Acyclic threoninol nucleic acid

Abstract

The present invention provides a nucleic acid having excellent cell membrane penetrating activity. The present invention pertains to: a nucleic acid having a structure represented by general formula (A1) or (A2) [in the formulas, R0 represents an alkyl group having 1-30 carbon atoms and being substituted with one or more fluorine atoms, a group having 1-5 ether-bonding oxygen atoms between carbon atoms of an alkyl group having 2-30 carbon atoms and being substituted with one or more fluorine atoms, an alkyl group having 10-30 carbon atoms and not being substituted with a fluorine atom, or a group having 1-5 ether-bonding oxygen atoms between carbon atoms of an alkyl group having 10-30 carbon atoms and not being substituted with a fluorine atom, n11 and n12 each independently represent an integer of 1 or more, B represents a nucleic acid base, and each black dot represents an atomic bond]; a cell membrane permeating agent containing said nucleic acid as an active ingredient; and a nucleic acid medicinal drug containing said nucleic acid as an active ingredient.

Description

[Technical Field]

[0001] This invention relates to an acyclic threoninol-type nucleic acid with excellent cell membrane permeability. This application claims priority based on Japanese Patent Application No. 2021-033170, filed in Japan on March 3, 2021, and the contents of that application are incorporated herein by reference. [Background technology]

[0002] Antibody drugs are superior as treatments for cancer and intractable diseases because they can be used to produce antibodies against proteins that cannot be targeted by small molecule drugs, thus enabling them to be used as medicine. Furthermore, antibody drugs have the advantage of high specificity for target molecules and fewer side effects. However, antibody drugs have difficulty passing through cell membranes and entering cells, making it difficult to target molecules other than those on the cell surface.

[0003] Research into nucleic acid drugs, which utilize oligonucleotides, is progressing as the next generation of drug discovery following antibody drugs. Nucleic acid drugs have advantages such as high specificity for target molecules and fewer side effects. However, like antibody drugs, nucleic acid drugs have low cell membrane permeability, making it difficult to reach target molecules present inside cells. In particular, because siRNA is double-stranded, both its molecular weight and negative charge are larger than those of antisense RNA, resulting in lower cell membrane permeability than antisense RNA, and thus requiring drug delivery via a carrier. Drug delivery agents that use lipid nanoparticles (Patent Document 1) or cationic polymer nanoparticles (Patent Document 2) are known. However, there is much room for improvement in terms of cell membrane permeability efficiency and toxicity concerns.

[0004] For example, compounds having polyfluoro structures are known to be stable and low-toxicity in vivo, and to be excellent at being taken up into cells and exiting endosomes (Non-Patent Literature 1). It has been reported that peptide dendrimers using lysine with perfluoroacylated amino groups in the side chain as constituent amino acids can be used for gene delivery by utilizing this property (Non-Patent Literature 2). Studies are also being conducted to introduce polyfluoro structures into oligonucleotides and peptide nucleic acids as a portion that has cell membrane permeability (Patent Literature 3, and Non-Patent Literature 3-6).

[0005] On the other hand, nucleic acid drugs face the challenge of stability when administered to the body, because the phosphodiester bond in nucleic acids is susceptible to degradation by nucleases. If stability is low, the drug may be degraded in the body before reaching the target tissue, and the desired therapeutic effect cannot be obtained. To improve the stability of nucleic acids, chimeric nucleic acids are used, which are artificial nucleic acids that have superior nuclease resistance and other properties compared to natural nucleic acids. Examples of such artificial nucleic acids include acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), acyclic threoninol nucleic acid (aTNA), and serinol nucleic acid (SNA) (Non-Patent Literature 7). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2011 / 036557 [Patent Document 2] International Publication No. 2017 / 212006 [Patent Document 3] International Publication No. 2012 / 130941 [Patent Document 4] Japanese Patent Publication No. 2006-321797 [Patent Document 5] International Publication No. 2000 / 056694 [Non-patent literature]

[0007] [Non-Patent Document 1] Zhang et al., MRS Communications, 2018, vol.8, p.303-313. [Non-Patent Document 2] Cai et al., ACS Applied Materials and Interfaces, 2016, vol. 8, p.5821-5832. [Non-Patent Document 3] Godeau et al., Medicinal Chemistry Communications, 2010, vol.1. p.76-78. [Non-Patent Document 4] Ellipilli et al., Chemical Communications, 2016, vol.52, p.521-524. [Non-Patent Document 5] Rochambeaua et al., Polymer Chemistry, 2016, vol.7, p. 4998-5003. [Non-Patent Document 6] Metelev et al., Theranostics, 2017, vol.7, p.3354-3368. [Non-Patent Document 7] Murayama et al., Chemistry A European Journal, 2013, vol.19, p.14151-14158. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention aims to provide nucleic acids with excellent cell membrane permeability. [Means for solving the problem]

[0009] The inventors of the present invention have discovered that introducing an alkyl group, which may be substituted with a fluorine atom, into the side chain of an acyclic threoninol-type nucleic acid (aTNA-type nucleic acid) improves cell membrane permeability, and have completed the present invention.

[0010] In other words, the present invention is as follows: [1] The following general formula (A1) or (A2)

[0011] [ka]

[0012] [In the formula, R 0 [These are alkyl groups having 1 to 30 carbon atoms substituted with one or more fluorine atoms, groups having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of alkyl groups having 2 to 30 carbon atoms substituted with one or more fluorine atoms, alkyl groups having 10 to 30 carbon atoms not substituted with fluorine atoms, or groups having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of alkyl groups having 10 to 30 carbon atoms not substituted with fluorine atoms; n11 and n12 are each independently integers of 1 or more; B is a nucleic acid base; black circles indicate bonds] A nucleic acid having the structure represented by . [2] The above R 0 The nucleic acid of [1], wherein the alkyl group having 1 to 30 carbon atoms is substituted with at least two fluorine atoms. [3] The aforementioned R 0 The nucleic acid of [2], wherein the group is a perfluoroalkyl group having 1 to 10 carbon atoms, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of a perfluoroalkyl group having 1 to 10 carbon atoms. [4] The aforementioned R 0 The nucleic acid of [1], wherein the nucleic acid is an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom. [5] Any of the nucleic acids from [1] to [4] above, wherein n11 or n12 is 5 or greater. [6] Any of the nucleic acids described in [1] to [5] above, which are permeable to the cell membrane. [7] A cell membrane permeable agent comprising any of the nucleic acids described in [1] to [6] above as an active ingredient. [8] A nucleic acid drug comprising any of the nucleic acids described in [1] to [6] above as an active ingredient. [Effects of the Invention]

[0013] The nucleic acid according to the present invention has excellent cell membrane permeability because an alkyl group is introduced into the side chain of the aTNA-type nucleic acid. For this reason, the nucleic acid is expected to be used in the pharmaceutical field as a physiologically active substance. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced in Example 1. [Figure 2] This figure shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced in Example 2. [Figure 3] This figure shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced in Example 3. [Figure 4] This figure shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced in Example 4. [Figure 5] This figure shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced in Example 5. [Figure 6] This figure shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced in Example 6. [Modes for carrying out the invention]

[0015] In the specification of the present invention and this application, "nucleic acid" means a molecule in which nucleotides are linked by phosphodiester bonds. The nucleotides include not only natural nucleotides such as DNA and RNA (nucleotides that exist in nature), but also artificial nucleotides obtained by modifying natural nucleotides and capable of forming phosphodiester bonds with natural nucleotides. Artificial nucleotides include those in which the side chain of a natural nucleotide is modified with a functional group such as an amino group, those in which the hydroxyl group at the 2'-position of the ribose backbone is substituted with a methoxy group, a fluoro group, a methoxyethyl group, etc., phosphorothioate nucleotides (in which the oxygen atom of the phosphate group is substituted with a sulfur atom), Morpholino nucleotides (in which ribose and deoxyribose are substituted with a morpholine ring), BNA (Bridged nucleic acid), HNA (Hexitol Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), TNA (Threose nucleic acid), GNA (Glycerol nucleic acid), CeNA (Cyclohexenyl nucleic acid), etc. Further, "nucleic acid" includes a molecule in which only one or more natural nucleotides are linked by phosphodiester bonds, a molecule in which one or more natural nucleotides and one or more artificial nucleotides are linked by phosphodiester bonds, and a molecule in which only one or more artificial nucleotides are linked by phosphodiester bonds.

[0016] In the specification of the present invention and this application, "C" p1-p2 "(where p1 and p2 are positive integers satisfying p1 < p2) means a group having a carbon number of p1 to p2.

[0017] In the specification of the present invention and this application, "C" 1-30 alkyl group" is an alkyl group having 1 to 30 carbon atoms, which may be linear or branched. "C" 2-30 alkyl group" is an alkyl group having 2 to 30 carbon atoms, which may be linear or branched. C 1-30Examples of the alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, heneicosyl group, docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group, and the like.

[0018] In the present invention and this specification, " 1-20 alkyl group" means an alkyl group having 1 to 20 carbon atoms, which may be linear or branched. " 2-20 alkyl group" means an alkyl group having 2 to 20 carbon atoms, which may be linear or branched. 1-20 Examples of the

[0019] In the present invention and this specification, " 1-10 alkyl group" means an alkyl group having 1 to 10 carbon atoms, which may be linear or branched. " 2-10 alkyl group" means an alkyl group having 2 to 10 carbon atoms, which may be linear or branched. 1-10 Examples of the alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, and the like.

[0020] In the present invention and this specification, "C 10-30 An alkyl group is an alkyl group having 10 to 30 carbon atoms, and may be a straight chain or a branched chain. 10-30 Examples of alkyl groups include undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, and triacontyl groups.

[0021] In the present invention and this specification, "C 1-6 An alkyl group is an alkyl group having 1 to 6 carbon atoms, and may be a straight chain or a branched chain. 1-6 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl groups.

[0022] In the present invention and this specification, "alkylene group" is a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, and may be a linear or branched chain. Examples of alkylene groups include methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, methylmethylene group, ethylmethylene group, methylethylene group, methylpropylene group, ethylethylene group, dimethylmethylene group, 1,2-dimethylethylene group, 1,1-dimethylethylene group, 1-ethylpropylene group, 2-ethylpropylene group, 1,2-dimethylpropylene Examples include the 2,2-dimethylpropylene group, 1-propylpropylene group, 2-propylpropylene group, 1-methyl-1-ethylpropylene group, 1-methyl-2-ethylpropylene group, 1-ethyl-2-methylpropylene group, 2-methyl-2-ethylpropylene group, 1-methylbutylene group, 2-methylbutylene group, 3-methylbutylene group, 2-ethylbutylene group, 1-methylpentylene group, 2-ethylpentylene group, and 1-methylhexylene group.

[0023] In the present invention and this specification, "C 1-20 A "perfluoroalkyl group" is a group in which all hydrogen atoms of an alkyl group with 1 to 20 carbon atoms are replaced by fluorine atoms. 1-10 Examples of perfluoroalkyl groups include perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluoroisopropyl, perfluorobutyl, perfluoroisobutyl, perfluorosec-butyl, perfluorotert-butyl, perfluoropentyl, perfluoroisopentyl, perfluoroneopentyl, perfluorotert-pentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, perfluorodecyl, perfluoroundecyl, perfluorododecyl, perfluorotridecyl, perfluorotetradecyl, perfluoropentadecyl, perfluorohexadecyl, perfluoroheptadecyl, perfluorooctadecyl, perfluorononadecyl, and perfluoroeicosyl groups.

[0024] In the present invention and this specification, a "perfluoroalkylene group" is a group in which all hydrogen atoms of an alkylene group are replaced with fluorine atoms. An example of a perfluoroalkylene group is the group in which all hydrogen atoms of the alkylene group mentioned above are replaced with fluorine atoms.

[0025] In the present invention and this specification, "ether-bonded oxygen atom" refers to an oxygen atom that links carbon atoms together, and does not include oxygen atoms linked in series. The maximum number of ether-bonded oxygen atoms that an alkyl group with Nc (where Nc is an integer of 2 or more) can have is Nc-1. Furthermore, "having ether-bonded oxygen atoms between carbon atoms, C 2-10 "Alkyl alkyl group" refers to C 2-10 This group has at least one ether-bonded oxygen atom between the carbon atoms of the alkyl group. Hereinafter, alkyl groups having an ether-bonded oxygen atom may be referred to as "ether-bonded alkyl groups."

[0026] In the present invention and this specification, "having an ether-bonded oxygen atom between carbon atoms, C 2-10 "Perfluoroalkyl group" is C 2-10 Ether bond-containing C having at least one ether-bonded oxygen atom between the carbon atoms of the alkyl group 2-10 This is a group in which all hydrogen atoms of the alkyl group are replaced with fluorine atoms. Hereinafter, perfluoroalkyl groups that have an ether-bonded oxygen atom may be referred to as "ether-bonded perfluoroalkyl groups."

[0027] In the present invention and this specification, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. "Halogen atom other than a fluorine atom" means a chlorine atom, a bromine atom, or an iodine atom. Examples of "halogen atoms other than a fluorine atom" are preferably chlorine atoms or bromine atoms, and chlorine atoms are particularly preferred.

[0028] Furthermore, hereafter, "compound (X)" refers to the compound represented by formula (X).

[0029] <Nucleic acid> The nucleic acid according to the present invention has an alkyl group at a specific site of the aTNA-type nucleic acid, which may be substituted with a fluorine atom. Like other artificial nucleic acids, this nucleic acid is expected to be used in the pharmaceutical field as a physiologically active substance. Specifically, the nucleic acid according to the present invention has a structure represented by the following general formula (A1) or (A2). Hereafter, "the structure represented by general formula (A1)" may be referred to as "structure (A1)," and "the structure represented by general formula (A2)" may be referred to as "structure (A2)."

[0030] [ka]

[0031] In general formulas (A1) and (A2), R 0 C is a carbon atom substituted with at least one fluorine atom. 1-30 Alkyl(C) 1-30 Fluoroalkyl groups, or C atoms not substituted with a fluorine atom 10-30 It is an alkyl group. If the alkyl group has two or more carbon atoms, it may have 1 to 5 ether-bonded oxygen atoms between carbon atoms. In the present invention and this specification, "C 1-30 "Fluoroalkyl group (if the alkyl group has two or more carbon atoms, the alkyl group may have 1 to 5 ether-bonded oxygen atoms between carbon atoms)" means "C 1-30 Fluoroalkyl groups, or C 2-30 This refers to a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of a fluoroalkyl group.

[0032] R 0 C 1-30 In the case of fluoroalkyl groups, one or more hydrogen atoms bonded to the carbon atom may be further substituted with halogen atoms other than fluorine atoms. Structure (A1) or structure (A2) is R 0C is a carbon atom substituted with at least two fluorine atoms. 1-30 It is preferable that it be a fluoroalkyl group. In particular, R 0 C 1-20 Fluoroalkyl groups are preferred, C 1-15 Fluoroalkyl groups are more preferred, C 2-15 Fluoroalkyl groups are more preferred, C 6-10 Fluoroalkyl groups are even more preferred. 0 C 1-30 In the case of fluoroalkyl groups, the number of hydrogen atoms substituted for fluorine atoms is not particularly limited as long as there is one or more; for example, three or more are preferred, six or more are more preferred, and seven or more are even more preferred.

[0033] R 0 C 1-30 In the case of fluoroalkyl groups, R 0 Specifically, a group represented by the following general formula (f-1) or (f-2) is preferred. Here, Rf P is a fully halogenated C 1-20 Alkyl(C) 1-20 A group in which all of the hydrogen atoms of an alkyl group are replaced by halogen atoms, and which has one or more fluorine atoms. Rf P If the number of carbon atoms is 2 or more, that is, fully halogenated C 2-20 In the case of alkyl groups, there may be 1 to 5 ether-bonded oxygen atoms between the carbon atoms. In the present invention and this specification, "a fully halogenated C which may have 1 to 5 ether-bonded oxygen atoms between the carbon atoms" is used. 2-20 "Alkyl alkyl group" refers to "fully halogenated C 2-20 Alkyl alkyl groups, or fully halogenated C 2-20 This refers to a group that has 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group. In the general formula (f-2), two Rf P These groups may be of the same kind or of different kinds. Rf P C 1-20 Perfluoroalkyl groups (C 1-20It is preferable that the alkyl group is a group in which all of the hydrogen atoms are replaced by fluorine atoms.

[0034] In the following general formulas (f-1) or (f-2), n1 is an integer between 0 and 10, and n2 is an integer between 0 and 9. When both n1 and n2 are 0, both represent a simple associativity. That is, when n1 is 0, the base represented by general formula (f-1) is -Rf P And when n2 is 0, the group represented by the general formula (f-2) is -CH(Rf P )2.

[0035] [ka]

[0036] R 0 If the group is represented by the general formula (f-1), then R 0 Rf P However, the group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 4. P However, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 2. P However, the group is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n1 is an integer from 0 to 2 (however, if n1 is 1, Rf P (excluding groups where the group is a trifluoromethyl group) is more preferably Rf PHowever, a group that is a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, where n1 is 0, is even more preferred.

[0037] R 0 If the group is represented by the general formula (f-2), then R 0 Rf P However, the group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 4. P However, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 2. P However, the group is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n2 is an integer from 0 to 2 (however, if n2 is 0 or 1, Rf P (excluding groups where the group is a trifluoromethyl group) is more preferably Rf P However, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n2 is 0, is even more preferred.

[0038] R 0 C 1-30 In the case of fluoroalkyl groups, R 0Examples include trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, perfluorodecyl group, difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1, Examples of nucleic acids according to the present invention include 1,2,2,3,3-hexafluoropropyl group, 1,1,2,3,3,3-hexafluoropropyl group, 1,1,2,2,3,3-hexafluorohexyl group, 1,1,2,2,3,3-hexafluorooctyl group, 1,1,2,2,3,3-hexafluorodecyl group, 1,1,2,2,3,3-hexafluorooctadecyl group, 1,1,2,2,3,3-hexafluorohexacosyl group, etc. In the nucleic acid according to the present invention, R is in general formula (A1) or (A2). 0 C 1-30 Nucleic acids having a perfluoroalkyl group structure are preferred, and in general formula (A1) or (A2), R 0 C 1-20 Nucleic acids having a perfluoroalkyl group structure are more preferred, and in general formula (A1) or (A2), R 0 C 1-10 Nucleic acids having a perfluoroalkyl group structure are more preferred.

[0039] R 0 C 10-30 If it is an alkyl group, the structure (A1) or structure (A2) is R 0 C 10-25 Alkyl alkyl groups are preferred, C 15-25 Alkyl alkyl groups are more preferred, C 15-23 Alkyl alkyl groups are even more preferred. Alkyl groups of sufficient length, like fluoroalkyl groups, are highly hydrophobic and contribute to the cell membrane permeability of nucleic acids having structure (A1) or structure (A2).

[0040] In general formula (A2), B is a nucleic acid base. Preferred nucleic acid bases include those found in natural nucleic acids, nucleic acid bases structurally similar to natural nucleic acid bases, and modified bases that have undergone various modifications. Modifications of the base include alkylation, hydroxylation, alkoxylation, acylation, dihydrolation, amination, formylation, halogenation, etc. Nucleic acid bases structurally similar to natural nucleic acid bases include triazole, imidazole, azapyrimidine, azapurine, etc. Specifically, the nucleic acid base of B is adenine, guanine, cytosine, thymine, uracil, 1-methyladenine, N6-methyladenine, 7-methylguanine, 5-methylcytosine, 1-methylthymine, 5-methyluracil, 5-hydroxymethylcytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, dihydrouracil, dihydrothymine, dihydrocytosine, 2,6-diaminoadenine, 2,6-diamino Examples include guanine, 6-thioguanine, 2-thioadenine, 2-thiocytosine, 4-thiouracil, 5-fluorouracil, 5-iodouracil, 5-halogenocytosine, 5-fluorocytosine, 5-trihalogenomethyluracil, 5-trifluoromethyluracil, 5-azathymine, 5-azacytosine, 6-azauracil, 8-azaadenine, 7-deazaadenine, 7-deazaguanine, and 3-deazauracil. The nucleic acid according to the present invention is preferably a nucleic acid having a structure in which B in general formula (A2) is adenine, guanine, cytosine, thymine, or uracil.

[0041] In general formulas (A1) and (A2), n11 and n12 are independent integers of 1 or more. n11 and n12 are the number of repeats per molecule of each structure, and the larger the number, the higher the hydrophobicity. This improves the cell membrane permeability of the nucleic acid. In the nucleic acid according to the present invention, it is preferable that n11 and n12 are 2 or more, and more preferably 5 or more. Furthermore, since it is easier to obtain structures similar to natural double-stranded nucleic acids and single-stranded nucleic acids, it is preferable that n11 and n12 are 10 or less, more preferably 8 or less, and even more preferably 6 or less. When n11 and n12 are 2 or more, the multiple structures (A1) or structures (A2) may be identical to each other or may be different structures.

[0042] In general formulas (A1) and (A2), black circles indicate binding sites. The nucleic acid according to the present invention may be any nucleic acid having structure (A1) or structure (A2), and the position in which structure (A1) or structure (A2) is introduced is not particularly limited, and it may be introduced at any site as long as it does not impair the function of the nucleic acid. For example, structure (A1) or structure (A2) may be directly or indirectly bound to the 5' or 3' end of the nucleic acid, or introduced between two nucleotides.

[0043] Among the nucleic acids according to the present invention, it is preferable that the nucleic acid having structure (A1) or structure (A2) at its 5' end has a bond extending from a carbon atom at the end of structure (A1) or structure (A2) that is bound to a hydroxyl group, and a bond extending from the oxygen atom of the phosphate group at the end of structure (A1) or structure (A2) that is bound to the sugar at the 5' end of the nucleic acid. Among the nucleic acids according to the present invention, it is preferable that the nucleic acid having structure (A1) or structure (A2) at its 3' end has a bond extending from a carbon atom at the end of structure (A1) or structure (A2) that is bound to the phosphate group at the 3' end of the nucleic acid, and a bond extending from the oxygen atom of the phosphate group at the end of structure (A1) or structure (A2) that is bound to a hydrogen atom.

[0044] Among the nucleic acids according to the present invention, as the nucleic acid in which the structure (A1) or the structure (A2) is introduced between two nucleotides, since it can be approximated by the structure of natural nucleic acids, the bond extending from the carbon atom at the end of the structure (A1) or the structure (A2) is preferably bonded to the phosphate group of another nucleotide, and the bond extending from the oxygen atom of the phosphate group at the end of the structure (A1) or the structure (A2) is preferably bonded to the sugar of another nucleotide.

[0045] When introducing a highly hydrophobic R 0 group into the aTNA-type nucleic acid according to the present invention, since it is introduced as the structure (A1) or the structure (A2), for example, when forming a DNA double helix structure, the R 0 group is exposed outside the helix structure, and a more stable double helix structure can be formed. In addition, the cell membrane permeability can be improved by the R 0 group exposed on the surface. That is, the nucleic acid according to the present invention is useful as a cell membrane permeabilizing agent. In addition, since the nucleic acid according to the present invention does not contain a nitrogen atom in the chain composed of the phosphodiester bond between the phosphate group and the sugar of the nucleic acid, for example, the intermediate when synthesized by the phosphoramidite method is relatively stable and the synthesis is also easy.

[0046] In the nucleic acid according to the present invention, the nucleic acid into which the structure (A1) or the structure (A2) is introduced is not particularly limited, and it may be a nucleic acid in which all the contained nucleotides are natural-type nucleotides, or a nucleic acid in which some or all are artificial nucleotides. It may also be a single-stranded nucleic acid or a double-stranded nucleic acid. Examples of such nucleic acids include genomic DNA, cDNA, mRNA, microRNA, siRNA, antisense oligonucleotides, nucleic acid aptamers, decoy nucleic acids, CpG (cytosine-phosphate-guanine) oligonucleotides, etc. It may also be an expression vector that expresses a target gene or siRNA in cells.

[0047] In the nucleic acid according to the present invention, structure (A1) or structure (A2) can be introduced into the target nucleic acid by various coupling reactions. For example, by performing the phosphoramidite method using a phosphoramidite containing structure (A1) or structure (A2) as a raw material, structure (A1) or structure (A2) can be easily introduced into the nucleic acid. Commonly used automated nucleic acid synthesizers utilize the phosphoramidite method. Therefore, by using a phosphoramidite containing structure (A1) or structure (A2) as a raw material, nucleic acids with structure (A1) or structure (A2) introduced at a desired position can be easily synthesized in an automated synthesizer for nucleic acids with various base sequences.

[0048] Examples of phosphoramidites containing structure (A1) or structure (A2) include, among the phosphoramidites commonly used in nucleic acid synthesis, compounds in which a sugar and a phosphate group are linked via structure (A1), and compounds in which the nucleoside portion is replaced with an organic group containing structure (A2).

[0049] The nucleic acid according to the present invention may be a nucleic acid having only structure (A1) or structure (A2). In this case, the binding sites at both ends of structure (A1) or structure (A2) are bound to a hydrogen atom or a hydroxyl group.

[0050] The synthesized target nucleic acid can be isolated and purified by various methods, such as ion chromatography, gel filtration chromatography, reverse-phase chromatography, and normal-phase chromatography.

[0051] The nucleic acids according to the present invention may be modified in various ways, provided that the function of the nucleic acid into which structure (A1) or structure (A2) is introduced is not impaired and the effects of the present invention are not impaired. Examples of such modifications include glycosylation, lipid modification, and peptide modification.

[0052] R 0 The group is highly hydrophobic and has a high affinity for cell membranes. Therefore, R 0 The nucleic acid according to the present invention, which has a structure (A1) or structure (A2) containing the group introduced, is R0 It has better cell membrane permeability than nucleic acids without the introduced group. Utilizing this property, the nucleic acid according to the present invention is particularly preferable as an active ingredient of nucleic acid pharmaceuticals. For example, by introducing the structure (A1) or the structure (A2) into a functional nucleic acid that exhibits some physiological activity when taken up into target cells in vivo so as not to impair its function, the uptake efficiency of the functional nucleic acid into target cells can be improved. For example, for a nucleic acid that has pharmacological activity but cannot reach the target cells in vivo, by introducing the structure (A1) or the structure (A2), the uptake efficiency of the nucleic acid into target cells can be improved. That is, by using the nucleic acid according to the present invention, a drug delivery system for delivering nucleic acid pharmaceuticals into cells can be easily constructed.

Examples

[0053] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0054] The NMR apparatus used for the analysis of the examples and comparative examples was JNM-ECZ400S (400 MHz) manufactured by JEOL Ltd., 1 In 1H NMR, tetramethylsilane was used as 0 PPM, 19 In 19F NMR, C6F6 was used as the reference value of -162 PPM.

[0055] <DNA Synthesis> In the subsequent experiments, DNA synthesis was performed using commercially available reagents, various phosphoramidites (acetonitrile solution, 0.1 M) and 5-ethylthio-1H-tetrazole (acetonitrile solution, 0.25 M) as an activator, by an NTS H-8 DNA / RNA synthesizer (manufactured by Nippon Techno Service Co., Ltd.).

[0056] The synthesis of 5'-aTNA-N[PFC8] modified DNA was performed as follows: First, DNA synthesis (tritil-off) was carried out on a 1000 Å CPG solid-supported column (1 μmole scale). Next, under a nitrogen atmosphere, aTNA-N[PFC8] amidite solution (0.1 M acetonitrile solution, 300 μL) and activator solution (300 μL) were mixed using a syringe in the presence of CPG. After 5 minutes, the resulting solution was removed from the column, and the DNA strands were capped, oxidized, and deblocked on a DNA synthesizer.

[0057] Deprotection of the synthesized 5'-aTNA-N[PFC8] modified DNA was performed as follows: First, DNA (trityl-off) supported on a CPG solid was treated with a 28% ammonium hydroxide aqueous solution at 50°C for 12 hours. Next, this crude product solution was separated from the solid support and concentrated under reduced pressure at 30°C. The resulting concentrate was filtered through a 0.45 μm centrifugal filter before HPLC purification. The resulting filtrate was filtered through a 0.45 μm centrifugal filter and then purified by HPLC. The resulting solution was quantified by absorbance at 260 nm.

[0058] The deprotected DNA was purified by HPLC. HPLC purification was performed under the following conditions.

[0059] Solvent (filtered through a 0.45 μm centrifuge): 100 mM triethylammonium acetate (TEAA) buffer (pH 7.0) and acetonitrile (HPLC grade) Elution gradient: 3-95% acetonitrile (40 minutes) Column: COSMOSIL packed column "5C18-MS-II" (4.6 ID x 150 nm, manufactured by Nacalai Tesque) Samples used for each analysis: A solution of crude DNA dissolved in 20-50 μL of ultrapure water was injected. Detection was performed using a diode array detector while monitoring the absorbance at 260 nm.

[0060] <Cell culture> In subsequent experiments, cell culture was performed as follows. HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific) supplemented with 10% FBS and 0.5% penicillin / streptomycin at 37°C in a humidified atmosphere (5% CO2 by volume). Cells for image analysis were cultured in 35 mm glass-bottom dishes (IWAKI).

[0061] <Confocal microscopy observation> In subsequent experiments, confocal microscopy observation of cells was performed as follows. Fluorescein-conjugated nucleic acid (500 μM, 5 μL) was added to HeLa cells and incubated at 37°C for 3 hours. Next, the nuclear stain hoechst33342 (2 μg / mL, 0.5 mL) was added to the cells and incubated for another hour. After incubation, the solution was removed from each dish, washed with PBS(-), and then DMEM (1 mL) was added. Each dish was then placed in a confocal laser scanning microscope, and fluorescence images were acquired using an excitation wavelength of 488 nm and an emission filter exceeding 505 nm.

[0062] <Flow Cytometry> In subsequent experiments, flow cytometry was performed as follows. HeLa cells were placed in a 12-well plate. 5Cells were seeded at a density of 1 cell per well and cultured. The day after seeding, the culture medium in each well was replaced with DMEM medium (1 mL) containing fluorescein-conjugated nucleic acid (2.5 μM), and incubated for 4 or 24 hours. Subsequently, the cell layer in the wells was washed twice with PBS, and the cells were detached by treatment with 0.05% (w / v) trypsin (200 μL) at 37°C for 5 minutes. The harvested cells were suspended in DMEM (600 μL). This cell suspension was separated by centrifugation (400 × g, 3 minutes), and PBS / 1% BSA (500 μL) was added. The percentage of fluorescent cells and the average fluorescence intensity of this cell suspension were analyzed using a flow cytometer ("guava easyCyte8", Luminex).

[0063] [Example 1] R 0 We synthesized nucleic acids having a structure (A1) in which the group is a perfluorooctyl group, and investigated their cell membrane permeability.

[0064] R 0 The phosphoramidite (aTNA-N[PFC8]) having a structure (A1) in which the group is a perfluorooctyl group was synthesized as follows.

[0065] (1) Amidite condensation

[0066] [ka]

[0067] D-threoninol (0.95 g, 9 mmol) was dissolved in dry methanol (10 mL) in an ice bath, and then ethyl heptadecafluorononanoate (4.8 g, 9.9 mmol, in a 5 mL methanol solution) was added dropwise. After stirring at 0°C for 2 hours, the solvent was removed by evaporation to obtain 4.9 g of the target intermediate (N-(1,3-dihydroxybutan-2-yl)nonanamide) (99% yield).

[0068] 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 6.9 Hz, 1H), δ 4.14-4.07 (m, 1H), δ 4.02 (t, J = 5.5 Hz, 1H), δ 3.95-3.88 (m, 1H), δ 3.76-3.64 (m, 2H) 19 F NMR (376 MHz, CDCl3) δ -81.6 (s, 3F), δ -119.6 (s, 2F), δ -122.0 (s, 2F), δ -122.4 (s, 2F), δ -112.8 (s, 2F), δ -123.4 (s, 2F), δ -126.8 (s, 2F)

[0069] (2)DMTr protection

[0070] [ka]

[0071] A 20 mL dry pyridine solution containing the intermediate (N-(1,3-dihydroxybutan-2-yl)nonanamide) (1.8 g, 8.8 mmol) and diisopropylethylamine (DIPEA) (1.7 mL, 1.4 g, 10.5 mmol) was cooled on ice under nitrogen. To this mixture, a 15 mL dry dichloromethane solution containing 4,4'-dimethoxytrityl chloride (3.6 g, 10.5 mmol) and 4-dimethylaminopyridine (0.16 g, 1.3 mmol) was added. The mixture was stirred at room temperature for 2.5 hours, the solvent was removed, and then silica gel column chromatography (hexane:ethyl acetate:Et3N = 80:20:3 (volume ratio)) was performed to obtain 3.6 g (7.2 mmol) of the DMTr-protected intermediate (60% yield).

[0072] 1H NMR (400 MHz, CDCl3) δ 7.36-7.20 (m, 8H), δ 7.06 (d, J = 8.7 Hz, 1H), δ 3.78 (s, 6H), δ 3.48 (dd, J = 4.2 Hz, 9.6Hz, 1H), δ 3.28 (dd, J = 3.7 Hz, 9.8Hz, 1H), δ 1.13 (d, J = 6.4Hz, 3H) 19 F NMR (376 MHz, CDCl3) δ -80.6 (s, 3F), δ -118.8 (dt, J=271 Hz, 14Hz, 1F), δ -119.7 (dt, J=272 Hz, 14Hz, 1F), δ -121.3 (s, 2F), δ -121.7 (s, 2F) , δ -122.2 (s, 2F), δ -122.6 (s, 2F), δ -126.0 (s, 2F)

[0073] (3) Amidite formation

[0074] [ka]

[0075] To a solution of 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (2.38 g, 7.9 mmol) dissolved in dry acetonitrile, 5-ethylthiotetrazole (ETT) (1.03 g, 7.9 mmol) was added under argon, and then a DMTr-protected intermediate (4.5 g, 5.3 mmol, dissolved in a mixed solvent of 2 mL of tetrahydrofuran (THF) and 10 mL of acetonitrile) was gradually added. The resulting reaction mixture was stirred under argon at room temperature for 14 hours. After evaporating the solvent under reduced pressure, the resulting crude product was purified under argon by column chromatography using degassed hexane / ethyl acetate (1:4 (volume ratio)) as the mobile phase. This isolated aTNA-N[PFC], a colorless oil (yield 28%).

[0076] 1H NMR (400 MHz, CDCl3) δ 7.41-7.13 (m, 9H), δ 6.84-6.79 (m, 4H), δ 4.33-4.20 (m, 1H), δ 4.14-4.07 (m, 1H), δ 3.82-3.72 (m, 6H) δ 3.63-3.43 (m, 4H) δ 3.33-3.13 (m, 2H), δ 2.57 (t, 6.4 Hz, 1H), δ 2.43-2.39 (m, 1H), δ 1.27-1.08 (m, 12H), δ 0.96 (d, J = 6.9 Hz, 3H) 19 F NMR (376 MHz, CDCl3) δ -80.6 (m, 2F), δ -118.8 (dt, J=278 Hz, 13Hz, 1F), δ -119.8 (dt, J=272 Hz, 13Hz, 1F), δ -121.3 (s, 2F), δ -121.7 (s, 2F), δ -122.1 (s, 2F), δ -122.6 (s, 2F) 31 P NMR (162 MHz, CDCl3), δ -149.6 (s) , δ -148.5 (s)

[0077] Using the synthesized aTNA-N[PFC8], as described above, nucleic acid (aTNA-N5[PFC8]) was synthesized by ligating five of the following structures (aTNA-N1[PFC8]: black circles in the formula indicate binding sites) to the 5' end of a control DNA (SEQ ID NO: 1), which is a natural single-stranded DNA.

[0078] [ka]

[0079] Nucleic acid (fluorescein-labeled aTNA-N5[PFC8]) with fluorescein conjugated to the 3' end of synthesized aTNA-N5[PFC8] was introduced into HeLa cells, and flow cytometry was performed to quantify the number of cells into which the fluorescein-labeled nucleic acid was introduced and the amount of fluorescein-labeled nucleic acid introduced into each cell, based on the fluorescence intensity of fluorescein. As a control, nucleic acid (fluorescein-labeled control DNA) with fluorescein conjugated to the 3' end of control DNA was similarly introduced into HeLa cells and analyzed by flow cytometry.

[0080] Table 1 shows the relative fluorescence intensity of cells introduced with each fluorescein-labeled nucleic acid ([fluorescence intensity of cells introduced with fluorescein-labeled nucleic acid] / [fluorescence intensity of cells introduced with fluorescein-labeled control DNA]), with the fluorescence intensity of cells introduced with fluorescein-labeled control DNA set as 1. Figure 1 shows the flow cytometry results of cells introduced with fluorescein-labeled control DNA and cells introduced with fluorescein-labeled aTNA-N5[PFC8]. In the figure, "none" indicates the results for cells that did not take up DNA, "DNA" indicates the results for cells introduced with fluorescein-labeled control DNA, and "aTNA-N5" indicates the results for cells introduced with fluorescein-labeled aTNA-N5[PFC8].

[0081] [Table 1]

[0082] As shown in Table 1, there were nearly twice as many cells that had been introduced with fluorescein-labeled aTNA-N5[PFC8] compared to cells that had been introduced with fluorescein-labeled control DNA. Furthermore, as shown in Figure 1, cells that had been introduced with fluorescein-labeled aTNA-N5[PFC8] had a higher fluorescence intensity per cell and a larger amount of fluorescein-labeled nucleic acid introduced per cell. These results indicate that aTNA-N5[PFC8] has higher cell membrane permeability than control DNA.

[0083] [Example 2] The fluorescein-labeled aTNA-N5[PFC8] prepared in Example 1 was hybridized with single-stranded DNA (control rDNA) consisting of a base sequence complementary to the control DNA (SEQ ID NO: 2) to create fluorescein-labeled double-stranded DNA (fluorescein-labeled aTNA-N5[PFC8] / rDNA) having a repeating structure (A1) at the 5' end and fluorescein bound to the 3' end. Similarly, fluorescein-labeled double-stranded DNA (fluorescein-labeled control DNA / rDNA) was prepared by hybridizing fluorescein-labeled control DNA and control rDNA. These fluorescein-labeled double-stranded DNAs were introduced into HeLa cells in the same manner as in Example 1, and then analyzed by flow cytometry.

[0084] Table 2 shows the relative fluorescence intensity of cells into which each fluorescein-labeled nucleic acid was introduced (fluorescence intensity of cells into which fluorescein-labeled control DNA / rDNA was introduced is set to 1). Figure 2 shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced. In the figure, "none" indicates the results of cells into which no DNA was incorporated, "DNA / rDNA" indicates the results of cells into which fluorescein-labeled control DNA / rDNA was introduced, and "aTNA-NF5" indicates the results of cells into which fluorescein-labeled aTNA-N5[PFC8] / rDNA was introduced.

[0085] [Table 2]

[0086] As shown in Table 2 and Figure 2, there were more cells introduced with fluorescein-labeled aTNA-N5[PFC8] / rDNA than with cells introduced with fluorescein-labeled control DNA / rDNA. These results confirm that even double-stranded DNA exhibits the same cell membrane permeability-improving effect due to its structure (A1) as single-stranded DNA.

[0087] [Example 3] R 0 We synthesized nucleic acids having a structure (A2) in which the group is a perfluorooctyl group.

[0088] R 0 The phosphoramidite (aTNA-C[PFC8]) having a structure (A2) in which the group is a perfluorooctyl group was synthesized as follows.

[0089] [ka]

[0090] (S)-Garner aldehyde (3.82 g, 7.0 mmol) is dissolved in dry ether (25 mL) in an oven-dried 50 mL two-necked round-bottom flask, followed by C8F 17 I (1.76 g, 7.7 mmol) was added and the mixture was cooled to -78°C. Next, MeLi (1.1 M, 7.7 mmol, 7 mL Et2O solution) in ether was added dropwise to the flask over 30 minutes, and the reaction mixture was stirred at -78°C for 4 hours. After that, the reaction was quenched with saturated NH4Cl, and the product was extracted with Et2O. The organic fractions were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum. The resulting crude product was purified by chromatography using a SiO2 / hexane (1:20 (volume ratio)) mixed solvent to obtain a white solid mixture of compound 1a and compound 1b (yield: compound 1a 25%, compound 1b 21%).

[0091] Compound 1a: 1 H NMR (400 MHz, CDCl3) δ 4.79 (m, 1H), 4.46 (m, 1H), 4.14 (m, 1H), 4.01 (dd, J = 10.0, 5.0 Hz, 1H), 3.90 (d, J = 9.6 Hz, 1H), 1.60 (s, 3H), 1.49 (m, 12H) 19 F NMR (376 MHz, CDCl3) δ -80.9 (s, 3F), -118.8 (d, JFF = 289.0 Hz, 1F), -121.5 - -123.7 (m, 10F), -125.3 - -127.1 (m, 2F), -127.5 (d, J FF (= 283.2 Hz, 1F)

[0092] Compound 1b: 1 H NMR (400 MHz, CDCl3) δ 4.60 (m, 1H), 4.27 (m, 2H), 4.03 (m, 1H), 3.82 (s) and 3.19 (s) (1H), 1.60 (m, 3H), 1.46 (m, 12H) 19 F NMR (376 MHz, CDCl3) δ -80.9 (s, 3F), -119.6 (d, J FF = 283.2 Hz) and (d, J FF = 283.2 Hz) (1F), -121.8 - -123.7 (m, 10F), -125.4 - -127.1 (m, 3F)

[0093] [ka]

[0094] Compound 1b (1.69 g, 2.6 mmol) was dissolved in MeOH (25 mL) in a 100 mL round-bottom flask in air and cooled to 0°C. Then, p-toluenesulfonic acid monohydrate (49.5 mg, 0.26 mmol) was added to the flask, and the reaction mixture in the flask was warmed to room temperature and stirred for a further 17 hours. After that, the reaction was quenched with saturated NaHCO3, and the product was extracted with ethyl acetate. The organic fractions were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum. The resulting crude product was purified by chromatography using a ethyl acetate / hexane (1:2 volume ratio) mixed solvent to obtain the target compound 2b as a white solid (yield 47%).

[0095] Compound 2b: 1 1H NMR (500 MHz, acetone-d6) δ 6.13 (d, J = 8.6 Hz, 1H), 5.58 (d, J = 6.9 Hz, 1H), 4.52 (d, J = 24.1 Hz, 1H), 4.18 (m, 1H), 4.04 (m, 1H), 3.92 (m, 1H), 3.81 (m, 1H), 1.41 (s, 9H) 19 19F NMR (470 MHz, acetone-d6) δ -81.6 (s, 3F), -118.7 (d, J FF = 278.8 Hz, 1F), -121.1 - -123.8 (m, 10F), -125.4 (d, J FF = 278.8 Hz, 1F), -126.5 (d, J FF = 264.1 Hz, 1F), -127.0 (d, J FF = 293.4, 1F)

[0096] Compound 2a was obtained in the same manner using compound 1a instead of compound 1b.

[0097] Compound 2a: 1 1H NMR (400 MHz, acetone-d6) δ 5.67 (dd, J = 16.9, 8.2 Hz, 1H), 4.60 (dt, J = 22.3, 5.3 Hz, 1H), 4.20 (t, J = 5.3 Hz, 1H), 4.03 (m, 1H), 3.55 (m, 2H), 1.35 (s, 9H) 19 19F NMR (376 MHz, acetone-d6) δ -81.5 (s, 3F), -119.3 (d, J FF = 286.1 Hz, 1F), -121.1 - -123.3 (m, 10F), -126.2 (d, J FF = 300.5 Hz, 1F), -126.8 (d, J FF = 283.2 Hz, 1F), -127.1 (d, JFF (= 289.0 Hz, 1F)

[0098] [ka]

[0099] Compound 2b (746 mg, 1.2 mmol) was dissolved in dry dichloromethane (DCM) (10 mL) in a 200 mL two-necked round-bottom flask and cooled to 0°C. Then, trifluoroacetic acid (TFA) (0.94 mL, 12.2 mmol) was added dropwise to the flask over 10 minutes, and the reaction mixture in the flask was warmed to room temperature and stirred for a further 18.5 hours. After that, the reaction was quenched with saturated NaHCO3, and the product was extracted with ethyl acetate. The organic fractions were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum. The resulting crude product was purified by chromatography using a ethyl acetate / hexane (1:2 volume ratio) mixed solvent to obtain the target compound 3b as a white solid (yield 98%).

[0100] Compound 3b: 1 H NMR (500 MHz, acetone-d6) δ 5.68 (s, 1H), 4.45 (d, J = 23.5 Hz, 1H), 3.86 (t, J = 10.9 Hz, 1H), 3.78 (d, J = 8.0 Hz, 1H), 3.76 (d, J = 7.5 Hz, 1H) 19 F NMR (470 MHz, acetone-d6) δ -81.6 (s, 3F), -118.6 (d, J FF = 278.8 Hz, 1F), -121.5 - -123.9 (m, 10F), -124.9 (d, J FF = 278.8 Hz, 1F), -126.3 (d, J FF = 293.4 Hz, 1F), -127.0 (d, J FF (= 293.4 Hz, 1F)

[0101] Compound 3a was obtained in the same manner by using compound 2a instead of compound 2b.

[0102] Compound 3a: 1 H NMR (400 MHz, acetone-d6) isomer I δ 4.25 (dd, J = 23.8, 6.9 Hz, 1H), 3.72 (m, 2H), 3.55 - 3.44 (m, 1H) isomer II δ 4.07 (dd, J = 24.9, 2.5 Hz, 1H), 3.84 (d, J = 4.1 Hz, 1H), 3.82 (d, J = 4.6 Hz, 1H), 3.35 (t, J = 6.6, 1H) 19 F NMR (376 MHz, acetone-d6) δ -81.7 (s, 3F), -120.2 (d, J FF = 280.3 Hz, 1F), -121.9 - -123.3 (m, 10F), -126.1 - -127.7 (m, 2F), -127.5 (d, J FF (= 283.2 Hz, 1F)

[0103] [ka]

[0104] Compound 3b (598 mg, 1.2 mmol) was dissolved in dry MeOH (4 mL) in a 25 mL two-necked round-bottom flask and cooled to 0°C. Then, trifluoroethyl acetate (287 μL, 340 mg, 2.4 mmol) was added dropwise to the flask over 10 minutes, and the reaction mixture in the flask was warmed to room temperature and stirred for a further 35 hours. After that, the solvent was evaporated under vacuum, and the resulting white oil was purified by chromatography using a toluene / hexane (1:2 volume ratio) mixed solvent to obtain the target compound 4b as a white solid (yield 72%).

[0105] Compound 4b: 1H NMR (400 MHz, acetone-d6) δ 8.50 (d, J = 8.7 Hz, 1H), 5.71 (d, J = 8.2 Hz, 1H), 4.62 (m, 1H), 4.43 (m, 1H), 4.34 (m, 1H), 3.98 (m, 1H), 3.89 (m, 1H) 19 F NMR (376 MHz, acetone-d6) δ -76.5 (s, 3F), -82.0 (s, 3F), -118.1 (d, J = 283.2 Hz, 1F), -120.9 - -124.4 (m, 10F), -125.5 (d, J = 283.2 Hz, 1F), -126.9 (d, J = 294.8 Hz, 1F), -127.5 (d, J = 289.0 Hz, 1F)

[0106] Compound 3b is the same as compound 3a, and compound 3a is obtained by using the same method as いて, and compound 4a is the same.

[0107] Compound 4a: 1 H NMR (400 MHz, acetone-d6) δ 8.00 (d, J = 8.7 Hz, 1H), 6.00 (s, 1H), 4.73 (dd, J = 21.3, 3.9 Hz, 1H), 4.49 (m, 2H), 3.69 (m, 2H) 19 F NMR (376 MHz, acetone-d6) δ -76.4 (s, 3F), -81.6 (s, 3F), -118.9 (d, J FF = 283.2 Hz, 1F), -121.3 - -123.2 (m, 10F), -125.8 - -127.5 (m, 3F)

[0108]

change

[0109] Compound 4b (526 mg, 0.87 mmol) was dissolved in dry pyridine (2 mL) in an oven-dried 25 mL two-necked round-bottom flask and cooled to 0°C. Next, DIPEA (167 μL, 124 mg, 0.96 mmol) was added to the flask, and 4,4'-dimethoxytrityl chloride (325 mg, 0.96 mmol, in a 3 mL solution of dry dichloromethane) and 4-dimethylaminopyridine (12.2 mg, 0.1 mmol) were added dropwise over 10 minutes. The reaction mixture in the flask was then warmed to room temperature and stirred for a further 22 hours. After that, the solvent was evaporated under vacuum, and the resulting reaction product was purified by chromatography using CHCl3 to obtain the target compound 5b as a yellow oil (yield 79%).

[0110] Compound 5b: 1 H NMR (400 MHz, acetone-d6) δ 8.83 (d, J = 8.7 Hz, 1H), 7.46 - 7.14 (m, 9H), 6.87 (dd, J = 8.9, 2.3 Hz, 4H), 5.83 (d, J = 8.7 Hz, 1H), 4.77 - 4.62 (m, 2H), 3.76 (s, 6H), 3.50 (m, 2H) 19 F NMR (376 MHz, acetone-d6) δ -76.2 (s, 3F), -81.5 (s, 3F), -117.4 (d, J FF = 289.0 Hz, 1F), -120.5 - -123.3 (m, 10F), -125.5 (d, J FF = 289.0 Hz, 1F), -126.2 (d, J FF = 289.0 Hz, 1F), -127.2 (d, J FF (= 289.0 Hz, 1F)

[0111] Compound 5a was obtained in the same manner by using compound 4a instead of compound 4b.

[0112] Compound 5a: 1H NMR (500 MHz, acetone-d6) δ 8.10 (s, 1H), 7.41 (d, J = 7.5 Hz, 2H) 7.29 - 7.13 (m, 7H), 6.84 (dd, J = 8.6, 1.7 Hz, 4H), 5.94 (s, 1H), 4.76 (dd, J = 20.6, 3.4 Hz, 1H), 4.70 (s, 1H), δ 3.74 (s, 6H), δ 3.34 (m, 2H) 19 F NMR (376 MHz, acetone-d6) δ -76.2 (s, 3F), -81.5 (s, 3F), -119.0 (d, J FF = 283.2 Hz, 1F), -121.3 - -123.2 (m, 10F), -125.8 - -127.5 (m, 3F)

[0113] [ka]

[0114] Compound 5b (623 mg, 0.68 mmol) was dissolved in ethanol (1.5 mL) in a 50 mL round-bottom flask, and 28% aqueous ammonia (3 mL) was added. The mixture was then stirred at room temperature for 4 days. The solvent was then evaporated from the reaction product in the flask under vacuum, and the target compound 6b was obtained as a yellow oil by chromatography using CHCl3 / MeOH (30:1 (volume ratio)) (yield 80%).

[0115] Compound 6b: 1 H NMR (500 MHz, acetone-d6) δ 7.49 - 7.19 (m, 9H), 6.89 (d, J = 8.0 Hz, 4H), 4.47 (dd, J = 29.8, 6.3 Hz, 1H), 4.05 (m, 1H), 3.78 (s, 6H), 3.58 - 3.48 (m, 2H) 19F NMR (376 MHz, acetone-d6) δ -81.6 (s, 3F), -119.0 (d, J FF = 294.8 Hz, 1F), -120.9 - -124.2 (m, 10F), -125.7 - -127.7 (m, 3F)

[0116] Compound 6a was obtained in the same manner by using compound 5a instead of compound 5b.

[0117] Compound 6a: 1 H NMR (500 MHz, acetone-d6) δ 7.44 (d, J = 8.0 Hz, 2H), 7.32 - 7.20 (m, 7H), 6.87 (d, J = 9.2 Hz, 4H), 4.28 (dd, J = 23.2, 6.5 Hz, 1H), 3.86 (m, 1H), 3.78 (s, 6H), 3.40 (dd, J = 10.0, 4.2 Hz, 1H), 3.33 (dd, J = 9.7, 4.0 Hz 1H), 2.80 (s, 2H) 19 F NMR (470 MHz, acetone-d6) δ -81.5 (s, 3F), -121.4 (d, J FF = 278.8 Hz, 1F), -122.1 - -123.9 (m, 10F), -125.9 - -127.3 (m, 3F)

[0118] [ka]

[0119] Compound 6b (332 mg, 0.41 mmol) and thymine-1-acetic acid (90 mg, 0.49 mmol) were dissolved in 10 mL of dry N,N-dimethylformamide (DMF) in a 25 mL two-necked round-bottom flask. Then, 285 μL, 2.04 mmol of dry triethylamine and the dehydrating condensation agent DMT-MM (CAS No: 3945-69-5) (170 mg, 0.61 mmol) were added to the flask, and the mixture was stirred at room temperature for 40 hours. The reaction was then quenched with saturated NaHCO3, and the product was extracted with CHCl3. The organic fractions were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum. The resulting crude product was purified by chromatography using a CHCl3 / MeOH (30:1 volume ratio) mixed solvent to obtain the target compound 7b as a yellow oil (yield 28%).

[0120] Compound 7b: 1 H NMR (400 MHz, acetone-d6) δ 10.2 (s, 1H), 8.02 (m, H), 7.49 - 7.20 (m, 10H), 6.89 (m, 4H), 4.59 (m, 1H), 4.58 (d, J = 16.0 Hz, 1H), 4.48 (d, J = 16.0 Hz, 1H), 3.76 (s, 6H), 3.56 (m, 2H), 3.42 (m, 1H), 1.80 (d, J = 0.9 Hz, 3H) 19 F NMR (376 MHz, acetone-d6) δ -81.5 (s, 3F), -117.8 (d, J FF = 294.8 Hz, 1F), -120.9 - -123.3 (m, 10F), -125.1 (d, J FF = 283.2 Hz, 1F), -126.2 (d, J FF = 294.8 Hz, 1F), -127.1 (d, J FF (= 289.0 Hz, 1F) LRMS (ESI-TOF): calcd for C 39 H 32 F17 N3NaO7[M+Na] + : 1000.19, found: 999.87

[0121] [ka]

[0122] 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (57 mg, 0.19 mmol) was dissolved in dry acetonitrile. To this solution, ETT (57 mg, 0.19 mmol) was added under argon, and then compound 7b (123 mg, 0.13 mmol, dissolved in a mixed solvent of 0.6 mL of THF and 0.6 mL of acetonitrile) was gradually added, and the mixture was stirred at room temperature for 2 days. After the solvent was evaporated under vacuum, the resulting crude product was purified by chromatography using siRNA / hexane (1:1 volume ratio) under argon and by chromatography using MeOH / HCl3 (1:100 volume ratio) under air to obtain the yellow oil aTNA-C[PFC8] (yield 74%).

[0123] 1 H NMR (500 MHz, acetone-d6) δ 10.2 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.49 - 7.21 (m, 10H), 6.91 - 6.87 (m, 4H), 4.84 - 4.47 (m, 3H), 4.00 - 3.91 (m, 1H), 3.78 (s, 6H), 3.60 - 3.31 (m, 7H), 2.93 - 2.67 (m, 2H), 1.81 (m, 3H), 1.31 - 1.00 (m, 12H) 19F NMR (470 MHz, acetone-d6) isomer I: δ -81.5 (s, 3F), -116.6 (d, JFF = 278.8 Hz, 1F), -120.0 (d, JFF = 249.4 Hz, 1F), -120.9 - -123.8 (m, 10F), -126.3 (d, JFF =308.1 Hz, 1F), δ -127.0 (d, JFF = 293.4 Hz, 1F) isomer II: δ -81.5 (s, 3F), -117.8 (d, JFF = 293.4 Hz, 1F), -120.9 - -123.8 (m, 10F), -125.1 (d, JFF = 278.8 Hz, 1F), -126.3 (d, JFF =308.1 Hz, 1F), -127.0 (d, JFF = 293.4 Hz, 1F) 31 P NMR (202 MHz, Acetone-d6), isomer I:δ -154.2 (s), isomer II:δ -152.1 (s) LRMS (ESI-TOF): calcd for C 48 H 49 F 17 N5NaO8P[M+Na] + : 1200.29, found: 1199.86

[0124] Using the synthesized aTNA-C[PFC8], nucleic acids (aTNA-C2[PFC8], aTNA-C5[PFC8]) were synthesized by ligating two or five of the following structures (aTNA-C1[PFC8]: black circles in the formula indicate binding sites) to the 5' end of the control DNA (SEQ ID NO: 1), which is the natural type of DNA, as described above.

[0125] [ka]

[0126] Nucleic acids (fluorescein-labeled aTNA-C2[PFC8] and fluorescein-labeled aTNA-C5[PFC8]) with fluorescein conjugated to the 3' end of synthesized aTNA-C2[PFC8] and aTNA-C5[PFC8] were introduced into HeLa cells, and flow cytometry was performed to quantify the number of cells into which the fluorescein-labeled nucleic acid was introduced and the amount of fluorescein-labeled nucleic acid introduced into each cell, based on the fluorescence intensity of fluorescein. As a control, fluorescein-labeled control DNA was introduced into HeLa cells in the same manner and analyzed by flow cytometry.

[0127] Table 3 shows the relative fluorescence intensity of cells into which each fluorescein-labeled nucleic acid was introduced (fluorescence intensity of cells into which fluorescein-labeled control DNA was introduced is set to 1). Figure 3 shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced. In the figure, "none" indicates the results of cells into which DNA was not incorporated, "DNA" indicates the results of cells into which fluorescein-labeled control DNA was introduced, "aTNA-C2" indicates the results of cells into which fluorescein-labeled aTNA-C2[PFC8] was introduced, and "aTNA-C5" indicates the results of cells into which fluorescein-labeled aTNA-C5[PFC8] was introduced.

[0128] [Table 3]

[0129] As shown in Table 3 and Figure 3, the number of cells into which fluorescein-labeled aTNA-C2[PFC8] and fluorescein-labeled aTNA-C5[PFC8] were introduced was higher than the number of cells into which fluorescein-labeled control DNA was introduced, indicating that these had higher cell membrane permeability than the control DNA. Furthermore, it was found that fluorescein-labeled aTNA-C5[PFC8] was introduced into a larger number of cells and exhibited higher cell permeability than fluorescein-labeled aTNA-C2[PFC8].

[0130] [Example 4] R0 We synthesized nucleic acids having a structure (A1) in which the heptadecyl group is located, and investigated their cell membrane permeability.

[0131] R 0 The phosphoramidite (aTNA-N[HC17]) having a structure (A1) in which the group is a heptadecyl group was synthesized as follows.

[0132] (1) Amidite condensation

[0133] [ka]

[0134] In a 200 mL two-necked round-bottom flask containing 10 mL of dried DMF, 6.62 mL of dried triethylamine (47.5 mmol), and 3.94 g of DMT-MM (14.3 mmol), D-threoninol (1 g, 9.5 mmol) and stearic acid (3.24 g, 11.4 mmol) were dissolved. After stirring at room temperature for 22 hours, the reaction was quenched with saturated NaHCO3, and the product was extracted with CHCl3. The organic fractions were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum. The resulting crude product was purified by chromatography using a CHCl3 / MeOH (20:1 volume ratio) mixed solvent to obtain the target intermediate 1 as a yellow oil (99% yield).

[0135] 1 H NMR (400 MHz, CDCl3) δ 6.20 (d, J = 6.9 Hz, 1H), δ 4.21-4.17 (m, 1H), δ 3.87-3.80 (m, 3H), δ 2.76-2.62 (m, 2H), δ 2.25 (t, 2H), δ 1.37-1.25 (m, 33H), δ 0.93-0.82 (m, 3H)

[0136] (2)DMTr protection

[0137] [ka]

[0138] In an oven-dried 200 mL two-necked round-bottom flask, intermediate 1 (2.23 g, 6.0 mmol) was dissolved in dry pyridine (20 mL) and cooled to 0°C. Diisopropylethylamine (1.15 mL, 6.6 mmol) was then added to this mixture, followed by the dropwise addition of 4,4'-dimethoxytrityl chloride (2.24 g, 6.6 mmol) and 4-dimethylaminopyridine (80.6 mg, 0.66 mmol), dissolved in 15 mL of dry dichloromethane, over 10 minutes. The mixture was heated to room temperature and stirred for 24 hours. The solvent was then removed under vacuum. The resulting crude product was purified by chromatography using an siRNA / hexane (1:4 volume ratio) mixed solvent to obtain DMTr-protected intermediate 2, a yellow oil (yield: 61%).

[0139] 1 H NMR (400 MHz, CDCl3) δ 7.37-7.19 (m, 9H), δ 6.82 (d, J = 9.2 Hz, 4H), δ 6.07 (d, J = 8.7 Hz, 1H), δ 4.09-4.04 (m, 1H), δ 3.94-3.90 (m, 1H), δ 3.78 (s, 6H), δ 3.42 (dd, J = 4.1 Hz, 9.6 Hz, 1H), δ 3.26 (dd, J = 3.2 Hz, 9.6 Hz, 1H), δ 3.10 (d, J = 2.3 Hz, 1H), δ 2.21 (t, J = 7.5 Hz, 2H), δ 1.30-1.20 (m, 30H), δ 1.11 (d, 3H), δ 0.87 (t, J = 6.5 Hz, 3H)

[0140] (3) Amidite formation

[0141] [ka]

[0142] To a solution of 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (983 mg, 3.26 mmol) dissolved in dry acetonitrile, ETT (424 mg, 3.26 mmol) was added under argon, and then DMTr-protected intermediate 3 (2.0 g, 2.97 mmol, dissolved in a mixed solvent of 5 mL of THF and 10 mL of acetonitrile) was gradually added. The resulting reaction mixture was stirred under argon at room temperature for 14 hours. After evaporating the solvent under reduced pressure, the resulting crude product was purified under argon by column chromatography using hexane / ethyl acetate (1:4 (volume ratio)) as the mobile phase. This isolated the yellow oil aTNA-N[HC17] (yield 77%).

[0143] 1 1H NMR (400 MHz, CDCl3) isomer I:δ 7.41-7.18 (m, 9H), δ 6.82-6.80 (m, 4H), δ 5.75 (d, 1H), δ 4.38-4.30 (m, 1H), δ 4.22-4.16 (m, 1H) δ 3.77 (m, 6H) δ 3.61-3.40 (m, 4H) δ 3.22-3.09 (m, 2H), δ 2.43-2.26 (m, 2H), δ 2.19-2.12 (m, 2H), δ 1.28-1.11 (m, 45H), δ 0.86 (d, J = 6.9 Hz, 3H) isomer II: δ 7.39 (d, 2H), δ 7.29-7.16 (m, 7H), δ 6.80 (dd, J = 1.4 Hz, 8.70 Hz, 4H), δ 5.59 (d, J = 8.7 Hz, 1H), δ 4.25-4.14 (m, 2H), δ 3.77 (m, 6H) δ 3.68-3.64 (m, 1H), δ 3.50-3.44 (m, 2H), δ 3.21 (dd, J = 6.4 Hz, 9.2 Hz, 1H), δ 3.21 (dd, J = 6.9 Hz, 9.2 Hz, 1H), δ2.57 (t, 2H), δ2.17-2.14 (m, 2H), δ 1.30-1.19 (m, 33H),δ 1.10 (d, J = 6.9 Hz, 6H) ,δ 0.97 (d, J = 6.9 Hz, 6H),δ 0.86 (d, J = 6.6 Hz, 3H)

[0144] Using the synthesized aTNA-N[HC17], nucleic acids (aTNA-N1[HC17] and aTNA-N2[HC17]) were synthesized by ligating one or two of the following structures (aTNA-N1[HC17]: black circles in the formula indicate binding sites) to the 5' end of the control DNA (SEQ ID NO: 1), which is the natural type of DNA, as described above.

[0145] [ka]

[0146] Nucleic acids (fluorescein-labeled aTNA-N1[HC17]] and fluorescein-labeled aTNA-N2[HC17]], in which fluorescein was conjugated to the 3' end of synthesized aTNA-N1[HC17] and aTNA-N2[HC17]] were introduced into HeLa cells. Specifically, 10 units per well were introduced into a 96-well plate. 4The procedure involved seeding HeLa cells to a single cell level, then replacing the medium with 1 mL of DMEM medium containing fluorescein-labeled nucleic acid (2.5 μM) and incubating for 4 hours. Subsequently, flow cytometry was performed in the same manner as described above, and the amount of fluorescein-labeled aTNA-N1[HC17], etc., introduced into the cells was quantified based on the fluorescence intensity of fluorescein. As a control, nucleic acid with fluorescein bound to the 3' end of control DNA (fluorescein-labeled control DNA) was introduced into HeLa cells in the same manner, and then flow cytometry was performed to quantify the amount of nucleic acid introduced into the cells.

[0147] Table 3 shows the relative fluorescence intensities of cells introduced with fluorescein-labeled aTNA-N1[HC17] and cells introduced with fluorescein-labeled aTNA-N2[HC17], with the fluorescence intensity of cells introduced with fluorescein-labeled control DNA set to 1. Figure 3 shows the flow cytometry results of cells introduced with fluorescein-labeled control DNA and cells introduced with fluorescein-labeled nucleic acid. In the figure, "none" indicates the results of cells that did not take up DNA, "DNA" indicates the results of cells introduced with fluorescein-labeled control DNA, "aTNA-NH1" indicates the results of cells introduced with fluorescein-labeled aTNA-N1[HC17], and "aTNA-NH2" indicates the results of cells introduced with fluorescein-labeled aTNA-N2[HC17].

[0148] [Table 4]

[0149] As shown in Table 4 and Figure 4, the number of cells into which fluorescein-labeled aTNA-N1[HC17] and fluorescein-labeled aTNA-N2[HC17] were introduced was greater than the number of cells into which fluorescein-labeled control DNA was introduced. Furthermore, the number of cells into which fluorescein-labeled aTNA-N2[HC17] was introduced was greater than that into which fluorescein-labeled aTNA-N1[HC17] was introduced. These results indicate that both aTNA-N1[HC17] and aTNA-N2[HC17] have higher cell membrane permeability than control DNA, and that the more repeats of the structure (A1) introduced into the nucleic acid, the easier it is for the DNA to be introduced into cells.

[0150] [Example 5] Fluorescein-labeled aTNA-N1[HC17] prepared in Example 4 was hybridized with control rDNA to produce fluorescein-labeled double-stranded DNA (fluorescein-labeled aTNA-N1[HC17] / rDNA) having a repeating structure (A1) at the 5' end and fluorescein bound to the 3' end. Similarly, fluorescein-labeled aTNA-N2[HC17] prepared in Example 4 was hybridized with control rDNA to produce fluorescein-labeled double-stranded DNA (fluorescein-labeled aTNA-N2[HC17] / rDNA). These fluorescein-labeled double-stranded DNAs and the fluorescein-labeled control DNA / rDNA used in Example 2 were introduced into HeLa cells in the same manner as in Example 1, and then analyzed by flow cytometry.

[0151] Table 5 shows the relative fluorescence intensity of cells into which each fluorescein-labeled nucleic acid was introduced (fluorescence intensity of cells into which fluorescein-labeled control DNA / rDNA was introduced is set to 1). Figure 5 shows the flow cytometry results of cells into which each fluorescein-labeled nucleic acid was introduced. In the figure, "none" indicates the results of cells into which no DNA was taken up, "DNA / rDNA" indicates the results of cells into which fluorescein-labeled control DNA / rDNA was introduced, "aTNA-N1 / rDNA" indicates the results of cells into which fluorescein-labeled aTNA-N1[HC17] / rDNA was introduced, and "aTNA-N2 / rDNA" indicates the results of cells into which fluorescein-labeled aTNA-N2[HC17] / rDNA was introduced.

[0152] [Table 5]

[0153] As shown in Table 5 and Figure 5, the number of cells into which fluorescein-labeled aTNA-N1[HC17] / rDNA and fluorescein-labeled aTNA-N2[HC17] / rDNA were introduced was greater than the number of cells into which fluorescein-labeled control DNA / rDNA was introduced. These results confirm that even double-stranded DNA exhibits the same cell membrane permeability-improving effect due to structure (A1) as single-stranded DNA.

[0154] [Example 6] R 0 However, in the group represented by the general formula (f-1), n1 is 2, and Rf P We synthesized a nucleic acid having a structure (A1) in which the group is a perfluorohexyl group.

[0155] R 0 In the group represented by the general formula (f-1), n1 is 2, and Rf P The phosphoramidite (aTNA-N[FC8]) having a structure (A1) in which the group is a perfluorohexyl group was synthesized as follows.

[0156] (1) Amidite condensation

[0157] [ka]

[0158] Compound 8 (0.44 g, 0.82 mmol), PyBOP (0.43 g, 0.82 mmol), and DIPEA (0.4 mL, 2.2 equivalents), dissolved in 10 mL of DMF, were dissolved in 15 mL of DMF under argon. 4,4,5,5,6,6,7,7,8,8,9,9,9-Tridecafluorononanoic acid (0.23 g, 0.59 mmol, 0.7 equivalents) was added to prepare the reaction solution. The resulting reaction solution was stirred at room temperature for 14 hours. The reaction mixture was then quenched with water (45 mL) and extracted twice with hexane / ethyl acetate (4:1 (volume ratio)). The organic fractions were combined, washed with water, dried over Na₂SO₄, filtered, and the solvent was removed under vacuum. The crude product obtained was purified by silica gel column chromatography (hexane:ethyl acetate:Et3N = 30:20:1 (volume ratio)) to obtain compound 9 (0.17 g, 0.22 mmol, yield 38%).

[0159] Compound 9: 1 H NMR (400 MHz, CDCl3) δ 7.39-7.21 (m, 9H), 6.84 (d, J = 5.9 Hz, 4H), 6.06 (d, J = 9.1 Hz, 1H), 4.13-4.10 (m, 1H), 3.92-3.90 (m, 1H), 3.78 (s, 6H), 3.41 (dd, J = 11.2, 5.7 Hz, 1H), 3.33 (dd, J = 9.6, 3.2 Hz, 1H), 2.90 (s, 1H), 2.53-2.44 (m, 4H), 1.12 (d, J = 6.4 Hz, 3H). 19 F NMR (376 MHz, CDCl3) δ -80.7 (s, 3F), -114.4 (s, 2F), -121.8 (s, 2F), -122.8 (s, 2F), -123.4 (s, 2F), -126.0 (s, 2F).

[0160] (2) DMTr Protection

[0161]

Chem.

[0162] A solution of 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (93 mg, 0.31 mmol) and ETT (40 mg, 0.31 mmol) in dry acetonitrile (8 mL) was added dropwise with a solution of Compound 9 (160 mg, 0.20 mmol) dissolved in a mixed solvent of THF / acetonitrile (2 mL / 2 mL) under argon. After stirring this reaction mixture at room temperature for 24 hours, the solvent was evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography using degassed hexane / ethyl acetate (3:1 (volume ratio)) as the mobile phase under argon. Thereby, Compound 10 (DMTr-protected aTNA-N[FC8]) as a colorless oil (yield 38%) was isolated.

[0163] Compound 10: 1 1H NMR (400 MHz, CDCl3) δ 7.41 - 7.20 (m, 9H), 6.82 - 6.79 (m, 4H), 5.94 (d, 9.1 Hz, 1H), 4.37 - 4.32 (m, 1H), 4.20 - 4.14 (m, 1H), 3.77 (s, 6H), 3.55 - 3.45 (m, 4H), 3.25 - 3.12 (m, 2H), 2.59 - 2.31 (m, 6H), 1.24 - 0.97 (m, 15H). 19 19F NMR (376 MHz, CDCl3) δ -80.7 (s, 3F), -114.5 (s, 2F), -121.8 (s, 2F), -122.8 (s, 2F), -123.4 (s, 2F), -126.0 (s, 2F).

[0164] Using the synthesized aTNA-N[FC8], as described above, nucleic acids (aTNA-N1[FC8] to aTNA-N5[FC8]) were synthesized by ligating one to five of the following structures (aTNA-N1[FC8]: black circles in the formula indicate binding sites) to the 5' end of the control DNA (SEQ ID NO: 1), which is the natural type of DNA.

[0165] [ka]

[0166] Nucleic acids (fluorescein-labeled aTNA-N1[FC8] to fluorescein-labeled aTNA-N5[FC8]) were formed by conjugating fluorescein to the 3' end of aTNA-N5[FC8] from synthesized aTNA-N1[FC8], and then introduced into HeLa cells. Specifically, 10 cells per well were introduced into a 96-well plate. 4 The procedure involved seeding HeLa cells to a concentration of 10 μM, then replacing the medium with 100 μL of DMEM medium containing fluorescein-labeled nucleic acid (2.0 μM) and incubating for 4 hours. Subsequently, flow cytometry was performed in the same manner as above, and the amount of fluorescein-labeled aTNA-N1[FC8], etc., introduced into the cells was quantified based on the fluorescence intensity of fluorescein. As a control, nucleic acid with fluorescein bound to the 3' end of control DNA (fluorescein-labeled control DNA) was introduced into HeLa cells in the same manner, and then flow cytometry was performed to quantify the amount of nucleic acid introduced into the cells. As another comparative measure, HeLa cells were incubated for 4 hours with a solution of lipofectamito prepared to a concentration of 10 μM of nucleic acid (fluorescein-labeled control DNA), diluted 10-fold using DMEM medium (100 μM). Subsequently, flow cytometry was performed in the same manner as above, and the amount of nucleic acid introduced into the cells was quantified based on the fluorescence intensity of fluorescein.

[0167] Table 6 shows the relative fluorescence intensity of cells introduced with fluorescein-labeled aTNA-N5[FC8], compared to the relative fluorescence intensity of cells introduced with fluorescein-labeled control DNA (set to 1). Figure 6 shows the flow cytometry results for cells introduced with fluorescein-labeled control DNA and cells introduced with fluorescein-labeled nucleic acid. In the figure, "none" indicates the results for cells that did not take up DNA, "DNA" indicates the results for cells introduced with fluorescein-labeled control DNA, and "Lipofectamine" indicates cells introduced with lipofectamine. The results shown are for cells introduced with fluorescein-labeled aTNA-N1[FC8] ("aTNA-NCF1"), fluorescein-labeled aTNA-N2[FC8] ("aTNA-NCF2"), fluorescein-labeled aTNA-N3[FC8] ("aTNA-NCF3"), fluorescein-labeled aTNA-N4[FC8] ("aTNA-NCF4"), and fluorescein-labeled aTNA-N5[FC8] ("aTNA-NCF5").

[0168] [Table 6]

[0169] As shown in Table 6 and Figure 6, more cells were introduced with fluorescein-labeled aTNA-N5[FC8] than with fluorescein-labeled control DNA than with fluorescein-labeled aTNA-N1[FC8]. Furthermore, more cells were introduced with fluorescein-labeled aTNA-N4[FC8] than with fluorescein-labeled aTNA-N1[FC8]. These results indicate that both aTNA-N1[FC8] and aTNA-N5[FC8] have higher cell membrane permeability than control DNA, although inferior to lipofectamine, and that the more repeats of the structure (A1) introduced into the nucleic acid, the easier it is for the proteins to be introduced into cells. [Industrial applicability]

[0170] This invention may involve C, which may be substituted with a fluorine atom, a highly hydrophobic group. 1-30 The present invention provides an aTNA-type nucleic acid containing an alkyl group. Because the nucleic acid according to the present invention has excellent cell membrane permeability, it is expected to be used in the pharmaceutical field as a physiologically active substance, for example, as a carrier for introducing pharmacoactive ingredients into target cells.

Claims

1. The following general formula (A1) or (A2) 【Chemistry 1】 [In the formula, R ０ n11 and n12 are each independent integers of 1 or more carbon atoms; B is a nucleic acid base; and the black circles indicate bonds. A nucleic acid having the structure represented by .

2. The aforementioned R ０ The nucleic acid according to claim 1, wherein the nucleic acid is an alkyl group having 8 to 30 carbon atoms substituted with 13 or more fluorine atoms.

3. The aforementioned R ０ The nucleic acid according to claim 2, wherein the nucleic acid is a perfluoroalkyl group having 8 to 10 carbon atoms.

4. The aforementioned R ０ The nucleic acid according to claim 1, wherein the alkyl group has 17 to 30 carbon atoms and is not substituted with a fluorine atom.

5. The nucleic acid according to any one of claims 1 to 4, wherein n11 or n12 is 5 or more.

6. The nucleic acid according to any one of claims 1 to 5, which is permeable to the cell membrane.

7. A nucleic acid drug comprising a nucleic acid according to any one of claims 1 to 6 as an active ingredient.

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