, 49.12, 36.09 ppm; HRMS calcd for MH ⁺ , C ₁₂ H ₁₄ N _{2 NaO 6} : 305.0744; found 305.0742; UV (H ₂ O, 25 ° C) ε ₂₆₀ = 3500 (L mol ^-1 cm ^-1 ).

(In the formula, Me is synonymous with compound 9.)

[Manufacturing Example 3]
Compound 8 (1.07 g, 1.86 mmol), 2- (N-phenoxyacetylamino) -5-iodopyridine (792 mg, 2.20 mmol), tetrakis (triphenylphosphine) palladium (0) (Pd (PPh ₃ )) ₄ ) (210 mg, 0.20 mmol) and CuI (10 mg, 0.053 mmol) were dissolved in acetonitrile (10 mL) and triethylamine (10 mL) under an argon atmosphere, and the mixture was stirred at 70 ° C. for 4 hours. After cooling to room temperature, the solvent was evaporated under reduced pressure, ethyl acetate was added to the residue, and the organic layer was washed with saturated sodium hydrogen carbonate and saturated brine. Then, the organic layer was dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. Purification by silica gel column chromatography (ethyl acetate: hexane = 1: 8 to 1: 5) gave compound 10 (1.0 g, yield 65%) represented by the following formula as a colorless foam substance.

(Compound 10)
Mp66-70 ° C; IR (KBr) 3535, 3396, 2952, 2929, 2857, 2359, 2045, 1707, 1601, 1510 cm ^-1 ; ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ8.94 (s, 1H), 8 .34 (d, J = 1.7Hz, 1H), 8.22 (d, J = 8.6Hz, 1H), 7.69 (dd, J = 8.6, 2.3Hz, 1H), 7. 50 (d, J = 7.4Hz, 2H), 7.40-7.33 (m, 6H), 7.24 (d, J = 8.0Hz, 2H), 7.19-7.16 (m) , 1H), 7.06 (t, J = 7.4Hz, 1H), 6.99 (d, J = 8.0Hz, 2H), 6.80-6.77 (m, 4H), 4.68 (D, J = 6.9Hz, 1H), 4.64 (d, J = 6.3Hz, 1H), 4.63 (s, 2H), 4.11 (q, J = 2.9Hz, 1H) , 4.08-4.06 (m, 1H), 3.76 (d, J = 1.7Hz, 6H), 3.44 (dd, J = 10.3, 2.9Hz, 1H), 3. 07 (dd, J = 10.3, 3.4Hz, 1H), 2.72 (d, J = 2.9Hz, 1H), 0.94 (s, 9H), 0.22 (s, 3H) ppm ¹³ C { ¹ H} NMR (101 _{MHz, CDCl 3} ) δ166.92, 158.52, 156.91, 150.87, 149.69, 145.02, 141.26, 136.20, 130.25 130, 23, 130.01, 129.21, 128.30, 127.90, 127.85, 126.84, 122.61, 116.08, 114.85, 113.50, 113.25, 113. 21,113.17,90.12.86.39,84.50,77.98,73.48,72.88,67.43,64.39,55.27,25.82,18.18, -4.26, -4.74 ppm; HRMS paid for MNa ⁺ , C ₄₇ H ₅₂ N _{2 NaO 8} Si: 823.3385; found 823.3392.

(In the formula, DMTr and TBS are synonymous with compound 8.)

[Manufacturing Example 4]
To a solution of the obtained compound 10 (500 mg, 0.50 mmol) in THF (10 mL) was added a 1 M TBAF / THF solution (1 mL), and the mixture was stirred at room temperature for 10 minutes. The solvent was evaporated under reduced pressure and purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1 to 2: 1). Methanol (30 mL) and 28% aqueous ammonia (30 mL) were added to the crude product 3, and the mixture was stirred at 55 ° C. for 13 hours. After cooling to room temperature, the solvent was evaporated under reduced pressure, dichloromethane was added to the residue, and the organic layer was washed with saturated brine. After drying the organic layer with sodium sulfate, the solvent was distilled off under reduced pressure. Dichloromethane (3 mL) was added to the residue to prepare a solution, trichloroacetic acid (300 mg, 1.8 mmol) was added thereto, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered, and the solid was washed with dichloromethane to obtain a crude product 4. This was purified by reverse phase HPLC (methanol) for preparative use to obtain Compound 2 (78.5 mg, yield 62%) represented by the following formula as a colorless solid.

(Compound 2)
Mp 69-74 ° C; IR (KBr) 3484, 3373, 2902, 2228, 1935, 1855, 1631, 1506, 1402 cm ^-1 ; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ7.98 (s, 1H), 7 .39 (d, J = 8.6Hz, 1H), 6.40 (d, J = 8.6Hz, 1H), 6.35 (s, 2H), 5.22 (s, 1H), 4.97 (S, 1H), 4.77 (s, 1H), 4.40 (d, J = 5.7Hz, 1H), 3.92-3.85 (m, 2H), 3.67 (q, J) ^{= 4.4Hz, 1H), 3.47-3.44 (} m, 2H) ppm; 13 C {1 H} NMR (126MHz, DMSO-d 6) δ159.71,151.69,140.03,108 .07, 106.34, 88.22, 85.29, 84.24, 76.69, 72.82, 71.63, 62.55 ppm; HRMS calcd for MH ⁺ , C ₁₂ H ₁₅ N ₂ O ₄ : 251.1206; found 251.1033; UV (H ₂ O, 25 ° C.) ε ₂₆₀ = 21000 (L mol ^-1 cm ^-1 ).

[Manufacturing Example 5]
Compound 9 (181 mg, 0.26 mmol) was dissolved in dichloromethane (3 mL) under an argon atmosphere, and i-Pr ₂ NP (Cl) O (CH ₂ ) ₂ CN (0.18 mL, 0.76 mmol) was added to the solution. 1-Methylimidazole (10 μL, 0.12 μmol) and N, N-diisopropylethylamine (1 mL) were added at room temperature and stirred for 2 hours. After stopping the reaction by adding a few drops of methanol, the solvent was evaporated under reduced pressure, ethyl acetate was added to the residue, and the organic layer was washed with saturated sodium hydrogen carbonate and saturated brine. After drying the organic layer with sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 2 to 2: 1) to prepare a diastereomeric mixture having the following formula. Compound 3 (142 mg, yield 61%) represented by (1) was obtained as a colorless foamy substance. Further purification was performed by preparative reverse phase HPLC (methanol).

(Compound 3)
Mp94-98 ° C; IR (KBr) 3201,3065,2962,2931,2857,2359,2251,1697,1509,1252cm ^-1 ; ^{1 1} H NMR (400 MHz, CDCl ₃ ) δ8.23 (s, 1H), 7 .53 (t, J = 6.6Hz, 2H), 7.45-7.37 (m, 4H), 7.24 (d, J = 3.2Hz, 2H), 7.21-7.17 ( m, 1H), 7.04 (d, J = 8.7Hz, 1H), 6.84-6.79 (m, 4H), 4.73-4.71 (m, 1H), 4.54 ( t, J = 4.6Hz, 0.5H), 4.49 (t, J = 4.4Hz, 0.5H), 4.46-4.42 (m, 1H), 4.21 (d, J) = 4.1Hz, 0.5H), 4.13 (d, J = 3.7Hz, 0.5H), 3.91-3.87 (m, 0.5H), 3.81-3.77 ( m, 6H), 3.67-3.61 (m, 0.5H), 3.57-3.42 (m, 3H), 3.05-3.02 (m, 3H), 2.65- 2.61 (m, 1H), 2.30 (t, J = 6.4Hz, 1H), 1.18-1.06 (m, 9H), 0.96 (d, J = 7.1Hz, 3H) ), 0.92 (t, J = 3.0Hz, 9H), 0.18-0.13 (m, 6H) ppm; 13 C {1 H} NMR (101MHz, CDCl 3) δ158.49,149. 78, 148.68, 148.61, 145.32, 130.37, 130.34, 130.26, 128.51, 128.44, 127.84, 126.78, 113.15, 113.13, 113.10, 100.00, 86.08, 82.54, 76.79, 76.65, 73.59, 63.77, 55.32, 55.30, 43.40, 43.28, 43. 06, 42.93, 36.10, 36.07, 25.96, 25.94, 24.79, 24.71, 24.63, 24.57, 20.16, 18.29, 0.09, -4.38, -4.42 ppm; HRMS calcd for MH ⁺ , C ₄₈ H ₆₃ N _{4 NaO 9} PSi: 921.3994; found 921.3998.

(In the formula, DMTr, TBS and Me are synonymous with compound 9.
i-Pr ₂ represents a diisopropyl group. )

[Manufacturing Example 6]
Compound 10 (250 mg, 0.31 mmol) was dissolved in dichloromethane (3 mL) under an argon atmosphere, and i-Pr ₂ NP (Cl) O (CH ₂ ) ₂ CN (0.18 mL, 0.76 mmol) was added to the solution. 1-Methylimidazole (10 μL, 0.12 μmol) and N, N-diisopropylethylamine (1 mL) were added at room temperature and stirred for 2 hours. After stopping the reaction by adding a few drops of methanol, the solvent was evaporated under reduced pressure, ethyl acetate was added to the residue, and the organic layer was washed with saturated sodium hydrogen carbonate and saturated brine. After drying the organic layer with sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 2 to 2: 1) to prepare a diastereomeric mixture having the following formula. Compound 4 (230 mg, yield 71%) represented by (2) was obtained as a colorless foamy substance. Further purification was performed by preparative reverse phase HPLC (methanol).

(Compound 4)
Mp48-52 ° C; IR (KBr) 3398, 2964, 2930, 2359, 2252, 1708, 1601, 1510, 1373 cm ^-1 ; ^{1 1} H NMR (400 MHz, CDCl ₃ ) δ8.96 (s, 1H), 8.37 (T, J = 2.3Hz, 1H), 8.23 (d, J = 8.7Hz, 1H), 7.73 (dt, J = 8.5, 2.4Hz, 1H), 7.53- 7.49 (m, 2H), 7.42-7.33 (m, 6H), 7.25-7.17 (m, 2H), 7.07 (t, J = 7.3Hz, 1H), 7.02-6.99 (m, 2H), 6.82-6.77 (m, 4H), 4.72 (t, J = 7.3Hz, 1H), 4.65-4.61 (m) , 3H), 4.55-4.52 (m, 0.5H), 4.25-4.16 (m, 1.5H), 3.93-3.86 (m, 1H), 3.77 -3.74 (m, 6H), 3.68-3.52 (m, 3H), 3.44 (ddd, J = 19.5, 10.5, 3.0Hz, 1H), 3.05- 3.00 (m, 1H), 2.65-2.61 (m, 1H), 2.31-2.27 (m, 1H), 1.29-1.11 (m, 9H), 0. 99 (d, J = 6.9Hz, 3H), 0.93 (td, J = 6.2, 3.1Hz, 9H), 0.19-0.14 (m, 6H) ppm; ¹³ C { ¹ H} NMR (101 _{MHz, CDCl 3} ) δ166.90, 158.49, 156.92, 150.91, 141.28, 130.24, 130.00, 128.39, 128.30, 127.89, 126 .83, 122.59, 116.26, 114.85, 113.48, 113.17, 72.55, 72.47, 67.42, 55.26, 42.86, 36.91, 26.00 , 25.95, 24.72, 24.67, 24.61, 18.46, 0.09, -3.10, -4.47 ppm; HRMS calcd for MH ⁺ , C ₅₆ H ₆₉ N ₄ NaO ₉ PSi : 1023.4464; found 1023.4472.

(In the formula, DMTr, TBS and i-Pr ₂ are synonymous with compound 3.)

[Manufacturing Example 7]
Using Compound 3, a 16-base artificial RNA oligomer was solid-phase synthesized using a 392 DNA / RNA Synthesizer (manufactured by Applied Biosystems) as an automatic DNA synthesizer. After completion of the automatic synthesis, an ammonia solution (28% aqueous ammonia: ethanol = 3: 1, 1 mL) was added to the solid phase, and the oligomer was cut out from the solid phase by shaking and stirring at 30 ° C. for 30 minutes. The solid phase was separated by filtration, and the solution was stirred at 40 ° C. for 8 hours and then lyophilized. Subsequently, dimethyl sulfoxide (DMSO) (100 μL) and triethylamine hydrofluorate (125 μL) were added to the residue, and the mixture was stirred with shaking at 65 ° C. for 2.5 hours. 3M sodium acetate solution (25 μL) and n-butanol (1 mL) were added, and the mixture was allowed to stand in a low temperature freezer for 30 minutes. After removing the supernatant, the remaining precipitate was washed with ethanol. Purification by reverse phase HPLC gave compound 5 (r (T ^* ) ₁₆ ).

(Compound 5)
MALDI-TOF MS: calcd for [MH ⁺ ], C ₁₉₂ H ₂₀₈ N ₃₂ O ₁₂₆ P ₁₅ : 5441.7; found 5449.5.

[Manufacturing Example 8]
A 16-base artificial RNA oligomer was solid-phase synthesized in the same manner as in Production Example 7 except that Compound 4 was used. After completion of the automatic synthesis, an ammonia solution (28% aqueous ammonia: ethanol = 3: 1, 1 mL) was added to the solid phase, and the oligomer was cut out from the solid phase by shaking and stirring at 30 ° C. for 30 minutes. The solid phase was separated by filtration, and the solution was shaken and stirred at 55 ° C. for 3 days, and then lyophilized. Subsequently, DMSO (100 μL) and triethylamine hydrofluorate (125 μL) were added to the residue, and the mixture was stirred with shaking at 65 ° C. for 2.5 hours. 3M sodium acetate solution (25 μL) and n-butanol (1 mL) were added, and the mixture was allowed to stand in a low temperature freezer for 30 minutes. After removing the supernatant, the remaining precipitate was washed with ethanol. Purification by reverse phase HPLC gave compound 6 (r ( ^Py A ^* ) ₁₆ ).

(Compound 6)
MALDI-TOF MS: calcd for [MH ⁺ ], C ₁₉₂ H ₂₀₈ N ₃₂ O ₉₄ P ₁₅ : 4929.9; found 4931.1.

[Reference example 1]
_{Compound 7 (d (A) 16} ), which is an oligomer having 16 bases of deoxyadenosine monophosphate (dAMP), which is a constituent of natural DNA, was obtained by requesting synthesis from Japan Genetic Research Institute.

[Reference example 2]
_{Compound 11 (d (T) 16} ), which is an oligomer having 16 bases of deoxythymidine monophosphate (dTMP), which is a constituent of natural DNA, was obtained by requesting synthesis from Japan Genetic Research Institute Co., Ltd.

[Reference example 3]
_{Compound 12 (r (U) 16} ), which is an oligomer having 16 bases of uridine monophosphate, which is a constituent of natural RNA, was obtained by requesting synthesis from Japan Genetic Research Institute Co., Ltd.

[Reference example 4]
J. Am. Chem. Soc. , 130, 8762-8768, 2008, ^{compound 13 (d (A *} ) ₁₆ ), which is an oligomer having 16 bases of ^{A *} , was synthesized as a constituent component of artificial DNA.

[Reference Example 5]
J. Am. Chem. Soc. , 130, 8762-8768, 2008, ^{compound 14 (d (T *} ) ₁₆ ), which is an oligomer having 16 bases of ^{T *} , was synthesized as a constituent component of artificial DNA.

<Base pair interaction analysis>
[Example 1]
A 1: 1 buffer solution of Compound 5 and Compound 7 (2 μM duplexes, 10 mM HEPES (pH 7.0), 10 mM MgCl ₂ , 100 mM NaCl) was prepared. The results of UV-vis spectrum measurement of the solution at 10 ° C. and 80 ° C. are shown in FIG. 1A. A V-560 UV / vis spectrophotometer (manufactured by JASCO Corporation) was used for the measurement of the UV-vis spectrum measurement. According to FIG. 1A, temperature-dependent and irreversible dark color effect and light color effect could be observed as in the case of natural nucleic acid. In addition, the results of circular dichroism (CD) spectrum measurements at 10, 20, 30, 40, 50, 60, 70, and 80 ° C. using the J-720WI spectrum spectrometer (manufactured by JASCO Corporation) are shown in FIG. 1B. Indicated. Further, the melting temperature curve was measured by monitoring the change in absorbance at 270 nm when the temperature was raised from 10 ° C. to 1.0 ° C. per minute. A V-560 UV / vis spectrophotometer (manufactured by JASCO Corporation) was used for the measurement of the melting temperature curve. _{The double chain melting temperature (Tm} value) was calculated from the maximum value obtained by first-derivating the melting temperature curve, and the result is shown in FIG. 1C. The T _m value was 71.0 ° C.

[Example 2]
The results of UV-vis spectrum measurement are shown in FIG. 2A and the results of CD spectrum measurement are shown in FIG. 2B in the same manner as in Example 1 except that compound 7 was changed to compound 13. Also, except for changing the compound 7 to compound 13, in the same manner as in Example, the results of the measurement of the melting temperature curve shown in FIG. 2C, was subjected to calculation in T _m value, T _m value It was 34.0 ° C.

[Example 3]
The results of UV-vis spectrum measurement are shown in FIG. 3A and the results of CD spectrum measurement are shown in FIG. 3B in the same manner as in Example 1 except that compound 7 was changed to compound 6. Also, except for changing the compound 7 to compound 6, in the same manner as in Example, the results of the measurement of the melting temperature curve shown in FIG. 3C, was subjected to calculation in T _m value, T _m value It was 55.0 ° C.

[Comparative Example 1]
_{When the T m} value was calculated in the same manner as in Example 1 except that the compound 5 was changed to the compound 11 _{, the T m} value was 48.5 ° C.

[Comparative Example 2]
_{When the T m} value was calculated in the same manner as in Example 1 except that the compound 5 was changed to the compound 12 _{, the T m} value was 39.0 ° C.

[Comparative Example 3]
_{When the T m} value was calculated in the same manner as in Example 1 except that the compound 5 was changed to the compound 14 _{, the T m} value was 40.5 ° C.

Since the polymer according to the present invention has an excellent affinity with natural DNA, even when the natural DNA forms a double chain, it forms a complementary hydrogen bond with one of the natural DNAs. It can form a base pair specifically with the polymer. For example, if it can be specifically bound to natural DNA or natural RNA related to a disease, it is expected to be applied to nucleic acid drugs such as antigene, antisense, and diagnostic agents.
In addition, the polymer according to the present invention can form a base pair with the polymer according to the present invention or other artificially synthesized nucleic acid. On the other hand, the base pair interaction is particularly excellent in thermal stability when it is a base pair of the polymer and natural nucleic acid (natural DNA) according to the present invention.
Currently, Peptide Nucleic Acid (PNA) or Locked Nucleic Acid (LNA) (also known as Bridged Nucleic Acid; BNA), which are typical artificially synthesized nucleic acids that are expected to be developed as anti-gene antisense drugs, There is a problem that each double strand is the most stable and difficult to handle, and the present invention may overcome the above problem.
Further, for example, if the polymer according to the present invention is immobilized on a DNA chip instead of the natural DNA, it is expected that the natural DNA as a sample can be efficiently captured, and a basic technique for increasing the detection sensitivity of the natural nucleic acid. It is also expected to be applied to DNA detection materials.

Claims (15)

1. The following formula (A)
   

   

   [In the formula (A), X is an aromatic ring group,
   R ¹ is -OR ^1a (in the formula, R ^1a represents a hydrogen atom or a substituent) or a phosphoramidite group.
   R ² is a hydrogen atom or a substituent and is
   R ³ is a halogen atom or −OR ^3a (in the formula, R ^3a represents a hydrogen atom or a substituent). ]
   The compound represented by.
2. The compound according to claim 1, wherein X is a heteroaromatic ring group.
3. The compound according to claim 1 or 2, wherein X is a pyridine ring group, a pyrimidine ring group or a purine ring group.
4. The compound according to any one of claims 1 to ³ , wherein R ³ is −OR 3a.
5. The compound according to any one of claims 1 to 4, wherein R ¹ and R ³ are hydroxy groups and R ^{2 is a hydrogen atom.}
6. The compound according to any one of claims 1 to 4, wherein R ¹ is a hydroxy group or a phosphoramidite group, R ³ is −OR ^3a , and R ² and R ^{3a are protecting groups.}
7. A polymer of the compound according to any one of claims 1 to 6.
8. The following formula (B)
   

   

   [In formula (B), X is an aromatic ring group,
   R ^a is −OR ^b (in the formula, R ^b represents a hydrogen atom or a substituent).
   R ³ is a halogen atom or −OR ^3a (in the formula, R ^3a represents a hydrogen atom or a substituent).
   n is 2 or more.
   A plurality of X, R ^a and R ³ may be be the same or different. ]
   The polymer according to claim 7, which has a structural unit represented by.
9. The polymer according to claim 7 or 8, which can form a base pair with nucleic acid.
10. Any of claims 7 to 9 in which the double-chain melting temperature of a base pair with a natural DNA is higher than that of a base pair between natural DNAs when a base pair is formed between nucleic acids having the same length. The polymer according to item 1.
11. The polymer according to any one of claims 8 to 10, wherein n is 10 or more.
12. A base pair containing the polymer according to any one of claims 7 to 11.
13. The base pair according to claim 12, which forms a double helix structure.
14. A nucleic acid detection material containing the polymer according to any one of claims 7 to 11.
15. A nucleic acid drug containing the compound, polymer or base pair according to any one of claims 1 to 13.

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