JP2021020883A - Photoresponsive nucleotide analog - Google Patents

Abstract

To provide a new photoreactive compound that can be used for a photoreaction technique of nucleic acids.SOLUTION: The present invention relates to an artificial nucleoside and an artificial nucleic acid, in which in a ribose skeleton, the 1st position is bound to a monovalent group of a nucleic acid base selected from the group consisting of a natural or non-natural nucleic acid base and a modified nucleic acid base obtained by modifying the natural or non-natural nucleic acid base with a protection group, and the 2nd position is bound to cyano vinyl carbazole via a single bond or a C1-C7 alkanediyl group.SELECTED DRAWING: None

Description

The present invention relates to photoresponsive nucleotide analogs (photoreactive nucleotide analogs) capable of photocrosslinking (photocrosslinking).

Basic techniques in the field of molecular biology include nucleic acid ligation and nucleic acid cross-linking. Nucleic acid ligation and cross-linking are used, for example, in combination with hybridization for gene transfer, base sequence detection, or, for example, inhibition of gene expression. To this end, nucleic acid ligation and cross-linking techniques are not limited to basic research in molecular biology, but include, for example, diagnosis and treatment in the medical field, or enzymes in the development and manufacture of therapeutic agents and diagnostic agents, industrial and agricultural fields. It is an extremely important technology used for the development and production of microorganisms.

As a nucleic acid photoreaction technique, a photolinking technique using 5-cyanovinyldeoxyuridine (Patent Document 1: Patent No. 3753938, Patent Document 2: Patent No. 3753942), modification having a 3-vinylcarbazole structure as a base site There is a photocrossing technique using a nucleoside (Patent Document 3: Patent No. 4814904, Patent Document 4: Patent No. 4940311).

3-cyanovinylcarbazole nucleoside (CNVK) is a molecule that can reversibly photocrosslink with pyrimidine bases in DNA and RNA in seconds (Non-Patent Document 1: Org. Lett., 2008). Examples of applications using this photocrosslinking reaction include optical control of antisense effect (Non-Patent Document 2: Biomater. Sci., 2014) and double doublex innovation for DNA (Non-Patent Document 3: Chem. Commun., 2017). It has been reported that it is a technology that can be used as a nucleic acid drug. In addition, stabilization of DNA structure (Non-Patent Document 4: J. Chem. Technology. Biotechnol., 2014) has also been reported, and it is a technology that can be used not only in the medical field but also in the field of nanotechnology. Has been reported.

Japanese Patent No. 3753938 Japanese Patent No. 3753942 Japanese Patent No. 4814904 Japanese Patent No. 4940311

Yoshimura Yoshimura, Kenzo Fujimoto, Ultrafast Reversible Photocrosslinking Reaction: Tower in Situ DNA Manipulation, Org. Lett. , 2008, 10 (15), 3227 Takashi Sakamoto, Atsoo Shigeno, Yuichi Ohtaki, Kenzo Fujimoto, Photo-regulation of genes expression exhibition inliving cells cells. Sci. , 2014, 2, 1154 Shigetaka Nakamura, Hayato Kawabata, Kenzo Fujimoto, Double duplex invasion of DNA integrated by ultrafast grafting method-cross-linking Commun. , 2017, 54, 7616 Shigetaka Nakamura, Kenzo Fujimoto, Creation of DNA Array Structure Equipped with heat resistance By Ultrafast Photocrossing. Chem. Technol. Biotechnol. , 2014, 89, 1086

Due to the importance of nucleic acid photochemical technology, new compounds that can be used in nucleic acid photochemical technology are further sought. An object of the present invention is to provide a novel photoreactive compound that can be used in a nucleic acid photoreaction technique.

The present inventor has enthusiastically searched for a photoreactive compound that can be used as a photoreactive cross-linking agent for a photoreactive technique for nucleic acids. For the carbon at the 2'position of the skeleton, a group having a 3-vinylcarbazole structure (X1) has a structure of -OL- (where X1 is a group described later and L is a linker part described later). We have arrived at the present invention by finding that the compound bound via the above is a photoreactive cross-linking agent that can be used in the photoreaction technique of such a nucleic acid.

Since the base portion of the nucleoside of the nucleoside has the structure of a conventional nucleobase, the compound thus obtained can form a hydrogen bond with the corresponding nucleobase in the same manner as the conventional nucleobase. That is, so-called base pairs can be formed.

On the other hand, the modified nucleoside having a conventional 3-vinylcarbazole structure at the base site cannot form a so-called base pair because the conventional nucleic acid base structure is not provided at the base portion. That is, the compound of the present invention is advantageous over the conventional modified nucleoside having a 3-vinylcarbazole structure at the base site in that it has the ability to form a base pair.

Since the compound according to the present invention has the ability to form a base pair depending on the nucleobase portion and at the same time has the photocrosslinking property due to the 3-vinylcarbazole structure, it can be variously modified and used for various purposes. Can be done. This characteristic structure of the compound according to the present invention can be an artificial nucleoside (nucleoside analog) because it has a structure in which a photocrosslinkable group is added to a natural nucleoside, and an artificial nucleotide (nucleoside analog) can be obtained. Nucleoside analogs), artificial nucleic acids (modified nucleic acids) containing such artificial nucleotides in their sequences, can be intermediates for their production, and of their synthesis. It can be a synthetic reagent used for this purpose. Then, when such an artificial nucleic acid forms a crosslink by a photoreaction, it becomes a photocrosslink (photocrosslink) formed from one strand of the double helix to the other strand, and thus is photoreactive. Nucleic acids can be used as a double helix photocrosslinking agent capable of specifically reacting with a desired sequence. When forming a double helix in this way, since the compound according to the present invention has the ability to form base pairs, accurate complementary chain formation without mismatch can be realized.

The compound according to the present invention is a photoreactive compound and initiates a photoreaction by light irradiation, but a compound that has been stable until then initiates a reaction in response to a signal of light irradiation. Photoreactivity is sometimes referred to as photoresponsiveness, emphasizing the meaning of doing so.

Therefore, the present invention includes the following (1) and the following.
(1)
Compound represented by the following formula I:
(I)
(However, in formula I,
X1 is a group represented by the following formula II:
(II)
(However, in Equation II,
R21 is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group of C2 to C7, or a hydrogen atom.
R22 and R23 are independently cyano groups, amide groups, carboxyl groups, C2-C7 alkoxycarbonyl groups, or hydrogen atoms.
R24 and R25 are independently hydrogen atom, halogen atom, -OH group, amino group, nitro group, methyl group, methyl fluoride group, ethyl group, ethyl fluoride group, and alkylsulfanyl group of C1 to C3, respectively. It is a group selected from the group consisting of. )

X2 is a nucleobase monovalent group selected from the group consisting of natural and non-natural nucleobases and modified nucleobases in which they are modified with protecting groups.
L is a single bond or an alkanediyl group of C1 to C7.

R11 is _one -OQ,
R12 is _two -OQ,

Q ₁ is
Hydrogen atom;
Phosphate group formed integrally with O bonded to Q ₁ ;
Nucleotides, nucleic acids or peptide nucleic acids linked via phosphodiester bonds formed by phosphate groups formed integrally with O that binds to Q ₁ ; and
Protecting groups selected from:
Trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, trimethylsilyl group, triethylsilyl group, t-butyldimethylsilyl group, acetyl group, benzoyl group;
A group selected from the group consisting of

Q ₂ is
Hydrogen atom;
Phosphate group formed integrally with O bonded to Q ₂ ;
Nucleotides, nucleic acids or peptide nucleic acids linked via phosphodiester bonds formed by phosphate groups formed integrally with O that binds to Q ₂ ; and
Protecting groups selected from:
2-Cyanoethyl-N, N-dialkyl (C1-C4) phosphoramidite group, methylphosphonamidite group, ethylphosphonamidite group, oxazaphosphoridine group, thiophosphite group, TEA salt of -PH (= O) OH, -PH (= O) OH DBU salt, -PH (= S) OH TEA salt, -PH (= S) OH DBU salt;
It is a group selected from the group consisting of. ).
(2)
The compound according to (1), wherein the group represented by the formula II is a group represented by the following formula IIa:
(IIa)
(However, in Formula IIa, R21, R22, R23 and R24 are the groups described above in Formula II).
(3)
The compound according to any one of (1) to (2), wherein X2 in the formula I is adenine, guanine, thymine, cytosine, or uracil.
(4)
An artificial nucleoside comprising the compound according to any one of (1) to (3), wherein R11 and R12 in the formula I are hydroxyl groups.
(5)
At least one of Q ₁ and Q ₂
Any of (1) to (3), which is a nucleotide or nucleic acid linked via a phosphate diester bond formed by a phosphate group formed integrally with O bound to Q ₁ or Q _2. An artificial nucleic acid consisting of the compounds described in.
(6)
A photoreactive cross-linking agent comprising the compound according to any one of (1) to (5).
(7)
A method for forming a photocrosslink with a nucleobase having a pyrimidine ring using the compound according to any one of (1) to (6).

The present invention includes a method of forming a photocrosslink between the compound and a nucleic acid base having a pyrimidine ring using the above compound, and includes a method of producing a photocrosslink by the method for forming the photocrosslink. The method for producing a photocrosslinked compound crosslinked by the photocrosslinking is included. When a photocrosslink is formed between a modified nucleic acid containing the above compound in the base sequence and a modified nucleic acid containing a nucleic acid base having a pyrimidine ring in the base sequence, the crosslink is formed by the above photocrosslink. The method for producing a photocrosslinked product includes a method for producing a photocrosslinked double-stranded nucleic acid.

The present invention provides a novel compound that serves as a photoreactive crosslinker that can be used in nucleic acid photoreaction techniques. Since the base portion of the nucleoside has the structure of a conventional nucleobase, the compound according to the present invention can form a hydrogen bond with the corresponding nucleobase in the same manner as the conventional nucleobase, that is, a so-called base. Can form a pair

FIG. 1-1 is an explanatory diagram showing the first half of the synthetic route of Scheme 1 for the synthesis of 2'- ^CNV K-C _n- A. FIG. 1-2 is an explanatory diagram showing the latter half of the synthesis route of Scheme 1 for the synthesis of 2'- ^CNV K-C _n- A. FIG. 2 is an MS spectrum of an oligonucleic acid containing 2'- ^CNV K. FIG. 3A is a chromatograph obtained by HPLC analysis after 0 sec of light irradiation (before light irradiation). FIG. 3B is a chromatograph obtained by HPLC analysis after 5 seconds of light irradiation. FIG. 4 shows the MS spectrum of the photocrosslinked product. FIG. 5-1 is a graph of the melting temperature curve of the duplex by the nucleic acid having A as the base at a specific position in the sequence. FIG. 5-2 is a graph of the melting temperature curve of the duplex by the nucleic acid having ^CNV K as the base at a specific position in the sequence. FIG. 5-3 is a graph of the melting temperature curve of a double strand with a nucleic acid having 2'- ^CNV K-C _6- A as a base at a specific position in the sequence. FIG. 6-1 is an explanatory diagram showing the first half of the synthetic route of Scheme 2 for the synthesis of 2'- ^CNV K-C _6- G. FIG. 6-2 is an explanatory diagram showing the latter half of the synthesis route of Scheme 2 for the synthesis of 2'- ^CNV K-C _6- G. FIG. 7-1 is an explanatory diagram showing the first half of the synthetic route of Scheme 3 for the synthesis of 2'- ^CNV K-C ₆ -C. FIG. 7-2 is an explanatory diagram showing the latter half of the synthesis route of Scheme 3 for the synthesis of 2'- ^CNV K-C ₆ -C. FIG. 8-1 is an explanatory diagram showing the first half of the synthetic route of Scheme 4 for the synthesis of 2'- ^CNV K-C _6- T. FIG. 8-2 is an explanatory diagram showing the latter half of the synthesis route of Scheme 4 for the synthesis of 2'- ^CNV K-C _6- T.

The present invention will be described in detail below with reference to specific embodiments. The present invention is not limited to the specific embodiments listed below.

[Compound structure]
The compounds of the present invention include compounds represented by the following formula I:
(I)

In formula I, X1 is a group represented by formula II:
(II)

In Formula II, R21 is a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom.

In Formula II, R22 and R23 are independently cyano groups, amide groups, carboxyl groups, C2-C7 alkoxycarbonyl groups, or hydrogen atoms.

In a preferred embodiment, as the alkoxycarbonyl group, for example, C2 to C7, preferably C2 to C6, C2 to C5, C2 to C4, more preferably C2 to C3, and particularly preferably C2 can be used.

In a preferred embodiment, R21 can be a cyano group, an amide group, a carboxyl group, or an alkoxycarbonyl group of C2 to C7, and R22 and R23 can be hydrogen atoms.

In Formula II, R24 and R25 independently represent a hydrogen atom, a halogen atom, a -OH group, an amino group, a nitro group, a methyl group, a methyl fluoride group, an ethyl group, an ethyl fluoride group, and C1 to C3. It is a group selected from the group consisting of alkylsulfanyl groups of.

Examples of the halogen atom include Br, Cl, F, and I atoms.

Examples of the methyl fluoride group include -CH ₂ F, -CHF ₂ , and -CF ₃ .

The fluorinated ethyl group, for example _{-CH 2 -CH 2 F, -CH 2} -CHF 2, -CH 2 -CF 3, -CHF-CH 3, -CHF-CH 2 F, -CHF-CHF 2, - _{CHF-CF 3, -CF 2 -CH} 3, -CF 2 -CH 2 F, -CF 2 -CHF 2, can be mentioned -CF ₂ -CF _3.

Examples of the alkylsulfanil group of C1 to C3 include -CH _2- SH group, -CH _2- CH _2- SH group, -CH (SH) -CH ₃ group, and -CH _2- CH _2- CH _2- SH group. , -CH _2- CH (SH) -CH ₃ groups, -CH (SH) -CH _2- CH ₃ groups can be mentioned.

In a preferred embodiment, R24 and R25 can each independently be a hydrogen atom, a halogen atom, -NH ₂ groups, -OH group, or -CH ₃ group, preferably a hydrogen atom. Can be done.

In a preferred embodiment, R24 can be the above-mentioned group and R25 can be the hydrogen atom at the same time.

In a preferred embodiment, R24 and R25 independently number the carbon atom to which the nitrogen atom is bonded in the leftmost 6-membered ring of Formula II as the C1 position and clock the carbon atom of the 6-membered ring. When the numbers are numbered C2, C3, C4, C5, and C6 in order, they can be used as substituents for carbon atoms at any of the C2, C3, C4, and C5 positions. it can.

In a preferred embodiment, R24 and R25 can be substituents at the C3 and C4 positions, respectively.

In a preferred embodiment, R24 can be a substituent at the C3 position and R25 can be a hydrogen atom at the C4 position. In this case, the group represented by the formula II becomes the group represented by the formula IIa described later.

In Formula I, X2 is a monovalent group of nucleobases selected from the group consisting of natural and unnatural nucleobases and modified nucleobases in which they are modified with protecting groups. Examples of the natural nucleobase include adenine, guanine, thymine, cytosine, uracil, inosin, and methylcytosine, and preferably adenine, guanine, thymine, cytosine, and uracil. Examples of the unnatural nucleic acid base include 2-aminopurine and nitroindole. The modified nucleobase modified by a protecting group means a modified nucleobase in which the amino group of the nucleobase is modified by a known protecting group. Examples of the amino group protecting group include an acetyl group, a benzoyl group, a phenoxyacetyl group, and an isopropylphenoxyacetyl group.

In a preferred embodiment, in Formula I, as the monovalent group of the natural nucleobase of X2, a group selected from the groups shown below and a group in which these are modified by a protecting group can be used:
(A)

(G)

(T)

(C)

(U)

In formula I, L represents the linker moiety and can be, for example, a single bond or an alkanediyl group of C1-C7. The case where L is a single bond means a state in which O and X1 to be bonded to L are bonded by a single bond.

As the alkanediyl group, for example, C1 to C7, preferably C2 to C6 can be used, and particularly preferably a pentyl group and a hexyl group.

In formula I, R11 is _one —OQ.

Q ₁ is
Hydrogen atom;
Phosphate group formed integrally with O bonded to Q ₁ ;
Nucleotides, nucleic acids or peptide nucleic acids linked via phosphodiester bonds formed by phosphate groups formed integrally with O that binds to Q ₁ ; and
Protecting groups selected from:
Trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, trimethylsilyl group, triethylsilyl group, t-butyldimethylsilyl group, acetyl group, benzoyl group;
It can be a group selected from the group consisting of.

In a preferred embodiment, Q ₁ can be a hydrogen atom.

In a preferred embodiment, Q ₁ can be a nucleotide or nucleic acid linked via a phosphodiester bond formed by a phosphate group formed integrally with O that binds to Q _1. ..

In a preferred embodiment, Q ₁ can be the protecting group described above, preferably a dimethoxytrityl group, a trityl group, a monomethoxytrityl group, a trimethoxytrityl group, and particularly preferably dimethoxy. It can be a trityl group.

In formula I, R12 is _two —OQ.

Q ₂ is
Hydrogen atom;
Phosphate group formed integrally with O bonded to Q ₂ ;
Nucleotides, nucleic acids or peptide nucleic acids linked via phosphodiester bonds formed by phosphate groups formed integrally with O that binds to Q ₂ ; and
Protecting groups selected from:
2-Cyanoethyl-N, N-dialkyl (C1-C4) phosphoramidite group, methylphosphonamidite group, ethylphosphonamidite group, oxazaphosphoridine group, thiophosphite group, TEA salt of -PH (= O) OH, -PH (= O) OH DBU salt, -PH (= S) OH TEA salt, -PH (= S) OH DBU salt;
It can be a group selected from the group consisting of.

The 2-cyanoethyl-N, N-dialkyl (C1-C4) phosphoramidite group has the following structure.
The R group and the R'group, which are the dialkyl groups, can be C1 to C4 alkyl groups, respectively. Examples of such 2-cyanoethyl-N, N-dialkyl (C1-C4) phosphoramidite groups include 2-cyanoethyl-N, N-dimethylphosphoromidite group, 2-cyanoethyl-N, N-diethylphosphoro. Examples include an amidite group and a 2-cyanoethyl-N, N-diisopropylphosphoromidite group.

The methylphosphonamidite group has the following structure and has the following structure.
The R group and the R'group can be hydrogen atoms or alkyl groups of C1 to C4, respectively.

The ethylphosphonamidite group has the following structure and has the following structure.
The R group and the R'group can be hydrogen atoms or alkyl groups of C1 to C4, respectively.

The oxazaphosphoridine group has the following structure and has the following structure.

In the above structure, a substituent in which a hydrogen atom is substituted with an alkyl group of C1 to C4 is also included.

The thiophosphite group has the following structure and
In the above structure, a substituent in which a hydrogen atom is substituted with an alkyl group of C1 to C4 is also included.

The TEA salt of −PH (= O) OH and the TEA salt of −PH (= S) OH are the respective triethylamine (TEA) salts.

The -PH (= O) OH DBU salt and the -PH (= S) OH DBU salt are the respective diazabicycloundecene (DBU) salts.

In a preferred embodiment, Q ₂ can be a hydrogen atom.

In a preferred embodiment, Q ₂ can be a nucleotide or nucleic acid linked via a phosphodiester bond formed by a phosphate group formed integrally with O that binds to Q _2. ..

In a preferred embodiment, Q ₂ can be the protecting group described above, preferably 2-cyanoethyl-N, N-dialkyl (C1-C4) phosphoramidite group, oxazaphosphoridine group, thio. It can be a phosphite group, particularly preferably a 2-cyanoethyl-N, N-diisopropylphosphoromidite group.

In a preferred embodiment, the group represented by the formula II can be the group represented by the following formula IIa:
(IIa)

In a preferred embodiment, in Formula IIa, R21, R22, R23 and R24 can be the groups described above in Formula II.

[Nucleoside analog (artificial nucleoside)]
In a preferable embodiment, in formula I, R11 is as -O-Q ₁ group, the Q ₁ may be a hydrogen atom, i.e. can be a R11 a hydroxyl group, the R12 simultaneously, -O- as Q ₂ groups, the Q ₂ can be a hydrogen atom, namely the R12 can be a hydroxyl group. In this case, the compound of formula I is a nucleoside analog (artificial nucleoside) represented by the following formula Ia:
(Ia)

In a preferred embodiment, in Formula Ia, X1, X2 and L can be the groups described above in Formula I.

That is, the compound represented by the above formula I can be a photoreactive nucleotide analog (photoreactive artificial nucleotide) having a characteristic structure.

[Nucleotide analog (artificial nucleotide)]
In a preferred embodiment, in Formula I, Q ₁ can be a phosphate group formed integrally with O bonded to Q ₁ , and Q ₂ can be a hydrogen atom. This structure becomes a nucleotide analog (artificial nucleotide) in which a phosphate group is added to a nucleotide analog (artificial nucleotide) represented by the formula Ia. That is, the compound represented by the above formula I can be a photoreactive nucleotide analog (photoreactive artificial nucleotide) having a characteristic structure.

[Modified nucleic acid]
In a preferred embodiment, Q _{1 is referred} to as Q ₂ as a nucleotide or nucleic acid linked via a phosphodiester bond formed by a phosphate group formed integrally with O that binds to Q _1. , Can be a nucleotide or nucleic acid linked via a phosphodiester bond formed by a phosphate group formed integrally with O that binds to Q ₂ . That is, the compound represented by the above formula I can be a modified nucleic acid or a modified oligonucleotide in which a photoreactive artificial nucleotide analog having a characteristic structure is incorporated into the sequence. In the present invention, the photoreactively modified nucleic acid and the photoreactively modified oligonucleotide thus prepared may be collectively referred to as a photoreactively modified nucleic acid.

In the modified nucleic acid according to the present invention, a photoreactive artificial nucleotide analog having a characteristic structure may be located at the end of the sequence, in which case only either side of Q ₁ or Q ₂ is located. It is a modified nucleotide or nucleic acid linked via a phosphodiester bond formed by a phosphate group formed integrally with O that binds to Q ₁ or Q ₂ . Further, a peptide nucleic acid can be used instead of the above nucleic acid to obtain a photoreactive modified peptide nucleic acid in which a photoreactive artificial nucleotide analog having a characteristic structure is incorporated into the sequence.

[Compound structure]
The compound according to the present invention has a skeletal structure represented by the formula I, and in the case of a natural nucleoside or nucleotide, the hydroxyl group is bonded to the carbon at the 2'position of ribose. It has a structure in which an -OL-X1 group is bonded instead of the hydroxyl group bonded to the C at the'position. The X1 group in this -OL-X1 group was so huge that at first glance it was completely unpredictable that any beneficial effect would be produced by arranging it in such a position. Rather, when the X2 group in formula I is a natural nucleobase such as A, T, C, G, and the natural nucleobase attempts to form a base pair by hydrogen bonding, the presence of a huge X1 group. Was expected to become a steric hindrance and hinder base pair formation. However, when this X1 group was bound as a structure represented by the formula I via the linker portion L, it was surprisingly found that it did not interfere with the base pairing by the nucleobases arranged as the X2 group. Furthermore, it was found that the X1 group bonded as a structure represented by the formula I via the linker portion L can exhibit photoreactivity.

Then, when the compound according to the formula I is formed as a single-stranded modified nucleic acid, a double helix can be formed with the single-stranded nucleic acid complementary thereto. Stabilized and sequence-specific, including base pairing with nucleobases located as X2 groups, and double helix formation not hindered by -OL-X1 groups. all right. Furthermore, when the double helix is formed in this way, the X1 group bonded as the structure represented by the formula I exhibits photoreactivity and is a complementary single strand that is the partner of the double helix formation. It was found that a crosslink can be formed by a photoreaction with a specific base in the sequence of the nucleic acid of the above, that is, a photocrosslink can be formed between the modified nucleic acid and the complementary nucleic acid.

[Reagent for producing modified nucleic acid]
In a preferable embodiment, the Q _1, may be a protective group described above, the Q _2, phosphate groups are formed in an O integral to bind to Q _2, and O that binds to Q ₂ It can be a nucleotide or nucleic acid linked via a phosphodiester bond formed by a phosphate group formed integrally, or the protecting group described above. That is, the compound represented by the above formula I can be used as a reagent for producing a photoreactive modified nucleic acid (reagent for synthesis).

In a preferred embodiment, Q ₁ can be the protecting group described above, and Q ₂ can be a phosphate group formed integrally with O bound to Q ₂ , or the protecting group described above. can do. As is well known, a compound having such a structure can be used as a monomer for nucleic acid synthesis, and can be a reagent that can be used by a known DNA synthesizer, for example, phosphoramidite. It can be a reagent for modifying nucleic acid synthesis (monomer for modifying nucleic acid synthesis) that can be used by the method and the H-phosphonate method.

Further, the Q _1, as a protecting group as described above, nucleotide or nucleic acid Q _2, are linked via a phosphodiester bond to be formed by a phosphate group which is formed by a O integral to bind to Q ₂ The structure is not a so-called monomer, but a modified nucleic acid. In such a case, it can be used as a production reagent (synthesis reagent) for synthesizing from there and extending the chain length.

[Photoreactive cross-linking agent]
In the compound according to the present invention, the X1 group portion can form a crosslink by a photoreaction. When the compound according to the present invention is formed as a single-stranded modified nucleic acid, a double helix can be formed with the single-stranded nucleic acid complementary thereto, and the X1 group portion forms a crosslink by a photoreaction. As a result, a photobridge (optical crosslink) between the chains formed from one chain of the double helix to the other can be formed. That is, the compound according to the present invention can be used as a photoreactive cross-linking agent.

[Formation of photocrosslinks]
When the compound according to the present invention is used as a modified nucleic acid and the modified nucleic acid according to the present invention is used as a single-stranded nucleic acid, it hybridizes with a single-stranded nucleic acid complementary thereto to form a double helix. Can be done. In the formation of the double helix, for the nucleic acid base at the position where the base pair should be formed in the complementary strand with respect to the X2 group portion in the formula I, a nucleic acid base that accurately causes the known base pair formation is used. Be selected. In other words, the exact complementary strand is selected with high specificity. When the formed double helix is irradiated with light, crosslinks can be formed by a photoreaction between the nucleic acid chains forming the double helix. This photocrosslinking occurs between the X1 group moiety in the modified nucleic acid and the nucleic acid base at a specific position in the base sequence of the companion complementary strand. The nucleobase in the complementary strand that is photocrosslinked as the counterparty to the X1 group portion may be referred to as a photocrosslinked nucleobase. The position of the photocrosslinked nucleic acid base in the base sequence of the complementary strand is based on the nucleic acid base located 5'-terminal by 1 base from the position of the nucleoside analog having the X2 group portion in the base sequence of the modified nucleic acid. This is the position to form a pair. In other words, this photocrosslink is located on the 3'end side of the base sequence of the complementary strand by one base from the nucleobase at which the base pair should be formed in the complementary strand with respect to the X2 group moiety. It is formed between the nucleobase and the X1 group moiety.

[Base specificity of photocrosslinking]
In the present invention, the base of the other party to which the X1 group portion can form a photocrosslink is a base having a pyrimidine ring. On the other hand, the pyranocarbazole structure does not form a photocrosslink with a base having a purine ring. That is, the photocrosslinkable compound according to the present invention forms photocrosslinks with respect to cytosine, uracil, and thymine as natural nucleic acid bases as counterparts of the X1 group moiety, while with respect to guanine and adenine. It has the peculiarity of not forming a photocrosslink.

[Sequence selectivity of photoreactive crosslinker]
In a preferred embodiment, when the compound according to the present invention is a photoreactive modified nucleic acid (photocrosslinkable modified nucleic acid), it is hybridized with a sequence having a base sequence complementary to the modified nucleic acid to form a double helix. Can be photocrosslinked after forming. As a result, the optical cross-linking reaction (photocrosslinking reaction) can be performed only on a specific sequence of interest. This sequence selectivity is very high because base pairing is required including the X2 group portion in the formula I. That is, the photoreactive cross-linking agent according to the present invention can impart extremely high base sequence selectivity by designing a sequence as desired.

The 3-cyanovinylcarbazole nucleoside ( ^CNV K), which has been conventionally known as a photocrosslinking element, does not have a structure in the molecule capable of forming a hydrogen bond in the molecule, and therefore has a hydrogen bond with a facing base in a DNA or RNA duplex. It was something that could not be assembled. Therefore, the facing base of ^CNV K photocrosslinks with the pyrimidine base on the 1 base 3'side of any of adenine, guanine, cytosine, and thymine (uracil). This property of ^CNV K is a very useful property in a sense, but on the other hand, it is a disadvantageous property from the viewpoint of performing accurate photocrosslinking because the information on the facing base of ^CNV K is ignored. Met. For example, reduced accuracy of photocrosslinking increases the risk of targeting non-targeted DNA or RNA, which can lead to toxic manifestations such as off-target effects. In contrast to ^CNV K having such properties, the 2'- ^CNV K series of photocrosslinking elements according to the present invention has adenine, guanine, cytosine, and thymine (adenine, guanine, cytosine, and thymine at the sugar portion 1'position, similar to natural nucleic acids. Since it retains a base portion such as uracil), it can form a hydrogen bond with a facing base. Then, while exhibiting high specificity by forming a hydrogen bond with a facing base, photocrosslinking can be performed by 3-cyanovinylcarbazole having a sugar portion at the 2'position, so that accurate photocrosslinking is performed. From the point of view, it is very advantageous.

[Conditions for photochemical reaction]
In a preferred embodiment, the following conditions can be used as the photoreaction for photocrosslinking. For example, the light emitted for photocrosslinking is generally in the range of 350 to 380 nm, preferably in the range of 360 to 370 nm, more preferably light containing a wavelength of 366 nm, and particularly preferably a single wavelength laser of 366 nm. It is light. The light irradiation time is several seconds, for example, 1 to 9 seconds, 1 to 7 seconds, and 1 to 5 seconds, so that the photoreaction can proceed and a photobridge can be formed. Since it is a photoreaction, various conditions can be widely used for the solvent and the like, including the use of an aqueous solution, a buffer solution, a physiological pH and a salt concentration.

[Synthesis of modified nucleic acid synthesis monomer and modified nucleic acid]
In a preferred embodiment, the method described in Scheme 1, Scheme 2, Scheme 3, and Scheme 4, which will be described later, or a method known to those skilled in the art is used to provide a hydroxyl group at the 2'-carbon of ribose. A monomer for synthesis (recipient for production) for obtaining a modified nucleic acid according to the present invention from a nucleoside (for example, compound 1 of scheme 1 described later) (for example, compound 5, compound 6, compound 7, compound 8 of scheme 1 described later). Can be obtained.

The structure of the monomer for synthesizing the modified nucleic acid according to the present invention is as described above, and if this is used as a nucleic acid synthesis reagent in known methods such as the phosphoramidite method and the H-phosphonate method, a nucleotide analog (artificial) can be used. A modified nucleic acid or a modified oligonucleotide in which the nucleotide) is incorporated into the sequence can be obtained. As described above, the modified nucleic acid synthesis monomer according to the present invention is excellent in that it can be used as a nucleic acid synthesis reagent in known methods such as the phosphoramidite method and the H-phosphonate method.

Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to the examples illustrated below.

[Synthesis of nucleoside analogs (photoreactive devices): Synthesis of 2'- ^CNV K-C _n- A]
Synthesize photoresponsive artificial nucleoside analog molecules (sometimes referred to as nucleoside analogs, or photoreactive elements or photocrosslinking elements), ie, 2'- ^CNV K-C _n- A, along the synthetic pathway shown in Scheme 1. Then, a modified nucleic acid synthesis monomer was further synthesized. The synthetic route of Scheme 1 is shown in FIG. 1 (FIGS. 1-1 and 1-2).

[Synthesis of Compound 2]
Compound 1 (3.0 g, 8.1 mmol) was dissolved in pyridine (80 mL). TIPDSCl (2.8 mL, 8.9 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 3.5 hours. Then, Ac2O (890 uL, 8.9 mmol) was added, and the mixture was stirred at room temperature for 13 hours. After completion of the reaction, water (10 mL) was added and the mixture was stirred for 5 min. The solvent was distilled off, and the mixture was extracted with AcOEt and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Finally, column chromatography (Hexane: AcOEt = 2: 3) was performed to obtain Compound 2 (2.8 g, 4.2 mmol, 53%):
¹ H-NMR: (400 MHz, CDCl ₃ ) δ1.02 ~ 1.11 (m, 28H), 2.18 (s, 3H), 4.07 (m, 2H) 4.16 (d, 1H, J = 7.9 Hz), 5.05 ~ 5.08 (m, 1H), 5.81 (d, 1H, J = 5.2 Hz), 6.09 (s, 1H), 7.44 ~ 7.62 (m, 3H), 8.02 (d, 2H, J = 7.2 Hz), 8.19 (s) , 1H), 8.76 (s, 1H),
SALDI-MS: Calc'd for C ₃₁ H ₄₅ N ₅ NaO ₇ Si ₂ [M + Na] ⁺ = 678.2755. Found 678.2742.

[Synthesis of Compound 3]
Compound 2 (3.87 g, 5.9 mmol) was dissolved in THF (50 mL). Next, AcOH (880 uL, 15.3 mmol) and 1 M TBAF (14.7 mL, 14.7 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the solvent was distilled off, the mixture was extracted with CHCl ₃ , and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain Compound 3 (2.2 g, 5.4 mmol, 92%):
¹ H-NMR: (400 MHz, DMSO-d ₆ ) δ 2.19 (s, 3H), 3.66 ~ 3.79 (m, 2H), 4.20 (s, 1H) 4.16 (d, 1H, J = 5.7 Hz), 5.05 ~ 5.08 (m, 2H), 5.81 (d, 1H, J = 5.2 Hz), 6.09 (s, 1H), 7.44 ~ 7.62 (m, 3H), 8.11 (d, 2H, J = 7.3 Hz), 8.81 (s, 2H), 11.32 (s, 1H),
SALDI-MS: Calc'd for C ₁₉ H ₁₉ N ₅ NaO ₆ [M + Na] ⁺ = 436.1233. Found 436.1226.

[Synthesis of Compound 4]
Compound 3 (1.58 g, 3.82 mmol) was dissolved in CH ₃ CN (40 mL). DHP (3.5 mL, 38.6 mmol) and p-TsOH (72 mg, 0.42 mmol) were added in this order, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, the solvent was distilled off, AcOEt was added, and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Then, the solvent was distilled off, MeOH (20 mL) and 28% NH ₃ aq (20 mL) were added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the solvent was distilled off, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 95: 5) was performed to obtain compound 4 (526 mg, 0.98 mmol, 25%):
SALDI-MS: Calc'd for C ₂₇ H ₃₃ N ₅ NaO ₇ [M + Na] ⁺ = 562.2278. Found 562.2270.

[Synthesis of Compound 5]
1-bromo-4-3-cyanovinylcarbazole (148 mg, 0.42 mmol) was dissolved in DMF (1 mL). 60% NaH (17 mg, 0.42 mmol) and NaI (63 mg, 0.42 mmol) were added, and the mixture was stirred at room temperature for 10 min. Compound 4 (150 mg, 0.28 mmol) dissolved in DMF (2 mL) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Then, the solvent was distilled off and it was dissolved in CH ₃ OH (3 mL). Next, p-TsOH (145 mg, 0.84 mmol) was added, and the mixture was stirred at room temperature for 8 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain compound 5 (33 mg, 0.05 mmol, 18%):
¹ H-NMR: (400 MHz, CDCl ₃ ) δ1.62 (t, 2H, J = 8.8 Hz), 1.93 (t, 2H, J = 8.8 Hz), 3.47 ~ 3.52 (m, 4H), 3.84 (d , 2H, J = 12.8 Hz), 4.00 (d, 2H, J = 4.2 Hz), 4.10 (d, 1H, J = 2.7 Hz), 4.29 (t, 2H, J = 6.8 Hz), 4.84 (t, 1H) , J = 6.0 Hz), 5.66 (d, 1H, J = 7.1 Hz), 5.74 (d, 1H, J = 16.5 Hz), 7.34 ~ 7.49 (m, 8H), 7.60 ~ 8.10 (m, 4H), 8.58 (s, 1H), 9.12 (s, 1H),
ESI-MS: Calc'd for C ₃₆ H ₃₃ N ₇ NaO ₅ [M + Na] ⁺ = 666.2441. Found 666.2452.

The structure of 1-bromo-4-3-cyanovinylcarbazole used in the synthesis of compound 4 to compound 5 is shown enlarged below.

[Synthesis of Compound 6]
1-bromo-6-3-cyanovinylcarbazole (1.1 g, 2.9 mmol) was dissolved in DMF (5 mL). 60% NaH (430 mg, 2.9 mmol) and NaI (115 mg, 2.9 mmol) were added, and the mixture was stirred at room temperature for 10 min. Compound 4 (515 mg, 0.96 mmol) dissolved in DMF (5 mL) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Then, the solvent was distilled off and dissolved in CH ₃ OH (10 mL). Next, p-TsOH (552 mg, 2.9 mmol) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain compound 6 (244 mg, 0.36 mmol, 38%):
¹ H-NMR: (400 MHz, DMSO-d ₆ ) δ1.20 ~ 1.56 (m, 8H), 3.63 ~ 3.75 (m, 2H), 4.01 ~ 4.10 (m, 2H) 4.39 (s, 1H), 4.48 (t, 2H, J = 7.1 Hz), 4.83 (d, 2H, J = 5.2 Hz) 5.24 (s, 2H), 5.57 (d, 1H, J = 6.3 Hz), 6.09 (d, 1H, J = 5.9) Hz), 6.45 (d, 1H, J = 16.6 Hz), 7.32 (t, 1H, J = 7.4 Hz), 7.56 ~ 7.85 (m, 8H), 8.08 ~ 8.22 (m, 3H), 8.54 (s, 1H) ), 8.81 (d, 1H, J = 17.1 Hz), 11.27 (s, 1H),
SALDI-MS: Calc'd for C ₃₈ H ₃₇ N ₇ NaO ₅ [M + Na] ⁺ = 694.2754. Found 694.2842.

The structure of 1-bromo-6-3-cyanovinylcarbazole used in the synthesis of compound 4 to compound 6 is shown enlarged below.

[Synthesis of Compound 7]
Compound 6 (75 mg, 0.11 mmol) was dissolved in pyridine (1.0 mL). DMAP (2 mg, 0.01 mmol) and DMTrCl (41 mg, 0.12 mmol) were added, and the mixture was stirred at room temperature for 5 hours. After completion of the reaction, water was added, and after completion of the reaction, the solvent was distilled off and extracted with CHCl ₃ , and sat. The organic layer was dried with NaHCO ₃ aq, Brine, wash, and Na ₂ SO ₄ . It was then azeotroped with toluene and finally column chromatographic (CHCl ₃ : MeOH = 97: 3 to 95: 5) to give compound 7 (60 mg, 0.06 mmol, 55%):
¹ H-NMR: (400 MHz, DMSO-d ₆ ) δ1.29 ~ 1.51 (m, 8H), 3.72 ~ 3.77 (m, 8H), 4.14 ~ 4.20 (m, 2H), 4.41 ~ 4.48 (m, 2H) ), 4.39 (s, 1H), 4.97 (d, 2H, J = 5.3 Hz), 4.83 (d, 2H, J = 5.2 Hz), 5.61 (d, 1H, J = 5.8 Hz), 6.08 (d, 1H) , J = 4.6 Hz), 6.44 (d, 1H, J = 16.6 Hz), 6.85 ~ 6.89 (m, 6H), 7.24 ~ 7.39 (m, 10H), 7.61 ~ 8.19 (m, 8H), 8.64 (s, 1H), 8.72 (s, 1H), 11.28 (s, 1H),
SALDI-MS: Calc'd for C ₅₉ H ₅₅ N ₇ NaO ₇ [M + Na] ⁺ = 996.4061. Found 996.4245.

[Synthesis of Compound 8]
Compound 7 (60 mg, 0.06 mmol) was dissolved in CH ₂ Cl ₂ (1 mL). 0.25 M Tetrazole (500 uL, 0.12 mmol), (iPr ₂ N) ₂ PO (CH ₂ ) ₂ CN (40 uL, 0.12 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, extraction with CH ₂ Cl ₂ was performed, and sat. The organic layer was dried with NaHCO ₃ aq, Brine, wash, and Na ₂ SO ₄ . Finally, column chromatography (Hexane: AcOEt = 2: 3) was performed to obtain Compound 8 (35 mg, 0.03 mmol, 50%).

[Synthesis of oligonucleic acid containing 2'- ^CNV K-C _6- A]
The following sequence (5'-CCAXAACC-3', X = 2'- ^CNV K-C _6- A) was synthesized using an oligo synthesizer. After completion of the reaction, 30 min was cut out using 28% aqueous ammonia (1 mL) (twice), and then deprotection was performed at 65 ° C. for 4 hours. Then, the solvent was distilled off by speedback, dissolved in 200 uL of purified water, and purified by HPLC. After that, analysis by MALDI-TOF-MS was performed to identify the target product. The MS spectrum of the oligonucleic acid containing 2'- ^CNV K is shown in FIG.
Calc'd for [M + H] ⁺ = 2976.683. Found 2976.573

[Investigation of photocrosslinking of oligonucleic acid containing 2'- ^CNV K-C _6- A]
[Photocrosslinking reaction]
Annealed 100 μM ODN1 (5'-CCAA2'- ^CNV KAACC-3'), 100 μM ODN2 (5'-GGTTTTTG-3'), 200 μM deoxyuridine, 50 mM cacodylic acid buffer (pH 7.4) containing 100 mM NaCl. It was allowed to stand still. Then, using a UV-LED (OmniCure, LX405-S), light irradiation at 366 nm was performed at 4 ° C. for 5 seconds.

[HPLC analysis]
50 μL of the crosslinked sample was analyzed by HPLC. 50 mM ammonium formate and acetonitrile were used for the analysis, and the solvent ratio was linearly changed so as to be 98% ammonium formate at the start of the analysis, 70% ammonium formate at 30 minutes, and 30% acetonitrile. The analysis was performed at a flow velocity of 1.0 mL / min, a column temperature of 60 ° C., and a detection wavelength of 260 nm.

3A and 3B show HPLC chromatograms before and after light irradiation obtained by HPLC analysis. FIG. 3A is a chromatograph after 0 sec of light irradiation (before light irradiation), and FIG. 3B is a chromatograph after 5 sec of light irradiation. After 5 s of light irradiation, the peak of ODN2 observed at 16 minutes and the peak of ODN1 observed at 27 minutes disappeared, and a new peak appeared at 19 minutes. This peak was sampled and analyzed by MALDI-TOF-MS to identify the target object:
Calc'd for [M + H] ⁺ = 5751.1675. Found = 5753.5033.

FIG. 4 shows the MS spectrum of the photocrosslinked product.

[Evaluation of face-to-face base recognition ability of oligonucleic acid containing 2'- ^CNV K-C _6- A]
A 50 mM cacodylic acid buffer (pH 7.4) containing 5 μM double strand and 100 mM NaCl was prepared, cooled from 85 ° C. to 4 ° C. at 1 ° C./min, and the absorbance at 260 nm at each temperature was measured. The obtained results are shown in FIG. 5 (FIGS. 5-1 and 5-2, 5-3). In FIG. 5 (FIGS. 5-1 and 5-2, 5-3), the horizontal axis of each graph is temperature (Celsius), and the vertical axis is absorbance.

The respective Tm values were obtained from the melting temperature curves of FIGS. 5 (5-1, 5-2, 5-3). The results are shown in Table 1.

From the results shown in FIGS. 5 (5-1, 5-2, 5-3) and Table 1, the newly developed 2'- ^CNV K-C _6- A recognizes face-to-face bases as compared with the conventional ^CNV K. It was confirmed that it has the ability.

[Synthesis of nucleoside analogs (photoreactive devices): Synthesis of 2'- ^CNV K-C _n- G]
Synthesize photoresponsive artificial nucleoside analog molecules (sometimes referred to as nucleoside analogs, or photoreactive elements or photocrosslinking elements), that is, 2'- ^CNV K-C _6- G, along the synthetic pathway shown in Scheme 2. Then, a modified nucleic acid synthesis monomer was further synthesized. The synthetic route of Scheme 2 is shown in FIG. 6 (FIGS. 6-1 and 6-2).

[Synthesis of Compound 10]
Compound 9 (1.0 g, 2.8 mmol) was dissolved in pyridine (28 mL). TIPDSCl (1.1 mL, 3.4 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 5 hours. Then, DMAP (34 mg, 0.28 mmol) and Ac ₂ O (530 uL, 5.6 mmol) were added, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction, water (5 mL) was added and the mixture was stirred for 5 min. The solvent was distilled off, and the mixture was extracted with AcOEt and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Finally, column chromatography (Hexane: AcOEt = 1: 2) was performed to obtain Compound 10 (1.6 g, 2.5 mmol, 89%). :
¹ H-NMR: (400 MHz, CDCl ₃ ) δ1.00 ~ 1.07 (m, 28H), 1.25 (d, 6H, J = 6.9 Hz), 2.15 (s, 3H), 2.60 (t, 1H, J = 6.9 Hz), 4.00 ~ 4.03 (m, 2H) 4.15 (d, 1H, J = 13.0 Hz), 4.62 ~ 4.65 (m, 1H), 5.59 (d, 1H, J = 5.1 Hz), 5.87 (s, 1H) ), 7.90 (s, 1H), 8.41 (s, 1H), 12.02 (s, 1H),
SALDI-MS: Calc'd for C ₂₈ H ₄₇ N ₅ NaO ₈ Si ₂ [M + Na] ⁺ = 660.2861. Found 660.2853.

[Synthesis of Compound 11]
Compound 10 (1.6 g, 2.5 mmol) was dissolved in THF (25 mL). Next, AcOH (430 uL, 7.5 mmol) and 1 M TBAF (7.5 mL, 7.5 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the solvent was distilled off, the mixture was extracted with CHCl ₃ , and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain Compound 11 (800 mg, 2.0 mmol, 80%). :
¹ H-NMR: (400 MHz, DMSO-d ₆ ) δ1.18 (d, 6H, J = 6.8 Hz), 1.34 ~ 1.40 (m, 1H), 1.62 (d, 1H, J = 7.4 Hz), 2.16 (s, 3H), 2.83 (t, 1H, J = 6.9 Hz), 3.22 (t, 1H, J = 8.3 Hz), 3.63 ~ 3.73 (m, 2H), 4.76 ~ 4.80 (m, 1H), 5.31 ( d, 1H, J = 4.0 Hz), 5.86 (d, 1H, J = 7.2 Hz), 8.34 (s, 1H), 11.75 (s, 1H), 12.16 (s, 1H),
SALDI-MS: Calc'd for C ₁₆ H ₂₁ N ₅ NaO ₇ [M + Na] ⁺ = 418.1339. Found 418.1333.

[Synthesis of Compound 12]
Compound 11 (800 mg, 2.0 mmol) was dissolved in CH ₃ CN (20 mL). DHP (2.5 mL, 27 mmol) and p-TsOH (40 mg, 0.20 mmol) were added in this order, and the mixture was stirred at room temperature for 20 hours. After completion of the reaction, the solvent was distilled off, AcOEt was added, and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Then, the solvent was distilled off, MeOH (10 mL) and 28% NH ₃ aq (10 mL) were added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, the solvent was distilled off, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 95: 5) was performed to obtain compound 12 (526 mg, 0.98 mmol, 49%):
SALDI-MS: Calc'd for C ₂₄ H ₃₅ N ₅ NaO ₈ [M + Na] ⁺ = 544.2383. Found 544.2378.

[Synthesis of Compound 13]
1-bromo-6-3-cyanovinylcarbazole (342 mg, 0.90 mmol) was dissolved in DMF (3 mL). 60% NaH (110 mg, 0.90 mmol) and NaI (135 mg, 0.90 mmol) were added, and the mixture was stirred at room temperature for 10 min. Compound 12 (313 mg, 0.60 mmol) dissolved in DMF (3 mL) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Then, the solvent was distilled off and dissolved in CH ₃ OH (6 mL). Next, p-TsOH (310 mg, 1.8 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain compound 13 (55 mg, 0.08 mmol, 14%):
^{1 1} H-NMR: (400 MHz, DMSO-d ₆ ) δ1.20 ~ 1.56 (m, 18H), 2.83 (t, 1H, J = 6.9 Hz), 3.26 ~ 3.65 (m, 6H), 3.84 ~ 4.20 ( m, 6H), 5.58 ~ 5.76 (m, 2H), 7.14 ~ 7.44 (m, 6H), 7.94 ~ 8.00 (m, 3H), 9.52 (s, 1H), 11.97 (s, 1H),
SALDI-MS: Calc'd for C ₃₃ H ₄₄ BrN ₅ NaO ₇ [M + Na] ⁺ = 724.2322. Found 724.2319.

An enlarged view of the structure of 1-bromo-6-3-cyanovinylcarbazole used in the synthesis of compound 12 to compound 13 is as already shown above.

[Synthesis of nucleoside analogs (photoreactive devices): Synthesis of 2'- ^CNV K-C _n -C]
Synthesize photoresponsive artificial nucleoside analog molecules (sometimes referred to as nucleoside analogs, or photoreactive elements or photocrosslinking elements), ie, 2'- ^CNV K-C ₆ -C, along the synthetic pathway shown in Scheme 3. Then, a modified nucleic acid synthesis monomer was further synthesized. The synthetic route of Scheme 3 is shown in FIG. 7 (FIGS. 7-1 and 7-2).

[Synthesis of Compound 15]
Compound 14 (1.0 g, 2.9 mmol) was dissolved in pyridine (29 mL). TIPDSCl (1.0 mL, 8.9 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 6.0 hours. Then, Ac ₂ O (547 uL, 5.8 mmol) was added, and the mixture was stirred at room temperature for 8.0 h. After completion of the reaction, water (10 mL) was added and the mixture was stirred for 5 min. The solvent was distilled off, and the mixture was extracted with AcOEt and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Finally, column chromatography (Hexane: AcOEt = 1: 3) was performed to obtain compound 15 (1.4.g, 2.2 mmol, 74%):
¹ H-NMR: (400 MHz, CDCl ₃ ) δ1.00 ~ 1.11 (m, 28H), 2.15 (s, 3H), 3.98 ~ 4.12 (m, 2H), 4.27 ~ 4.36 (m, 2H), 5.47 ( d, 1H, J = 4.6 Hz), 5.95 (s, 1H), 7.49 ~ 7.63 (m, 4H), 7.89 (d, 2H, J = 7.3 Hz), 8.24 (d, 1H, J = 7.3 Hz), 8.72 (s, 1H),
SALDI-MS: Calc'd for C ₃₁ H ₄₅ N ₅ NaO ₇ Si ₂ [M + Na] ⁺ = 654.2643. Found 654.2634.

[Synthesis of Compound 16]
Compound 15 (1.36 g, 2.2 mmol) was dissolved in THF (22 mL). Next, AcOH (370 uL, 6.6 mmol) and 1 M TBAF (6.6 mL, 6.6 mmol) were added, and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, the solvent was distilled off, the mixture was extracted with CHCl ₃ , and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain compound 16 (725 mg, 1.8 mmol, 80%):
¹ H-NMR: (400 MHz, CDCl ₃ ): δ2.13 (s, 3H), 3.89 ~ 4.02 (m, 4H), 4.27 (s, 1H), 4.54 ~ 4.58 (m, 1H), 4.69 (t) , 1H, J = 5.3 Hz), 5.32 (t, 1H, J = 4.6 Hz), 5.37 (d, 1H, J = 4.6 Hz), 6.00 (d, 1H, J = 5.4 Hz), 7.39 ~ 7.53 (m) , 4H), 7.86 (d, 2H, J = 7.3 Hz), 8.57 (d, 1H, J = 7.5 Hz), 8.89 (d, 1H, J = 7.5 Hz),
SALDI-MS: Calc'd for C ₁₈ H ₁₉ N ₃ NaO ₇ [M + Na] ⁺ = 412.1121. Found 412.332.

[Synthesis of Compound 17]
Compound 16 (1.0 g, 2.6 mmol) was dissolved in CH ₃ CN (26 mL). DHP (2.4 mL, 26 mmol) and p-TsOH (44 mg, 0.26 mmol) were added in this order, and the mixture was stirred at room temperature for 8 hours. After completion of the reaction, the solvent was distilled off, AcOEt was added, and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Then, the solvent was distilled off, MeOH (13 mL) and 28% NH ₃ aq (13 mL) were added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, the solvent was distilled off, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 95: 5) was performed to obtain compound 17 (600 mg, 1.1 mmol, 45%):
SALDI-MS: Calc'd for C ₂₆ H ₃₃ N ₃ NaO ₈ [M + Na] ⁺ = 538.2165. Found 538.2160.

[Synthesis of Compound 18]
1-bromo-6-3-cyanovinylcarbazole was dissolved in DMF (3 mL). 60% NaH (27 mg, 0.68 mmol) and NaI (102 mg, 0.68 mmol) were added, and the mixture was stirred at room temperature for 10 min. Compound 17 (233 mg, 0.45 mmol) dissolved in DMF (4 mL) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Then, the solvent was distilled off and it was dissolved in CH ₃ OH (7 mL). Next, p-TsOH (232 mg, 1.35 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain compound 18 (47 mg, 0.07 mmol, 16%):
¹ H-NMR: (400 MHz, CDCl ₃ ) δ1.25 ~ 1.45 (m, 8H), 3.35 ~ 3.45 (m, 2H), 3.62 ~ 3.78 (m, 2H), 3.80 ~ 4.19 (m, 3H), 5.62 ~ 5.74 (m, 2H), 7.15 ~ 7.44 (m, 10H), 7.74 (d, 2H, J = 7.6 Hz), 7.95 ~ 8.00 (m, 3H),
SALDI-MS: Calc'd for C ₃₃ H ₄₄ BrN ₅ NaO ₇ [M + Na] ⁺ = 724.2322. Found 724.2319.

An enlarged view of the structure of 1-bromo-6-3-cyanovinylcarbazole used in the synthesis of compound 17 to compound 18 is as already shown above.

[Synthesis of nucleoside analogs (photoreactive devices): Synthesis of 2'- ^CNV K-C _n- T]
Synthesize photoresponsive artificial nucleoside analog molecules (sometimes referred to as nucleoside analogs, or photoreactive elements or photocrosslinking elements), ie, 2'- ^CNV K-C _6- T, along the synthetic pathway shown in Scheme 4. Then, a modified nucleic acid synthesis monomer was further synthesized. The synthetic route of Scheme 4 is shown in FIG. 8 (FIGS. 8-1 and 8-2).

[Synthesis of Compound 20]
Compound 19 (2.0 g, 5.4 mmol) was dissolved in pyridine (54 mL). TIPDSCl (1.9 mL, 5.9 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 9 hours. Then, Ac ₂ O (1.0 mL, 10.8 mmol) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water (10 mL) was added and the mixture was stirred for 5 min. The solvent was distilled off, and the mixture was extracted with AcOEt and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Finally, column chromatography (Hexane: AcOEt = 1: 3) was performed to obtain compound 20 (1.28 g, 2.3 mmol, 42%).
^{1 1} H-NMR: (400 MHz, CDCl ₃ ) δ0.95 ~ 1.05 (m, 28H), 1.87 (s, 3H), 2.10 (s, 3H), 3.92 ~ 3.97 (m, 2H), 4.17 (d, 1H, J = 12.4 Hz), 4.33 ~ 4.37 (m, 1H), 5.36 (d, 1H, J = 5.3 Hz), 5.83 (s, 1H), 7.34 (s, 1H), 9.78 (s, 1H),
SALDI-MS: Calc'd for C ₂₄ H ₄₂ N ₂ NaO ₈ Si ₂ [M + Na] ⁺ = 565.2377. Found 565.2371.

[Synthesis of Compound 21]
Compound 20 (2.7 g, 4.9 mmol) was dissolved in THF (50 mL). Next, AcOH (700 uL, 14.7 mmol) and 1 M TBAF (14.7 mL, 14.7 mmol) were added, and the mixture was stirred at room temperature for 1 h. After completion of the reaction, the solvent was distilled off, the mixture was extracted with CHCl ₃ , and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100: 0 to 95: 5) was performed to obtain compound 21 (1.4 g, 4.7 mmol, 96%):
¹ H-NMR: (400 MHz, DMSO-d ₆ ) δ1.84 (s, 3H), 2.15 (s, 3H), 3.66 (d, 2H, J = 9.1 Hz), 4.06 (d, 1H, J = 2.8 Hz), 4.33 (d, 1H, J = 6.0 Hz), 5.17 (d, 1H, J = 2.8 Hz), 5.35 (s, 1H), 5.74 (d, 1H, J = 5.9 Hz), 5.87 (d , 1H, J = 7.0 Hz), 7.78 (s, 1H), 11.43 (s, 1H)
SALDI-MS: Calc'd for C ₁₂ H ₁₆ N ₂ NaO ₇ [M + Na] ⁺ = 323.0855. Found 323.0851.

[Synthesis of Compound 22]
Compound 21 (500 mg, 1.6 mmol) was dissolved in CH ₃ CN (16 mL). DHP (1.4 mL, 16.0 mmol) and p-TsOH (27 mg, 0.16 mmol) were added in this order, and the mixture was stirred at room temperature for 3.5 hours. After completion of the reaction, the solvent was distilled off, AcOEt was added, and sat. It was washed with NaHCO ₃ aq (twice) and Brine, and the organic layer was dried with Na ₂ SO ₄ . Then, the solvent was distilled off, MeOH (8 mL) and 28% NH ₃ aq (8 mL) were added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the solvent was distilled off, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 95: 5) was performed to obtain compound 22 (400 mg, 0.94 mmol, 59%):
SALDI-MS: Calc'd for C ₂₀ H ₃₀ N ₂ NaO ₈ [M + Na] ⁺ = 449.1900. Found 449.1894.

[Synthesis of Compound 23]
1-bromo-6-3-cyanovinylcarbazole (137 mg, 0.36 mmol) was dissolved in DMF (2 mL). 60% NaH (15 mg, 0.36 mmol) and NaI (54 mg, 0.36 mmol) were added, and the mixture was stirred at room temperature for 10 min. Compound 22 (100 mg, 0.24 mmol) dissolved in DMF (2 mL) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Then, the solvent was distilled off and dissolved in CH ₃ OH (4 mL). Next, p-TsOH (124 mg, 0.72 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, water was added, the mixture was extracted with AcOEt, and the organic layer was dried with water, Brine with wash, and Na ₂ SO ₄ . Finally, column chromatography (CHCl ₃ : MeOH = 100% to 95: 5) was performed to obtain compound 23 (27 mg, 0.05 mmol, 20%):
^{1 1} H-NMR: (400 MHz, CDCl ₃ ) δ1.20 ~ 1.56 (m, 11H), 3.41 ~ 3.65 (m, 2H), 3.89 ~ 4.05 (m, 2H), 4.17 ~ 4.22 (m, 2H), 5.62 ~ 5.74 (m, 2H), 5.79 (d, 1H, J = 16.5 Hz), 7.20 ~ 7.50 (m, 8H), 7.99 ~ 8.07 (m, 1H) ,,
SALDI-MS: Calc'd for C ₃₃ H ₄₄ BrN ₅ NaO ₇ [M + Na] ⁺ = 724.2322. Found 724.2319.

An enlarged view of the structure of 1-bromo-6-3-cyanovinylcarbazole used in the synthesis of compound 12 to compound 13 is as already shown above.

The present invention provides a novel compound that serves as a photoreactive crosslinker that can be used in nucleic acid photoreaction techniques. The present invention is an industrially useful invention.

Claims (7)

Compound represented by the following formula I:
(I)
(However, in formula I,
X1 is a group represented by the following formula II:
(II)
(However, in Equation II,
R21 is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group of C2 to C7, or a hydrogen atom.
R22 and R23 are independently cyano groups, amide groups, carboxyl groups, C2-C7 alkoxycarbonyl groups, or hydrogen atoms.
R24 and R25 are independently hydrogen atom, halogen atom, -OH group, amino group, nitro group, methyl group, methyl fluoride group, ethyl group, ethyl fluoride group, and alkylsulfanyl group of C1 to C3, respectively. It is a group selected from the group consisting of. )

X2 is a nucleobase monovalent group selected from the group consisting of natural and non-natural nucleobases and modified nucleobases in which they are modified with protecting groups.
L is a single bond or an alkanediyl group of C1 to C7.

R11 is _one -OQ,
R12 is _two -OQ,

Q ₁ is
Hydrogen atom;
Phosphate group formed integrally with O bonded to Q ₁ ;
Nucleotides, nucleic acids or peptide nucleic acids linked via phosphodiester bonds formed by phosphate groups formed integrally with O that binds to Q ₁ ; and
Protecting groups selected from:
Trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, trimethylsilyl group, triethylsilyl group, t-butyldimethylsilyl group, acetyl group, benzoyl group;
A group selected from the group consisting of

Q ₂ is
Hydrogen atom;
Phosphate group formed integrally with O bonded to Q ₂ ;
Nucleotides, nucleic acids or peptide nucleic acids linked via phosphodiester bonds formed by phosphate groups formed integrally with O that binds to Q ₂ ; and
Protecting groups selected from:
2-Cyanoethyl-N, N-dialkyl (C1-C4) phosphoramidite group, methylphosphonamidite group, ethylphosphonamidite group, oxazaphosphoridine group, thiophosphite group, TEA salt of -PH (= O) OH, -PH (= O) OH DBU salt, -PH (= S) OH TEA salt, -PH (= S) OH DBU salt;
It is a group selected from the group consisting of. ). The compound according to claim 1, wherein the group represented by the formula II is a group represented by the following formula IIa:
(IIa)
(However, in Formula IIa, R21, R22, R23 and R24 are the groups described above in Formula II). The compound according to any one of claims 1 and 2, wherein X2 in the formula I is adenine, guanine, thymine, cytosine, or uracil. An artificial nucleoside comprising the compound according to any one of claims 1 to 3, wherein R11 and R12 in the formula I are hydroxyl groups. At least one of Q ₁ and Q ₂
Any of claims 1 to 3, wherein the nucleotide or nucleic acid is linked via a phosphodiester bond formed by a phosphate group formed integrally with O bound to Q ₁ or Q _2. An artificial nucleic acid consisting of the compounds described. A photoreactive cross-linking agent comprising the compound according to any one of claims 1 to 3. A method for forming a photocrosslink with a nucleobase having a pyrimidine ring using the compound according to any one of claims 1 to 3.