Nucleic acid containing perfluoroalkyl and method for producing the same

Abstract

The present invention provides a nucleic acid having excellent cell membrane permeability and a method for producing the same. A nucleic acid comprising a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonded oxygen atoms between carbon atoms. In the nucleic acid described above, the perfluoroalkyl group is directly or indirectly bonded to the 5' end or 3' end of the nucleic acid or the perfluoroalkyl group is indirectly introduced between two nucleotides. In addition, a nucleic acid pharmaceutical comprises a nucleic acid comprising a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonded oxygen atoms between carbon atoms as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a nucleic acid into which a perfluoroalkyl group has been introduced and a method for producing the same. Background Art

[0002] If antibodies can be made by the immune system for proteins that cannot be targeted by low-molecular drugs, they can become pharmaceuticals, so antibody drugs are excellent as therapeutic drugs for cancer and difficult and complicated diseases. In addition, antibody drugs have the advantages of high specificity for target molecules and few side effects. However, antibody drugs are difficult to enter cells through cell membranes, so it is difficult to use molecules other than those on the cell surface as target molecules.

[0003] As the next generation of drug development of antibody pharmaceuticals, nucleic acid pharmaceuticals using oligonucleotides are being studied. Nucleic acid pharmaceuticals have a high specificity to target molecules and few side effects. However, nucleic acid pharmaceuticals, like antibody pharmaceuticals, have low cell membrane permeability and are difficult to reach the target molecule present in the cell. In particular, siRNA is double-stranded, so compared with antisense RNA, molecular weight and negative charge are both enlarged, and cell membrane permeability is lower than antisense RNA, and it is necessary to utilize the drug delivery of a carrier. As a drug delivery agent, it is known to have a preparation using lipid nanoparticles (patent document 1) and a preparation using cationic polymer nanoparticles (patent document 2). However, it is mostly necessary to improve the efficiency and toxicity of cell membrane permeability.

[0004] On the other hand, it is known that compounds having a polyfluorinated structure are stable in vivo and have low toxicity, and are excellent in being introduced into cells and being released from endosomes (Non-patent Document 1). It has been reported that this property can be utilized to use peptide dendrimers in gene delivery, using ricin toxin in which the amino groups constituting the side chains of the amino acids are perfluorinated (Non-patent Document 2). In addition, the introduction of a polyfluorinated structure into oligonucleotides and peptide nucleic acids as a portion having cell membrane permeability has also been studied (Patent Document 3 and Non-patent Documents 3 to 6).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2011 / 036557

[0008] Patent Document 2: International Publication No. 2017 / 212006

[0009] Patent Document 3: International Publication No. 2012 / 130941

[0010] Patent Document 4: Japanese Patent Application Publication No. 2006-321797

[0011] Patent Document 5: International Publication No. 2000 / 056694

[0012] Non-patent literature

[0013] Non-patent literature 1: Zhang et al., MRS Communications, 2018, vol. 8, p. 303-313.

[0014] Non-patent literature 2: Cai et al., ACS Applied Materials and Interfaces, 2016, vol. 8, p. 5821-5832.

[0015] Non-patent document 3: Godeau et al., Medicinal Chemistry Communications, 2010, vol. 1. p. 76-78.

[0016] Non-patent document 4: Ellipilli et al., Chemical Communications, 2016, vol. 52, p. 521-524.

[0017] Non-patent document 5: Rochambeaua et al., Polymer Chemistry, 2016, vol. 7, p. 4998-5003.

[0018] Non-patent document 6: Metelev et al., Theranostics, 2017, vol. 7, p. 3354-3368. Summary of the invention

[0019] An object of the present invention is to provide a nucleic acid having excellent cell membrane permeability and a method for producing the same.

[0020] The present inventors have found that the cell membrane permeability of nucleic acid is improved by introducing a perfluoroalkyl group, particularly a perfluoroalkyl group having an ether-bonded oxygen atom, and have completed the present invention.

[0021] That is, the present invention is as follows.

[0022] [1] A nucleic acid comprising a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0023] [2] The nucleic acid according to [1] above, wherein the perfluoroalkyl group is directly or indirectly bonded to the 5' end or 3' end of the nucleic acid.

[0024] [3] The nucleic acid according to [1] above, wherein the perfluoroalkyl group is indirectly introduced between two nucleotides.

[0025] [4] The nucleic acid according to any one of [1] to [3] above, which has a structure represented by the following general formula (A1).

[0026]

[0027] Here, the groups in the formula have the following meanings.

[0028] R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, and a black dot represents a bonding site.

[0029] [5] The nucleic acid according to any one of [1] to [4] above, which is a cell membrane-permeable nucleic acid.

[0030] [6] A method for producing a perfluoroalkyl group-containing nucleic acid, comprising synthesizing the perfluoroalkyl group-containing nucleic acid according to any one of [1] to [5] above by a phosphoramidite method using a compound represented by the following general formula (A2) as a raw material.

[0031]

[0032] Here, the groups in the formula have the following meanings.

[0033] R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonded oxygen atoms between carbon atoms, na is an integer of 1 to 10, DMTr is 4,4'-dimethoxytriphenylmethyl, and i-Pr is isopropyl.

[0034] [7] A nucleic acid pharmaceutical comprising, as an active ingredient, a nucleic acid containing a perfluoroalkyl group having 2 to 10 carbon atoms which may have 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0035] [8] A method for improving the cell membrane permeability of nucleic acid, comprising introducing into the nucleic acid a perfluoroalkyl group having 2 to 10 carbon atoms which may have 1 to 5 ether-bonded oxygen atoms between carbon atoms to improve the cell membrane permeability.

[0036] [9] The method for improving the cell membrane permeability of nucleic acid according to [8] above, wherein the perfluoroalkyl group is a group having 1 to 5 ether-bonded oxygen atoms between carbon atoms.

[0037]

[10] The method for improving the cell membrane permeability of nucleic acid according to [8] above, wherein the perfluoroalkyl group is a group having no ether-bonded oxygen atom between carbon atoms.

[0038]

[11] The method for improving the cell membrane permeability of a nucleic acid according to any one of [8] to

[10] , wherein the perfluoroalkyl group is directly or indirectly bonded to the 5' end or 3' end of the nucleic acid.

[0039] In addition, the nucleic acid according to the present invention has excellent cell membrane permeability due to the introduction of a perfluoroalkyl group, and therefore, it is expected that the nucleic acid can be used as a physiologically active substance in the field of medicines.

[0040] According to the production method of the present invention, a nucleic acid into which a perfluoroalkyl group has been introduced can be produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A This is a graph showing the results of flow cytometry of cells into which fluorescein-modified nucleic acid modified with N[C8-PFC] at the 5′ end in Experimental Example 1 has been introduced.

[0042] Figure 1B This is a graph showing the results of flow cytometry of cells into which fluorescein-modified nucleic acid modified with N[C8-PFC] at the 3′ end in Experimental Example 1 has been introduced.

[0043] Figure 2A This is a graph showing the results of flow cytometry of cells into which the fluorescein-modified double-stranded nucleic acids annealed in each combination in Experimental Example 1 were introduced after incubation for 4 hours.

[0044] Figure 2B This is a graph showing the results of flow cytometry of cells into which the fluorescein-modified double-stranded nucleic acids annealed in each combination in Experimental Example 1 were introduced after incubation for 24 hours.

[0045] Figure 3 This is a graph showing the results of flow cytometry of cells into which fluorescein-modified nucleic acid modified with N[C5-PFPE] at the 5′ end in Experimental Example 2 has been introduced.

[0046] Figure 4 This is a graph showing the results of flow cytometry of cells into which fluorescein-modified nucleic acids of cont-DNA, FE5, and FE10 were introduced in Experimental Example 2.

[0047] Figure 5 This is a graph showing the results of flow cytometry of cells into which fluorescein-modified nucleic acids of cont-DNA, FE(2)1, FE(2)2, and FE(2)5 were introduced in Experimental Example 3. DETAILED DESCRIPTION

[0048] In the present invention and the specification of this application, "nucleic acid" refers to a molecule formed by phosphodiester bonding of nucleotides. The nucleotides include not only natural nucleotides (naturally occurring nucleotides) such as DNA and RNA, but also artificial nucleotides that are modified from natural nucleotides and can form phosphodiester bonds with natural nucleotides. Examples of artificial nucleotides include artificial nucleotides in which the side chains of natural nucleotides are modified with functional groups such as amino groups, artificial nucleotides 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, or the like, phosphorothioate nucleotides (nucleotides in which the oxygen atom of the phosphate group is substituted with a sulfur atom), morpholino nucleotides (nucleotides in which ribose or deoxyribose is 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), and the like. In addition, "nucleic acid" also includes any of the following molecules: molecules such as DNA and RNA that are composed of only one or more natural nucleotides bonded by phosphodiester bonds, molecules that are composed of one or more natural nucleotides and one or more artificial nucleotides bonded by phosphodiester bonds, and molecules that are composed of only one or more artificial nucleotides bonded by phosphodiester bonds.

[0049] In the present invention and the specification of this application, “C p1-p2 "(p1 and p2 are positive integers satisfying p1<p2) refers to a group having a carbon atom number of p1~p2.

[0050] In the present invention and the specification of this application, “C 1-10 "Alkyl" refers to an alkyl group having 1 to 10 carbon atoms, which may be a straight chain or a branched chain. 2-10 "Alkyl" refers to an alkyl group having 2 to 10 carbon atoms, which may be a straight chain or a branched chain. 1-10 Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl and decyl.

[0051] In the present invention and the specification of this application, “C 1-6 "Alkyl" refers to an alkyl group having 1 to 6 carbon atoms, which may be a straight chain or a branched chain. 1-6Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl and hexyl.

[0052] In the present invention and the specification of this application, an "alkylene group" is a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, and may be a linear or branched group. Examples of the alkylene group include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, methylmethylene, ethylmethylene, methylethylene, methylpropylene, ethylethylene, dimethylmethylene, 1,2-dimethylethylene, 1,1-dimethylethylene, 1-ethylpropylene, 2-ethylpropylene, 1,2-dimethylpropylene, 2,2-dimethylpropylene, 1-propylpropylene, 2-propylpropylene, 1-methyl-1-ethylpropylene, 1-methyl-2-ethyl-propylene, 1-ethyl-2-methyl-propylene, 2-methyl-2-ethyl-propylene, 1-methylbutylene, 2-methylbutylene, 3-methylbutylene, 2-ethylbutylene, 1-methylpentylene, 2-ethylpentylene, and 1-methylhexylene.

[0053] In the present invention and the specification of this application, “C 1-10 A "perfluoroalkyl group" is a group in which all hydrogen atoms of an alkyl group having 1 to 10 carbon atoms are substituted with fluorine atoms. 1-10 Examples of the perfluoroalkyl group include perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluoroisopropyl, perfluorobutyl, perfluoroisobutyl, perfluorosec-butyl, perfluorotert-butyl, perfluoropentyl, perfluoroisopentyl, perfluoroneopentyl, perfluorotert-pentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, and perfluorodecyl.

[0054] In the present invention and the specification of the present application, "perfluoroalkylene" is a group in which all hydrogen atoms of an alkylene group are substituted with fluorine atoms. Examples of perfluoroalkylene include groups in which all hydrogen atoms of the alkylene group listed above are substituted with fluorine atoms.

[0055] In the present invention and the specification of this application, "ether-bonded oxygen atoms" refer to oxygen atoms connecting carbon atoms, and do not include oxygen atoms connected in series. An alkyl group having Nc carbon atoms (Nc is an integer greater than 2) may have a maximum of Nc-1 ether-bonded oxygen atoms. 2-10 "Alkyl" refers to 2-10 A group having at least one ether-bonded oxygen atom between carbon atoms of the alkyl group. Hereinafter, this may be referred to as an "alkyl group containing an ether bond".

[0056] In the present invention and the specification of this application, "C 2-10"Perfluoroalkyl" refers to 2-10 The alkyl group has at least one ether-bonded oxygen atom between carbon atoms. 2-10 A group in which all hydrogen atoms of an alkyl group are substituted with fluorine atoms. Hereinafter, a "perfluoroalkyl group having an ether-bonded oxygen atom between carbon atoms" may be referred to as an "ether-bonded perfluoroalkyl group".

[0057] In addition, hereinafter, "compound n" means a compound represented by formula (n).

[0058] <Nucleic acid containing a perfluoroalkyl group containing an ether bond>

[0059] The nucleic acid involved in the present invention is a nucleic acid containing a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonded oxygen atoms between carbon atoms. Since the nucleic acid contains a perfluoroalkyl group, it is expected to be used as a physiologically active substance in the field of medicines like other fluorine-containing compounds.

[0060] The perfluoroalkyl group is not particularly limited as long as it has 2 to 10 carbon atoms and 1 to 5 ether-bonded oxygen atoms between carbon atoms. The perfluoroalkyl group possessed by the nucleic acid of the present invention is preferably a group represented by any one of the following general formulas (F1) to (F3).

[0061] -R F1 -OR F2 (F1)

[0062] -R F3 -(O-CF2-CF2-)n1-OR F4 (F2)

[0063] -R F5 -(O-CF2-CF2-CF2-)n2-OR F6 (F3)

[0064] In the general formula (F1), R F1 is a perfluoroalkylene group having q1 carbon atoms, R F2 The perfluoroalkyl group has a carbon number of q2. The sum of q1 and q2 is a natural number of 2 to 10.

[0065] In the general formula (F2), R F3 is a perfluoroalkylene group having q3 carbon atoms, R F4 It is a perfluoroalkyl group having q4 carbon atoms. n1, q3 and q4 are natural numbers (2×n1+q3+q4) of 4 to 10.

[0066] In the general formula (F3), R F5 is a perfluoroalkylene group having q5 carbon atoms, R F6It is a perfluoroalkyl group having q6 carbon atoms. n2, q5 and q6 are natural numbers of 5 to 10 (3×n2+q5+q6).

[0067] As R in the general formula (F1) F1 , R in the general formula (F2) F3 and R in the general formula (F3) F5 , may be a linear perfluoroalkylene group or a branched perfluoroalkylene group. F1 , R F3 and R F5 , preferably C 1-3 The perfluoroalkylene group is more preferably a group in which all the hydrogen atoms of methylene, ethylene, methylmethylene, ethylmethylene, dimethylmethylene and methylethylene are substituted with fluorine atoms, further preferably a group in which all the hydrogen atoms of methylene, methylmethylene, ethylmethylene and dimethylmethylene are substituted with fluorine atoms, and still more preferably a group in which all the hydrogen atoms of methylene and methylmethylene are substituted with fluorine atoms.

[0068] As R in the general formula (F1) F2 , R in the general formula (F2) F4 and R in the general formula (F3) F6 , may be a linear perfluoroalkyl group or a branched perfluoroalkyl group. F2 , R F4 and R F6 , preferably a linear or branched C 1-5 Perfluoroalkyl, more preferably linear C 1-5 Perfluoroalkyl, more preferably a linear C 2-4 Perfluoroalkyl.

[0069] The perfluoroalkyl group contained in the nucleic acid of the present invention is preferably R F1 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, R F2 A straight chain C 1-5 A perfluoroalkyl compound; R in the general formula (F2) F3 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n1 is 1, 2 or 3, R F4 A straight chain C 1-7 A perfluoroalkyl compound; R in the general formula (F3) F5 is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n2 is 1 or 2, R F6 A straight chain C 1-6 A perfluoroalkyl compound, more preferably R in the general formula (F1) F1is a methylene group or a methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, R F2 A straight chain C 1-5 A perfluoroalkyl compound; R in the general formula (F2) F3 is a methylene or methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n1 is 1, R F4 A straight chain C 1-6 A perfluoroalkyl compound; R in the general formula (F2) F3 is a methylene or methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n1 is 2, R F4 A straight chain C 1-4 A perfluoroalkyl compound; R in the general formula (F2) F3 is a methylene or methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n1 is 3, R F4 A straight chain C 1-3 A perfluoroalkyl compound; R in the general formula (F3) F5 is a methylene or methylmethylene group in which all hydrogen atoms are replaced by fluorine atoms, n2 is 1, R F6 A straight chain C 1-6 Perfluoroalkyl compounds.

[0070] In the present invention, as the ether bond-containing perfluoroalkyl group introduced into the nucleic acid, for example, the ether bond-containing perfluoroalkyl group possessed by the fluorine compound described together with the synthesis method thereof in Patent Document 4 or 5 can be used.

[0071] In the nucleic acid involved in the present invention, the position of introducing the ether bond perfluoroalkyl group is not particularly limited, and it can be introduced into any part as long as the function of the nucleic acid is not impaired. For example, the ether bond perfluoroalkyl group can be directly or indirectly bonded to the 5' end or 3' end of the nucleic acid, or it can be introduced between two nucleotides. When the ether bond perfluoroalkyl group is indirectly introduced into the nucleic acid via a linking group, the linking group is not particularly limited as long as the effect of the present invention is not impaired, and any divalent or trivalent organic group can be used. Examples of the linking group include alkylene, alkenylene, carbonyl, amino, ether bond, thioether bond, ester bond, amide bond, polyethylene glycol group (PEG: -(C2H4O) n -), siloxane bonds, silyl ether bonds, sugars, peptides. In addition, from pyrrole rings, pyrazole rings, imidazole rings, triazole rings, pyridine rings, pyrimidine rings, pyrazine rings, A group obtained by removing two or three hydrogen atoms from a ring such as an azole ring, a thiazole ring, a furan ring, a thiophene ring, or a benzene ring can also be used as a linking group.

[0072] In the nucleic acid involved in the present invention, the nucleic acid containing ether bond perfluoroalkyl is not particularly limited, and may be a nucleic acid in which all the nucleotides contained are natural nucleotides, or may be a nucleic acid in which part or all of them are artificial nucleotides. In addition, it may be a single-stranded nucleic acid or a double-stranded nucleic acid. As the nucleic acid, for example, genomic DNA, cDNA, mRNA, microRNA, siRNA, antisense oligonucleotide, nucleic acid aptamer, decoy nucleic acid, CpG (cytosine-phosphate-guanine) oligonucleotide, etc. may be cited. In addition, it may be an expression vector for expressing a target gene or siRNA in a cell.

[0073] In the nucleic acid involved in the present invention, the ether bond perfluoroalkyl group can be introduced into the nucleic acid of the object to be introduced by various coupling reactions. For example, by using a phosphoramidite containing a perfluoroalkyl group containing a ether bond as a raw material and performing a phosphoramidite method, the ether bond perfluoroalkyl group can be easily introduced into the nucleic acid. The general nucleic acid automatic synthesis device utilizes the phosphoramidite method. Therefore, by using a phosphoramidite containing a perfluoroalkyl group containing a ether bond as a raw material, a nucleic acid containing a perfluoroalkyl group containing a ether bond can be easily synthesized by an automatic synthesis device into which a desired position for nucleic acids of various base sequences is introduced.

[0074] As the phosphoramidite containing an ether bond perfluoroalkyl group, for example, a compound in which the nucleoside portion is replaced with an organic group containing an ether bond perfluoroalkyl group in the phosphoramidite commonly used for nucleic acid synthesis. As such a compound, for example, a compound represented by the following general formula (A2) can be cited.

[0075]

[0076] In the general formula (A2), R FE is the above-mentioned ether bond-containing perfluoroalkyl group. In addition, na is an integer of 1 to 10. i-Pr represents an isopropyl group, and DMTr represents a 4,4'-dimethoxytriphenylmethyl group.

[0077] The nucleic acid synthesized by the phosphoramidite method using compound (A2) has the structure of the following general formula (A1). In general formula (A1), R FE is the above-mentioned ether bond-containing perfluoroalkyl group. In addition, na is an integer of 1 to 10. The black dots represent the bonding sites.

[0078]

[0079] The synthesized target nucleic acid can be separated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, etc.

[0080] The nucleic acid involved in the present invention may be modified in various ways as long as the function of the nucleic acid into which the ether-bonded perfluoroalkyl group is introduced and the effects of the present invention are not impaired. Examples of such modifications include sugar chain modification, lipid modification, and peptide modification.

[0081] The perfluoro structure has a high affinity for cell membranes. Therefore, the nucleic acid of the present invention into which the ether bond perfluoroalkyl group is introduced has excellent cell membrane permeability compared to the nucleic acid into which the group is not introduced. Utilizing this property, the nucleic acid of the present invention is particularly preferably used as an effective ingredient of nucleic acid pharmaceuticals. For example, by introducing the ether bond perfluoroalkyl group into a functional nucleic acid that is introduced into a target cell in a living body and shows certain physiological activities in a manner that does not damage its function, the efficiency of introduction of the functional nucleic acid into the target cell can be improved.

[0082] <Method for improving cell membrane permeability of nucleic acid>

[0083] By introducing into nucleic acid a C-terminal moiety which may have 1 to 5 ether-bonded oxygen atoms between carbon atoms, 2-10 Perfluoroalkyl groups can improve the cell membrane permeability of nucleic acids. 2-10 Perfluoroalkyl is a group consisting of a C-1 to C-2 ether-bonded oxygen atom. 2-10 Perfluoroalkyl" and "C 2-10 Perfluoroalkyl" structure.

[0084] "C having 1 to 5 ether-bonded oxygen atoms between carbon atoms 2-10 The "perfluoroalkyl group" is the above-mentioned ether bond-containing perfluoroalkyl group. The introduction of the above-mentioned ether bond-containing perfluoroalkyl group into nucleic acid can be carried out by the phosphoramidite method using a phosphoramidite containing an ether bond-containing perfluoroalkyl group as described above.

[0085] C without ether-bonded oxygen atom 2-10 The introduction of perfluoroalkyl groups into nucleic acids can be carried out in the same manner, and C-containing groups having no ether-bonded oxygen atoms can be used. 2-10 The phosphoramidite of perfluoroalkyl group is carried out by the phosphoramidite method. In addition, the C-terminal group without ether-bonded oxygen atom can be introduced into nucleic acid by the method described in Non-Patent Documents 3 to 6. 2-10 Perfluoroalkyl.

[0086] A perfluorinated structure can be introduced into nucleic acids, and a C-terminal having 1 to 5 ether-bonded oxygen atoms between carbon atoms can be used. 2-10 Perfluoroalkyl groups can be introduced into any position of nucleic acid. 2-10 The position of the perfluoroalkyl group in the nucleic acid can be arbitrarily determined within the limit not impairing the function of the nucleic acid.

[0087] By introducing C 2-10Nucleic acids having increased cell membrane permeability by introducing a perfluoroalkyl group are preferably used as active ingredients of nucleic acid pharmaceuticals, similarly to nucleic acids into which a perfluoroalkyl group containing an ether bond is introduced.

[0088] For nucleic acids that have pharmacological activity but cannot reach cells, the efficiency of introducing the nucleic acid into target cells can be improved by implementing the method for improving the cell membrane permeability of nucleic acids involved in the present invention. That is, by the method for improving the cell membrane permeability of nucleic acids involved in the present invention, a drug delivery system for delivering nucleic acid pharmaceuticals into cells can be easily constructed.

[0089] Example

[0090] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0091] The NMR apparatus used in the analysis of the Examples and Comparative Examples was JNM-ECZ400S (400 MHz) manufactured by JEOL Ltd. 1 In HNMR, tetramethylsilane was set to 0 PPM. 19 In F NMR, the standard value of C6F6 is -162 PPM.

[0092] <Tm measurement>

[0093] In the following experiments, the melting temperature (Tm) of nucleic acids was determined by measuring the change in absorbance at 260 nm as a function of temperature using a UV-Vis spectrophotometer (UV-2550, manufactured by SHIMADZU). The melted sample was denatured at 100°C and slowly lowered to room temperature for annealing. The absorbance at 260 nm was measured and detected every 0.5°C from 20°C to 90°C. The concentration of double-stranded DNA was 4 μM in a buffer (10 mM sodium phosphate, pH 7.0, 100 mM NaCl). Tm was calculated using the neutral method (neutral method).

[0094] <Cell culture>

[0095] In the following experiments, cell culture was performed as follows.

[0096] HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fischer Scientific) supplemented with 10% FBS and 0.5% penicillin / streptomycin at 37°C in a humidified atmosphere (5% CO2). Cells used for image analysis were cultured in a 35 mm glass bottom dish (IWAKI).

[0097] <Confocal microscopy observation>

[0098] In the following experiments, confocal microscopy observation of cells was performed as follows.

[0099] Fluorescein-bound N[PFC] or N[PFPE] (500 μM, 5 μL) was added to HeLa cells and incubated at 37°C for 3 hours. Next, Hoechst 33342 (2 μg / mL, 0.5 mL) was added to the cells as a nuclear stain and further incubated for 1 hour. The solution was removed from each incubated disk, washed with PBS (-), and DMEM (1 mL) was added. Next, each disk was placed on a confocal laser scanning microscope, and a fluorescence image was obtained using an excitation wavelength of 488 nm and a light emission filter greater than 505 nm.

[0100] <Flow cytometry>

[0101] In the following experiments, flow cytometry was performed as follows.

[0102] HeLa cells were cultured to become 10 5 The cells were inoculated in a 12-well plate at 100 μg / well for culture. On the next day after inoculation, the culture medium of each well was replaced with DMEM culture medium (1 mL) containing DNA (2.5 μM) modified with N[PFC] or N[PFPE] bonded with fluorescein, and incubated for 4 hours or 24 hours. Thereafter, the cell layer in the well was washed twice with PBS, and then the cells were detached by treating with 0.05% (w / v) trypsin (200 μL) at 37°C for 5 minutes. The recovered cells were suspended in DMEM (600 μL). The cell suspension was separated by centrifugation (400G, 3 minutes), and PBS / 1% BSA (500 μL) was added. The proportion of fluorescent cells and the average fluorescence intensity of the cell suspension were analyzed by flow cytometry ("guava easyCyte8", manufactured by Luminex).

[0103] In the case of double-stranded DNA, the sample was denatured at 100°C and then slowly lowered to room temperature for annealing.

[0104] [Example 1]

[0105] A phosphoramidite containing a perfluoroalkyl group having 5 carbon atoms and two ether-bonded oxygen atoms (CF3CF2O(CF2)2O(CF2)-: hereinafter referred to as C5-PFPE group) was synthesized and used to prepare a nucleic acid containing a C5-PFPE group by the phosphoramidite method.

[0106] (1) Synthesis of C5-PFPE carboxylic acid

[0107] The carboxylate containing a C5-PFPE group is synthesized by the method described in Patent Document 4.

[0108] CH3CH2O(CH2)2O(CH2)2OH (300 g) was added to a 2 L autoclave made of Hastelloy C, the reactor was cooled, and CF3CF2CF2OCF(CF3)CF2OCF(CF3)COF (1339 g) was slowly introduced under closed stirring in such a way that the internal temperature was kept below 30°C. After the entire amount was introduced, stirring was further performed at 30°C for 3 hours, and the HF generated in the reaction was expelled to the outside of the system by bubbling nitrogen to obtain the product. The product was analyzed by GC (gas chromatography), and the result showed that 99.6% of CH3CH2O(CH2)2O(CH2)2OCOCF(CF3)OCF2CF(CF3)OCF2CF2CF3 was generated, and no unreacted CH3CH2O(CH2)2O(CH2)2OH was detected. The product was used in the next step without purification.

[0109] R-113 (312 g) was added to a nickel autoclave of 500 mL, and the mixture was stirred and maintained at 25°C. A cooler maintained at 20°C, a NaF particle filling layer, and a cooler maintained at -10°C were arranged in series at the gas outlet of the autoclave. In addition, a liquid return line was provided for returning the condensed liquid from the cooler maintained at -10°C to the autoclave. After nitrogen was blown into the autoclave at room temperature for 1 hour, fluorine gas diluted with nitrogen to 20% (hereinafter referred to as 20% diluted fluorine gas) was blown at room temperature at a flow rate of 17.04 L / h for 1 hour. Next, while blowing 20% ​​diluted fluorine gas at the same flow rate, a solution prepared by dissolving the above-obtained product (10 g) in R-113 (150 g) was injected over 4.1 hours.

[0110] Next, the pressure in the autoclave was raised to 0.15MPaG while blowing 20% ​​diluted fluorine gas at the same flow rate. The R-113 solution with a benzene concentration of 0.01g / mL was heated from 25°C to 40°C and 9mL was injected. The benzene solution injection port of the autoclave was closed, and stirring was continued for 0.3 hours. Next, while maintaining the pressure in the reactor at 0.15MPaG and the temperature in the reactor at 40°C, 6mL of the above benzene solution was injected. The benzene solution injection port of the autoclave was closed, and stirring was continued for 0.3 hours. Repeat the same operation once more. The total amount of benzene injected was 0.22g, and the total amount of R-113 injected was 21mL. Thereafter, stirring was continued for 1 hour while blowing 20% ​​diluted fluorine gas at the same flow rate. Next, the pressure in the reactor was brought to normal pressure, and nitrogen was blown for 1 hour. Use 19 The product was analyzed by F NMR, and the result showed that the target compound (CF3CF2O(CF2)2O(CF2)2OCOCF(CF3)OCF2CF(CF3)OCF2CF2CF3) was contained in a yield of 99%. 1As a result of H NMR and GC-MS analysis, no compound having a CH bond was confirmed.

[0111] The above-obtained CF3CF2O(CF2)2O(CF2)2OCOCF(CF3)OCF2CF(CF3)OCF2CF2CF3 (4273 g) was added to the kettle (2 L volume) of the distillation tower equipped with a reflux at 10°C, and potassium fluoride (12.6 g) was added to heat and stir (heat medium temperature: 100-130°C), and the fraction was recovered by reactive distillation. 1273 g of a fraction with a purity of more than 99% was recovered as the main fraction. The boiling point was 66.5°C and the yield was 84.5%. The fraction was used 1 As a result of H NMR and GC-MS analysis, no compound having a CH bond was confirmed.

[0112] The obtained CF3CF2O(CF2)2OCF2COF (107 g) was put into a 200 mL Hastelloy autoclave, and water (6 g) was slowly added dropwise under ice cooling while stirring vigorously for hydrolysis. After the addition, the temperature was slowly raised to room temperature, and stirring was continued for 5 hours. Thereafter, the HF generated in the reaction was expelled out of the system by bubbling nitrogen, and single distillation was continued to obtain 84 g of a fraction with a boiling point of 74°C / (30×133.322 Pa). The purity was 99.4%, and the yield was 79%.

[0113] (2) Synthesis of C5-PFPE alcohol

[0114] The C5-PFPE carboxylic acid obtained in the above (1) is reduced to obtain an alcohol containing a C5-PFPE group.

[0115] A carboxylate containing a C5-PFPE group (17.3 g, 50 mmol) and anhydrous tetrahydrofuran (50 mL) were added to a three-necked flask equipped with a magnetic stirrer, a thermometer and a cooler. Next, sodium borohydride (2.84 g, 75 mmol) was added to the flask in an ice bath below 15°C, and then boron trifluoride ether complex (9.42 mL, 75 mmol) was further added dropwise. After the reaction mixture was refluxed for 24 hours, it was cooled to 5°C and distilled water was added until no gas was generated. Next, the reaction mixture was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous Na2SO4, filtered, and the solvent was distilled off to obtain the alcohol as the target compound (yield 67%).

[0116] (3) Synthesis of trifluoromethanesulfonate

[0117] Synthesis of trifluoromethanesulfonates containing C5-PFPE groups.

[0118]

[0119] The fluorinated alcohol (4.98 g, 15.0 mmol) was dissolved in dry dichloromethane (75 mL), followed by the addition of dry triethylamine (15 mL) and the mixture was cooled to -78°C. Trifluoromethanesulfonic anhydride (6.34 g, 22.5 mmol) was added dropwise to the reaction solution over 30 minutes, resulting in the reaction mixture turning dark. The reaction mixture was stirred at 0°C for 1 hour, warmed to room temperature, and further stirred for 3 hours. Thereafter, saturated NaHCO3 was added to stop the reaction. NaHCO3 (50 mL) was added to the reaction product, and then extracted three times with dichloromethane (50 mL). All organic phases were combined, washed with brine, and dried over MgSO4. The solvent was removed under vacuum to obtain the target compound as a dark black oily substance (yield 27.3%). The obtained crude product was purified by 1 H NMR analysis and used in the next step without further purification.

[0120] 1 HNMR (400MHz, CDCl3) δ4.70 (t, J=7.8Hz, 2H). 19 FNMR (376MHz, CDCl3) δ-74.1 (s, 3F), δ-77.6 (s, 2F), δ-86.6 (s, 3F), δ-88.2--88.7 (m, 6F).

[0121] (4) Diethanol amination

[0122] The trifluoromethanesulfonate obtained in the above (3) is subjected to diethanol amination.

[0123]

[0124] Add a solution of the trifluoromethanesulfonate (1.6 g, 3.5 mmol) and diethanolamine (0.74 g, 7.0 mmol) obtained in (3) above to dry DMF (N,N-dimethylformamide) (5 mL). Place the resulting reaction mixture in an oil bath at 100°C and stir for 21 hours. Next, cool the reactant, dissolve it in water (20 mL), and extract the product three times with dichloromethane (20 mL). Combine all the organic phases, wash with brine, dry with MgSO4, and remove the solvent under vacuum to obtain the target compound. The resulting crude product is purified by 1 H NMR analysis and used in the next step without further purification.

[0125] 1 HNMR (400MHz, CDCl3) δ3.58 (t, J=5.0Hz, 4H), δ3.24 (t, J=11.0Hz, 2H), δ2.86 (t, J=5.5Hz, 4H).

[0126] (5)DMTr protection

[0127] One of the two hydroxyl groups of the diethanolamine obtained in the above (4) is protected with DMTr (4,4'-dimethoxytriphenylmethyl).

[0128]

[0129] The diethanolamine (0.92 g, 2.2 mmol) obtained in (4) above was dissolved in dry dichloromethane (7.5 mL) and triethylamine (1 mL). DMTrCl (0.75 g, 2.2 mmol) was added little by little to the reaction mixture and stirred at room temperature for 2 hours. Next, the solvent was removed from the reactant under vacuum to obtain a dark brown oily substance. The oily substance was purified by column chromatography using a silica gel column pre-treated with triethylamine and a mixture of EtOAc / hexane (volume ratio 1: 4) to obtain the target compound as a yellow oily substance (yield 238 mg, yield 15.2%).

[0130] 1 HNMR (400MHz, CDCl3) δ7.40 (t, J=8.2Hz, 2H), δ7.33-7.20 (m, 7H), δ6.82 (d, J=8.7Hz, 4H), δ3.78 (s, 6H), δ3.50 (dd , J=5.5Hz, 5.0Hz, 2H), δ3.26-3.20 (m, 4H), δ2.93 (t, J=5.5Hz, 2H), δ2.81 (t, J=5.0Hz, 2H), δ2.44 (t, J=6.0Hz, 1H). 19 FNMR (376MHz, CDCl3) δ-73.6 (s, 2F), δ-86.5 (s, 3F), δ-88.3--88.4 (m, 4F), δ-88.6--88.7 (m, 2F).

[0131] (6) Phosphoramidite

[0132] The hydroxyl groups in the diethanolamine obtained in the above (5) which are not protected by the DMTr group are phosphoramiditized.

[0133]

[0134] 3-((Bis(diisopropylamino)phosphino)oxy)propionitrile was dissolved in dry acetonitrile. 5-(Ethylthiotetrazole) (ETT) (65 mg, 0.5 mmol) was added to the reaction solution under argon, and then a solution obtained by dissolving the DMTr-protected diethanolamine (0.24 mg, 0.33 mmol) obtained in (5) above in THF (1 mL) and acetonitrile (1 mL) was added little by little. Next, the reaction mixture was stirred at room temperature for 4 hours under argon, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using degassed hexane / ethyl acetate (volume ratio 1:4) as the mobile phase under argon, and the target compound (N[C5-PFPE] phosphoramidite) was isolated as a colorless oily substance (yield 130 mg, yield 43%).

[0135] 1 HNMR (400MHz, Acetone-d6) δ7.45 (t, J=7.3Hz, 2H), δ7.33-7.17 (m, 7H), δ6. 85 (td, J=2.7Hz, 9.1Hz, 4H), δ3.84-3.56 (m, 6H), δ3.76 (s, 6H), δ3.50 (t, J=1 1.4Hz, 2H), δ3.20 (t, 6.0Hz, 2H), δ3.00 (t, J=5.5Hz, 2H), δ2.95 (t, J=5.0Hz, 2H), δ2.67 (t, J=6.0Hz, 2H), δ1.16 (d, J=6.9Hz, 6H), δ1.12 (d, J=6.9Hz, 6H). 19 FNMR (376MHz, Acetone-d6) δ-75.5 (m, 2F), δ-87.4 (s, 3F), δ-88.9 (m, 2F), δ-89.1 (m, 2F), δ-89.3 (m, 2F). 31 PNMR(162MHz, Acetone-d6)δ-148.0(m).

[0136] (7)DNA synthesis

[0137] 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, using NTSH-8 DNA / RNA synthesizer (manufactured by NIHON TECHNO SERVICE).

[0138] The synthesis of 5'-N[C5-PFPE] modified DNA in which an N[C5-PFPE] group was bonded to the 5' end of an oligonucleotide consisting of sequence number 1 (5'-TTTTTCAGTTGACCATATA-3') was performed as follows. DNA synthesis (detritylation) was performed on a CPG solid support column (1 μmole specification). Next, under a nitrogen atmosphere, the N[C5-PFPE]phosphoramidite synthesized in (6) above (0.1 M acetonitrile solution, 300 μL) and the activator solution (300 μL) were mixed in the presence of CPG using a syringe. After mixing for 5 minutes, the solution was removed from the column and chain capping, oxidation and deprotection were performed using a DNA synthesizer (yield 11.0%).

[0139] 5'-N[C5-PFPE] modified DNA

[0140] Sequence: 5'-(N[C5-PFPE])TTTTTCAGTTGACCATATA-3'LRMS(MALDI)[MH] - :6245.0(calcd), 6244.2(found)

[0141] The synthesis of 3'-N[C5-PFPE] modified DNA in which an N[C5-PFPE] group is bonded to the 3' end of an oligonucleotide consisting of sequence number 2 (5'-TATATGGTCAACTGAAAAA-3'), which is a complementary base sequence to sequence number 1, is carried out as follows. First, in a nitrogen atmosphere, the N[C5-PFPE] phosphoramidite synthesized in (4) above (0.1 M acetonitrile solution, 300 μL) and an activator solution (300 μL) are added to a 50% flask. After mixing for 5 minutes, the solution was removed from the column and chain capping, oxidation and deprotection were performed using a DNA synthesizer. Next, a DNA oligomer was synthesized by the conventional phosphoramidite method using a DNA synthesizer (yield 7.9%).

[0142] 3'-N[C5-PFPE] modified DNA

[0143] Sequence: 5'-TATATGGTCAACTGAAAAA(N[C5-PFPE])-3'LRMS(MALDI)[MH] - :6321.1(calcd), 6321.3(found)

[0144] The deprotection of nucleic acid synthesized by DNA synthesizer is carried out as follows. First, the DNA supported by CPG solid (detritylated) is treated in an aqueous solution of ammonium hydroxide (28%) at 50°C for 12 hours. Next, the crude product solution is separated from the solid support and concentrated at 30°C under reduced pressure. Next, the obtained concentrate is filtered using a 0.45 μm centrifugal filter and purified by HPLC. The obtained solution is quantified by absorbance at 260 nm.

[0145] HPLC purification was performed under the following conditions.

[0146] Solvent (filtered through a 0.45 μm centrifugal filter): 100 mM triethylammonium acetate (TEAA) buffer (pH 7.0) and HPLC grade acetonitrile, elution gradient: 3-95% acetonitrile (40 minutes), chromatographic column: COSMOSIL packed chromatographic column "5C18-MS-II" (4.6 ID×150 nm, manufactured by Nacalai Tesque Co., Ltd.), test crude DNA: injection of a solution dissolved in 20 to 50 μL ultrapure water, detection: measuring the absorbance at 260 nm using a diode array detector.

[0147] The Tm value of the synthesized nucleic acid was measured. RNA having a base sequence corresponding to the DNA before N[C5-PFPE] modification was used for the measurement of the Tm value. The nucleic acids used in the measurement are shown in Table 1, and the results of the Tm value measurement are shown in Table 2.

[0148] [Table 1]

[0149] name Base sequence FE1 <![CDATA[5’-(N[c5-PFPE])TTTTTCAGTTGACCATATA-3’]]> FE2 <![CDATA[5’-(N[C5-PFPE])2TTTTTCAGTTGACCATATA-3’]]> rFE <![CDATA[5’-TATATGGTCAACTGAAAAA(N[C5-PFPE])-3’]]> rFE <![CDATA[5’-TATATGGTCAACTGAAAAA(N[C5-PFPE])2-3’]]> cont-DNA 5'-TTTTTCAGTTGACCATATA-3' cont-rDNA 5'-TATATGGTCAACTGAAAAA-3' cont-RNA 5'-UUUUUCAGUUGACCAUAUA-3' cont-rRNA 5'-UAUAUGGUCAACUGAAAAA-3'

[0150] [Table 2]

[0151] Sequence 1 Sequence 2 Tm cont-DNA cont-rDNA 55.5℃ FE1 rFE 56.5℃ FE1 cont-rDNA 56.5℃ cont-DNA rFE 55.1℃ cont-DNA cont-rRNA 52.9℃ FE1 cont-rRNA 56.8℃ cont-RNA cont-rDNA 48.3℃ cont-RNA rFE 49.5℃

[0152] The Tm value of N[C5-PFPE]-modified DNA was almost the same as that of DNA before modification. When annealing with RNA, the Tm value increased slightly.

[0153] [Example 2]

[0154] Synthesis of a perfluoroalkyl group (-C8F) containing 8 carbon atoms and no ether-bonded oxygen atom 17 : Hereinafter referred to as C8-PFC group) is used to produce a nucleic acid containing a C8-PFC group by the phosphoramidite method.

[0155] The C8-PFC group-containing phosphoramidite represented by the following formula was synthesized by the method described in Non-Patent Document 5.

[0156]

[0157] A 5'-N[C8-PFC] modified DNA having an N[C8-PFC] group bonded to the 5' end of an oligonucleotide consisting of sequence number 1 and a 3'-N[C8-PFC] modified DNA having an N[C8-PFC] group bonded to the 3' end of an oligonucleotide consisting of sequence number 2 were synthesized in the same manner as in Example 1 except that the obtained phosphoramidite containing a C8-PFC group was used.

[0158] The Tm value of the synthesized nucleic acid was measured. The Tm value was measured in the same manner as in Example 1, using RNA having a base sequence corresponding to the DNA before N[C8-PFC] modification. The nucleic acids used in the measurement are shown in Table 3, and the results of the Tm value measurement are shown in Table 4.

[0159] [Table 3]

[0160] name Base sequence F1 <![CDATA[5’-(N[C8-PFC])TTTTTCAGTTGACCATATA-3’ <!-- 12 -->]]> F2 <![CDATA[5’-(N[C8-PFc])2TTTTTcAGTTGACCATATA-3’]]> F5 <![CDATA[5’-(N[C8-PFC])5TTTTTCAGTTGACCATATA-3’]]> rF1 <![CDATA[5’-TATATGGTCAACTGAAAAA(N[C8-PFC])-3’]]> rF2 <![CDATA[5’-TATATGGTCAACTGAAAAA(N[c8-PFC])2-3’]]> rF5 <![CDATA[5’-TATATGGTCAACTGAAAAA(N[C8-PFC])5-3’]]>

[0161] [Table 4]

[0162] Sequence 1 Sequence 2 Tm cont-DNA cont-rDNA 55.8℃ F1 rF1 59.0℃ F2 rF2 72.0℃ F3 rF3 7g.9℃ F1 cont-rDNA 57.6℃ cont-DNA rFE 55.5℃ F2 cont-rDNA 57.6℃ cont-DNA rFE 56.7℃ F5 cont-rDNA 50.6℃ cont-DNA rFE 50.8℃ cont-DNA cont-rRNA 52.9℃ F1 cont-rRNA 57.5℃ F2 cont-rRNA 54.6℃ F5 cont-rRNA 48.5℃ cont-RNA cont-rDNA 48.3℃ cont-RNA rF1 49.3℃ cont-RNA rF2 50.3℃ cont-RNA rF5 45.4℃

[0163] When DNA modified with N[C8-PFC] was annealed to each other, it was observed that the Tm value tended to increase as the number of introduced N[C8-PFC] increased. When DNA modified with N[C8-PFC] was annealed to DNA or RNA, it was observed that the Tm value tended to increase when two N[C8-PFC] were introduced compared to the case where one N[C8-PFC] was introduced, but when five N[C8-PFC] were introduced, the Tm value decreased compared to the DNA or RNA before modification.

[0164] [Test Example 1]

[0165] The nucleic acid in which fluorescein was bonded to the 3' end of the N[C8-PFC] modified DNA synthesized in Example 2 was kept in a single-stranded state or double-stranded, added to the culture medium of HeLa cells and incubated, and the efficiency of introduction into the cells was analyzed by flow cytometry.

[0166] Figure 1A The results of flow cytometry of cells into which the fluorescein-modified nucleic acids of cont-DNA, F1, F2, and F5 were introduced are shown. Figure 1B The results of flow cytometry of cells into which fluorescein-modified nucleic acids of cont-rDNA, rF1, rF2, and rF5 were introduced are shown. For single-stranded nucleic acids modified with N[C8-PFC] at the 3' end, the amount of N[C8-PFC]-modified nucleic acids introduced into cells was greater than that of unmodified nucleic acids (cont-rDNA) ( Figure 1B). For single-stranded nucleic acids modified with N[C8-PFC] at the 5' end, there was no difference between F1 and cont-DNA fluorescein-modified nucleic acids with one N[C8-PFC] introduced, but the amount of fluorescein-modified nucleic acids F2 and F5 with two or five N[C8-PFC] introduced into cells was greater than that of cont-DNA fluorescein-modified nucleic acids ( Figure 1A ).

[0167] FIG. 2 shows the results of flow cytometry of cells into which fluorescein-modified double-stranded nucleic acids annealed in various combinations were introduced. Figure 2A Results for cells incubated for 4 hours, Figure 2B The results are for cells incubated for 24 hours. The amount of double-stranded nucleic acid composed of F2 and rF2 incorporated into the cells was the largest, and the amount of double-stranded nucleic acid composed of cont-DNA and rF2 incorporated into the cells was greater than that composed of cont-DNA and cont-rDNA.

[0168] These results show that modification of nucleic acid with N[C8-PFC] increases the amount of nucleic acid introduced into cells and improves the efficiency of introduction. The improvement in the efficiency of introduction into cells is due to the fact that modification with N[C8-PFC] increases the cell membrane permeability of nucleic acid.

[0169] [Test Example 2]

[0170] The nucleic acid in which fluorescein was bonded to the 3' end of the N[C5-PFPE]-modified DNA synthesized in Example 1 was kept in a single-stranded state and added to a culture medium of HeLa cells for incubation, and the efficiency of introduction into the cells was analyzed by flow cytometry.

[0171] Figure 3 The results of flow cytometry of cells into which fluorescein-modified nucleic acids of cont-DNA, FE1, and FE2 were introduced are shown. The single-stranded nucleic acids modified with N[C5-PFPE] at the 5' end were introduced into cells at a higher amount than the unmodified nucleic acids (cont-rDNA).

[0172] In the same manner as above, a 5'-N[C5-PFPE] modified DNA was synthesized in which an N[C5-PFPE] group was bonded to the 5' end of an oligonucleotide consisting of SEQ ID NO: 1. The synthesized nucleic acid is shown in Table 5.

[0173] [Table 5]

[0174] name Base sequence FE5 <![CDATA[5’-(N[C5-PFPE])5TTTTTCAGTTGACCATATA-3’]]> FE10 <![CDATA[5’-(N[C5-PFPE]) 10 TTTTTCAGTTGACCATATA-3’]]>

[0175] Figure 4The results of flow cytometry of cells into which fluorescein-modified nucleic acids of cont-DNA, FE5, and FE10 were introduced are shown. The single-stranded nucleic acids modified with N[C5-PFPE] at the 5' end were introduced into cells at a higher amount than the unmodified nucleic acid (cont-rDNA).

[0176] [Example 3]

[0177] A phosphoramidite containing a perfluoroalkyl group having 7 carbon atoms and 3 ether-bonded oxygen atoms (CF3CF2O(CF2)2O(CF2)2O(CF2)-: hereinafter referred to as C7-PFPE group) was synthesized and used to produce a nucleic acid containing a C7-PFPE group by the phosphoramidite method.

[0178] (1) Synthesis of C7-PFPE carboxylic acid

[0179] Using CH3CH2(OCH2CH2)3OH and F(CF2)3OCF(CF3)CF2OCF(CF3)COF as starting materials, CF3CF2(OCF2CF2)2OCF2COF was obtained by the liquid phase fluorination method and KF thermal decomposition method described in International Publication WO2000 / 056694.

[0180] The obtained CF3CF2(OCF2CF2)2OCF2COF is synthesized in the same manner as the above-mentioned C5-PFPE carboxylic acid to obtain CF3CF2(OCF2CF2)2OCF2COOH (C7-PFPE carboxylic acid).

[0181] (2) Synthesis of C7-PFPE alcohol

[0182] CF3CF2(OCF2CF2)2OCF2CH2OH (C7-PFPE alcohol) was obtained from C7-PFPE carboxylic acid in the same manner as the synthesis of the above-mentioned C5-PFPE alcohol.

[0183] (3) Synthesis of trifluoromethanesulfonate

[0184] Synthesis of trifluoromethanesulfonates containing C7-PFPE groups.

[0185]

[0186] The above C7-PFPE alcohol (11.2 g, 25.0 mmol) was dissolved in water (5 mL), followed by the addition of triethylamine (5 mL) and the mixture was cooled to 0°C. Trifluoromethanesulfonic acid chloride (4.42 g, 26.3 mmol) was added dropwise to the reaction solution over 30 minutes. The reaction mixture was stirred at room temperature for 1.5 hours. The product was extracted three times with dichloromethane (10 mL). All organic phases were combined, washed with brine, dried with MgSO4, and the solvent was removed under vacuum to obtain the target compound as an oily substance. The obtained crude product was purified by filtration. 1 H NMR analysis, without further purification, used in the next step. (Yield 64%)

[0187] 1 HNMR (400MHz, CDCl3) δ4.67 (t, J = 8.2Hz, 2H), 19 FNMR(376MHz, CDCl3)δ-74.1(s,3F),δ-77.7(s,2F),δ-86.6(s,3F),δ-88.4--88.7(m,10F)

[0188] (4) Diethanol amination

[0189] The trifluoromethanesulfonate containing a C7-PFPE group obtained in the above (3) is subjected to diethanolamination.

[0190]

[0191] Add the solution of trifluoromethanesulfonate (9.3 g, 16 mmol) containing C7-PFPE group and diethanolamine (3.36 g, 32 mmol) obtained in (3) above to dry DMF (N,N-dimethylformamide) (12 mL). Place the obtained reaction mixture in an oil bath at 100°C and stir for 15 hours. Next, cool the reactant, dissolve it in water (20 mL), and extract the product three times with dichloromethane (20 mL). Combine all the organic phases, wash with brine, dry with MgSO4, and remove the solvent under vacuum to obtain the target compound. The obtained crude product is used 1 H NMR analysis and used in the next step without further purification.

[0192] 1 HNMR (400MHz, CDCl3) δ3.34 (m, 4H), δ3.09 (dt, J = 2.3, 11.0Hz, 2H), δ2.68 (m, 4H), 19 FNMR(376MHz, CDCl3)δ-74.0(s,2F),δ-87.4(s,3F),δ-89.0--89.6(m,10F)

[0193] (5)DMTr protection

[0194] One of the two hydroxyl groups of the C7-PFPE group-containing diethanolamine obtained in the above (4) was protected with DMTr (4,4'-dimethoxytriphenylmethyl).

[0195]

[0196] The diethanolamine (6.58 g, 12.3 mmol) containing a C7-PFPE group obtained in (4) above was dissolved in dry dichloromethane (20 mL) and triethylamine (6 mL). DMTrCl (4.58 g, 13.5 mmol) was added little by little to the reaction mixture and stirred at room temperature for 2 hours. Next, the solvent was removed from the reactant under vacuum to obtain a dark brown oily substance. The oily substance was purified by column chromatography using a silica gel column pre-treated with triethylamine and a mixture of EtOAc / hexane (volume ratio 1:4) to obtain the target compound as a yellow oily substance (yield 37%).

[0197] 1 HNMR (400MHz, CDCl3) δ7.41 (d, J=7.3Hz, 2H), δ7.33-7.15 (m, 7H), δ6.81 (dt, J=9.1, 3.7Hz, 4H), δ3.78 (s, 6H), δ3.50 ( dd, J=6.0Hz, 10.8Hz, 2H), δ3.24-3.19 (m, 4H), δ2.91 (t, J=5.5Hz, 2H), δ2.79 (t, J=5.0Hz, 2H), δ2.43 (t, J=6.0Hz, 1H) 19 FNMR(376MHz, CDCl3)δ-74.4(m,2F),δ-86.7(s,3F),δ-88.1--88.6(m,10F)

[0198] (6) Phosphoramidite

[0199] The hydroxyl groups not protected by the DMTr groups in the C7-PFPE group-containing diethanolamine obtained in the above (5) are phosphoramiditized.

[0200]

[0201] 3-((Bis(diisopropylamino)phosphino)oxy)propionitrile (1.76 g, 5.9 mmol) was dissolved in dry acetonitrile. 5-(Ethylthiotetrazolyl) (ETT) (7.61 g, 5.9 mmol) was added to the reaction solution under argon, and then a solution obtained by dissolving the DMTr-protected diethanolamine containing a C7-PFPE group obtained in (5) above (3.26 g, 3.9 mmol) in THF (5 mL) and acetonitrile (5 mL) was added little by little. Next, the reaction mixture was stirred at room temperature for 4 hours under argon. The solvent was evaporated under reduced pressure. The crude product obtained was purified by column chromatography using degassed hexane / ethyl acetate (volume ratio 1:4) as the mobile phase under argon, and the target compound (N[C7-PFPE] phosphoramidite) (yield 78%) was isolated as a colorless oily substance.

[0202] 1 HNMR(400MHz, acetone-d6)δ87.41(d,J=8.7Hz,2H),δ7.32-7.14(m,7H),δ6.83-6.79(m,4H),δ3.80-3.51(m,6H)δ3.77(s,6H)δ3.50(t,J=11.4Hz,2 H), δ3.25(t,11.4Hz,2H), δ3.15(t,J=6.0Hz,2H), δ2.94-2.89(m,4H),2.54(t,J=6.4Hz,2H), δ1.16(d,J=6.9Hz,6H), δ1.12(d,J=6.9Hz,6H), 19 FNMR (376MHz, acetone-d6) δ-75.0 (m, 2F), δ-86.6 (s, 3F), δ-88.3--88.8 (m, 10F), 31 PNMR (162MHz, acetone-d6), δ-148.4(s)

[0203] A 5'-N[C7-PFPE] modified DNA in which an N[C7-PFPE] group was bonded to the 5' end of the oligonucleotide consisting of SEQ ID NO: 1 was synthesized.

[0204] The synthesis of 5'-N[C7-PFPE] modified DNA in which an N[C7-PFPE] group is bonded to the 5' end of the oligonucleotide consisting of sequence number 1 is carried out as follows. DNA synthesis (detritylation) was performed on a CPG solid support column (1 μmole specification). Next, N[C7-PFPE]phosphoramidite synthesized in (6) above (0.1 M acetonitrile solution, 300 μL) and activator solution (300 μL) were mixed in the presence of CPG using a syringe under a nitrogen atmosphere. After mixing for 5 minutes, the solution was removed from the column and chain capping, oxidation and deprotection were performed using a DNA synthesizer.

[0205] Deprotection of the nucleic acid and HPLC purification were performed in the same manner as above. The synthesized nucleic acids are shown in Table 6.

[0206] [Table 6]

[0207] name Base sequence FE(2)1 <![CDATA[5’-(N[C7-PFPE])TTTTTCAGTTGACCATATA-3’]]> FE(2)2 <![CDATA[5’-(N[C7-PFPE])2TTTTTCAGTTGACCATATA-3’]]> FE(2)5 <![CDATA[5’-(N[C7-PFPE])5TTTTTCAGTTGACCATATA-3’]]>

[0208] Figure 5 The results of flow cytometry of cells into which fluorescein-modified nucleic acids of cont-DNA, FE(2)1, FE(2)2, and FE(2)5 were introduced. Single-stranded nucleic acids modified with N[C7-PFPE] at the 5' end were introduced into cells at higher levels than unmodified nucleic acids (cont-rDNA).

[0209] [Example 4]

[0210] A phosphoramidite containing a perfluoroalkyl group having less than 4 carbon atoms and having no ether-bonded oxygen atom (-C4F9: hereinafter referred to as C4-PFC group) is synthesized, and a nucleic acid containing a C4-PFC group is produced by the phosphoramidite method using the synthesized phosphoramidite.

[0211] (1) Synthesis of trifluoromethanesulfonate

[0212] Synthesis of trifluoromethanesulfonates containing C4-PFC groups.

[0213]

[0214] Fluorinated alcohol (C4F9CH2OH) (500 mg, 2.0 mmol) was dissolved in dry dichloromethane (1 mL), followed by addition of dry triethylamine (1 mL) and cooling to -78°C. Trifluoromethanesulfonic anhydride (8.46 g, 3 mmol) was added dropwise to the reaction solution over 30 minutes, resulting in the reaction mixture turning dark. The reaction mixture was stirred at 0°C for 1 hour, warmed to room temperature, and further stirred for 18 hours. Thereafter, saturated NaHCO3 (10 mL) was added to stop the reaction, and the mixture was extracted three times with dichloromethane (50 mL). All organic phases were combined, washed with brine, and dried over MgSO4. The solvent was removed under vacuum to obtain the target compound as a dark black oily substance. The obtained crude product was purified by 1H NMR analysis showed that the product was used in the next step without further purification (yield 61%).

[0215] 1 HNMR (400MHz, CDCl3) δ4.91 (t, J=12.3Hz, 2H), 19 FNMR (376MHz, CDCl3) δ-74.3 (s, 3F), δ-81.2 (s, 3F), δ-120.1 (s, 2F), δ-124.0 (s, 2F), δ-126.4 (s, 2F)

[0216] (2) Diethanol amination

[0217] The trifluoromethanesulfonate containing a C4-PFC group obtained in the above (1) is subjected to diethanol amination.

[0218]

[0219] The solution of the trifluoromethanesulfonate containing C4-PFC group (459 mg, 1.2 mmol) and diethanolamine (254 mg, 2.4 mmol) obtained in (1) above was added to dry DMF (5 mL). The obtained reaction mixture was placed in an oil bath at 100°C and stirred for 18 hours. Next, the reactant was cooled, dissolved in water (10 mL), and the product was extracted 3 times with dichloromethane (20 mL). All organic phases were combined, washed with brine, dried with MgSO4, and the solvent was removed under vacuum to obtain the target compound. The obtained crude product was used 1 H NMR analysis and used in the next step without further purification.

[0220] 1 HNMR(400MHz, CDCl3)δ3.61(t,J=5.0Hz,4H), δ3.21(t,J=17.4Hz,2H), δ2.89(t,J=5.5Hz,4H), 19 FNMR(376MHz, CDCl3)δ-81.2(s,3F),δ-117.0(s,2F),δ-124.7(s,2F),δ-126.2(s,2F)

[0221] (3)DMTr protection

[0222] One of the two hydroxyl groups of the C4-PFC group-containing diethanolamine obtained in the above (2) is protected with DMTr (4,4'-dimethoxytriphenylmethyl).

[0223]

[0224] The diethanolamine (3.20 g, 9.5 mmol) containing the C4-PFC group obtained in (2) above was dissolved in dry dichloromethane (20 mL) and triethylamine (5 mL). DMTrCl (3.21 g, 9.5 mmol) was added little by little to the reaction mixture and stirred at room temperature for 2.5 hours. Next, the solvent was removed from the reactant under vacuum to obtain a dark brown oily substance. The oily substance was purified by column chromatography using a silica gel column pre-treated with triethylamine and a mixture of EtOAc / hexane (volume ratio 1:4) to obtain the target compound as a yellow oily substance (yield 54%).

[0225] 1 HNMR (400MHz, CDCl3) δ7.41 (d, J = 7.3Hz, 2H), δ7.33-7.18 (m, 7H), δ6.82 (d, J = 9.2Hz, 4H), δ3.78 (s, 6H), δ3.52 (dd, J = 5.5Hz, 10. 5Hz, 2H), δ3.31 (t, J = 16.9Hz, 2H), δ3.23 (t, J = 5.5Hz, 2H) δ2.94 (t, J = 5.5Hz, 2H), δ2.81 (t, J = 5.0Hz, 2H), δ2.44 (t, J = 6.2Hz, 1H) 19 FNMR(376MHz, CDCl3)δ-79.9(s,3F),δ-117.1(s,2F),δ-125.0(s,2F),δ-125.9(s,2F)

[0226] (4) Phosphoramidite

[0227] The hydroxyl group not protected by the DMTr group in the C4-PFC group-containing diethanolamine obtained in the above (3) is phosphoramiditized.

[0228]

[0229] 3-((Bis(diisopropylamino)phosphino)oxy)propionitrile (1.84 g, 6.1 mmol) was dissolved in dry acetonitrile. 5-(Ethylthiotetrazolyl) (ETT) (1.05 g, 7.7 mmol) was added to the reaction solution under argon, and then a solution obtained by dissolving the DMTr-protected diethanolamine containing a C4-PFC group obtained in (3) above (3) (3.26 g, 5.1 mmol) in THF (5 mL) and acetonitrile (5 mL) was added little by little. Next, the reaction mixture was stirred at room temperature for 4 hours under argon, and the solvent was evaporated under reduced pressure. Under argon, the obtained crude product was purified by column chromatography using degassed hexane / ethyl acetate (volume ratio 1:4) as the mobile phase, and the target compound (N[C4-PFC] phosphoramidite) was isolated as a colorless oily substance (yield 45%).

[0230] 1 HNMR (400MHz, acetone-d6) δ7.41 (t, J = 8.7Hz, 2H), δ7.33-7.17 (m, 7H), δ6.85 (t d,J=2.3Hz,8.7Hz,4H),δ3.84-3.56(m,6H)δ3.76(s,6H)δ3.37(t,J=16.5H z,2H),δ3.20(t,6.0Hz,2H),δ2.94(t,J=5.5Hz,2H),δ2.90(t,J=6.4Hz,2H ), δ2.56(t,J=6.4Hz,2H), δ1.16(dd,J=6.9Hz,6H), δ1.12(d,J=6.9Hz,6H), 19 FNMR(376MHz, acetone-d6)δ-80.9(m,2F),δ-117.7(s,2F),δ-124.3(s,2F),δ-126.0(s,2F), 31 PNMR (162MHz, acetone-d6), δ-148.4(s)

[0231] Industrial Applicability

[0232] The present invention provides a perfluoroalkyl group-containing nucleic acid and a method for producing the same. The nucleic acid of the present invention has excellent cell membrane permeability and is therefore expected to be used in the pharmaceutical field as a physiologically active substance such as a carrier for introducing a drug component into a target cell.

[0233] It should be noted that the entire contents of the specification, claims, drawings, and abstract of Japanese Patent Application No. 2019-177622 filed on September 27, 2019 are cited herein and adopted as the disclosed contents of the specification of the present invention.

Claims

1. A nucleic acid comprising a perfluoroalkyl group having 2 to 10 carbon atoms, wherein the perfluoroalkyl group has 1 to 5 oxygen atoms in the form of ether bonds between carbon atoms. Having a structure represented by the following general formula (A1), in, The groups in the formula represent the following meanings: R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 oxygen atoms with ether bonds between carbon atoms. na is an integer of 1 to 10, and a black dot represents a bonding site.

2. The nucleic acid according to claim 1, wherein The perfluoroalkyl group is directly or indirectly bonded to the 5' end or 3' end of the nucleic acid.

3. The nucleic acid according to claim 1, wherein The perfluoroalkyl group is indirectly introduced between two nucleotides. The nucleic acid according to any one of claims 1 to 3, which is a cell membrane permeable nucleic acid.

5. A method for producing a perfluoroalkyl group-containing nucleic acid, comprising synthesizing the perfluoroalkyl group-containing nucleic acid according to any one of claims 1 to 4 by a phosphoramidite method using a compound represented by the following general formula (A2) as a raw material, in, The groups in the formula represent the following meanings: R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 oxygen atoms with ether bonds between carbon atoms, na is an integer of 1 to 10, DMTr is 4,4'-dimethoxytriphenylmethyl, and i-Pr is isopropyl.

6. A nucleic acid pharmaceutical comprising as an active ingredient a nucleic acid containing a perfluoroalkyl group having 2 to 10 carbon atoms, wherein the perfluoroalkyl group has 1 to 5 oxygen atoms in the form of ether bonds between carbon atoms. The nucleic acid has a structure represented by the following general formula (A1): in, The groups in the formula represent the following meanings: R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 oxygen atoms with ether bonds between carbon atoms. na is an integer of 1 to 10, and a black dot represents a bonding site.

7. A method for improving the cell membrane permeability of nucleic acid, comprising introducing a perfluoroalkyl group having 2 to 10 carbon atoms into the nucleic acid to improve the cell membrane permeability, wherein the perfluoroalkyl group has 1 to 5 oxygen atoms in the form of ether bonds between carbon atoms. The nucleic acid has a structure represented by the following general formula (A1): in, The groups in the formula represent the following meanings: R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 oxygen atoms with ether bonds between carbon atoms. na is an integer of 1 to 10, and a black dot represents a bonding site.

8. The method for improving the cell membrane permeability of nucleic acid according to claim 7, wherein: The perfluoroalkyl group is directly or indirectly bonded to the 5' end or 3' end of the nucleic acid.

9. A compound represented by the following general formula (A2), in, The groups in the formula represent the following meanings: R FE It is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 oxygen atoms with ether bonds between carbon atoms, na is an integer of 1 to 10, DMTr is 4,4'-dimethoxytriphenylmethyl, and i-Pr is isopropyl.

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