Methods for producing chimeric molecules

Abstract

Provided is a method for producing a chimeric molecule by a liquid-phase synthesis method, the method including linking an anionic main-chain backbone nucleic acid including DNA to a neutral or cationic main-chain backbone nucleic acid including PNA. The method for producing a chimeric molecule according to one aspect of the present invention is a method for producing a chimeric molecule in which a first nucleic acid is fused to a second nucleic acid, the method including the production of a chimeric molecule by preparing a second chimeric molecule precursor by the introduction of a second nucleic acid through a hydroxyl group in a first chimeric molecule precursor, optionally performing deprotection, and introducing a first nucleic acid into the second chimeric molecule precursor, in which the first chimeric molecule precursor includes a lipophilic anchor, the second nucleic acid has an amino group that may be protected, the deprotection of the second chimeric molecule precursor is the deprotection of a protected amino group in the second nucleic acid introduced into the second chimeric molecule precursor, the introduction of the first nucleic acid is the introduction through the amino group in the second nucleic acid, and each of the steps is performed by a liquid-phase synthesis method.

Description

[Technical Field]

[0001] The present invention relates to a method for producing chimeric molecules. This application claims priority from Japanese Patent Application No. 2022-039293, filed on March 14, 2022, the contents of which are incorporated herein by reference. [Background technology]

[0002] In recent years, nucleic acid drugs, like antibody drugs, have been attracting attention as next-generation molecular targeted drugs. One known drug strategy for nucleic acid medicines is antisense nucleic acids (ASOs), which target messenger RNA (mRNA), microRNA (miRNA), and small interfering RNA (siRNA), which are involved in disease progression, and which selectively recognize their base sequence and form complexes to suppress the function of the target RNA, thereby exerting a therapeutic effect. For these nucleic acid medicines to effectively exert their pharmacological effects, 1) high in vivo stability and 2) high specificity for the target nucleic acid and complex stability are required, and the development of modified oligonucleotides / artificial oligonucleotides, which are made by chemically modifying natural DNA / RNA, is being actively researched.

[0003] Reducing the dosage of nucleic acid drugs has been proposed as a methodology for overcoming the broad off-target effects of nucleic acid drugs (toxicity specific to nucleic acid drugs that does not depend on target nucleic acid recognition). However, reducing the dosage naturally results in a decrease in the amount of complex formed with the target RNA, and effective drug efficacy cannot be expected. As a solution, attention has been focused on nucleic acid drugs with catalytic functions that utilize RNase H, which catalytically cleaves target RNA with a small amount of ASO (Non-Patent Document 1).

[0004] As a nucleic acid drug with catalytic-like function utilizing RNase H, a chimeric molecule has been reported that focuses on the dissociation process after RNA cleavage and contributes to the construction of an oligonucleic acid system that can rapidly dissociate from the target RNA complex after cleavage, and can inhibit the function of the target nucleic acid at low concentrations and suppress off-target effects (Patent Document 1).

[0005] Solid-phase synthesis is widely used for the synthesis of oligonucleotides and peptides. Although solid-phase synthesis has been optimized and automated, it is essentially a heterogeneous system, and to compensate for its low reactivity, it is necessary to use a large excess of reagents, for example, 6 to 10 equivalents or more, leaving room for improvement in terms of cost. Furthermore, advanced technology is required for isolation and purification, and scaling up is difficult due to limitations on the amount of functional groups supported on the solid-phase resin for reaction. While suitable for laboratory scale, solid-phase synthesis is difficult to apply from the perspective of industrialization.

[0006] In an attempt to overcome the drawbacks of solid-phase synthesis, while taking advantage of the advantage of solid-phase synthesis in that isolation and purification after the reaction can be performed simply by filtration and washing, methods have been proposed in which only specific components dissolved in the liquid phase are precipitated and isolated as a solid, thereby facilitating isolation and purification after the reaction (Patent Documents 2 to 7). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2021 / 015234 [Patent Document 2] International Publication No. 2012 / 157723 [Patent Document 3] International Publication No. 2014 / 189142 [Patent Document 4] International Publication No. 2010 / 104169 [Patent Document 5] International Publication No. 2010 / 113939 [Patent Document 6] International Publication No. 2011 / 078295 [Patent Document 7] International Publication No. 2016 / 117663 [Non-patent literature]

[0008] [Non-Patent Document 1] Liang,X.et al.,Mol.Ther., 2017, 25(9), 2075 Summary of the Invention [Problem to be solved by the invention]

[0009] For example, as shown in the above-mentioned Patent Documents 2 to 7, there have been reports of the production by liquid phase synthesis of oligomers that are not formed by fusion of heterogeneous molecules, such as oligoribonucleic acids, oligodeoxyribonucleic acids, or oligopeptides. However, as reported in Patent Document 1, the only known method for producing chimeric molecules, typically fused in any combination of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) with peptide nucleic acid (PNA) or peptide ribonucleic acid (PRNA) having an oligopeptide backbone, is solid-phase synthesis.

[0010] In particular, there have been no reports using liquid phase synthesis methods for introducing PNA or PRNA into oligoRNA or oligoDNA via the terminal nitrogen atom of the oligoRNA or oligoDNA, or for introducing RNA or DNA into oligoPNA or oligoPRNA via the terminal nitrogen atom of the oligoPNA or oligoPRNA. In other words, although there have been reports of methods for linking molecules of the same type using liquid-phase synthesis, at present, the only option is to rely on solid-phase synthesis for at least some of the steps in the construction of PNA-DNA structures in which PNA or pRNA is fused to RNA or DNA, or DNA-PNA structures in which RNA or DNA is fused to PNA or pRNA.

[0011] Furthermore, in solid-phase synthesis, the method of further extending DNA using DNA as a starting point can proceed almost quantitatively at each step using an automated synthesizer, maintaining sufficient yields even when extending multiple DNA fragments. On the other hand, when constructing PNA-DNA structures using DNA as a starting point using solid-phase synthesis, the yield is only about 70% at most, and the yield decreases by several percent as the PNA elongation proceeds. As a result, even with solid-phase synthesis, not only is the construction of PNA-DNA structures insufficient in yield, but the reaction system is complicated by numerous side reactions, requiring a great deal of effort for isolation.

[0012] In view of the above problems, the present invention aims to provide a method for producing a chimeric molecule by liquid phase synthesis, which involves introducing a nucleic acid or a derivative thereof having a neutral or cationic main chain skeleton, such as PNA or pRNA, into a nucleic acid or a derivative thereof having an anionic main chain skeleton, such as RNA or DNA, including the production of a second chimeric molecule precursor described below and the production of a chimeric molecule using the second chimeric molecule precursor. Another object of the present invention is to provide a method for producing a chimeric molecule by liquid phase synthesis, which involves introducing a nucleic acid having an anionic main chain skeleton, such as RNA or DNA, or a derivative thereof, into a nucleic acid having a neutral or cationic main chain skeleton, such as PNA or pRNA, or a derivative thereof, including the production of a chimeric molecule using a third chimeric molecule precursor described below. [Means for solving the problem]

[0013] The present invention has the following gist. A method for producing a chimeric molecule in which at least one first nucleic acid or a derivative thereof having a neutral or cationic main chain backbone and at least one second nucleic acid or a derivative thereof having an anionic main chain backbone are fused together, the method comprising: the production method includes preparing a second chimera molecule precursor by introducing the second nucleic acid or a derivative thereof via a hydroxyl group of a first chimera molecule precursor having a hydroxyl group; optionally, deprotecting the second chimeric molecule precursor; and introducing the first nucleic acid or a derivative thereof into the second chimeric molecule precursor or a deprotected second chimeric molecule precursor; the first chimera molecule precursor having a hydroxyl group comprises a lipid-soluble anchor; the second nucleic acid or a derivative thereof has an amino group which may be protected, the deprotection of the second chimeric molecule precursor is the deprotection of the protected amino group of the second nucleic acid or a derivative thereof introduced into the second chimeric molecule precursor; the introduction of the first nucleic acid or a derivative thereof is introduction of the second nucleic acid or a derivative thereof via the amino group; A production method characterized in that the preparation of the second chimera molecule precursor, the deprotection of the second chimera molecule precursor, and the production of the chimera molecule are all carried out by liquid phase synthesis.

[0014] It is preferable that the first nucleic acid is PNA, PRNA, PNA / PRNA, or LNA, and the second nucleic acid is RNA or DNA. Preferably, the first nucleic acid is PNA and the second nucleic acid is DNA. It is preferred that the chimeric molecule has a portion where the first nucleic acid or a derivative thereof is linked to the 5' end of the second nucleic acid or a derivative thereof. The first chimera molecule precursor is preferably a compound represented by a specific formula described below. The second nucleic acid or a derivative thereof is preferably a compound represented by a specific formula described below. It is preferred that the optionally protected amino group is a protected amino group, and the protecting group of the protected amino group is an optionally substituted trityl group. The first nucleic acid or a derivative thereof is preferably a compound represented by a specific formula described below.

[0015] Another aspect of the present invention is as follows. A method for producing a chimeric molecule in which at least one first nucleic acid or a derivative thereof having a neutral or cationic main chain backbone and at least one second nucleic acid or a derivative thereof having an anionic main chain backbone are fused together, the method comprising: the production method includes introducing the second nucleic acid or a derivative thereof into a third chimera molecule precursor having an amino group via the amino group of the third chimera molecule precursor, the third chimera molecule precursor having an amino group comprises a partial structure derived from a first nucleic acid or a derivative thereof, an amino group derived from the first nucleic acid or a derivative thereof, and a lipid-soluble anchor; A production method characterized in that the introduction of the second nucleic acid or a derivative thereof into the third chimera molecule precursor having an amino group is carried out by liquid phase synthesis.

[0016] It is preferable that the first nucleic acid is PNA, PRNA, PNA / PRNA, or LNA, and the second nucleic acid is RNA or DNA. Preferably, the first nucleic acid is PNA and the second nucleic acid is DNA. It is preferred that the chimeric molecule has a portion where the first nucleic acid or a derivative thereof is linked to the 3' end of the second nucleic acid or a derivative thereof. The third chimera molecule precursor is preferably a compound represented by a specific formula described below. The second nucleic acid or a derivative thereof is preferably a compound represented by a specific formula described below.

[0017] Yet another aspect of the present invention is as follows. [1] A method for producing a chimeric molecule in which at least one first nucleic acid, which is PNA, PRNA, PNA / PRNA, or LNA, or a derivative thereof, is fused with at least one second nucleic acid, which is RNA or DNA, or a derivative thereof, the method comprising: the derivative of the first nucleic acid is a halogenated derivative, a deaminated derivative, or a derivative having a sulfur atom instead of an oxygen atom at the base moiety bound to the first nucleic acid, the derivative of the second nucleic acid is a halogenated derivative, a deaminated derivative, or a derivative having a sulfur atom instead of an oxygen atom at the base moiety bound to the second nucleic acid, or a phosphorothioate-type DNA or RNA, a phosphorodithioate-type DNA or RNA, or a morpholino-type nucleic acid; The base moiety refers to uracil, cytosine, thymine, adenine, guanine, a purine ring, or a pyrimidine ring bound to a nucleic acid; the production method includes: preparing a second chimera molecule precursor by introducing the second nucleic acid or a derivative thereof via a hydroxyl group at the 5'-position of RNA or DNA of a first chimera molecule precursor having a hydroxyl group; oxidation of the phosphorus atom of the second chimeric molecule precursor with peroxide; optionally, deprotecting the second chimeric molecule precursor; and introducing the first nucleic acid or a derivative thereof into the second chimeric molecule precursor or a deprotected second chimeric molecule precursor; The first chimeric molecule precursor is represented by formula (1):

[0018] [ka]

[0019] (In formula (1), Ar * represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; each R independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Base represents the base moiety; and deoxyribose in the formula may have an optionally protected hydroxyl group at the 2'-position. The linker is -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, -NH-C(=O)-, -S-, -S(=O)-, or -S(=O)2-.), or a structure represented by formula (2)

[0020] [ka]

[0021] (In formula (2), Ar * represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; each R independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Base represents the base moiety; X represents a single bond or represents one or more first nucleic acids or derivatives thereof and / or second nucleic acids or derivatives thereof via a bond between an oxygen atom bonded to X and a phosphorus atom; deoxyribose in the formula may have an optionally protected hydroxyl group at the 2'-position. The linker is -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, -NH-C(=O)-, -S-, -S(=O)-, or -S(=O)2-. The second nucleic acid is represented by formula (4):

[0022] [ka]

[0023] (in formula (4), p represents an integer of 0 or more; AG represents an amino group which may be protected; Base represents the base moiety; NR2 represents a dialkylamino group; LV represents a 2-cyanoethyl group, an allyl group, or a benzyl group; and each deoxyribose in the formula may independently have a hydroxyl group which may be protected at the 2'-position), the deprotection of the second chimeric molecule precursor is the deprotection of the protected amino group of the second nucleic acid or a derivative thereof introduced into the second chimeric molecule precursor; the introduction of the first nucleic acid or a derivative thereof is introduction of the second nucleic acid or a derivative thereof via the amino group; The first nucleic acid is represented by formula (5):

[0024] [ka]

[0025] (in formula (5), q represents an integer of 0 or more; PG represents a protecting group for the adjacent amino group; and Base represents the base moiety), A production method characterized in that the preparation of the second chimera molecule precursor, the deprotection of the second chimera molecule precursor, and the production of the chimera molecule are all carried out by liquid phase synthesis. [2] The method of [1], wherein the first nucleic acid is PNA and the second nucleic acid is DNA. [3] The method of producing according to [1] or [2], wherein the first nucleic acid or a derivative thereof is PNA, and the second nucleic acid or a derivative thereof is DNA. [4] Any of the manufacturing methods [1] to [3], wherein the peroxide is tert-butyl hydroperoxide or metachloroperbenzoic acid, and the amount of the peroxide used is 2 to 5 moles per mole of the second chimeric molecule precursor used, or 2 to 5 moles per mole of the first chimeric molecule precursor used in preparing the second chimeric molecule precursor. [5] The production method according to any one of [1] to [4], wherein the linker in the formula (1) and the formula (2) is -O-, the aromatic hydrocarbon ring having 6 to 14 carbon atoms is a benzene ring, and R is a hydrogen atom. [6] The method according to any one of [1] to [5], wherein LV in the formula (4) is a 2-cyanoethyl group. [7] The production method according to any one of [1] to [6], wherein the optionally protected amino group in AG of the formula (4) is a protected amino group, and the protecting group of the protected amino group is a trityl group, a p-methoxyphenyldiphenylmethyl group, or a di(p-methoxyphenyl)phenylmethyl group. [8] The method of any of [1] to [7], wherein the protecting group of the adjacent amino group in the PG of the formula (5) is a fluorenylmethoxycarbonyl group in which the fluorenyl group may be substituted with a group selected from the group consisting of halogen, nitro, cyano, trifluoromethyl, carboxy, alkyloxycarbonyl, dialkylcarbamoyl, and alkylcarbonyl; a trityl group; a p-methoxyphenyldiphenylmethyl group; a di(p-methoxyphenyl)phenylmethyl group; or a phthalimide-type protecting group. [9] The production method of [1], wherein the optionally protected hydroxyl group that can be substituted at the 2'-position of deoxyribose in the formula (1), the formula (2), and the formula (4) is an unprotected hydroxyl group.

[10] A method for producing a chimeric molecule in which at least one first nucleic acid, which is PNA, PRNA, PNA / PRNA, or LNA, or a derivative thereof, is fused with at least one second nucleic acid, which is RNA or DNA, or a derivative thereof, the method comprising: the derivative of the first nucleic acid is a halogenated derivative, a deaminated derivative, or a derivative having a sulfur atom instead of an oxygen atom at the base moiety bound to the first nucleic acid, the derivative of the second nucleic acid is a halogenated derivative, a deaminated derivative, or a derivative having a sulfur atom instead of an oxygen atom at the base moiety bound to the second nucleic acid, or a phosphorothioate-type DNA or RNA, a phosphorodithioate-type DNA or RNA, or a morpholino-type nucleic acid; The base moiety refers to uracil, cytosine, thymine, adenine, guanine, a purine ring, or a pyrimidine ring bound to a nucleic acid; the production method includes introducing the second nucleic acid or a derivative thereof into a third chimera molecule precursor having an amino group via the amino group of the third chimera molecule precursor, The third chimeric molecule precursor is represented by formula (11):

[0026] [ka]

[0027] (In the formula, Ar * represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; and Base represents the base moiety. The linker is -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, -NH-C(=O)-, -S-, -S(=O)-, or -S(=O)2-.), or a structure represented by formula (12)

[0028] [ka]

[0029] (In the formula, Ar * represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; each R independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Base represents the base moiety; Z represents a single bond or represents one or more first nucleic acids or derivatives thereof and / or second nucleic acids or derivatives thereof via a bond between a nitrogen atom bonded to Z and a carbonyl carbon atom; each deoxyribose in the formula may independently have an optionally protected hydroxyl group at the 2'-position. The linker is -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, -NH-C(=O)-, -S-, -S(=O)-, or -S(=O)2-. The second nucleic acid is represented by formula (14):

[0030] [ka]

[0031] (wherein p represents an integer of 0 or more; AG / HG represent an amino group or a hydroxyl group which may be protected; Base represents the base moiety; LV represents a 2-cyanoethyl group, an allyl group, or a benzyl group; and each deoxyribose in the formula may independently have a hydroxyl group which may be protected at the 2'-position), A production method characterized in that the introduction of the second nucleic acid or a derivative thereof into the third chimera molecule precursor having an amino group is carried out by liquid phase synthesis. [Effects of the Invention]

[0032] According to the present invention, there is provided a method for producing a chimeric molecule by liquid phase synthesis, in which a nucleic acid having a neutral or cationic main chain skeleton, such as PNA or pRNA, or a derivative thereof, is introduced into a nucleic acid having an anionic main chain skeleton, such as RNA or DNA, or a derivative thereof. By using this method, it is possible to produce, by liquid phase synthesis, PNA-DNA structures in which a PNA structure is fused to an oligo-RNA or oligo-DNA, or in which a PNA structure is further bound to such structures; or PNA-DNA-PNA structures in which a PNA structure is fused to a DNA-PNA structure, or in which a PNA structure is further bound to such structures.

[0033] Furthermore, according to another aspect of the present invention, there is provided a method for producing a chimeric molecule by liquid phase synthesis, in which a nucleic acid having an anionic main chain skeleton, such as RNA or DNA, or a derivative thereof, is introduced into a nucleic acid having a neutral or cationic main chain skeleton, such as PNA or pRNA, or a derivative thereof. By using this method, it is possible to produce by liquid phase synthesis DNA-PNA structures in which a DNA structure or an RNA structure is fused to an oligo-PNA or oligo-PRNA, or in which a DNA structure or an RNA structure is further bound to such structures; or DNA-PNA-DNA structures in which a PNA-DNA structure is fused to a DNA structure or an RNA structure, or in which a DNA structure or an RNA structure is further bound to such structures. DETAILED DESCRIPTION OF THE INVENTION

[0034] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments and can be practiced with various modifications within the scope of the gist thereof.

[0035] The chimeric molecule that is the target of the production method of the present invention is a compound in which at least one first nucleic acid or derivative thereof (hereinafter, including derivatives, simply referred to as "first nucleic acid") having a neutral or cationic main chain backbone and at least one second nucleic acid or derivative thereof (hereinafter, including derivatives, simply referred to as "second nucleic acid") having an anionic main chain backbone are fused together. In other words, the chimeric molecule that is the target of the production method of the present invention may have a partial structure in which the first nucleic acid and the second nucleic acid are fused together. The chimeric molecule, which is the target of the production method of the present invention, has multiple nucleic acids linked together, any part of which may be a first nucleic acid and the remaining part may be a second nucleic acid.

[0036] In a chimeric molecule in which multiple nucleic acids are linked, the lower limit of the total number of nucleic acids may be 2, 3, 5, 10, or 16. The upper limit of the total number of nucleic acids may be 80, 40, 30, 23, 15, 10, or 5. The above upper and lower limits can be combined arbitrarily. For example, the number may be 2 to 80, 2 to 40, 2 to 15, 2 to 10, 2 to 5, 3 to 80, 3 to 40, 3 to 10, 5 to 40, 10 to 30, or 16 to 23. In a chimeric molecule in which multiple nucleic acids are linked, the ratio of the number of first nucleic acids to the number of second nucleic acids in the total number of nucleic acids is not particularly limited, but may be 1:10 to 10:1, preferably 1:3 to 3:1, and more preferably 1:2 to 2:1, in terms of the number of first nucleic acids:the number of second nucleic acids.

[0037] In the present invention, the first nucleic acid and the second nucleic acid each preferably have the ability to bind to a target nucleic acid. The target nucleic acid is not particularly limited as long as it is a nucleic acid or a derivative thereof having a target sequence to which the chimeric molecule, which is the target of the manufacturing method of the present invention, can bind, but RNA or DNA is preferred. When the chimeric molecule is used as an active ingredient of a pharmaceutical composition, the target nucleic acid is preferably RNA or DNA encoding a protein that causes the disease to be treated using the pharmaceutical composition, or mRNA, miRNA, or siRNA related to the disease.

[0038] In the present invention, the term "nucleic acid" refers to nucleic acids in a broad sense, including RNA and DNA, which are generally defined as nucleic acids, as well as so-called artificial nucleic acids such as PNA, PRNA, and LNA (bridged artificial nucleic acid). Nucleic acid derivatives are not particularly limited, but include, for example, halogenated derivatives of the base moiety (uracil, cytosine, thymine, adenine, guanine, or a purine ring or pyrimidine ring) bound to the nucleic acid, deaminated derivatives, and derivatives having sulfur atoms instead of oxygen atoms in each nucleic acid base. Other nucleic acid derivatives include phosphorothioate-type DNA and RNA, phosphorodithioate-type DNA and RNA, and, for example, morpholino-type nucleic acids as disclosed in the aforementioned Patent Document 3. In the present specification, "halogen" means fluoro, chloro, bromo or iodo, and is preferably fluoro, chloro or bromo.

[0039] The ribose or deoxyribose in RNA, DNA, and pRNA may be ribose or deoxyribose in which the carbon atoms at positions 2 and 4 of LNA or the like are bound by a divalent organic group, and examples of such ribose or deoxyribose include ribose or deoxyribose having the structure described in the aforementioned Patent Document 7. The ribose or deoxyribose in RNA, DNA, and pRNA is preferably ribose or deoxyribose.

[0040] In the present invention, the first nucleic acid or derivative thereof having a neutral or cationic main chain backbone preferably has a neutral main chain backbone. The neutral backbone is not particularly limited, and examples thereof include an amide backbone (typically, a backbone having N-(2-aminoethyl)glycine units). Examples of nucleic acids or derivatives thereof having an amide backbone include PNA or derivatives thereof, PRNA or derivatives thereof, and a combination of PNA and PRNA (hereinafter also referred to as "PNA / PRNA") or derivatives thereof. In the PNA / PRNA combination, the PRNA may be bound to any position in the PNA or may be bound to any part of the PNA, for example, a combination such as PNA-PRNA-PNA.

[0041] The cationic main chain skeleton is not particularly limited, but examples thereof include an imino skeleton, a phosphoric acid amide skeleton, a phosphoramidite skeleton, and a skeleton having a cationic side chain such as an amino group or a guanidium group on the side chain of the amide skeleton.

[0042] In the second nucleic acid or derivative thereof of the present invention, the anionic main chain backbone is not particularly limited, and examples thereof include a sugar-phosphate backbone and a sugar-thiophosphate backbone. Examples of nucleic acids or derivatives thereof having a sugar-phosphate backbone or a sugar-thiophosphate backbone include RNA or derivatives thereof, and DNA or derivatives thereof.

[0043] In the chimeric molecule, the first nucleic acid or a derivative thereof is preferably any one selected from the group consisting of PNA, PRNA, PNA / PRNA, and LNA, and derivatives thereof, more preferably any one selected from the group consisting of PNA and derivatives thereof. Also, in the chimeric molecule, the second nucleic acid or a derivative thereof is preferably any one selected from the group consisting of RNA, DNA, and derivatives thereof. In particular, in a chimeric molecule, it is preferred that the first nucleic acid or a derivative thereof is any one selected from the group consisting of PNA, PRNA, PNA / PRNA, and LNA, and derivatives thereof, and the second nucleic acid or a derivative thereof is any one selected from the group consisting of RNA and DNA, and derivatives thereof; it is more preferred that the first nucleic acid or a derivative thereof is any one selected from the group consisting of PNA and derivatives thereof, and the second nucleic acid or a derivative thereof is any one selected from the group consisting of RNA and DNA, and derivatives thereof; it is even more preferred that the first nucleic acid or a derivative thereof is any one selected from the group consisting of PNA and derivatives thereof, and the second nucleic acid or a derivative thereof is any one selected from the group consisting of DNA and derivatives thereof. When a chimeric molecule contains multiple first nucleic acids or derivatives thereof or multiple second nucleic acids or derivatives thereof, the multiple first nucleic acids or derivatives thereof may be the same or different from each other, and the multiple second nucleic acids or derivatives thereof may be the same or different from each other. In the chimeric molecule, the first nucleic acid may be linked to either the 3' end or the 5' end of the second nucleic acid. In one embodiment of the present invention, it is preferable that the second nucleic acid has at least one portion at its 5' end to which the first nucleic acid is bound, or alternatively, it is necessary that the second nucleic acid has at least one portion at its 5' end to which the first nucleic acid is bound. In another embodiment of the present invention, it is preferred that the second nucleic acid has at least one portion at its 3' end to which the first nucleic acid is bound, or alternatively, it is necessary that the second nucleic acid has at least one portion at its 3' end to which the first nucleic acid is bound.

[0044] Examples of chimeric molecules in which at least one first nucleic acid and one second nucleic acid are fused include a fusion of a first nucleic acid, PNA (or a derivative thereof; the same applies below), with a second nucleic acid, DNA, and a fusion of a first nucleic acid, PNA / PRNA, with a second nucleic acid, DNA. Fusion products of PNA and DNA include chimeric molecules in which PNA is fused to the 5'-position of DNA (hereinafter also referred to as "PNA-DNA chimeric molecules") and chimeric molecules in which PNA is fused to the 3'-position of DNA (hereinafter also referred to as "DNA-PNA chimeric molecules"). In one embodiment of the present invention, PNA-DNA chimeric molecules are preferred. In another embodiment of the present invention, DNA-PNA chimeric molecules are preferred. The fusion of PNA / PRNA and DNA includes a chimeric molecule in which PNA / PRNA is fused to the 5'-position of DNA (hereinafter referred to as "PNA / PRNA-DNA chimeric molecule" or "P R PD), chimeric molecules in which PNA / PRNA is fused to the 3'-side of DNA (hereinafter referred to as "DNA-PNA / PRNA chimeric molecules" or "DP R In one embodiment of the present invention, P R PD is preferred. In another aspect of the present invention, DP R P is preferred.

[0045] The chimeric molecule, which is the target of the production method of the present invention, has multiple nucleic acids linked together, any part of which may be a first nucleic acid and the remaining part may be a second nucleic acid. - elongation of the first nucleic acid (preferably, elongation that generates a bond between the N-terminus of the first nucleic acid and the C-terminus of the first nucleic acid) using the first nucleic acid as a starting point (preferably, via the N-terminus of the first nucleic acid); - extension of a second nucleic acid (preferably via the N-terminus of the first nucleic acid) using the first nucleic acid as a starting point (preferably via the N-terminus of the first nucleic acid) (preferably an extension that generates a bond between the N-terminus of the first nucleic acid and the 3'-position of the second nucleic acid), - extension of the first nucleic acid (preferably via the 5'-position of the second nucleic acid) via the second nucleic acid (preferably an extension that generates a bond between the 5'-position of the second nucleic acid and the C-terminus of the first nucleic acid), and - extension of the second nucleic acid (preferably via the 5'-position of the second nucleic acid) (preferably an extension that generates a bond between the 5'-position of the second nucleic acid and the 3'-position of the second nucleic acid), By combining the above, it is possible to produce a chimeric molecule in which the first nucleic acid and the second nucleic acid are combined in any order. Of these, known methods including solid-phase synthesis can be used for the extension of a first nucleic acid using a first nucleic acid as a clue, and for the extension of a second nucleic acid using a second nucleic acid as a clue.

[0046] For example, the extension of the first nucleic acid using the first nucleic acid as a clue can be performed using the methods described in Patent Documents 4 to 6 mentioned above, or K. Ogami, et al., Chem. Lett., 2018, 47, 138-140, or methods similar thereto. For example, the second nucleic acid can be elongated using the second nucleic acid as a clue by employing the methods described in the aforementioned Patent Documents 2 and 7 or methods similar thereto.

[0047] The extension of a first nucleic acid using a second nucleic acid as a clue, particularly the extension that generates a bond with the C-terminus of the first nucleic acid via the 5'-position of the second nucleic acid, can be carried out using the manufacturing method of the present invention described below (hereinafter also referred to as the first aspect of the present invention). The extension of a second nucleic acid using a first nucleic acid as a starting point, particularly the extension of the second nucleic acid to the N-terminus of the first nucleic acid via the 3'-position, can be achieved by employing the manufacturing method of the present invention described below (hereinafter also referred to as the second aspect of the present invention).

[0048] [First aspect of the present invention] The method for producing a chimeric molecule of the first aspect comprises the following steps (A1) to (A3). (A1) introduction of a second nucleic acid via a hydroxyl group of a first chimeric molecule precursor having a hydroxyl group; (A2) optionally deprotecting the second chimeric molecule precursor; and (A3) Introduction of the first nucleic acid into the second chimeric molecule precursor or the deprotected second chimeric molecule precursor. The production method of the first aspect of the present invention will be described below, taking as an example the production of a PNA-DNA chimeric molecule (a chimeric molecule in which PNA is fused to the 5'-side of DNA) using PNA as the first nucleic acid and DNA as the second nucleic acid as necessary.

[0049] (A1) Introduction of a second nucleic acid via a hydroxyl group of a first chimeric molecule precursor having a hydroxyl group (hereinafter simply referred to as "first chimeric molecule precursor") (preparation of a second chimeric molecule precursor) This step involves introducing a second nucleic acid into the first chimera molecule precursor using a hydroxyl group present in the first chimera molecule precursor, resulting in the production of a second chimera molecule precursor. The first chimeric molecule precursor has a hydroxyl group for introducing the second nucleic acid, and also contains a lipid-soluble anchor that solidifies the target substance by adding a specific solvent to the solution after the reaction, thereby facilitating its recovery. After the introduction of the second nucleic acid, the first nucleic acid is introduced using the introduced second nucleic acid as a guide, and the second nucleic acid has an amino group at the 5'-position that may be protected.

[0050] In this step, the second nucleic acid is introduced by nucleophilic attack from the hydroxyl group of the first chimera molecule precursor, resulting in a bond between the oxygen atom derived from the hydroxyl group of the first chimera molecule precursor and the phosphorus atom of the second nucleic acid, DNA, preferably the phosphorus atom connected to the 3'-position of deoxyribose in the DNA. The second nucleic acid may be a combination of two or more second nucleic acids. The phosphorus atom of the second nucleic acid that is subjected to nucleophilic attack is preferably trivalent. In this case, one bond is substituted with the oxygen atom at the 3'-position of deoxyribose (ribose in the case of RNA), another bond is substituted with a leaving group, and the remaining bond is finally substituted with -O- is substituted with a group that can give rise to

[0051] The leaving group may be any leaving group that is used in a nucleophilic substitution reaction of an oxygen atom with a phosphorus atom under weakly acidic conditions. For example, a dialkylamino group may be used. As the dialkylamino group, a diisopropylamino group is preferably used, and for example, a diethylamino group or an ethylisopropylamino group may also be used.

[0052] Finally -O - The group capable of generating the formula (I) may be any group in which a group that is detached from an oxygen atom is substituted for the oxygen atom under conditions that do not affect other structures in the chimeric molecule or chimeric molecule precursor. For example, under weakly basic conditions, -O - Examples of suitable ethoxy groups include an ethoxy group substituted with an electron-withdrawing group at the 2-position, which can produce -O - The allyloxy group can be reduced to -O by catalytic hydrogenation in the presence of palladium on carbon. - Examples of suitable aryl groups include a benzyloxy group, which can give Under weak basic conditions, -O - The group that can generate the above can be finally eliminated during cleavage of the lipophilic anchor. Furthermore, the group that is eliminated from the oxygen atom under weakly basic conditions is a functional group necessary for nucleophilic attack from the first chimera molecule precursor in this step, but in other steps, it may or may not be eliminated, and may be eliminated intentionally or may be eliminated due to the reaction conditions.

[0053] The optionally protected amino group of the second nucleic acid (the first nucleic acid is introduced via this amino group, as described below) includes an amino group and a protected amino group. A protected amino group is preferred for smooth progress of this step. The amino-protecting group is not particularly limited as long as it can be removed under acidic conditions, and examples thereof include those described in "Greene's Protective Groups in Organic Synthesis" by P.G.W. Utz and T.W. Greene (4th ed., 2006), with an optionally substituted trityl group being preferred. Examples of the optionally substituted trityl group include a trityl group (Tr), a p-methoxyphenyldiphenylmethyl group (MMTr), and a di(p-methoxyphenyl)phenylmethyl group (DMTr), with MMTr being more preferred. When a compound having two or more second nucleic acids bound thereto is used as the second nucleic acid in this step, the optionally protected amino group possessed by the second nucleic acid only needs to be possessed by the second nucleic acid located at the 5'-end of the compound having two or more second nucleic acids bound thereto, and the second nucleic acids other than the second nucleic acid located at the 5'-end may each be bound via an oxygen atom.

[0054] For example, the second nucleic acid used in this step has the following structure:

[0055] [ka]

[0056] In the formula, p represents an integer of 0 or more; AG represents an optionally protected amino group; Base represents a base moiety (which may be the same or different); NR2 represents a leaving group used in the nucleophilic substitution reaction of oxygen atom with phosphorus atom under weak acidic conditions; LV represents a group that leaves from the oxygen atom. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position.

[0057] p is preferably 0 to 40, more preferably 0 to 20, even more preferably 0 to 10, still more preferably 0 to 5, particularly preferably 0 to 1, and particularly preferably 0.

[0058] Examples of the optionally protected hydroxyl group that deoxyribose is allowed to have at the 2'-position include 2'-modified groups such as a methoxy group and a methoxyethyloxy group, as well as groups that can be selectively removed after synthesis to generate a hydroxyl group, such as a tert-butyldimethylsilyloxy group, a triisopropyloxymethyloxy group, a tetrahydropyranyloxy group, a bis(2-acetylmethoxy)methyloxy group, and a levulinyloxy group. Hereinafter, the same applies to deoxyribose that is allowed to have an optionally protected hydroxyl group at the 2'-position in this specification.

[0059] The second nucleic acid used in this step, for example, has one bond of the phosphorus atom substituted with the oxygen atom at the 3'-position of deoxyribose, another bond substituted with a leaving group, and finally, the remaining bond substituted with -O - The second nucleic acid substituted with a group capable of generating the above can be produced, for example, by the method described in U.S. Patent Application Publication US2020 / 0399304, Tetrahedron Letters, 39(24), 4215-4218, 1998, or Journal of Carbohydrate Chemistry, 24(2), 145-160, 2005, or a method similar thereto.

[0060] The lipophilic anchor of the first chimera molecule precursor may be a partial structure that solidifies the target substance by adding a specific solvent to the solution after the reaction, thereby facilitating recovery. Examples of the partial structures include those disclosed in Patent Documents 2 to 6, and in particular, the pseudo-solid-phase protecting group disclosed in Patent Document 2 can be used. In the method for producing a chimeric molecule of the present invention, a starting material containing a lipid-soluble anchor as part of its structure is used, and therefore a chimeric molecule containing a lipid-soluble anchor as part of its structure can be produced. A chimeric molecule not containing a lipid-soluble anchor as part of its structure may be produced by cleaving the lipid-soluble anchor from a chimeric molecule containing a lipid-soluble anchor as part of its structure produced by the method for producing a chimeric molecule of the present invention. It is preferable that the lipid-soluble anchor is not cleaved during the production of the chimeric molecule, but can be cleaved at any stage. It is preferable that the lipid-soluble anchor is not removed under acidic conditions but can be removed under basic conditions.

[0061] The lipophilic anchor may be an organic group having an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which one or more aliphatic hydrocarbon groups having 10 to 40 carbon atoms are bonded via a single bond or a linker.

[0062] The aliphatic hydrocarbon group having 10 to 40 carbon atoms may be linear, branched, or cyclic, or may be a mixture of these. The aliphatic hydrocarbon group may have one or two unsaturated bonds. In the aliphatic hydrocarbon group, one or two methylene carbon atoms may be replaced with an etheric oxygen atom. The aliphatic hydrocarbon group is preferably linear; more preferably linear and free of unsaturated bonds; and even more preferably linear and free of unsaturated bonds and etheric oxygen atoms. The aliphatic hydrocarbon group having 10 to 40 carbon atoms preferably has 10 to 30 carbon atoms, more preferably 12 to 28 carbon atoms, further preferably 14 to 22 carbon atoms, and particularly preferably 16 to 20 carbon atoms. As the aliphatic hydrocarbon group having 10 to 40 carbon atoms, a linear alkyl group having 14 to 22 carbon atoms is preferred, a linear alkyl group having 16 to 20 carbon atoms is more preferred, a linear alkyl group having 17 to 19 carbon atoms is even more preferred, and a linear alkyl group having 18 carbon atoms is particularly preferred.

[0063] The aliphatic hydrocarbon group having 10 to 40 carbon atoms and the aromatic hydrocarbon ring having 6 to 14 carbon atoms are preferably bonded via a linker. Examples of the linker include -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, -NH-C(=O)-, -S-, -S(=O)-, and -S(=O)-, with -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, and -NH-C(=O)- being preferred, and -O- being more preferred.

[0064] Examples of the aromatic hydrocarbon ring having 6 to 14 carbon atoms include a benzene ring, a naphthalene ring, an anthracene ring, and a phenanthrene ring, with a benzene ring and a naphthalene ring being preferred, and a benzene ring being more preferred.

[0065] In the aromatic hydrocarbon ring having 6 to 14 carbon atoms, 1 to 5 aliphatic hydrocarbon groups having 10 to 40 carbon atoms may be bonded via a single bond or a linker, preferably 1 to 4, more preferably 1 to 3, even more preferably 2 to 3, and particularly preferably 3.

[0066] Examples of the fat-soluble anchor include an organic group having a benzene ring in which 1 to 5 aliphatic hydrocarbon groups having 14 to 22 carbon atoms are bonded via an -O-linker; an organic group having a benzene ring in which 2 to 3 aliphatic hydrocarbon groups having 16 to 20 carbon atoms are bonded via an -O-linker is preferred; an organic group having a benzene ring in which 3 aliphatic hydrocarbon groups having 18 carbon atoms are bonded via an -O-linker is more preferred; and an organic group having 3,4,5-tri(n-octadecanyloxy)phenyl is even more preferred.

[0067] Examples of the organic group having an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker include organic groups having the following structures, and an organic group having the structure represented by the following formula (i) is preferred.

[0068] [ka]

[0069] In the formula, Ar * represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; NA each independently represents a position at which the first nucleic acid or the second nucleic acid is bound to the structure containing the first nucleic acid or the second nucleic acid.

[0070] The alkyl group having 1 to 6 carbon atoms in R may be linear, branched, or cyclic, or may be a mixture of these. Examples include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group, with a methyl group and an ethyl group being preferred, and a methyl group being more preferred. R is preferably a hydrogen atom.

[0071] For example, as in formula (i), when the position binding to the structure containing the first nucleic acid or second nucleic acid represented by NA is a carbonyl carbon, it is preferred that it is bound to the oxygen atom at the 3'- or 5'-position, preferably the 3'-position, of the deoxyribose or ribose of the nucleoside.

[0072] The hydroxyl groups of the first chimera molecule precursor are preferably selected from the group consisting of hydroxyl groups at the 3'- and 5'-positions of deoxyribose and ribose of nucleosides and hydroxyl groups at the 3'- and 5'-positions of deoxyribose and ribose of DNA and RNA, which are second nucleic acids; more preferably, they are selected from the group consisting of hydroxyl groups at the 5'-positions of deoxyribose and ribose of nucleosides and hydroxyl groups at the 5'-positions of deoxyribose and ribose of DNA and RNA, which are second nucleic acids.

[0073] When the hydroxyl group possessed by the first chimera molecule precursor is the hydroxyl group at the 5'-position of deoxyribose or ribose of a nucleoside, the first chimera molecule precursor A may be a first chimera molecule precursor A in which the oxygen atom at the 3'-position of deoxyribose or ribose of the nucleoside is directly substituted at the NA position of the lipid-soluble anchor represented by formula (i). The first chimera molecule precursor A has, for example, the following structure:

[0074] [ka]

[0075] In the formula, Ar * and R has the above meaning; Base indicates the base moiety. The deoxyribose may have an optionally protected hydroxyl group at the 2'-position.

[0076] When the hydroxyl group of the first chimera molecule precursor is a hydroxyl group at the 5'-position of deoxyribose or ribose of the second nucleic acid, DNA or RNA, the first chimera molecule precursor B may be such that the oxygen atom of the hydroxyl group of the first chimera molecule precursor A is directly bonded to the 3'-position phosphate moiety of deoxyribose or ribose of the second nucleic acid, DNA or RNA, or the oxygen atom of the hydroxyl group of the first chimera molecule precursor A is bonded to the 3'-position phosphate moiety of deoxyribose or ribose of the second nucleic acid, DNA or RNA, via one or more first nucleic acids and / or second nucleic acids (which may be a combination of a first nucleic acid and a second nucleic acid). The first chimera molecule precursor B has, for example, the following structure:

[0077] [ka]

[0078] In the formula, Ar * , R and Base have the same meanings as above; X represents a single bond or represents one or more first nucleic acids and / or second nucleic acids via a bond between an oxygen atom and a phosphorus atom bonded to X. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position.

[0079] When the hydroxyl group possessed by the first chimera molecule precursor is a hydroxyl group at the 5'-position of deoxyribose or ribose of the second nucleic acid, DNA or RNA, the first chimera molecule precursor C may be a first chimera molecule precursor C in which the N-terminus of the first nucleic acid, PNA or pRNA, whose C-terminus is bound to the NA position of the lipid-soluble anchor represented by formula (ii), is directly linked to the 3'-position phosphate site of deoxyribose or ribose of the second nucleic acid, DNA or RNA, or the N-terminus of the first nucleic acid, PNA or pRNA, whose C-terminus is bound to the NA position of the lipid-soluble anchor represented by formula (ii), is linked to the 3'-position phosphate site of deoxyribose or ribose of the second nucleic acid, DNA or RNA, via one or more first nucleic acids and / or second nucleic acids (which may be a combination of the first nucleic acid and the second nucleic acid). The first chimera molecule precursor C has, for example, the following structure:

[0080] [ka]

[0081] In the formula, Ar * , R and Base have the same meanings as above; Y represents a single bond or represents one or more first nucleic acids and / or second nucleic acids via a bond between a nitrogen atom and a phosphorus atom bonded to Y. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position.

[0082] In X of the first chimera molecule precursor B and Y of the first chimera molecule precursor C, the one or more first nucleic acids and / or second nucleic acids are linked via a bond between an oxygen atom (or a nitrogen atom) bound to X (or Y) and a phosphorus atom, and the total number of nucleic acids may be 1 to 70, preferably 1 to 31, more preferably 1 to 21, even more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.

[0083] Since the aforementioned fat-soluble anchor solidifies the target substance by adding a specific solvent to the solution after the reaction, facilitating recovery, the molecular weight of the first chimera molecule precursor is preferably 300 to 6,000, more preferably 300 to 5,000, and even more preferably 300 to 4,000.

[0084] The first chimera molecule precursor used in this step can be produced, for example, by the methods described in the aforementioned patent documents, particularly Patent Documents 2 and 7, or the methods described in the Examples of the present application, or by methods similar thereto.

[0085] This step is carried out using an excess amount of the second nucleic acid relative to the first chimeric molecule precursor in a solvent inert to the reaction.

[0086] It is preferable to use a non-polar solvent as the reaction solvent. Examples of non-polar solvents include halogenated hydrocarbons such as chloroform, dichloromethane, and 1,2-dichloroethane; aromatic hydrocarbons such as benzene, toluene, xylene, and mesitylene; fatty acid esters such as ethyl acetate and isopropyl acetate; aliphatic hydrocarbons such as hexane, pentane, heptane, octane, and cyclohexane; non-polar ethers such as diethyl ether and cyclopentyl methyl ether; and any combination thereof. Halogenated hydrocarbons are preferred. In addition, a polar solvent may be used in combination as long as the proportion of the nonpolar solvent in the total reaction solvent is 50% by volume or more. Examples of polar solvents include nitriles such as acetonitrile and propionitrile, polar ethers such as 1,4-dioxane and tetrahydrofuran, amides such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone and dimethyl sulfoxide, and any combination thereof.

[0087] For example, a combination of a halogenated hydrocarbon and a nitrile is preferred, and a combination of dichloromethane and acetonitrile is more preferred. In this case, the mixing ratio (volume %) may be 50:50 to 99:1, preferably 80:20 to 99:1, and more preferably 80:20 to 95:5.

[0088] The second nucleic acid used in excess relative to the first chimera molecule precursor can be used in an amount of 1.5 to 10 equivalents, preferably 1.6 to 8 equivalents, more preferably 1.8 to 5 equivalents, and even more preferably 2 to 4 equivalents relative to the number of moles of the first chimera molecule precursor used.

[0089] To facilitate the smooth progress of this step, an activator such as 1H-tetrazole or 4,5-dicyanoimidazole may be used. When using such an activator, 1 to 25 equivalents, preferably 5 to 20 equivalents, more preferably 8 to 15 equivalents, and even more preferably 8 to 12 equivalents, based on the number of moles of the first chimera molecule precursor used.

[0090] The reaction temperature and reaction time can be appropriately changed depending on the types and amounts of reagents used. For example, the mixture may be stirred at 0 to 100°C, preferably 15 to 50°C, and more preferably 20 to 30°C for 5 minutes to 24 hours. The substrate concentration in the reaction can be appropriately adjusted depending on the types and amounts of reagents used. For example, the amount of solvent used can be adjusted so that the concentration of the first chimera molecule precursor in the reaction solution is 0.01 to 0.2 mol / L, preferably 0.02 to 0.1 mol / L, more preferably 0.025 to 0.08 mol / L, and even more preferably 0.03 to 0.05 mol / L.

[0091] Following this step, the phosphorus atom of the second chimeric molecule precursor may be oxidized or thioxidized, if necessary. The oxidation or thioxidation of the phosphorus atom can be carried out by a conventional method, for example, see the process of converting a phosphite triester bond to a phosphate triester bond or a thiophosphate triester bond in the aforementioned Patent Document 7.

[0092] For the oxidation of phosphorus atoms, a peroxide is preferably used. Preferred peroxides are tert-butyl hydroperoxide and metachloroperbenzoic acid. Tert-butyl hydroperoxide is available commercially as a decane or toluene solution, which can be used directly or after being diluted 1 to 3 times. The amount of the oxidizing agent or thio-oxidizing agent used is, for example, 1 to 50 moles, preferably 1 to 10 moles, and more preferably 2 to 5 moles per mole of the second chimera molecule precursor or the first chimera molecule precursor used in the previous step.

[0093] The oxidation or thioxidation of the phosphorus atom may be carried out in a so-called one-pot manner following the step of introducing the second nucleic acid, without first isolating and purifying the second chimeric molecule precursor after the introduction of the second nucleic acid.

[0094] After the reaction is complete, a polar solvent may be added to the reaction solution to solidify and recover the target product (second chimera molecule precursor). Because the first chimera molecule precursor used in the production method of the present invention contains a lipid-soluble anchor, the target product of this step (second chimera molecule precursor) solidifies and precipitates upon addition of a polar solvent. Therefore, the target product of this step can be recovered by filtration. As the polar solvent, acetonitrile and methanol are preferably used in terms of versatility and cost, and methanol is particularly preferred. The amount of polar solvent added to the reaction solution after completion of the reaction in order to recover the target product by filtration depends on the substrate concentration in the reaction, but is, for example, 5 to 50 times, preferably 5 to 30 times, more preferably 8 to 20 times, and even more preferably 10 to 15 times the total amount (volume) of the reaction solvent used.

[0095] (A2) Optionally, deprotecting the second chimeric molecule precursor. The second chimeric molecule precursor obtained in the above step has an optionally protected amino group derived from the introduced second nucleic acid. Since the first nucleic acid is introduced via this amino group in the next step, if the optionally protected amino group is a protected amino group, it is necessary to deprotect the amino group. The conditions for deprotecting the amino group can be appropriately changed depending on the protecting group used. Specifically, the method described in the aforementioned "Greene's Protective Groups in Organic Synthesis (4th edition, 2006)" can be adopted.

[0096] For example, when the protecting group for the amino group is MMTr, the reaction is carried out using an acid in a solvent inert to the reaction. The acid that can be used is not particularly limited, but examples thereof include halogenoacetic acids such as trichloroacetic acid, trifluoroacetic acid, and dichloroacetic acid; and sulfonic acids such as methanesulfonic acid, trifluoromethanesulfonic acid, and p-toluenesulfonic acid. Since good results can be obtained, halogenoacetic acids are preferred, and trichloroacetic acid is particularly preferred. The amount of acid used is, for example, 1 to 100 mol, preferably 5 to 80 mol, more preferably 10 to 60 mol, and even more preferably 20 to 50 mol per mole of the second chimera molecule precursor used.

[0097] As in the previous step, after the reaction is completed, a polar solvent may be added to the reaction solution to solidify and recover the target product (deprotected second chimera molecule precursor).

[0098] The deprotected second chimera molecule precursor may be subjected to the next step as is, or may be subjected to the next step after removing the group that leaves from the oxygen atom under weakly basic conditions, as shown by LV in the above formula. The removal of the group that leaves from the oxygen atom can be performed by a conventional method. For the removal of the group that leaves from the oxygen atom under weakly basic conditions, see, for example, De Napoli et al., Chem. Commun., 2005, 2586-2588 and U. Pradere et al., Chem. Rev., 2014, 114, 9154-9218.

[0099] (A3) Introduction of the first nucleic acid into the second chimeric molecule precursor or the deprotected second chimeric molecule precursor (hereinafter simply referred to as the "second chimeric molecule precursor") (production of the chimeric molecule) In this step, the first nucleic acid is introduced using the amino group of the second chimeric molecule precursor as a foothold, resulting in the production of a chimeric molecule.

[0100] In this step, the introduction of the first nucleic acid involves the formation of an amide bond between the amino group of the second chimera molecule precursor and the carboxyl group of the first nucleic acid by an amidation reaction or a reaction equivalent thereto. The first nucleic acid may be one to which one or more first nucleic acids are bound. As the first nucleic acid, an N-terminal protected PNA monomer, such as an Fmoc-type PNA monomer, can be used. The fluorenyl group in Fmoc (fluorenylmethoxycarbonyl) in the Fmoc-type PNA monomer may have one or two substituents at any substitution position to adjust the reactivity during Fmoc removal. Preferred substituents are electron-withdrawing groups, such as halogen, nitro, cyano, trifluoromethyl, and carbonyl (carboxy, alkyloxycarbonyl, dialkylcarbamoyl, and alkylcarbonyl).

[0101] For example, the first nucleic acid used in this step has the following structure:

[0102] [ka]

[0103] In the formula, q represents an integer of 0 or more; PG represents a protecting group for the adjacent amino group; Base indicates the base moiety (which may be the same or different).

[0104] q is preferably 0 to 40, more preferably 0 to 30, even more preferably 0 to 20, even more preferably 0 to 10, and particularly preferably 0.

[0105] The first nucleic acid used in this step may be a commercially available compound, or a compound in which two or more first nucleic acids are bound can be prepared by subjecting commercially available monomers to a conventional amidation reaction.

[0106] As the protecting group for the amino group, preferably optionally substituted Fmoc, more preferably Fmoc, can be used. Alternatively, trityl, including Tr, MMTr, and DMTr, more preferably MMTr, or a phthalimide-type protecting group that can be removed using hydrazine can also be used.

[0107] This step is carried out using an excess amount of the first nucleic acid relative to the second chimeric molecule precursor, in the presence of a condensing agent as needed, in a solvent inert to the reaction.

[0108] Examples of the solvent used here include, but are not limited to, halogenated hydrocarbons, aromatic hydrocarbons, fatty acid esters, non-polar / polar ethers, nitriles, amides, and any combination thereof. For example, tetrahydrofuran is preferred.

[0109] The first nucleic acid used in excess relative to the second chimera molecule precursor can be used in an amount of 1.01 to 5 equivalents, preferably 1.05 to 3 equivalents, more preferably 1.1 to 2.5 equivalents, and even more preferably 1.1 to 2.2 equivalents relative to the number of moles of the second chimera molecule precursor used.

[0110] A condensing agent may be used to facilitate the progress of this step. Condensing agents include, but are not limited to, dicyclohexylcarbodiimide, 1,1'-carbonyldiimidazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), diphenylphosphoryl azide, and phosphorus oxychloride. For example, COMU is preferred.

[0111] When a condensing agent, preferably COMU, is used as the condensing agent, it can be used in an amount of 1 to 10 equivalents, preferably 1.5 to 8 equivalents, more preferably 2 to 6 equivalents, even more preferably 2.5 to 5 equivalents, and particularly preferably 3 to 4.5 equivalents relative to the number of moles of the second chimera molecule precursor used.

[0112] The carboxyl group in the first nucleic acid can also be converted to a reactive derivative before reacting with the second chimeric molecule precursor. Examples of reactive derivatives of the carboxyl group include acid halides obtained by reaction with halogenating agents such as phosphorus oxychloride and thionyl chloride; mixed acid anhydrides obtained by reaction with isobutyl chloroformate; and activated esters obtained by condensation with 1-hydroxybenzotriazole. The reaction of these reactive derivatives with the second chimeric molecule precursor is carried out in a solvent inert to the reaction, such as halogenated hydrocarbons, aromatic hydrocarbons, or ethers. The use of an organic base such as triethylamine, N,N-diisopropylethylamine, or N-methylmorpholine may be advantageous for smooth reaction.

[0113] In this step, the use of an additive (for example, 1-hydroxybenzotriazole) may be preferable for the reaction. In some cases, it may be advantageous to carry out the reaction in the presence of an organic base or an inorganic base such as potassium carbonate, sodium carbonate, or potassium hydroxide, for example, N,N-diisopropylethylamine, in order to ensure smooth reaction. When an organic base, preferably N,N-diisopropylethylamine, is used as the organic base, it can be used in an amount of 1 to 10 equivalents, preferably 1.5 to 8 equivalents, more preferably 1.5 to 5 equivalents, even more preferably 2 to 4 equivalents, and particularly preferably 2 to 3 equivalents, relative to the number of moles of the second chimera molecule precursor used. Furthermore, it can be used in an amount of 1 to 10 times, preferably 1.5 to 5 times, more preferably 2 to 4 times, and even more preferably 2.5 to 3 times the number of phosphate backbones in the second chimera molecule precursor.

[0114] The reaction temperature and reaction time can be appropriately changed depending on the types and amounts of reagents used. For example, the mixture may be stirred at 0 to 100°C, preferably 15 to 50°C, and more preferably 20 to 30°C for 5 minutes to 24 hours. The substrate concentration in the reaction can be appropriately adjusted depending on the types and amounts of reagents used. For example, the amount of solvent used can be adjusted so that the concentration of the first chimera molecule precursor in the reaction solution is 0.01 to 0.2 mol / L, preferably 0.02 to 0.1 mol / L, more preferably 0.025 to 0.08 mol / L, and even more preferably 0.03 to 0.05 mol / L.

[0115] As in the previous step, after the reaction is completed, a polar solvent may be added to the reaction solution to solidify and recover the target product (chimera molecule).

[0116] The chimeric molecule obtained in the above step may further have a first nucleic acid or a second nucleic acid linked to it via the N-terminus of the introduced first nucleic acid. In this case, the protecting group of the amino group of the introduced first nucleic acid can be removed, and the first nucleic acid or the second nucleic acid can be bound using the amino group as a clue.

[0117] The method for removing the protecting group of the amino group of the introduced first nucleic acid may be selected depending on the type of protecting group, and the method described in the aforementioned "Greene's Protective Groups in Organic Synthesis (4th edition, 2006)" can be used. When the protecting group of the amino group of the introduced first nucleic acid is Fmoc, for example, a method of reacting with a solution of 2 volume % 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 2 volume % piperidine, for example, a tetrahydrofuran (THF) solution, can be used.

[0118] Introduction of an additional first nucleic acid into a chimeric molecule can be carried out in accordance with the above-mentioned (3) introduction of a first nucleic acid into a second chimeric molecule precursor or a deprotected second chimeric molecule precursor (hereinafter simply referred to as "second chimeric molecule precursor") (production of a chimeric molecule). The second nucleic acid can be introduced by the method described in F. Bergmann, et al., Tetrahedron Lett., 1995, 36(38), 6823-6826 or a method similar thereto, or the method described in WO 2017 / 086397 or a method similar thereto.

[0119] By cleaving the lipid-soluble anchor from the chimeric molecule thus produced that contains the lipid-soluble anchor as part of its structure, it is possible to produce a chimeric molecule that does not contain the lipid-soluble anchor as part of its structure. As described above, it is preferable that the lipid-soluble anchor is not cleaved during the production of the chimeric molecule, but can be cleaved at any stage. It is preferable that the lipid-soluble anchor is not removed under acidic conditions, but can be removed under basic conditions. When the lipophilic anchor is represented by formula (i) or (ii), it can be cleaved by the methods described in the aforementioned Patent Documents 2 and 7, or methods similar thereto. For example, the lipophilic anchor residue is precipitated by the action of aqueous ammonium hydroxide in a polar solvent, preferably ethanol, and the target chimeric molecule can be isolated from the supernatant. Note that if a group that is cleaved from an oxygen atom under weakly basic conditions (a group indicated by LV) remains at the phosphate site of the introduced second nucleic acid without being cleaved, or if a protecting group (e.g., Fmoc) for the amino group of the introduced first nucleic acid remains without being removed, a chimeric molecule can be obtained from which these groups have also been cleaved.

[0120] [Second Aspect of the Present Invention] The method for producing a chimeric molecule of the second aspect comprises the following step (B1): (B1) Introduction of a second nucleic acid via an amino group of a third chimeric molecule precursor having an amino group. The production method of the second aspect of the present invention will be described below, taking as an example the production of a DNA-PNA chimeric molecule (a chimeric molecule in which PNA is fused to the 3'-side of DNA) using PNA as the first nucleic acid and DNA as the second nucleic acid as necessary.

[0121] (B1) Introduction of a second nucleic acid via an amino group of a third chimeric molecule precursor having an amino group (hereinafter simply referred to as "third chimeric molecule precursor") (production of a chimeric molecule) In this step, the second nucleic acid is introduced using the amino group of the third chimeric molecule precursor as a foothold, resulting in the production of a chimeric molecule. The third chimera molecule precursor has an amino group for introducing the second nucleic acid and also a lipid-soluble anchor that solidifies the target product by adding a specific solvent to the solution after the reaction, thereby facilitating recovery. The third chimera molecule precursor also has a partial structure derived from the first nucleic acid, and the amino group of the third chimera molecule precursor is derived from the first nucleic acid. The third chimera molecule precursor having a partial structure derived from the first nucleic acid means that the first nucleic acid, preferably PNA or pRNA, more preferably PNA, is bound to another portion of the third chimera molecule precursor at its C-terminus, and the N-terminus of the first nucleic acid, preferably PNA or pRNA, more preferably PNA, is exposed. The exposed N-terminus of the PNA may be present, for example, as a quaternary ammonium salt. The exposed N-terminus of the first nucleic acid, preferably PNA, is an amino group possessed by the third chimera molecule precursor, and serves as a foothold for introducing the second nucleic acid.

[0122] In this step, the second nucleic acid is subjected to nucleophilic attack by the amino group of the third chimeric molecule precursor, resulting in a bond between the nitrogen atom derived from the amino group of the third chimeric molecule precursor and a phosphorus atom of the second nucleic acid, DNA, preferably the phosphorus atom connected to the 3'-position of deoxyribose in the DNA. The second nucleic acid may be a combination of two or more second nucleic acids. The phosphorus atom of the second nucleic acid that is subjected to nucleophilic attack is preferably an oxidized pentavalent phosphorus atom. In this case, one bond is substituted with the oxygen atom at the 3'-position of deoxyribose (ribose in the case of RNA), and the other bond is finally substituted with -O - It is substituted with a group that can produce a bond, and the remaining bond is bonded to a hydrogen atom.

[0123] Finally -O - As the group capable of generating the above, the embodiments explained in the production method of the first embodiment can be adopted.

[0124] For example, the second nucleic acid used in this step has the following structure:

[0125] [ka]

[0126] In the formula, p represents an integer of 0 or more; AG / HG represent an optionally protected amino group or hydroxyl group; Base represents a base moiety (which may be the same or different); LV represents a group that leaves from the oxygen atom. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position.

[0127] p is preferably 0 to 40, more preferably 0 to 20, even more preferably 0 to 10, still more preferably 0 to 5, particularly preferably 0 to 1, and particularly preferably 0.

[0128] The optionally protected amino or hydroxyl group in AG / HG serves as a clue for further nucleic acid elongation from the chimeric molecule produced in this step. To ensure smooth progress of this step, a protected amino or hydroxyl group is preferred. In another embodiment, the optionally protected amino or hydroxyl group in AG / HG is preferably an optionally protected hydroxyl group, and more preferably a protected hydroxyl group. The protecting group for the amino group is not particularly limited as long as it is a group that can be removed under acidic conditions. In the first embodiment, a protecting group that is acceptable for the optionally protected amino group of the second nucleic acid introduced via the hydroxyl group of the first chimera molecule precursor can be used. The hydroxyl-protecting group is not particularly limited as long as it is a group that can be removed under acidic conditions, and examples thereof include groups described in "Greene's Protective Groups in Organic Synthesis" by P.G.W. Uts and T.W. Greene (4th ed., 2006). An optionally substituted trityl group is preferred, and in another embodiment, an optionally substituted fluorenylmethoxycarbonyl group (Fmoc) is preferred. Examples of the optionally substituted trityl group include a trityl group (Tr), a p-methoxyphenyldiphenylmethyl group (MMTr), and a di(p-methoxyphenyl)phenylmethyl group (DMTr), with DMTr being more preferred. The optionally substituted fluorenylmethoxycarbonyl group includes a fluorenylmethoxycarbonyl group in which the fluorenyl group in the fluorenylmethoxycarbonyl group is substituted with halogen, nitro, cyano, trifluoromethyl, and / or carbonyl (carboxy, alkyloxycarbonyl, dialkylcarbamoyl, alkylcarbonyl).

[0129] The second nucleic acid used in this step can be produced, for example, from the compound of formula (4) above (the second nucleic acid used in step A1 of the first aspect) by a known method, a method described in the Examples of the present application, or a method similar thereto.

[0130] The lipid-soluble anchor possessed by the third chimera molecule precursor can be the same as that described in the production method of the first embodiment. Examples of the organic group having an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which the aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker include organic groups having the following structures, and an organic group having the structure represented by the following formula (ii) is preferred.

[0131] [ka]

[0132] The symbols in the formula are as defined above.

[0133] For example, as in formula (ii), when the position at which the structure containing the first nucleic acid or second nucleic acid represented by NA is bound is a nitrogen atom, it is preferable that the nitrogen atom binds to the carbonyl carbon of the first nucleic acid, PNA or pRNA, to form an amide bond.

[0134] The third chimera molecule precursor may be a third chimera molecule precursor A in which a partial structure derived from the first nucleic acid contained in the third chimera molecule precursor is directly substituted at the NA position of the lipid-soluble anchor represented by formula (ii). The third chimera molecule precursor A has, for example, the following structure:

[0135] [ka]

[0136] In the formula, Ar * represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Base indicates the base moiety.

[0137] The third chimera molecule precursor may be a third chimera molecule precursor B in which a partial structure derived from a first nucleic acid contained in the third chimera molecule precursor is linked to the NA position of the lipid-soluble anchor represented by formula (ii) via one or more first nucleic acids and / or second nucleic acids (which may be a combination of the first nucleic acid and the second nucleic acid). The third chimeric molecule precursor B has, for example, the following structure:

[0138] [ka]

[0139] In the formula, Ar * , R and Base have the same meanings as above; Z represents a single bond, or represents one or more first nucleic acids and / or second nucleic acids via a bond between the nitrogen atom bonded to Z and the carbonyl carbon atom. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position.

[0140] The third chimera molecule precursor may be a third chimera molecule precursor C in which the 5'-hydroxyl group of deoxyribose or ribose of a nucleoside having an oxygen atom at the 3'-position bound to the NA position of the lipid-soluble anchor represented by formula (i) is directly linked to a partial structure derived from a first nucleic acid contained in the third chimera molecule precursor, or the 5'-hydroxyl group of deoxyribose or ribose of a nucleoside having an oxygen atom at the 3'-position bound to the NA position of the lipid-soluble anchor represented by formula (i) is linked to a partial structure derived from a first nucleic acid contained in the third chimera molecule precursor via one or more first nucleic acids and / or second nucleic acids (which may be a combination of the first nucleic acid and the second nucleic acid). The third chimeric molecule precursor C has, for example, the following structure:

[0141] [ka]

[0142] In the formula, Ar * , R and Base have the same meanings as above; W represents a single bond, or represents one or more first nucleic acids and / or second nucleic acids via a bond between an oxygen atom bonded to Z and a carbonyl carbon atom. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position.

[0143] In Z of the third chimera molecule precursor B and W of the third chimera molecule precursor C, in one or more first nucleic acids and / or second nucleic acids connected via a bond between a nitrogen atom (or oxygen atom) bound to Z (or W) and a carbonyl carbon atom, the total number of nucleic acids may be 1 to 70, preferably 1 to 31, more preferably 1 to 21, even more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.

[0144] Since the aforementioned fat-soluble anchor solidifies the target substance by adding a specific solvent to the solution after the reaction, facilitating its recovery, the molecular weight of the third chimera molecule precursor is preferably 300 to 6,000, more preferably 300 to 5,000, and even more preferably 300 to 4,000.

[0145] The third chimera molecule precursor used in this step can be produced, for example, by the methods described in the aforementioned patent documents, the methods described in the Examples of the present application, or methods based thereon. Alternatively, it may be produced according to the method described in K. Ogami, et al., Chem. Lett., 2018, 47, 138-140.

[0146] This step is carried out using an excess amount of the second nucleic acid relative to the third chimeric molecule precursor in a solvent inert to the reaction.

[0147] Examples of the solvent used herein include, but are not limited to, halogenated hydrocarbons; aromatic hydrocarbons; non-polar / polar ethers; nitriles; amides; and any combination thereof.

[0148] For example, a combination of a halogenated hydrocarbon and a nitrile is preferred, and a combination of dichloromethane and acetonitrile is more preferred. In this case, the mixing ratio (volume %) may be 50:50 to 99:1, preferably 80:20 to 99:1, and more preferably 80:20 to 95:5.

[0149] The second nucleic acid used in excess relative to the third chimera molecule precursor can be used in an amount of 1.5 to 50 equivalents, preferably 3 to 30 equivalents, more preferably 5 to 20 equivalents, and even more preferably 8 to 15 equivalents relative to the number of moles of the third chimera molecule precursor used.

[0150] This reaction involves activating the phosphorus atom of the second nucleic acid used in this step by oxidative halogenation, preferably chlorination, and forming a bond between the phosphorus atom and the amino group derived from the first nucleic acid in the third chimeric molecule precursor used in this step. This reaction can be carried out under reaction conditions known as the Atherton-Todd reaction, for example, as described in Atherton, FR et al., J. Chem. Soc., 1945, 660.

[0151] Specifically, this step can be carried out in a solvent inert to the reaction, using a reagent for oxidatively halogenating the phosphorus atom of the second nucleic acid used in this step, and a base to prevent acidification of the reaction system. Reagents used in this step for oxidatively halogenating the phosphorus atom of the second nucleic acid include, for example, carbon tetrachloride, sulfuryl chloride, trichloroisocyanuric acid, iodine, and iodoform, with carbon tetrachloride being preferred for ease of handling. As the base to prevent the reaction system from becoming acidic, various organic and inorganic bases can be used, but it is preferable to use a tertiary amine such as triethylamine or diisopropylethylamine.

[0152] To ensure smooth reaction, these reagents may be used in excess relative to the third chimeric molecule precursor used in this step. For example, the reagent for oxidatively halogenating the phosphorus atom of the second nucleic acid and the base for preventing acidification in the reaction system may each be used in an amount of 1.5 to 50 equivalents, preferably 3 to 30 equivalents, more preferably 5 to 20 equivalents, and even more preferably 8 to 15 equivalents, relative to the moles of the third chimeric molecule precursor used. The base for preventing acidification in the reaction system may be used in an amount equal to or greater than the amount of the reagent for oxidatively halogenating the phosphorus atom of the second nucleic acid, in order to prevent acidification in the reaction system.

[0153] The reaction temperature and reaction time can be appropriately changed depending on the types and amounts of reagents used. For example, the mixture may be stirred at 0 to 100°C, preferably 15 to 50°C, and more preferably 20 to 30°C for 5 minutes to 24 hours. The substrate concentration in the reaction can be appropriately adjusted depending on the types and amounts of reagents used. For example, the amount of solvent used can be adjusted so that the concentration of the third chimera molecule precursor in the reaction solution is 0.01 to 0.2 mol / L, preferably 0.02 to 0.1 mol / L, more preferably 0.025 to 0.08 mol / L, and even more preferably 0.03 to 0.05 mol / L.

[0154] After the reaction is complete, a polar solvent may be added to the reaction solution to solidify and recover the target product (chimera molecule). Because the third chimera molecule precursor used in the production method of the present invention contains a lipid-soluble anchor, the target product (chimera molecule) of this step solidifies and precipitates upon addition of a polar solvent. Therefore, the target product of this step can be recovered by filtration. As the polar solvent, acetonitrile and methanol are preferably used in terms of versatility and cost, and methanol is particularly preferred. The amount of polar solvent added to the reaction solution after completion of the reaction in order to recover the target product by filtration depends on the substrate concentration in the reaction, but is, for example, 5 to 50 times, preferably 5 to 30 times, more preferably 8 to 20 times, and even more preferably 10 to 15 times the total amount (volume) of the reaction solvent used. When a polar solvent is added to a reaction solution in order to recover the target product by filtration, if the amount of the target product recovered is small, the amount of polar solvent added may be adjusted, preferably increased, or the reaction solution after the addition of the polar solvent may be cooled.

[0155] The chimeric molecule obtained in the above step may further be linked to a first nucleic acid or a second nucleic acid via the amino group or hydroxy group at the 5'-position of the introduced second nucleic acid. In this case, the protecting group of the amino or hydroxy group at the 5'-position of the introduced second nucleic acid is removed, and the first or second nucleic acid can be bound using the amino or hydroxy group as a clue.

[0156] The method for removing the protecting group of the amino group or hydroxy group of the introduced second nucleic acid may be selected depending on the type of protecting group, and the method described in the aforementioned "Greene's Protective Groups in Organic Synthesis (4th edition, 2006)" can be used. When the protecting group for the hydroxy group of the introduced second nucleic acid is an optionally substituted trityl group, for example, DMTr, it can be removed using an acid in a solvent inert to the reaction. The acid that can be used is not particularly limited, but examples thereof include halogenoacetic acids such as trichloroacetic acid, trifluoroacetic acid, and dichloroacetic acid; and sulfonic acids such as methanesulfonic acid, trifluoromethanesulfonic acid, and p-toluenesulfonic acid. Since good results can be obtained, halogenoacetic acids are preferred, and trichloroacetic acid is particularly preferred. The amount of acid used is, for example, 1 to 100 mol, preferably 5 to 80 mol, more preferably 10 to 60 mol, and even more preferably 20 to 50 mol per mole of chimeric molecule used.

[0157] By cleaving the lipid-soluble anchor from the chimeric molecule thus produced that contains the lipid-soluble anchor as part of its structure, it is possible to produce a chimeric molecule that does not contain the lipid-soluble anchor as part of its structure. As described above, it is preferable that the lipid-soluble anchor is not cleaved during the production of the chimeric molecule, but can be cleaved at any stage. It is preferable that the lipid-soluble anchor is not removed under acidic conditions, but can be removed under basic conditions. When the lipophilic anchor is represented by formula (i) or (ii), it can be cleaved by the methods described in the aforementioned Patent Documents 2 and 7, or methods similar thereto. For example, the lipophilic anchor residue is precipitated by the action of aqueous ammonium hydroxide in a polar solvent, preferably ethanol, and the target chimeric molecule can be isolated from the supernatant. Note that if a group that is cleaved from an oxygen atom under weakly basic conditions (a group indicated by LV) remains at the phosphate site of the introduced second nucleic acid without being cleaved, or if a protecting group (e.g., Fmoc) for the amino group of the introduced first nucleic acid remains without being removed, a chimeric molecule can be obtained from which these groups have also been cleaved. [Example]

[0158] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0159] Preparation Example A1 (Alkylation)

[0160] [ka]

[0161] A mixture of methyl gallate (15.0 g, 81.5 mmol), 1-bromooctadecane (89.7 g, 269 mmol), and potassium carbonate (67.2 g, 486 mmol) in DMF (N,N-dimethylformamide) (300 mL) was stirred at 80 °C for 1 day. The reaction mixture was cooled to room temperature and diluted with toluene. This was washed with water. The organic layer was partially concentrated and suspended in methanol. The resulting precipitate was collected by filtration, and the solid was dried under reduced pressure to give the target compound (80.1 g, quantitative) as a white solid.

[0162] 1H NMR (600 MHz, CDCl3) δ 7.24 (s, 2H), 4.00 (td, J = 6.5, 3.4 Hz, 7H), 3.91-3.86 (m, 4H), 1.84-1.69 (m, 7H), 1.45 (td, J = 8.9, 5.1 Hz, 7H), 1.24 (s, 98H), 0.87 (t, J = 7.0 Hz, 11H) ppm. The NMR data were consistent with those reported in existing literature (S. Kim, et al., Eur. J. Chem., 2013, 19, 8615-8620).

[0163] Production example A2 (LAH reduction)

[0164] [ka]

[0165] To a solution of LiAlH (1.00 g, 21.1 mmol) in THF (81.0 mL) was added dropwise a solution of the methyl ester (5.01 g, 5.32 mmol) in THF (96.0 mL) via cannulation at room temperature. After stirring at room temperature for 3 h, the reaction mixture was cooled in an ice bath and quenched by adding water (1.00 mL) followed by 15% aqueous NaOH (3.00 mL). The mixture was warmed to room temperature and stirred for 1 h. After adding water (3.00 mL), the mixture was dried over NaSO and filtered. The filtrate was concentrated, and the crude product was dissolved in dichloromethane (30.0 mL). The product was recrystallized by adding methanol (200 mL), and the precipitate was collected by filtration. The solid was dried under reduced pressure to give the target compound (4.55 g, 93.6% yield) as a white powder.

[0166] 1H NMR (400 MHz, CDCl3) δ 6.54 (s, 2H), 4.58 (s, 2H), 3.94 (dt, J = 15.4, 6.6 Hz, 6H), 1.84-1.66 (m, 6H), 1.46 (d, J = 7.9 Hz, 7H), 1.24 (s, 67H), 0.91-0.82 (m, 9H) ppm. The NMR data were consistent with the data reported in the existing literature (H. Tamiaki, et al., Bull. Chem. Soc. Jpn., 2001, 74, 733-738).

[0167] Production Example A3 (Linker Introduction)

[0168] [ka]

[0169] To a solution of 5'-O-DMTr-thymidine (3.00 g, 5.51 mmol) and succinic anhydride (1.10 g, 11.0 mmol) in dichloromethane (8.26 mL) was added triethylamine (2.79 g, 3.83 mL, 27.6 mmol). After stirring at room temperature for 7.5 h, the reaction mixture was diluted with dichloromethane and washed with saturated aqueous NH4Cl followed by brine. The aqueous phase was extracted twice with dichloromethane. The combined organic layers were dried over Na2SO4 and concentrated to give the target compound (4.40 g, quantitative) as a pale yellow foam.

[0170] 1H NMR (600 MHz, CDCl3) δ 7.61 (s, 1H), 7.38-7.33 (m, 2H), 7.30-7.25 (m, 4H), 7.25-7.18 (m, 6H), 6.84-6.78 (m, 4H), 6.36 (dd, J = 9.3, 5.2 Hz, 1H), 5.41 (d, J = 5.3 Hz, 1H), 5.29 (s, 1H), 4.17 (s, 1H), 3.76 (d, J = 2.6 Hz, 6H), 3.44 (dd, J = 10.2, 2.5 Hz, 1H), 3.39 (dd, J = 10.5, 2.8 Hz, 1H), 3.02 (q, J = 7.3 Hz, 4H), 2.57 (s, 3H), 2.50 (dd, J = 13.6, 5.0 Hz, 1H), 2.40-2.35 (m, 1H), 1.35-1.32 (m, 3H), 1.27 (t, J = 7.3 Hz, 5H) ppm. The NMR data were consistent with those reported in existing literature (P. Kumar, et al., Nucleosides Nucleotides, 1993, 12, 565-584; C. Johnston, et al., Chemistry-Methods, 2021, 1, 1-8).

[0171] Production Example A4 (Immobilization of nucleosides onto anchors)

[0172] [ka]

[0173] To a solution of the benzyl alcohol derivative (100 mg, 0.109 mmol) in THF (2.00 mL) were added nucleoside-3'-O-succinate (122 mg, 0.164 mmol), EDC hydrochloride (52.3 mg, 0.273 mmol), and DMAP (4-dimethylaminopyridine) (33.4 mg, 0.273 mmol). After stirring at room temperature for 3 h, the reaction mixture was suspended in methanol. The resulting precipitate was collected by filtration and rinsed with methanol. The pellet was dried under reduced pressure to give the target compound (154 mg, 91.7% yield) as a white solid.

[0174] 1 H NMR (600 MHz, CDCl3) δ 8.19 (s, 1H), 7.60 (d, J = 1.7 Hz, 1H), 7.39-7.34 (m, 2H), 7.32-7.20 (m, 9H), 6.83 (d, J = 8.8 Hz, 4H), 6.52 (s, 2H), 6.42 (dd, J = 8.5, 6.1 Hz, 1H), 5.47 (dd, J = 4.4, 2.7 Hz, 1H), 5.00 (s, 2H), 4.13 (q, J = 2.4 Hz, 1H), 3.94 (d, J = 6.5 Hz, 3H), 3.93-3.88 (m, 3H), 3.78 (s, 6H), 3.50-3.41 (m, 2H), 2.66 (dq, J = 8.5, 5.9 Hz, 4H), 2.48-2.40 (m, 2H), 1.81-1.75 (m, 4H), 1.72 (dt, J = 15.0, 7.6 Hz, 3H), 1.50-1.38 (m, 6H), 1.24 (s, 96H), 0.87 (t, J = 7.1 Hz, 9H) ppm. 13C NMR (151 MHz, CDCl3) δ 172.10, 171.91, 163.42, 158.87, 153.32, 150.28, 144.25, 138.32, 135.58, 135.31, 135.19, 130.53, 130.18, 128.23, 128.16, 127.35, 113.42, 111.71, 107.11, 87.32, 84.43, 84.05, 75.95, 73.53, 69.22, 67.29, 63.81, 55.35, 37.94, 32.03, 30.43, 29.87, 29.83, 29.77, 29.73, 29.55, 29.51, 29.47, 29.16, 29.03, 26.23, 22.79, 14.23, 11.65 ppm. HRESIMS calcd. for C 96 H 15 0N2NaO 13 , 1562.1030 [M+Na] + ; found 1562.1060.

[0175] Preparation Example A5 (Trityl Removal; Preparation of First Chimeric Molecule Precursor)

[0176] [ka]

[0177] To a cooled solution of the starting material (156 mg, 101 mmol) in chloroform (5.49 mL) was added 184 mM trichloroacetic acid / dichloromethane (2.74 mL, trichloroacetic acid: 505 mmol) in an ice bath. The mixture was stirred at 0 °C for 1.5 h and then warmed to room temperature. After stirring at room temperature for 4.5 h, the reaction mixture was diluted with methanol. The resulting precipitate was collected by filtration and rinsed with methanol. The solid was dried under reduced pressure to give the target compound (117 mg, 93.6% yield) as a white foam.

[0178] 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 7.49 (t, J = 1.3 Hz, 1H), 6.52 (s, 2H), 6.18 (dd, J = 8.3, 6.1 Hz, 1H), 5.26 (dt, J = 6.3, 2.4 Hz, 1H), 5.01 (s, 2H), 4.03 (q, J = 2.5 Hz, 1H), 4.01-3.75 (m, 9H), 2.74-2.61 (m, 4H), 2.56 (t, J = 4.9 Hz, 1H), 2.47-2.27 (m, 2H), 1.91 (d, J = 1.2 Hz, 3H), 1.86-1.66 (m, 7H), 1.51-1.38 (m, 7H), 1.24 (s, 97H), 0.91-0.82 (m, 10H) ppm. 13 C NMR (101 MHz, CDCl3) δ 172.11, 172.03, 163.46, 153.29, 150.33, 138.23, 136.48, 130.64, 111.45, 107.16, 86.26, 85.08, 77.31, 75.19, 73.57, 69.24, 67.20, 62.65, 37.16, 32.02, 30.41, 29.86, 29.82, 29.76, 29.72, 29.54, 29.50, 29.47, 29.18, 29.14, 26.22, 22.79, 14.22, 12.68 ppm. HRESIMS calcd. for C 75 H 132 N2NaO 11 , 1259.9724 [M+Na] + ; found 1259.9734.

[0179] Production example A6 (elongation of the second nucleic acid)

[0180]

change

[0181] A dichloromethane solution (1.62 mL) of 3'-O-lipophilic anchor-modified thymidine (20.0 mg, 16.2 μmol) and 5'-O-DMTr-thymidine phosphoramidite (24.1 mg, 32.4 μmol) was combined with a 0.25 M acetonitrile solution (162 μL, 40.5 μmol) of 5-benzylthio-1H-tetrazole (BTT). After 8 hours of reaction at room temperature, 21.5 mg (81 μmol) of metachloroperbenzoic acid (mCPBA) was added and the mixture was allowed to react for 1 hour at room temperature. The reaction solution was diluted with methanol to form a suspension. The resulting suspension was filtered to collect the precipitate, and the solid on the filter paper was washed with methanol. The solid was then dried overnight under vacuum to obtain the desired dinucleotide as a white powder (17.9 mg, 58.3% yield).

[0182] 1H NMR (600 MHz, CDCl3) δ 9.26-8.55 (m, 2H), 7.51 (t, J = 12.3 Hz, 1H), 7.43 (d, J = 4.0 Hz, 1H), 7.38-7.27 (m, 9H), 7.27-7.19 (m, 12H), 6.83 (dd, J = 8.5, 3.7 Hz, 6H), 6.52 (d, J = 3.4 Hz, 6H), 6.46-6.33 (m, 1H), 6.33-6.09 (m, 4H), 5.38-5.24 (m, 3H), 5.17 (s, 3H), 5.09-4.90 (m, 6H), 4.40-4.00 (m, 17H), 4.00-3.89 (m, 20H), 3.85 (s, 4H), 3.77 (dd, J = 3.9, 1.7 Hz, 9H), 3.61-3.45 (m, 1H), 3.37 (d, J = 10.3 Hz, 1H), 3.32-3.13 (m, 1H), 2.84-2.73 (m, 4H), 2.73-2.59 (m, 17H), 2.59-2.21 (m, 10H), 1.96-1.85 (m, 14H), 1.85-1.64 (m, 45H), 1.50-1.37 (m, 27H), 1.24 (s, 294H), 0.86 (td, J = 7.0, 1.5 Hz, 31H) ppm. 13C NMR (151 MHz, CDCl3) δ 172.11, 163.85, 163.65, 158.94, 153.33, 150.49, 144.05, 138.34, 136.60, 135.85, 135.73, 135.19, 135.04, 130.51, 130.22, 128.24, 128.17, 127.44, 116.59, 116.44, 116.26, 113.45, 111.88, 111.71, 111.45, 107.15, 87.43, 86.40, 86.27, 85.67, 85.10, 84.39, 82.48, 79.12, 75.17, 74.00, 73.88, 73.58, 73.56, 69.27, 67.69, 67.30, 63.30, 62.61, 62.48, 62.08, 55.38, 39.01, 38.47, 37.16, 36.76, 36.53, 32.02, 30.44, 29.86, 29.82, 29.76, 29.73, 29.55, 29.52, 29.46, 29.18, 29.02, 26.23, 22.78, 19.81, 19.71, 19.67, 14.20, 12.61, 12.53, 11.78 ppm. 31 P NMR (243 MHz, CDCl3) δ -1.86, -1.94, -2.09, -2.15 ppm. HRESIMS calcd. for C 109 H 166 N5NaO 20 P, 1920.1790 [M+Na] + ; found 1920.1878.

[0183] Preparation Example A7 (Trityl Removal; Preparation of First Chimeric Molecule Precursor)

[0184] [ka]

[0185] To a solution of the starting material (17.9 mg, 9.43 μmol) in dichloromethane (2.00 mL) was added 184 mM trichloroacetic acid / dichloromethane solution (2.00 mL, trichloroacetic acid: 368 μmol) on an ice bath. The mixture was stirred at 0°C for 4 minutes, then allowed to warm to room temperature and stirred for an additional hour. The reaction mixture was diluted with methanol. The resulting precipitate was collected by filtration, and the resulting solid was washed with methanol. The solid was dried under reduced pressure to give the target compound (16.9 mg, yield >99.0%) as a white powder.

[0186] 1 H NMR (600 MHz, CDCl3) δ 9.37-8.78 (m, 1H), 7.49 (d, J = 1.3 Hz, 1H), 7.43 (q, J = 2.5 Hz, 1H), 7.37-7.29 (m, 1H), 7.25 (s, 2H), 6.52 (s, 3H), 6.26-6.09 (m, 2H), 5.40-5.24 (m, 1H), 5.17 (td, J = 6.0, 3.0 Hz, 1H), 5.01 (d, J = 3.9 Hz, 3H), 4.42-4.25 (m, 5H), 4.21 (dq, J = 5.4, 2.8 Hz, 1H), 4.15 (t, J = 4.1 Hz, 1H), 4.00-3.89 (m, 9H), 3.85 (s, 3H), 3.28-3.04 (m, 1H), 2.79 (td, J = 6.1, 3.3 Hz, 2H), 2.68 (dh, J = 15.8, 4.2 Hz, 6H), 2.58-2.27 (m, 5H), 1.97-1.84 (m, 8H), 1.81-1.63 (m, 17H), 1.52-1.38 (m, 10H), 1.24 (s, 98H), 0.86 (t, J = 7.0 Hz, 15H) ppm. 13C NMR (151 MHz, CDCl3) δ 172.14, 163.76, 153.33, 150.47, 138.35, 136.67, 135.87, 130.51, 116.59, 111.87, 111.41, 107.12, 86.47, 86.34, 85.70, 82.56, 79.14, 74.02, 73.88, 73.58, 69.27, 67.65, 67.30, 62.61, 62.10, 62.00, 38.46, 37.15, 36.53, 32.02, 30.44, 29.86, 29.82, 29.76, 29.73, 29.55, 29.52, 29.46, 29.04, 26.23, 22.78, 19.86, 19.82, 14.20, 12.66, 12.61, 12.53, 1.10 ppm. 31 P NMR (243 MHz, CDCl3) δ -1.86, -1.94 ppm. HRESIMS calcd. for C 88 H 148 N5NaO 18 P, 1617.0450 [M+Na] + ; found 1617.0467.

[0187] Example A1 (Preparation of second chimeric molecule precursor)

[0188] [ka]

[0189] Acetonitrile (43.6 μL) was added to a dichloromethane solution (463 μL) of 3'-O-lipophilic anchor-modified thymidine (20.0 mg, 16.2 μmol). Next, 5'-MMTr-aminated thymidine phosphoramidite (23.1 mg, 32.4 μmol) and 1H-tetrazole (11.3 mg, 162 μmol) were added, and the mixture was allowed to react at room temperature for 5 hours. Dichloromethane (200 μL) was added to the reaction solution, followed by the addition of 5'-MMTr-aminated thymidine phosphoramidite (11.6 mg, 16.2 μmol) and 1H-tetrazole (5.65 mg, 81.0 μmol). After 6.5 hours of reaction at room temperature, a 5-6 M tert-butyl hydroperoxide / decane solution (13.0 μL, 64.8 μmol) was added to the reaction solution and the mixture was allowed to react at room temperature for 1 hour. The reaction solution was diluted with methanol to form a suspension. The resulting suspension was transferred to a centrifuge tube and centrifuged at 3,500 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dried overnight under vacuum to obtain the desired dinucleotide as a white solid (23.3 mg, 77.2% yield).

[0190] 31 P NMR (243 MHz, CDCl3) δ -1.84, -1.95 ppm.

[0191] Example A2 (Preparation of second chimeric molecule precursor)

[0192] [ka]

[0193] Acetonitrile (40.3 μL) was added to a dichloromethane solution (403 μL) of 3'-O-lipophilic anchor-modified thymidine (17.4 mg, 14.1 μmol). Next, 5'-MMTr-aminated thymidine phosphoramidite (20.1 mg, 28.2 μmol) and 4,5-dicyanoimidazole (16.7 mg, 141 μmol) were added and the mixture was allowed to react at room temperature for 6 hours. An additional 5'-MMTr-aminated thymidine phosphoramidite (20.1 mg, 28.2 μmol) was added to the reaction solution, and the mixture was allowed to react at room temperature for 3 hours. After the reaction was complete, a 5-6 M tert-butyl hydroperoxide / decane solution (11.3 μL, 56.4 μmol) was added to the reaction solution, and the mixture was allowed to react at room temperature for 1 hour. The reaction solution was diluted with methanol to form a suspension. The resulting precipitate was collected by filtration, and the resulting solid was washed with methanol and dried under reduced pressure to give the target compound (22.9 mg, yield 75.6%) as a white solid.

[0194] 1 H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 8.0 Hz, 3H), 7.33 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 4.4 Hz, 3H), 7.05 (s, 1H), 6.87 - 6.75 (m, 2H), 6.52 (s, 2H), 6.22 (s, 2H), 5.29 (s, 2H), 5.06 - 4.93 (m, 3H), 4.41 - 4.07 (m, 7H), 3.93 (d, J = 9.6 Hz, 7H), 3.76 (d, J = 3.6 Hz, 3H), 2.86 - 2.45 (m, 10H), 2.45 - 2.20 (m, 4H), 2.16 (s, 4H), 1.90 (d, J = 3.5 Hz, 4H), 1.87 - 1.67 (m, 11H), 1.45 (s, 10H), 1.24 (s, 92H), 0.87 (t, J = 6.8 Hz, 11H) ppm. 31 P NMR (162 MHz, CDCl3) δ -1.95 ppm.

[0195] Example A3 (Deprotection of second chimeric molecule precursor)

[0196] [ka]

[0197] To a solution of the starting material (23.3 mg, 12.5 μmol) in dichloromethane (2.70 mL) was added 184 mM trichloroacetic acid / dichloromethane solution (2.70 mL, trichloroacetic acid: 488 μmol) on an ice bath. The mixture was stirred at 0°C for 8 minutes, then allowed to warm to room temperature and stirred for an additional hour. The reaction mixture was diluted with methanol. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dried overnight under vacuum to obtain the desired dinucleotide as a white solid (8.80 mg, 44.2% yield).

[0198] 31 P NMR (162 MHz, CDCl3) δ -2.02 ppm.

[0199] Example A4 (Introduction of a first nucleic acid into a second chimeric molecule precursor; production of a chimeric molecule)

[0200] [ka]

[0201] To a solution of the starting material (8.80 mg, 5.52 μmol) in tetrahydrofuran (552 μL), Fmoc-PNA-T monomer (3.35 mg, 6.62 μmol), COMU (7.60 mg, 17.8 μmol), and N,N-diisopropylethylamine (1.78 mg, 2.38 μL, 13.8 μmol) were added in that order and reacted at room temperature for 20 hours. After completion of the reaction, the reaction solution was suspended in methanol. The suspension was diluted 10-fold with acetonitrile, transferred to a centrifuge tube, and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the solid was dried overnight under vacuum to obtain a white solid PNA-DNA chimeric molecule (7.90 mg, 70.5% yield).

[0202] 31 P NMR (243 MHz, CDCl3) δ -1.64 ppm. MALDI-TOF-MS calculation for C 111 H 169 N9O 23 P, 2028.587 [MH] - ; found 2028.657.

[0203] Example A5 (Deprotection of Chimeric Molecule)

[0204] [ka]

[0205] To a tetrahydrofuran solution (500 μL) of the starting material (7.90 mg, 3.89 μmol), a tetrahydrofuran solution (500 μL) of 2% piperidine / 2% DBU was added, and the mixture was stirred at room temperature for 1 hour. The reaction was quenched by adding 1 M hydrochloric acid dropwise until the pH of the reaction solution reached 5. The reaction mixture was then suspended in methanol (5.00 mL), and the resulting suspension was transferred to a centrifuge tube. The suspension was diluted with acetonitrile (34.0 mL) and centrifuged at 3,500 rpm for 20 minutes. The resulting precipitate was washed three times with acetonitrile, and the resulting solid was dried overnight under vacuum to obtain a white solid PNA-DNA chimeric molecule (6.80 mg, 96.7% yield).

[0206] MALDI-TOF-MS calculation for C 96 H 159 N9O 21 P, 1806.3443 [MH] - ; found 1806.446.

[0207] Example A6 (Introduction of amino acids into chimeric molecules)

[0208] [ka]

[0209] To a solution of the starting material (6.80 mg, 3.76 μmol) in tetrahydrofuran (376 μL), Fmoc-glycine-OH (2.24 mg, 7.52 μmol), COMU (6.42 mg, 15.0 μmol), and N,N-diisopropylethylamine (1.46 mg, 1.95 μL, 11.3 μmol) were added in that order and reacted at room temperature for 17.5 hours. After the reaction was complete, the reaction solution was suspended in methanol. The suspension was diluted 10-fold with acetonitrile, transferred to a centrifuge tube, and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the solid was dried overnight under vacuum to obtain the Fmoc-Gly-PNA-DNA chimeric molecule as a white solid (4.60 mg, 58.5% yield).

[0210] MALDI-TOF-MS calculation for C 113 H 172 N 10 O 24 P, 2085.639 [MH] - ; found 2086.052.

[0211] Example A7 (Cleavage of lipid-soluble anchor from chimeric molecule)

[0212] [ka]

[0213] A PNA-DNA chimera with a protecting group / anchor (4.60 mg, 2.20 μmol) was suspended in ethanol (400 μL) and 28% aqueous ammonia (1.20 mL) and heated at 80°C for 5 hours. The reaction solution was returned to room temperature and then concentrated to dryness. The resulting white solid was suspended in methanol, and the resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 15 minutes. The supernatant was collected, concentrated to dryness, and then dissolved in ultra-deionized water. Purity analysis was performed by LC-MS and absorbance measurement was performed using a NanoDrop. The purity of the target chimeric molecule, N-Gly-T(PNA)TT(DNA)-3', was 63.4%, and the yield was 26.8% (0.59 μmol, ε 260 = 26,160 M -1 ·cm -1 ) was.

[0214] HRESIMS calcd. for C 33 H 44 N 10 O 16 P, 867.2680 [MH] - ; found 867.2758.

[0215] Preparation Example A8 (elongation of second nucleic acid, oxidation of phosphorus atom, removal of trityl; preparation of first chimeric molecule precursor)

[0216] [ka]

[0217] To a solution of 3'-O-lipophilic anchor-modified thymidine (108 mg, 87.3 μmol) in dichloromethane (2.49 mL) and acetonitrile (249 μL), 5'-O-DMTr-thymidine phosphoramidite (195 mg, 262 μmol) and 1H-tetrazole (61.2 mg, 874 μmol) were added and reacted at room temperature for 7.5 hours. A 5-6 M tert-butyl hydroperoxide / decane solution (69.8 μL, 349 μmol) was added and reacted at room temperature for 1 hour. The reaction solution was diluted with methanol to form a suspension. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dissolved in dichloromethane (4.74 mL). The resulting dichloromethane solution was cooled in an ice bath, and then 184 mM trichloroacetic acid / dichloromethane solution (4.74 mL) was added and stirred for 2 minutes on the ice bath. The reaction solution was returned to room temperature and stirred for an additional hour. After the reaction was completed, the reaction solution was diluted with methanol to suspend the reaction solution. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dissolved in dichloromethane. After azeotroping with benzene three times, the resulting solid was dried under vacuum overnight to obtain the dinucleotide as a white solid (136 mg, yield 97.8%).

[0218] MALDI-TOF-MS calculation for C 88 H 148 N5NaO 18 P, 1617.045 [M+Na] + ; found 1617.119.

[0219] Preparation Example A9 (elongation of second nucleic acid, oxidation of phosphorus atom, removal of trityl; preparation of first chimeric molecule precursor)

[0220] [ka]

[0221] To a solution of 3'-O-lipophilic anchor-modified dinucleotide (136 mg, 85.3 μmol) in dichloromethane (2.44 mL) and acetonitrile (244 μL), 5'-O-DMTr-thymidine phosphoramidite (191 mg, 256 μmol) and 1H-tetrazole (59.8 mg, 853 μmol) were added and allowed to react at room temperature for 5 hours. A 5-6 M tert-butyl hydroperoxide / decane solution (68.2 μL, 341 μmol) was added and allowed to react at room temperature for 1 hour. The reaction solution was diluted with methanol to form a suspension. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dissolved in dichloromethane (4.74 mL). The resulting dichloromethane solution was cooled in an ice bath, and then 184 mM trichloroacetic acid / dichloromethane solution (4.74 mL) was added and stirred for 2 minutes on the ice bath. The reaction solution was returned to room temperature, and 2-3 drops of trifluoroacetic acid were added and stirred for 1 hour. After the reaction was completed, the reaction solution was diluted with methanol to suspend the reaction solution. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dissolved in dichloromethane. After azeotropy with benzene three times, the resulting solid was dried under vacuum overnight to obtain the trinucleotide as a white solid (82.6 mg, yield 49.5%).

[0222] MALDI-TOF-MS calculation for C 101 H 164 N8NaO 25 P2, 1975.391 [M+Na] + ; found 1975.886.

[0223] Example A8 (Introduction of a second nucleic acid into a first chimeric molecule precursor: Preparation of a second chimeric molecule precursor / deprotection)

[0224] [ka]

[0225] To a solution of 3'-O-lipophilic anchor-modified trinucleotide (82.6 mg, 42.3 μmol) in dichloromethane (1.21 mL) and acetonitrile (121 μL), 5'-MMTrNH-thymidine phosphoramidite (90.7 mg, 127 μmol) and 1H-tetrazole (29.6 mg, 423 μmol) were added and reacted at room temperature for 10 hours. A 5-6 M tert-butyl hydroperoxide / decane solution (33.8 μL, 169 μmol) was added to the reaction solution and reacted at room temperature for 1 hour. The reaction solution was diluted with methanol to form a suspension. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dissolved in dichloromethane (2.00 mL). To the resulting dichloromethane solution, 184 mM trichloroacetic acid / dichloromethane solution (10.0 mL) was added and stirred at room temperature for 45 minutes. After the reaction was complete, the reaction solution was diluted with methanol to form a suspension. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dissolved in dichloromethane. After three azeotropic distillations with benzene, the resulting solid was dried overnight under vacuum to obtain a 5'-aminated tetranucleotide as a brown solid (75.4 mg, 77.2% yield). MALDI-TOF-MS analysis confirmed that the resulting tetranucleotide consisted of a mixture of tetranucleotides with all three cyanoethyl groups on the phosphate backbone, those with one missing, and those with two missing.

[0226] MALDI-TOF-MS calculation for C 114 H 182 N 12 O 31 P3, 2309.684 [M+H] + ; found 2310.708 (with all three cyanoethyl groups), C 111 H 179 N 11 O31 P3, 2256.620 [M+H] + ; found 2257.655 (one cyanoethyl group removed), C 108 H 176 N 10 O 31 P3, 2203.566 [M+H] + ; found 2204.544 (two cyanoethyl groups removed).

[0227] Example A9 (Removal of cyanoethyl group)

[0228] [ka]

[0229] To a tetrahydrofuran solution (1.00 mL) of 5'-aminated tetranucleotide (75.4 mg, 32.7 μmol), a tetrahydrofuran solution (1.00 mL) of 2% piperidine / 2% DBU was added, and the mixture was stirred at room temperature for 1 hour. Methanol (45.0 mL) was added to the reaction solution to form a suspension, and the resulting suspension was transferred to a centrifuge tube and centrifuged at 4,000 rpm for 20 minutes. The resulting precipitate was washed three times with methanol, and the resulting solid was dissolved in dichloromethane. After three azeotropic distillations with benzene, the resulting solid was dried under vacuum overnight to give the decyanoethylated product as a brown solid (62.7 mg, 89.2% yield).

[0230] MALDI-TOF-MS calculation for C 105 H 171 N9O 31 P3, 2148.477 [MH] - ; found 2148.571.

[0231] Production Example B1 (Esterification)

[0232] [ka]

[0233] The benzyl alcohol derivative (1) (1.00 g, 1.09 mmol) prepared in Preparation Example A2 was dissolved in tetrahydrofuran (20.0 mL), followed by the addition of Fmoc-glycine (488 mg, 1.64 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (523 mg, 2.73 mmol), and 4-dimethylaminopyridine (334 mg, 2.73 mmol). The resulting mixture was stirred at room temperature for 20 hours. Methanol (150 mL) was added to the reaction solution, and the resulting precipitate was collected by suction filtration. The resulting solid was washed with methanol and dried under reduced pressure to give compound 2 as a white powder (1.29 g, 99.2% yield).

[0234] 1 H NMR (600 MHz,CDCl3) δ 7.77 (d, J = 7.6 Hz, 1.5H), 7.60 (d, J = 7.4 Hz, 1.5H), 7.40 (t, J = 7.4 Hz, 2H), 7.34 - 7.28 (m, 2H), 6.54 (s, 2H), 5.32 - 5.27 (m, 0.5H), 5.12 - 5.04 (m, 2H), 4.41 (d, J = 7.1 Hz, 1.5H), 4.24 (t, J = 7.1 Hz, 0.5H), 4.05 (d, J = 5.6 Hz, 1.5H), 3.94 (ddq, J = 9.4, 6.6, 3.6 Hz, 6H), 1.82 - 1.69 (m, 6H), 1.49 - 1.42 (m, 6H), 1.26 (s, 87H), 0.88 (t, J = 7.1 Hz, 9H)ppm. 13C NMR (151 MHz, CDCl3) δ 170.07, 156.41, 153.42, 153.39, 143.92, 141.44, 138.59, 130.07, 127.88, 127.22, 125.22, 120.14, 107.28, 73.58, 69.32, 67.85, 67.40, 47.24, 43.02, 32.08, 30.48, 29.91, 29.87, 29.81, 29.77, 29.58, 29.55, 29.52, 26.27, 26.25, 22.84, 14.26 ppm. ESI-TOF-MS C 78 H 129 NNa2O7(M + 2Na) 2+ calcd. m / z 618.9777, found m / z 619.4417.

[0235] Production example B2 (Fmoc removal)

[0236] [ka]

[0237] Compound 2 (500 mg, 419 μmol) was dissolved in tetrahydrofuran (21.0 mL), and then a 2% piperidine / 2% diazabicycloundecene (DBU) / tetrahydrofuran solution (21.0 mL) was added and stirred at room temperature for 3 hours. The reaction solution was adjusted to pH 5 by dropwise addition of 1 M hydrochloric acid. Subsequently, acetonitrile (200 mL) was added to precipitate the reaction product. The resulting precipitate was collected by suction filtration. The resulting solid was washed with acetonitrile (200 mL) and then dried under reduced pressure to obtain compound 3 as a white solid (429 mg, yield 99.2%).

[0238] 1H NMR (600 MHz, CDCl3) δ 8.74 (s, 2H), 6.50 (s, 2H), 5.06 (s, 2H), 4.00 (s, 1H), 3.90 (q, J = 6.6 Hz, 6H), 1.81 - 1.68 (m, 9H), 1.44 (dp, J = 12.2, 7.0 Hz, 7H), 1.25 (d, J = 4.1 Hz, 80H), 0.90 - 0.85 (m, 10H) ppm. ESI-TOF-MS C 63 H 120 NO5(M + H) + calcd. m / z 970.9161, found m / z 970.9318.

[0239] Preparation Example B3 (Immobilization of First Nucleic Acid on Anchor)

[0240] [ka]

[0241] Compound 3 (200 mg, 199 μmol) was dissolved in tetrahydrofuran (19.9 mL), and Fmoc-PNA(Thymine)-OH (202 mg, 398 μmol), ethyl 2-cyano-2-((dimethylimino)(morpholino)methoxyimino)acetate hexafluorophosphate (COMU) (256 mg, 597 μmol), and diisopropylethylamine (77.2 mg, 103 μL, 597 μmol) were added and stirred at room temperature for 6 hours. Acetonitrile (100 mL) was added to the reaction solution, and the resulting precipitate was collected by suction filtration. The resulting solid was washed with acetonitrile (200 mL) and dried under reduced pressure to give compound 4 as a light brown solid (281 mg, 96.9% yield).

[0242] 1H NMR (600 MHz, CDCl3) δ 8.37 (s, 0.4H), 8.13 (s, 0.6H), 7.74 (dd, J = 7.9, 4.4 Hz, 2H), 7.60 (dd, J = 16.0, 7.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.29 (dd, J = 14.9, 7.5 Hz, 2H), 7.05 (s, 0.6H), 6.94 (s, 0.3H), 6.77 (s, 0.5H), 6.60 - 6.54 (m, 0.5H), 6.49 (s, 2H), 5.07 (s, 1H), 5.01 (s, 0.5H), 4.44 (s, 1H), 4.38 (d, J = 7.4 Hz, 1.5H), 4.22 - 4.15 (m, 1H), 4.08 - 4.02 (m, 3H), 3.93 - 3.88 (m, 6H), 3.63 (s, 1H), 3.43 (d, J = 4.6 Hz, 1H), 2.16 (s, 0.2H), 2.00 (s, 0.8H), 1.82 (s, 2H), 1.78 - 1.68 (m, 7H), 1.57 (s, 7H), 1.43 (d, J = 7.7 Hz, 7H), 1.24 (s, 80H), 0.87 (t, J = 7.1 Hz, 10H) ppm. ESI-TOF-MS C 89 H 144 N5O 11 (M + H) + calcd. m / z 1460.1545, found m / z 1460.2759.

[0243] Production Example B4 (Production of the third molecular precursor)

[0244]

change

[0245] Compound 4 (250 mg, 171 μmol) was dissolved in tetrahydrofuran (17.1 mL), and then a 2% piperidine / 2% diazabicycloundecene (DBU) / tetrahydrofuran solution (17.1 mL) was added and stirred at room temperature for 3 hours. The reaction solution was adjusted to pH 5 by dropwise addition of 1 M hydrochloric acid. Subsequently, acetonitrile (150 mL) was added to precipitate the reaction product. The resulting precipitate was collected by suction filtration. The resulting solid was washed with acetonitrile (150 mL) and then dried under reduced pressure to obtain compound 5 as a white solid (207 mg, yield 95.0%).

[0246] 1 H NMR (600 MHz, CDCl3) δ 10.49 (br, 0.5H), 8.82 (br, 0.5H), 8.53 (m, 1H), 7.91 (m, 1.5H), 7.14 (m, 0.5H), 6.61 - 6.43 (m, 2H), 4.97 (s, 1.8H), 4.70 (m, 0.6H), 4.58 (s, 0.2H), 4.55 - 4.32 (m, 1.4H), 4.06 (m, 2H), 3.97 - 3.86 (m, 6.4H), 3.66 (m, 0.6H), 3.30 (m, 1.8H), 3.13 (m, 0.2H), 2.00 (d, J = 8.4 Hz, 1H), 1.87 - 1.70 (m, 11H), 1.42 (d, J = 15.3 Hz, 6H), 1.33 - 1.22 (m, 85H), 0.89 - 0.83 (m, 9H) ppm. ESI-TOF-MS C 74 H 134 NO(M + H) + calcd. m / z 1237.0176, found m / z 1237.0181.

[0247] Preparation Example B5 (Preparation of second nucleic acid to be introduced)

[0248] [ka]

[0249] 5'-O-DMTr-thymidine 3'-O-phosphoramidite (1.00 g, 1.34 mmol) was dissolved in acetonitrile (22.3 mL) and then ultra-deionized water (2.48 mL) was added. Subsequently, 0.25 M 5-(benzylthio)-1H-tetrazole / acetonitrile solution (10.7 mL, 2.68 mmol) was added and stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (150 mL) and washed three times with saturated aqueous sodium bicarbonate (150 mL) and once with saturated brine (150 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated on a rotary evaporator to give compound 7 as a white foamy solid (980 mg, >99%).

[0250] 1 H NMR (600 MHz, CDCl3) δ 9.30 - 9.14 (m, 1H), 7.45 - 7.41 (m, 1.5H), 7.29 - 7.26 (m, 2H), 7.23 - 7.14 (m, 7H), 6.75 (dq, J = 7.6, 1.0 Hz, 4H), 6.36 (ddd, J = 8.8, 5.5, 1.4 Hz, 1H), 6.22 (d, J = 11.2 Hz, 0.5H), 5.20 - 5.10 (m, 1H), 4.23 - 3.99 (m, 3H), 3.69 (d, J = 0.7 Hz, 6H), 3.45 (td, J = 10.8, 3.0 Hz, 1H), 3.31 (ddd, J = 10.7, 3.9, 2.6 Hz, 1H), 2.67 (t, J = 6.1 Hz, 1H), 2.61 - 2.47 (m, 2H), 2.37 (dtd, J = 14.3, 8.1, 5.9 Hz, 1H), 1.33 (t, J = 1.0 Hz, 3H) ppm. 31 P NMR (243 MHz, CDCl3) δ 7.56 ppm. ESI-TOF-MS C 34 H 36 N3NaO9P (M + Na) +calcd. m / z 684.2081, found m / z 684.2104.

[0251] Example B1 (Introduction of a second nucleic acid into a third chimeric molecule precursor: production of a chimeric molecule)

[0252] [ka]

[0253] Compound 5 (20.0 mg, 15.7 μmol) was suspended in dichloromethane (449 μL) and acetonitrile (45.0 μL), followed by the addition of compound 7 (104 mg, 157 μmol), carbon tetrachloride (24.1 mg, 15.1 μL, 157 μmol), and triethylamine (15.9 mg, 21.8 μL, 157 μmol). The resulting solution was stirred at room temperature for 6 hours, after which methanol (13.0 mL) was added to the reaction mixture. The resulting suspension was transferred to a centrifuge tube. The mixture was centrifuged at 3,500 rpm for 7 minutes, and the supernatant was removed. The precipitate was then resuspended in methanol (13.0 mL), centrifuged, and the supernatant was removed. This procedure was repeated three times. The precipitate was dried under reduced pressure to obtain compound 8 as a white powder (25.0 mg, 83.9% yield).

[0254] 1H NMR (400 MHz, CDCl3) δ 7.50 (br, 0.6H), 7.35 (s, 0.4H), 7.30 - 7.25 (m, 3H), 7.18 - 7.14 (m, 3H), 7.03 (br, 0.5H), 6.84 - 6.80 (m, 3.5H), 6.55 - 6.49 (m, 2H), 6.18 (br, 1H), 5.29 (s, 6H), 5.11 (br, 1H), 5.05 - 5.02 (m, 2H), 4.29 - 4.19 (m, 4H), 4.09 - 4.03 (m, 2.5H), 3.95 - 3.90 (m, 8H), 3.79 - 3.77 (m, 6H), 2.77 (t, J = 4 Hz, 2H), 2.51 (br, 1H), 1.89 - 1.86 (m, 4H), 1.79 - 1.68 (m, 10H), 1.44 - 1.43 (m, 6.5H), 1.28 - 1.24 (m, 90H), 0.88 - 0.85 (m, 10H) ppm. 31 P NMR (162 MHz, CDCl3) δ 9.75, 9.55 ppm. ESI-TOF-MS C 108 H 167 KN8NaO 18 P (M + K + Na) 2+ calcd. m / z 979.0847, found m / z 980.9728.

[0255] Example B2 (deprotection of キメラ molecule)

[0256]

change

[0257] Compound 8 (14.6 mg, 7.70 μmol) was dissolved in dichloromethane (1.00 mL), and then 184 mM trichloroacetic acid / dichloromethane solution (2.00 mL) was added and stirred at room temperature for 15 minutes. Methanol (10.0 mL) was added to the reaction solution, and the resulting suspension was transferred to a centrifuge tube. The mixture was centrifuged at 3,500 rpm for 7 minutes, and the supernatant was removed to recover the precipitate. The precipitate was resuspended in methanol (13.0 mL), centrifuged, and the supernatant was removed. This procedure was repeated three times. The precipitate was dried under reduced pressure to obtain compound 9 as a white powder (8.60 mg, 69.9% yield).

[0258] 1 H NMR (400 MHz, CDCl3) δ 9.21 (m, 2H), 7.51 - 7.49 (m, 1.5H), 7.04 (m, 2H), 6.56 - 6.52 (m, 2H), 6.20 (m, 1.5H), 5.30 (s, 1.5H), 5.07 - 5.01 (m, 3H), 4.70 -4.47 (m, 3H), 4.30 - 4.05(m, 6H), 3.97 - 3.85 (m, 8H), 3.49 (m, 4H), 3.38 - 3.26 (m, 4H), 2.78 (m, 2H), 2.58 - 2.23 (m, 3H), 2.17 (s, 1H), 2.01 (s, 0.5H), 1.90 (m, 4H), 1.80 - 1.71 (m, 6H), 1.57 (m, 3H, overlapping with water signal), 1.46 (m, 7H), 1.30 - 1.25 (m, 73H), 1.11 (s, 2H), 0.89 -0.86 (m, 9H) ppm. 31 P NMR (162 MHz, CDCl3) δ 9.73, 9.54 ppm. ESI-TOF-MS C 87 H 149 N8NaO 16 P (M + Na) + calcd. m / z 1616.0721, found m / z 1616.0783.

[0259] Example B3 (Cleavage of lipid-soluble anchor from chimeric molecule)

[0260] [ka]

[0261] Compound 9 (5.00 mg, 3.14 μmol) was suspended in ethanol (400 μL), and then 28% aqueous ammonia (1.20 mL) was added. The resulting suspension was incubated at 80°C for 10.5 hours, returned to room temperature, and concentrated. The residue was suspended in methanol (8.00 mL) and transferred to a centrifuge tube. The supernatant was collected by centrifugation at 3,500 rpm for 10 minutes, and the precipitate was removed. The collected supernatant was concentrated to give compound 10 as a white solid (318 μg, 0.480 μmol, 15.3% yield).

[0262] 1 H NMR (400 MHz, D2O) δ 7.48 - 7.42 (m, 1H), 7.26 - 7.14 (m, 1H), 6.08 (dt, J = 13.3, 7.0 Hz, 1H), 4.74 (s, 1H), 4.56 - 4.45 (m, 1.5H), 4.28 - 4.16 (m, 1H), 3.97 (s, 2H), 3.81 - 3.68 (m, 1.5H), 3.68 - 3.51 (m, 3H), 3.41 - 3.26 (m, 2H), 3.15 (td, J = 6.5, 1.2 Hz, 2.5H), 3.04 - 2.88 (m, 2H), 2.75 (td, J = 6.7, 1.1 Hz, 1.5H), 2.35 - 2.12 (m, 2H), 1.67 (tt, J = 3.1, 1.2 Hz, 6H) ppm. 31 P NMR (161 MHz, D2O) δ 8.53, 8.35 ppm. ESI-TOF-MS C 23 H 31 N7O 13 P (MH) -calcd. m / z 644.1723, found m / z 644.1963. [Industrial Applicability]

[0263] According to the present invention, there is provided a method for producing a chimeric molecule by liquid phase synthesis, in which a nucleic acid having a neutral or cationic main chain skeleton, such as PNA or pRNA, or a derivative thereof, is introduced into a nucleic acid having an anionic main chain skeleton, such as RNA or DNA, or a derivative thereof.

Claims

1. A method for producing a chimeric molecule in which at least one first nucleic acid or a derivative thereof having a neutral or cationic main chain backbone and at least one second nucleic acid or a derivative thereof having an anionic main chain backbone are fused together, the method comprising: the production method includes introducing the second nucleic acid or a derivative thereof into a third chimera molecule precursor having an amino group via the amino group of the third chimera molecule precursor; the third chimera molecule precursor having an amino group comprises a partial structure derived from a first nucleic acid or a derivative thereof, an amino group derived from the first nucleic acid or a derivative thereof, and a lipid-soluble anchor; A method for producing a chimeric molecule, comprising the step of introducing the second nucleic acid or a derivative thereof into the third chimeric molecule precursor having an amino group by liquid phase synthesis.

2. The method of claim 1 , wherein the first nucleic acid is PNA, PRNA, PNA / PRNA, or LNA, and the second nucleic acid is RNA or DNA.

3. The method of claim 2 , wherein the first nucleic acid is PNA and the second nucleic acid is DNA.

4. The method according to claim 2, wherein the chimeric molecule has a portion where the first nucleic acid or a derivative thereof is bound to the 3' end of the second nucleic acid or a derivative thereof.

5. The third chimeric molecule precursor is represented by formula (11): 【Chemical 1】 (In the formula, Ar ＊ represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Base indicates the base portion. Formula (12) 【Chemistry 2】 (In the formula, Ar ＊ represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Base represents the base moiety; Z represents a single bond, or represents one or more first nucleic acids and / or second nucleic acids via a bond between a nitrogen atom bonded to Z and a carbonyl carbon atom. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position. and formula (13) 【Chemistry 3】 (In the formula, Ar ＊ represents an aromatic hydrocarbon ring having 6 to 14 carbon atoms to which an aliphatic hydrocarbon group having 10 to 40 carbon atoms is bonded via a single bond or a linker; R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Base represents the base moiety; W represents a single bond or represents one or more first nucleic acids and / or second nucleic acids via a bond between an oxygen atom bonded to Z and a carbonyl carbon atom. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position. The method according to claim 1, wherein the compound is selected from the group consisting of compounds represented by the formula:

6. The second nucleic acid or a derivative thereof is represented by formula (14): 【Chemistry 4】 (wherein p represents an integer of 0 or more; AG / HG represent an optionally protected amino group or hydroxyl group; Base represents a base moiety (which may be the same or different); LV represents a group that leaves from the oxygen atom. Each deoxyribose may independently have an optionally protected hydroxyl group at the 2'-position. The method according to claim 1, wherein the compound is represented by the formula:

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