Perfluoroalkyl group-containing nucleic acid and method for producing the same

Abstract

To provide a nucleic acid with excellent cell membrane permeability and a production method therefor.SOLUTION: The present invention provides a nucleic acid containing a C2-10 perfluoroalkyl group having 1 to 5 ether-bonding oxygen atoms between carbon atoms, the nucleic acid having a structure represented by the following general formula (A1), where the groups in the formula have the following meanings: RFE is a C2-10 perfluoroalkyl group having 1 to 5 ether-bonding oxygen atoms between carbon atoms, na is an integer of 1 to 10, and the black circle indicates a bonding site.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid having a perfluoroalkyl group introduced therein and a method for producing the same. [Background technology]

[0002] Antibody drugs are excellent as treatments for cancer and intractable diseases because they can be made into drugs by using the immune system to create antibodies against proteins that cannot be targeted by small molecule drugs. Antibody drugs also have the advantage of being highly specific to their target molecules and having few 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 using oligonucleotides is underway as the next generation of drug discovery after antibody drugs. Nucleic acid drugs have the advantages of high specificity for target molecules and minimal side effects. However, like antibody drugs, nucleic acid drugs have low cell membrane permeability, making it difficult to deliver them to target molecules present within cells. In particular, because siRNA is double-stranded, it has a larger molecular weight and negative charge than antisense RNA, making its cell membrane permeability lower than that of antisense RNA, necessitating drug delivery using a carrier. Known drug delivery agents include those using lipid nanoparticles (Patent Document 1) and those using cationic polymer nanoparticles (Patent Document 2). However, there is much that needs to be improved in terms of cell membrane permeability efficiency and toxicity concerns.

[0004] On the other hand, compounds with polyfluoro structures are known to be stable and low-toxic in vivo, and to have excellent cellular uptake and endosome escape properties (Non-Patent Document 1). Taking advantage of this property, it has been reported that peptide dendrimers using lysine, whose side chain amino group is perfluoroacylated as a constituent amino acid, can be used for gene delivery (Non-Patent Document 2). Furthermore, studies are also being conducted on the introduction of polyfluoro structures into oligonucleotides and peptide nucleic acids as moieties capable of penetrating cell membranes (Patent Document 3, and Non-Patent Documents 3-6). [Prior art documents] [Patent documents]

[0005] [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 Application Laid-Open No. 2006-321797 [Patent Document 5] International Publication No. 2000 / 056694 [Non-patent literature]

[0006] [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. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a nucleic acid having excellent cell membrane permeability and a method for producing the same. [Means for solving the problem]

[0008] The present inventors have discovered that the cell membrane permeability of nucleic acids can be improved by introducing a perfluoroalkyl group, particularly a perfluoroalkyl group having an ether-bonded oxygen atom, and have completed the present invention.

[0009] That is, the present invention is as follows. [1] 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. [2] The nucleic acid according to [1] above, wherein the perfluoroalkyl group is directly or indirectly bound to the 5' or 3' end of the nucleic acid. [3] The nucleic acid of [1], wherein the perfluoroalkyl group is indirectly introduced between two nucleotides. [4] The nucleic acid according to any one of [1] to [3] above, which has a structure represented by the following general formula (A1):

[0010] [ka]

[0011] However, the groups in the formula have the following meanings. R FE is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms, na is an integer of 1 to 10, and a black circle represents a bond. [5] The nucleic acid according to any one of [1] to [4] above, which is a cell membrane-permeable nucleic acid. [6] A method for producing a nucleic acid containing a perfluoroalkyl group, which comprises synthesizing a nucleic acid containing a perfluoroalkyl group of any one of the above [1] to [5] by a phosphoramidite method using a compound represented by the following general formula (A2) as a raw material:

[0012] [ka]

[0013] However, the groups in the formula have the following meanings. R FE is a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms, na is an integer of 1 to 10, DMTr is a 4,4'-dimethoxytriphenylmethyl group, and i-Pr is an isopropyl group. [7] A nucleic acid drug, the active ingredient of which is a nucleic acid containing a perfluoroalkyl group having 2 to 10 carbon atoms, which may have 1 to 5 ether-bonding oxygen atoms between carbon atoms. [8] A method for improving the cell membrane permeability of nucleic acids, which comprises introducing a perfluoroalkyl group having 2 to 10 carbon atoms, which may have 1 to 5 ether-bonding oxygen atoms between carbon atoms, into a nucleic acid to improve the cell membrane permeability. [9] The method for improving cell membrane permeability of nucleic acid according to [8] above, wherein the perfluoroalkyl group is a group having 1 to 5 ether-bonding oxygen atoms between carbon atoms.

[10] The method for improving cell membrane permeability of nucleic acid according to [8] above, wherein the perfluoroalkyl group is a group that does not have an ether-bonding oxygen atom between carbon atoms.

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

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

[0014] The nucleic acid according to the present invention has excellent cell membrane permeability due to the introduction of a perfluoroalkyl group, and is therefore expected to be used as a physiologically active substance in the pharmaceutical field. According to the production method of the present invention, a nucleic acid into which a perfluoroalkyl group has been introduced can be produced. [Brief explanation of the drawings]

[0015] [Figure 1A] FIG. 1 shows the results of flow cytometry of cells that were taken up with a fluorescein-modified nucleic acid modified with N[C8-PFC] at the 5′ end in Test Example 1. [Figure 1B] FIG. 1 shows the results of flow cytometry of cells that were taken up with a fluorescein-modified nucleic acid modified with N[C8-PFC] at the 3' end in Test Example 1. [Figure 2A] FIG. 1 shows the results of flow cytometry of cells that were incubated for 4 hours to incorporate fluorescein-modified double-stranded nucleic acids annealed in each combination in Test Example 1. [Figure 2B] FIG. 1 shows the results of flow cytometry of cells that were incubated for 24 hours to incorporate fluorescein-modified double-stranded nucleic acids annealed in each combination in Test Example 1. [Figure 3] FIG. 10 shows the results of flow cytometry of cells that have been incorporated with fluorescein-modified nucleic acid modified with N[C5-PFPE] at the 5′ end in Test Example 2. [Figure 4]FIG. 1 shows the results of flow cytometry of cells into which fluorescein-modified nucleic acids of cont-DNA, FE5, and FE10 were incorporated in Test Example 2. [Figure 5] FIG. 1 shows the results of flow cytometry of cells that had been loaded with fluorescein-modified nucleic acids cont-DNA, FE(2)1, FE(2)2, and FE(2)5 in Experimental Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0016] In the present invention and this specification, the term "nucleic acid" refers to a molecule in which nucleotides are linked by phosphodiester bonds. Such 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. Artificial nucleotides include natural nucleotides in which the side chains have been modified with functional groups such as amino groups; nucleotides in which the hydroxyl group at the 2' position of the ribose backbone has been replaced with a methoxy group, a fluoro group, a methoxyethyl group, etc.; phosphorothioate nucleotides (nucleotides in which the oxygen atom of the phosphate group has been replaced with a sulfur atom); morpholino nucleotides (nucleotides in which ribose or deoxyribose has been replaced with a morpholine ring); bridged nucleic acid (BNA), hexitol nucleic acid (HNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), and cyclohexenyl nucleic acid (CeNA). Furthermore, "nucleic acid" includes molecules such as DNA and RNA in which only one or more naturally occurring nucleotides are linked by phosphodiester bonds, molecules in which one or more naturally occurring nucleotides are linked by phosphodiester bonds with one or more artificial nucleotides, and molecules in which only one or more artificial nucleotides are linked by phosphodiester bonds.

[0017] In the present invention and the specification of the present 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.

[0018] In the present invention and the specification of the present application, "C 1-10 alkyl group" is an alkyl group having 1 to 10 carbon atoms, which may be linear or branched. "C 2-10 alkyl group" is an alkyl group having 2 to 10 carbon atoms, which may be linear or branched. Examples of C 1-10 alkyl groups 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.

[0019] In the present invention and the specification of the present application, "C 1-6 alkyl group" is an alkyl group having 1 to 6 carbon atoms, which may be linear or branched. Examples of C 1-6 alkyl groups 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 and the like.

[0020] In the present invention and this specification, an "alkylene group" is a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, and may be straight-chain or branched. Examples of the alkylene group include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a methylmethylene group, an ethylmethylene group, a methylethylene group, a methylpropylene group, an ethylethylene group, a dimethylmethylene group, a 1,2-dimethylethylene group, a 1,1-dimethylethylene group, a 1-ethylpropylene group, a 2-ethylpropylene group, and a 1,2-dimethylpropylene group. Examples of the alkyl group include a 2,2-dimethylpropylene group, a 1-propylpropylene group, a 2-propylpropylene group, a 1-methyl-1-ethylpropylene group, a 1-methyl-2-ethylpropylene group, a 1-ethyl-2-methylpropylene group, a 2-methyl-2-ethylpropylene group, a 1-methylbutylene group, a 2-methylbutylene group, a 3-methylbutylene group, a 2-ethylbutylene group, a 1-methylpentylene group, a 2-ethylpentylene group, and a 1-methylhexylene group.

[0021] In the present invention and the present specification, "C 1-10 A "perfluoroalkyl group" is a group in which all hydrogen atoms of an alkyl group having 1 to 10 carbon atoms have been substituted with fluorine atoms. 1-10 Examples of perfluoroalkyl groups include a perfluoromethyl group, a perfluoroethyl group, a perfluoropropyl group, a perfluoroisopropyl group, a perfluorobutyl group, a perfluoroisobutyl group, a perfluorosec-butyl group, a perfluorotert-butyl group, a perfluoropentyl group, a perfluoroisopentyl group, a perfluoroneopentyl group, a perfluorotert-pentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, and a perfluorodecyl group.

[0022] In the present invention and this specification, a "perfluoroalkylene group" is a group in which all hydrogen atoms of an alkylene group have been substituted with fluorine atoms. Examples of perfluoroalkylene groups include the above-mentioned alkylene groups in which all hydrogen atoms have been substituted with fluorine atoms.

[0023] In the present invention and the present specification, the term "ether-bonded oxygen atom" refers to an oxygen atom that connects carbon atoms, and does not include oxygen atoms in which oxygen atoms are connected in series. The maximum number of ether-bonded oxygen atoms that an alkyl group having Nc carbon atoms (Nc is an integer of 2 or more) can have is Nc-1. In addition, "a C alkyl group having an ether-bonded oxygen atom between carbon atoms" refers to an alkyl group having Nc carbon atoms (Nc is an integer of 2 or more). 2-10 "Alkyl group" means C 2-10 A group having at least one ether bond-forming oxygen atom between carbon atoms of the alkyl group. Hereinafter, this may be referred to as an "ether bond-containing alkyl group."

[0024] In the present invention and the present specification, "C 2-10 "Perfluoroalkyl group" is C 2-10 Ether bond-containing C having at least one ether bond oxygen atom between the carbon atoms of the alkyl group 2-10 It is a group in which all hydrogen atoms of an alkyl group have been substituted with fluorine atoms. Hereinafter, a "perfluoroalkyl group having an ether-bonded oxygen atom between carbon atoms" may be referred to as an "ether bond-containing perfluoroalkyl group."

[0025] In the following description, "compound n" means a compound represented by formula (n).

[0026] <Nucleic acid containing an ether bond-containing perfluoroalkyl group> The nucleic acid according to the present invention is a nucleic acid containing a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms. Because the nucleic acid contains a perfluoroalkyl group, it is expected to be used in the pharmaceutical field as a physiologically active substance, similar to other fluorine-containing compounds.

[0027] The perfluoroalkyl group is not particularly limited as long as it has 2 to 10 carbon atoms and has 1 to 5 ether-bonded oxygen atoms between the carbon atoms. The perfluoroalkyl group contained in the nucleic acid according to the present invention is preferably, for example, a group represented by any one of the following general formulas (F1) to (F3).

[0028] [ka]

[0029] In general formula (F1), R F1 is a perfluoroalkylene group having q1 carbon atoms, and R F2 is a perfluoroalkyl group having q2 carbon atoms. q1 and q2 are natural numbers whose sum is 2 or more and 10 or less. In general formula (F2), R F3 is a perfluoroalkylene group having q3 carbon atoms, and R F4 is a perfluoroalkyl group having q4 carbon atoms. n1, q3, and q4 are natural numbers such that 2×n1+q3+q4 is 4 or more and 10 or less. In general formula (F3), R F5 is a perfluoroalkylene group having q5 carbon atoms, and R F6 is a perfluoroalkyl group having q6 carbon atoms. n2, q5, and q6 are natural numbers such that 3×n2+q5+q6 is 5 or more and 10 or less.

[0030] R in general formula (F1) F1 , R in general formula (F2) F3 and R in general formula (F3) F5 R may be a linear perfluoroalkylene group or a branched perfluoroalkylene group. F1 , R F3 , and R F5 As for C 1-3A perfluoroalkylene group is preferred, and a methylene group, an ethylene group, a methylmethylene group, an ethylmethylene group, a dimethylmethylene group, or a group in which all hydrogen atoms of a methylethylene group have been substituted with fluorine atoms is more preferred, a methylene group, a methylmethylene group, an ethylmethylene group, or a dimethylmethylene group in which all hydrogen atoms have been substituted with fluorine atoms is even more preferred, and a methylene group or a methylmethylene group in which all hydrogen atoms have been substituted with fluorine atoms is even more preferred.

[0031] R in general formula (F1) F2 , R in general formula (F2) F4 and R in general formula (F3) F6 R may be a linear perfluoroalkyl group or a branched perfluoroalkyl group. F2 , R F4 , and R F6 As for the C 1-5 Perfluoroalkyl groups are preferred, and linear C 1-5 Perfluoroalkyl groups are more preferred, and linear C 2-4 Perfluoroalkyl groups are more preferred.

[0032] The perfluoroalkyl group contained in the nucleic acid according to the present invention is, for example, R F1 is a methylene group or a methylmethylene group in which all hydrogen atoms have been substituted with fluorine atoms, and R F2 However, linear C 1-5 Compounds that are perfluoroalkyl groups; R in the general formula (F2) F3 is a group in which all hydrogen atoms of a methylene group or a methylmethylene group are substituted with fluorine atoms, n1 is 1, 2, or 3, and R F4 However, linear C 1-7 Compounds that are perfluoroalkyl groups; R in general formula (F3) F5 is a group in which all hydrogen atoms of a methylene group or a methylmethylene group are substituted with fluorine atoms, n2 is 1 or 2, and R F6 However, linear C 1-6 A compound in which R in the general formula (F1) is a perfluoroalkyl group is preferred.F1 is a methylene group or a methylmethylene group in which all hydrogen atoms have been substituted with fluorine atoms, and R F2 However, linear C 1-5 Compounds that are perfluoroalkyl groups; R in the general formula (F2) F3 is a group in which all hydrogen atoms of a methylene group or a methylmethylene group are substituted with fluorine atoms, n1 is 1, and R F4 However, linear C 1-6 Compounds that are perfluoroalkyl groups; R in the general formula (F2) F3 is a group in which all hydrogen atoms of a methylene group or a methylmethylene group are substituted with fluorine atoms, n1 is 2, and R F4 However, linear C 1-4 Compounds that are perfluoroalkyl groups; R in the general formula (F2) F3 is a group in which all hydrogen atoms of a methylene group or a methylmethylene group are substituted with fluorine atoms, n1 is 3, and R F4 However, linear C 1-3 Compounds that are perfluoroalkyl groups; R in general formula (F3) F5 is a group in which all hydrogen atoms of a methylene group or a methylmethylene group are substituted with fluorine atoms, n2 is 1, and R F6 However, linear C 1-6 Compounds which are perfluoroalkyl groups are more preferred.

[0033] In the present invention, the ether bond-containing perfluoroalkyl group introduced into the nucleic acid can be, for example, an ether bond-containing perfluoroalkyl group contained in a fluorine compound described in Patent Document 4 or 5 together with its synthesis method.

[0034] In the nucleic acid of the present invention, the position at which the ether bond-containing perfluoroalkyl group is introduced is not particularly limited, and it may be introduced at any site as long as the function of the nucleic acid is not impaired. For example, the ether bond-containing perfluoroalkyl group may be directly or indirectly bound to the 5'-end or 3'-end of the nucleic acid, or may be introduced between two nucleotides. When the ether bond-containing perfluoroalkyl group is indirectly introduced into the nucleic acid via a linking group, the linking group is not particularly limited as long as it does not impair the effects of the present invention, and any divalent or trivalent organic group can be used. Examples of the linking group include alkylene groups, alkenylene groups, carbonyl groups, amino groups, ether bonds, thioether bonds, ester bonds, amide bonds, polyethylene glycol groups (PEG:-(CHO)-), siloxane bonds, silyl ether bonds, sugars, and peptides. Furthermore, groups in which two or three hydrogen atoms have been removed from a ring such as a pyrrole ring, a pyrazole ring, an imidazole ring, a triazole ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, an oxazole ring, a thiazole ring, a furan ring, a thiophene ring, or a benzene ring can also be used as the linking group. Furthermore, these can also be used in appropriate combinations.

[0035] In the nucleic acid according to the present invention, the nucleic acid into which the ether bond-containing perfluoroalkyl group is introduced is not particularly limited, and may be a nucleic acid in which all nucleotides contained therein are natural nucleotides, or a nucleic acid in which some or all of the nucleotides contained therein are artificial nucleotides. Furthermore, the nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. Examples of the nucleic acid include genomic DNA, cDNA, mRNA, microRNA, siRNA, antisense oligonucleotide, nucleic acid aptamer, decoy nucleic acid, and CpG (cytosine-phosphate-guanine) oligonucleotide. Furthermore, the nucleic acid may be an expression vector that expresses a target gene or siRNA in a cell.

[0036] In the nucleic acids according to the present invention, the ether bond-containing perfluoroalkyl group can be introduced into the target nucleic acid by various coupling reactions. For example, the ether bond-containing perfluoroalkyl group can be easily introduced into the nucleic acid by performing the phosphoramidite method using a phosphoramidite containing an ether bond-containing perfluoroalkyl group as a starting material. Widely used automated nucleic acid synthesizers utilize the phosphoramidite method. Therefore, by using a phosphoramidite containing an ether bond-containing perfluoroalkyl group as a starting material, nucleic acids with ether bond-containing perfluoroalkyl groups introduced at desired positions can be easily synthesized using an automated synthesizer.

[0037] Examples of phosphoramidites containing an ether bond-containing perfluoroalkyl group include compounds in which the nucleoside moiety of phosphoramidites generally used in nucleic acid synthesis is substituted with an organic group containing an ether bond-containing perfluoroalkyl group, such as the compound represented by the following general formula (A2):

[0038] [ka]

[0039] In general formula (A2), R FE is the above-mentioned ether bond-containing perfluoroalkyl group, and na is an integer of 1 to 10. i-Pr represents an isopropyl group, and DMTr represents a 4,4'-dimethoxytriphenylmethyl group.

[0040] The nucleic acid synthesized by the phosphoramidite method using the compound (A2) has a structure represented by the following general formula (A1): FE is the above-mentioned ether bond-containing perfluoroalkyl group, and na is an integer of 1 to 10. The black circle represents a bond.

[0041] [ka]

[0042] 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.

[0043] The nucleic acid of the present invention may be modified in various ways, such as sugar chain modification, lipid modification, and peptide modification, as long as the modification does not impair the function of the nucleic acid to which the ether bond-containing perfluoroalkyl group is introduced and does not impair the effects of the present invention.

[0044] The perfluoro structure has a high affinity for cell membranes. Therefore, the nucleic acid according to the present invention, into which an ether bond-containing perfluoroalkyl group has been introduced, has better cell membrane permeability than nucleic acids without such a group. Utilizing this property, the nucleic acid according to the present invention is particularly preferred as an active ingredient in nucleic acid medicines. For example, by introducing an ether bond-containing perfluoroalkyl group into a functional nucleic acid that exhibits some physiological activity upon being taken up into target cells in the body without impairing its function, the uptake efficiency of the functional nucleic acid into the target cells can be improved.

[0045] <Method for improving cell membrane permeability of nucleic acids> The nucleic acid may have 1 to 5 ether-bonding oxygen atoms between carbon atoms, C 2-10 By introducing a perfluoroalkyl group, the cell membrane permeability of the nucleic acid can be improved. 2-10 "Perfluoroalkyl group" means "a group having 1 to 5 ether-bonded oxygen atoms between carbon atoms, 2-10 perfluoroalkyl group" and "C 2-10 and a "perfluoroalkyl group."

[0046] "C has 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 a nucleic acid can be carried out by the phosphoramidite method using a phosphoramidite containing an ether bond-containing perfluoroalkyl group, as described above.

[0047] C, which does not have an ether-bonded oxygen atom 2-10 Similarly, perfluoroalkyl groups can be introduced into nucleic acids by using C 2-10 It can be carried out by the phosphoramidite method using a phosphoramidite containing a perfluoroalkyl group. In addition, it is also possible to form a C 2-10 Perfluoroalkyl groups can be introduced.

[0048] It is sufficient that a perfluoro structure is introduced into the nucleic acid, and 1 to 5 ether-bonding oxygen atoms may be present between carbon atoms. 2-10 The perfluoroalkyl group may be introduced into any position of the nucleic acid. 2-10 The position of the nucleic acid where the perfluoroalkyl group is introduced can be determined arbitrarily as long as the function of the nucleic acid is not impaired.

[0049] C, which does not have an ether-bonded oxygen atom 2-10 Nucleic acids in which cell membrane permeability has been improved by the introduction of a perfluoroalkyl group are also preferred as active ingredients of nucleic acid medicines, similar to nucleic acids in which an ether bond-containing perfluoroalkyl group has been introduced.

[0050] By applying the method of improving the cell membrane permeability of nucleic acids according to the present invention to nucleic acids that have pharmacological activity but cannot reach the interior of cells, the uptake efficiency of the nucleic acids into target cells can be improved. In other words, the method of improving the cell membrane permeability of nucleic acids according to the present invention makes it possible to easily construct a drug delivery system that delivers nucleic acid drugs into cells.

Example

[0051] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0052] 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 a reference value of -162 ppm.

[0053] <Tm measurement> In the subsequent experiments, the melting temperature (Tm) of the nucleic acid 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 cooled to room temperature for annealing. The absorbance at 260 nm was measured and detected at 0.5 °C intervals 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 by the midpoint method.

[0054] <Cell culture> In the subsequent experiments, cell culture was performed as follows. HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, manufactured by Thermo Fischer Scientific) supplemented with 10% FBS and 0.5% penicillin / streptomycin in a humidified atmosphere at 37 °C (5% CO2 by volume). Cells for image analysis were cultured in 35 mm glass-bottom dishes (manufactured by IWAKI).

[0055] <Confocal microscopy observation> In the subsequent experiments, confocal microscopy observation of cells was performed as follows. Fluorescein-conjugated N[PFC] or N[PFPE] (500 μM, 5 μL) was added to HeLa cells and incubated at 37°C for 3 hours. The nuclear stain Hoechst 33342 (2 μg / mL, 0.5 mL) was then added to the cells and incubated for an additional hour. After incubation, the solution was removed from each dish, washed with PBS (-), and DMEM (1 mL) was added. Each dish was then placed on a confocal laser scanning microscope, and fluorescence images were acquired using an excitation wavelength of 488 nm and an emission filter above 505 nm.

[0056] <Flow cytometry> In the following experiments, flow cytometry was performed as follows. HeLa cells were plated at 10 5 Cells were seeded at 1000p / well and cultured. The day after seeding, the medium in each well was replaced with 1 mL of DMEM medium containing fluorescein-conjugated N[PFC] or N[PFPE]-modified DNA (2.5 μM) and incubated for 4 or 24 hours. The cell layer in the well was then washed twice with PBS and detached by treatment with 0.05% (w / v) trypsin (200 μL) at 37°C for 5 minutes. The recovered cells were suspended in 600 μL of DMEM. The cell suspension was separated by centrifugation (400×G, 3 minutes) and PBS / 1% BSA (500 μL) was added. The percentage of fluorescent cells and mean fluorescence intensity of the cell suspension were analyzed using a flow cytometer (guava easyCyte8, Luminex). For double-stranded DNA, samples were denatured at 100°C and slowly cooled to room temperature to anneal.

[0057] [Example 1] A phosphoramidite containing a five-carbon perfluoroalkyl group (CF3CF2O(CF2)2O(CF2)-: hereinafter referred to as C5-PFPE group) with two ether-bonded oxygen atoms was synthesized, and nucleic acids containing C5-PFPE groups were produced using this by the amidite method.

[0058] (1) Synthesis of C5-PFPE carboxylic acid The carboxylate containing a C5-PFPE group was synthesized by the method described in Patent Document 4. A 2-L Hastelloy C autoclave was charged with 300 g of CH3CHO(CH2)2O(CH2)2OH. The reactor was cooled, and 1339 g of CF3CF2CF2OCF(CF3)CF2OCF(CF3)COF was slowly introduced into the autoclave while stirring, keeping the internal temperature below 30°C. After the entire amount was introduced, the mixture was stirred at 30°C for an additional 3 hours. The HF generated during the reaction was then purged by bubbling nitrogen gas through the system to obtain the product. GC (gas chromatographic) analysis of the product revealed that CH3CHO(CH2)2O(CH2)2OCOCF(CF3)OCF2CF(CF3)OCF2CF2CF3 was 99.6% formed, with no unreacted CH3CHO(CH2)2O(CH2)2OH detected. This product was used in the next step without purification.

[0059] R-113 (312 g) was added to a 500 mL nickel autoclave and stirred to maintain the temperature at 25°C. A condenser maintained at 20°C, a NaF pellet packed bed, and a condenser maintained at -10°C were installed in series at the gas outlet of the autoclave. A liquid return line was also installed from the condenser maintained at -10°C to return the condensed liquid to the autoclave. Nitrogen gas was blown into the autoclave at room temperature for 1 hour, and then fluorine gas diluted to 20% with nitrogen gas (hereinafter referred to as 20% diluted fluorine gas) was blown into the autoclave at a flow rate of 17.04 L / h for 1 hour at room temperature. Next, while blowing the 20% diluted fluorine gas at the same flow rate, a solution of the product (10 g) obtained above in R-113 (150 g) was injected over 4.1 hours.

[0060] Next, the pressure inside the autoclave was increased to 0.15 MPaG while blowing in 20% diluted fluorine gas at the same flow rate. 9 mL of an R-113 solution with a benzene concentration of 0.01 g / mL was injected while raising the temperature from 25°C to 40°C, the benzene solution inlet of the autoclave was closed, and stirring was continued for 0.3 hours. Next, while maintaining the pressure inside the reactor at 0.15 MPaG and the temperature inside the reactor at 40°C, 6 mL of the benzene solution was injected, the benzene solution inlet of the autoclave was closed, and stirring was continued for 0.3 hours. The same operation was repeated once more. The total amount of benzene injected was 0.22 g, and the total amount of R-113 injected was 21 mL. Thereafter, stirring was continued for 1 hour while blowing in 20% diluted fluorine gas at the same flow rate. Next, the pressure inside the reactor was returned to normal pressure, and nitrogen gas was blown in for 1 hour. The product was 19 F NMR analysis revealed that the target compound (CF3CF2O(CF2)2O(CF2)2OCOCF(CF3)OCF2CF(CF3)OCF2CF2CF3) was contained in a yield of 99%. 1 As a result of analysis by 1 H NMR and GC-MS, no compound having a C—H bond was confirmed.

[0061] The 2 L distillation column vessel equipped with a reflux condenser at 10°C was charged with the CF3CF2O(CF2)2O(CF2)2OCOCF(CF3)OCF2CF(CF3)OCF2CF2CF3 (4273 g) obtained above, and potassium fluoride (12.6 g) was added. The mixture was heated and stirred (heat medium temperature: 100-130°C), and a fraction was recovered by reactive distillation. As the main fraction, 1273 g of a fraction with a purity of 99% or more was recovered. The boiling point was 66.5°C, and the yield was 84.5%. 1 As a result of analysis by 1 HNMR and GC-MS, no compound having a C—H bond was confirmed.

[0062] The resulting CF3CF2O(CF2)2OCF2COF (107 g) was placed in a 200 mL Hastelloy autoclave, and under ice cooling and vigorous stirring, water (6 g) was slowly added dropwise to carry out hydrolysis. After the addition, the temperature was gradually raised to room temperature, and stirring was continued for an additional 5 hours. The HF generated by the reaction was then expelled from the system by bubbling nitrogen gas, and simple distillation was continued, yielding 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%.

[0063] (2) Synthesis of C5-PFPE alcohol The C5-PFPE carboxylic acid obtained in (1) above was reduced to obtain an alcohol containing a C5-PFPE group. A carboxylate salt 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 condenser. Sodium borohydride (2.84 g, 75 mmol) was then added to the flask in an ice bath below 15°C, followed by the dropwise addition of boron trifluoride etherate (9.42 mL, 75 mmol). The reaction mixture was refluxed for 24 hours, cooled to 5°C, and distilled water was added until gas evolution ceased. The reaction mixture was then extracted three times with dichloromethane. All organic phases were combined, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated to obtain the target alcohol compound (yield 67%).

[0064] (3) Synthesis of trifluoromethanesulfonate Trifluoromethanesulfonates containing C5-PFPE groups were synthesized.

[0065] [ka]

[0066] 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 cooling to -78 °C. Trifluoromethanesulfonic anhydride (6.34 g, 22.5 mmol) was added dropwise to the reaction mixture over 30 minutes, causing the reaction mixture to turn dark. The reaction mixture was stirred at 0 °C for 1 hour, then warmed to room temperature and stirred for an additional 3 hours. The reaction was then quenched by the addition of saturated NaHCO3. The reaction product was added with NaHCO3 (50 mL) and extracted three times with dichloromethane (50 mL). All organic phases were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum to give the desired compound as a thick black oil (yield 27.3%). The resulting crude product was 1 Analyzed by 1 H NMR and used in the next step without further purification.

[0067] 1 H NMR(400MHz, CDCl3) δ4.70(t,J=7.8Hz,2H). 19 F NMR(376MHz,CDCl3) δ-74.1(s,3F),δ-77.6(s,2F),δ-86.6(s,3F),δ-88.2--88.7(m,6F).

[0068] (4) Diethanolamine The trifluoromethanesulfonate obtained in (3) above was converted to diethanolamine.

[0069] [ka]

[0070] A solution of trifluoromethanesulfonate (1.6 g, 3.5 mmol) obtained in (3) above and diethanolamine (0.74 g, 7.0 mmol) was added to dry DMF (N,N-dimethylformamide) (5 mL). The resulting reaction mixture was placed in an oil bath at 100°C and stirred for 21 hours. The reaction mixture was then cooled and dissolved in water (20 mL), and the product was extracted three times with dichloromethane (20 mL). All organic phases were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum to obtain the target compound. The resulting crude product was 1 Analyzed by 1 H NMR and used in the next step without further purification.

[0071] 1 H NMR(400MHz, CDCl3) δ3.58(t,J=5.0Hz,4H),δ3.24(t,J=11.0Hz,2H),δ2.86(t,J=5.5Hz,4H).

[0072] (5)DMTr protection One of the two hydroxyl groups of the diethanolamine obtained in (4) above was protected with DMTr (4,4'-dimethoxytriphenylmethyl group).

[0073] [ka]

[0074] 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 portionwise to the reaction mixture, which was then stirred at room temperature for 2 hours. The solvent was then removed under vacuum to give a dark brown oil. This oil was purified by column chromatography using a silica column pretreated with triethylamine and a mixture of EtOAc and hexane (volume ratio 1:4) to give the target compound as a yellow oil (yield 238 mg, 15.2%).

[0075] 1 H NMR(400MHz,CDCl3) δ7.40(t,J=8.2Hz,2H), δ7.33-7.20(m,7H),δ 6.82(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),δ 2.81(t,J=5.0Hz,2H),δ2.44(t,J=6.0Hz,1H). 19 F NMR(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).

[0076] (6) Amidite formation In the diethanolamine obtained in (5) above, the hydroxyl group not protected with a DMTr group was converted into an amidite.

[0077] [ka]

[0078] 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile was dissolved in dry acetonitrile. 5-(Ethylthiotetrazole) (ETT) (65 mg, 0.5 mmol) was added to the reaction mixture under argon, followed by the addition in portions of a solution of the DMTr-protected diethanolamine (0.24 mg, 0.33 mmol) obtained in (5) above dissolved in THF (1 mL) and acetonitrile (1 mL). The reaction mixture was stirred at room temperature under argon for 4 hours, and the solvent was evaporated under reduced pressure. The resulting crude product was purified by column chromatography under argon using degassed hexane / ethyl acetate (1:4 volume ratio) as the mobile phase, and the target compound (N[C5-PFPE] amidite) was isolated as a colorless oil (yield 130 mg, 43%).

[0079] 1H NMR(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=11.4Hz,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 F NMR(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 P NMR(162MHz,Acetone-d6) δ-148.0(m).

[0080] (7) DNA synthesis DNA synthesis was carried out 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 an NTS H-8 DNA / RNA synthesizer (manufactured by Nippon Techno Service Co., Ltd.).

[0081] 5'-N[C5-PFPE]-modified DNA, in which an N[C5-PFPE] group was attached to the 5' end of an oligonucleotide consisting of SEQ ID NO: 1 (5'-TTTTTCAGTTGACCATATA-3'), was synthesized as follows. First, DNA synthesis (trityl-off) was performed on a 1000 Å CPG solid support column (1 μmole scale). Next, under a nitrogen atmosphere, the N[C5-PFPE] amidite (0.1 M acetonitrile solution, 300 μL) synthesized in (6) above and the activator solution (300 μL) were mixed using a syringe in the presence of CPG. After 5 minutes of mixing, the solution was removed from the column, and the strands were capped, oxidized, and deblocked using a DNA synthesizer (yield 11.0%).

[0082] 5'-N[C5-PFPE] modified DNA Sequence: 5'-(N[C5-PFPE])TTTTTCAGTTGACCATATA-3'LRMS(MALDI)[MH] - :6245.0(calcd), 6244.2(found)

[0083] 3'-N[C5-PFPE]-modified DNA was synthesized by attaching an N[C5-PFPE] group to the 3' end of an oligonucleotide consisting of SEQ ID NO: 2 (5'-TATATGGTCAACTGAAAAA-3'), which is the complementary base sequence of SEQ ID NO: 1, as follows. First, under a nitrogen atmosphere, the N[C5-PFPE] amidite (0.1 M acetonitrile solution, 300 μL) synthesized in (4) above and an activator solution (300 μL) were mixed using a syringe in the presence of 1000 Å of Glen UnySupport® (1 μmole scale). After 5 minutes of mixing, the solution was removed from the column, and the strands were capped, oxidized, and deblocked using a DNA synthesizer. Next, DNA oligomers were synthesized using the standard phosphoramidite method using a DNA synthesizer (7.9% yield).

[0084] 3'-N[C5-PFPE] modified DNA Sequence: 5'-TATATGGTCAACTGAAAAA(N[C5-PFPE])-3'LRMS(MALDI)[MH] - :6321.1(calcd), 6321.3(found)

[0085] Deprotection of nucleic acids synthesized using a DNA synthesizer was carried out as follows. First, DNA (trityl-off) supported on a CPG solid was treated with an aqueous ammonium hydroxide solution (28%) at 50°C for 12 hours. The crude product solution was then separated from the solid support and concentrated under reduced pressure at 30°C. The resulting concentrate was then filtered through a 0.45 μm centrifugal filter and purified by HPLC. The resulting solution was quantified by absorbance at 260 nm.

[0086] HPLC purification was carried out under the following conditions. 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 min), Column: COSMOSIL packed column "5C18-MS-II" (4.6 ID x 150 nm, manufactured by Nacalai Tesque), Crude DNA to be tested: A solution dissolved in 20 to 50 μL of ultrapure water was injected. Detection: The absorbance at 260 nm was measured with a diode array detector.

[0087] The Tm values ​​of the synthesized nucleic acids were measured. For Tm value measurements, RNA with a base sequence corresponding to the DNA before N[C5-PFPE] modification was used. The nucleic acids used for the measurements are shown in Table 1, and the Tm value measurement results are shown in Table 2.

[0088] [Table 1]

[0089] [Table 2]

[0090] The Tm value of the N[C5-PFPE]-modified DNA was almost the same as that of the unmodified DNA. When annealed with RNA, the Tm value increased slightly.

[0091] [Example 2] A perfluoroalkyl group having 8 carbon atoms and no ether-bonded oxygen atom (-CF 17 A phosphoramidite containing a C8-PFC group (hereinafter referred to as a C8-PFC group) was synthesized, and a nucleic acid containing a C8-PFC group was produced using this by the amidite method.

[0092] The phosphoramidite containing a C8-PFC group represented by the following formula was synthesized by the method described in Non-Patent Document 5.

[0093] [ka]

[0094] [ka]

[0095] A 5'-N[C8-PFC] modified DNA in which an N[C8-PFC] group was attached to the 5' end of an oligonucleotide consisting of SEQ ID NO: 1, and a 3'-N[C8-PFC] modified DNA in which an N[C8-PFC] group was attached to the 3' end of an oligonucleotide consisting of SEQ ID NO: 2, were synthesized in the same manner as in Example 1, except that the obtained phosphoramidite containing a C8-PFC group was used.

[0096] The Tm values ​​of the synthesized nucleic acids were measured. For the Tm value measurement, RNA having a base sequence corresponding to the DNA before N[C8-PFC] modification was used, as in Example 1. The nucleic acids used for the measurement are shown in Table 3, and the Tm value measurement results are shown in Table 4.

[0097] [Table 3]

[0098] [Table 4]

[0099] When N[C8-PFC]-modified DNA was annealed to itself, the Tm tended to increase with the number of N[C8-PFC] introduced. When N[C8-PFC]-modified DNA was annealed to DNA or RNA, the Tm tended to be higher when two N[C8-PFC] moieties were introduced rather than one. However, when five N[C8-PFC] moieties were introduced, the Tm was lower than that of unmodified DNA or unmodified RNA.

[0100] [Test Example 1] The nucleic acid synthesized in Example 2, in which fluorescein was bound to the 3' end of the N[C8-PFC] modified DNA, was added to the culture medium of HeLa cells either as a single strand or as a double strand, and incubated. The efficiency of uptake into the cells was examined by flow cytometry.

[0101] Figure 1A shows the results of flow cytometry of cells that had been transfected with cont-DNA, F1, F2, and F5 fluorescein-modified nucleic acids, and Figure 1B shows the results of flow cytometry of cells that had been transfected with cont-rDNA, rF1, rF2, and rF5 fluorescein-modified nucleic acids. For single-stranded nucleic acids with N[C8-PFC] modification at the 3' end, all N[C8-PFC]-modified nucleic acids were transfected into cells at higher levels than unmodified nucleic acids (cont-rDNA) (Figure 1B). Among single-stranded nucleic acids modified with N[C8-PFC] at the 5' end, F1, which had one N[C8-PFC] introduced, showed no difference from the fluorescein-modified nucleic acid of cont-DNA, but F2 and F5, which had two or five N[C8-PFC] introduced, showed higher intracellular uptake than the fluorescein-modified nucleic acid of cont-DNA (Figure 1A).

[0102] Figure 2 shows the results of flow cytometry of cells that were incubated with fluorescein-modified double-stranded nucleic acids annealed in each combination. Figure 2A shows the results for cells incubated for 4 hours, and Figure 2B shows the results for cells incubated for 24 hours. The double-stranded nucleic acid composed of F2 and rF2 was taken up most significantly into the cells, and the double-stranded nucleic acid composed of cont-DNA and rF2 was taken up more significantly than the double-stranded nucleic acid composed of cont-DNA and cont-rDNA.

[0103] These results indicate that modifying nucleic acids with N[C8-PFC] increases the amount of nucleic acid taken up into cells and improves its uptake efficiency. This improvement in the efficiency of uptake into cells is due to the improved cell membrane permeability of nucleic acids resulting from the N[C8-PFC] modification.

[0104] [Test Example 2] The nucleic acid synthesized in Example 1, in which fluorescein was bound to the 3' end of the N[C5-PFPE]-modified DNA, was added as a single strand to the culture medium of HeLa cells and incubated, and the efficiency of uptake into the cells was examined by flow cytometry.

[0105] Figure 3 shows the results of flow cytometry of cells that had been loaded with fluorescein-modified nucleic acids (cont-rDNA, FE1, and FE2). The single-stranded nucleic acids modified with N[C5-PFPE] at the 5' end were loaded into cells in greater amounts than unmodified nucleic acids (cont-rDNA).

[0106] In the same manner as above, a 5'-N[C5-PFPE]-modified DNA was synthesized by attaching an N[C5-PFPE] group to the 5' end of an oligonucleotide consisting of SEQ ID NO: 1. The synthesized nucleic acids are shown in Table 5.

[0107] [Table 5]

[0108] Figure 4 shows the results of flow cytometry of cells that had been loaded with fluorescein-modified nucleic acids (cont-rDNA, FE5, and FE10). The single-stranded nucleic acids modified with N[C5-PFPE] at the 5' end were all loaded into cells in greater amounts than unmodified nucleic acids (cont-rDNA).

[0109] [Example 3] A phosphoramidite containing a seven-carbon perfluoroalkyl group (CF3CF2O(CF2)2O(CF2)2O(CF2)-: hereinafter referred to as C7-PFPE group) with three ether-bonded oxygen atoms was synthesized, and this was used to produce a nucleic acid containing a C7-PFPE group by the amidite method.

[0110] (1) Synthesis of C7-PFPE carboxylic acid 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 pyrolysis method described in International Publication WO2000 / 056694. The obtained CF3CF2(OCF2CF2)2OCF2COF was treated in the same manner as in the synthesis of the C5-PFPE carboxylic acid to obtain CF3CF2(OCF2CF2)2OCF2COOH (C7-PFPE carboxylic acid).

[0111] (2) Synthesis of C7-PFPE alcohol In the same manner as in the synthesis of the C5-PFPE alcohol, CF3CF2(OCF2CF2)2OCF2CH2OH (C7-PFPE alcohol) was obtained from C7-PFPE carboxylic acid.

[0112] (3) Synthesis of trifluoromethanesulfonate Trifluoromethanesulfonates containing C7-PFPE groups were synthesized.

[0113] [ka]

[0114] The C7-PFPE alcohol (11.2 g, 25.0 mmol) was dissolved in water (5 mL), followed by the addition of triethylamine (5 mL) and cooling to 0° C. To this reaction mixture, trifluoromethanesulfonic acid chloride (4.42 g, 26.3 mmol) was added dropwise 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 over MgSO4, and the solvent was removed under vacuum to give the desired compound as an oil. The resulting crude product was 1 It was analyzed by H NMR and used in the next step without further purification (yield 64%).

[0115] 1 H NMR (400 MHz, CDCl3) δ 4.67 (t, J = 8.2 Hz, 2H) 19 F NMR (376 MHz, CDCl3) δ -74.1 (s, 3F), δ -77.7 (s, 2F), δ -86.6 (s, 3F), δ -88.4 - -88.7 (m, 10F)

[0116] (4) Diethanolamine The trifluoromethanesulfonate containing the C7-PFPE group obtained in (3) above was converted to diethanolamine.

[0117] [ka]

[0118] A solution of the C7-PFPE group-containing trifluoromethanesulfonate (9.3 g, 16 mmol) obtained in (3) above and diethanolamine (3.36 g, 32 mmol) was added to dry DMF (N,N-dimethylformamide) (12 mL). The resulting reaction mixture was placed in an oil bath at 100°C and stirred for 15 hours. The reaction mixture was then cooled and dissolved in water (20 mL), and the product was extracted three times with dichloromethane (20 mL). All organic phases were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum to obtain the target compound. The resulting crude product was 1 Analyzed by 1 H NMR and used in the next step without further purification.

[0119] 1 H NMR (400 MHz, CDCl3) δ 3.34 (m, 4H), δ 3.09 (dt, J = 2.3, 11.0 Hz, 2H), δ 2.68 (m, 4H) 19 F NMR (376 MHz, CDCl3) δ -74.0 (s, 2F), δ -87.4 (s, 3F), δ -89.0 - -89.6 (m, 10F)

[0120] (5)DMTr protection One of the two hydroxyl groups of the diethanolamine containing the C7-PFPE group obtained in (4) above was protected with DMTr (4,4'-dimethoxytriphenylmethyl group).

[0121] [ka]

[0122] The C7-PFPE-containing diethanolamine (6.58 g, 12.3 mmol) obtained in (4) above was dissolved in dry dichloromethane (20 mL) and triethylamine (6 mL). DMTrCl (4.58 g, 13.5 mmol) was added portionwise to the reaction mixture, which was then stirred at room temperature for 2 hours. The solvent was then removed under vacuum to give a dark brown oily substance. This oily substance was purified by column chromatography using a silica column pretreated with triethylamine and a mixture of EtOAc and hexane (volume ratio 1:4) to give the target compound as a yellow oily substance (yield 37%).

[0123] 1 H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.3 Hz, 2H), δ 7.33-7.15 (m, 7H), δ 6.81 (dt, J = 9.1, 3.7 Hz, 4H), δ 3.78 (s, 6H), δ 3.50 (dd, J = 6.0 Hz, 10.8 Hz, 2H), δ 3.24-3.19 (m, 4H), δ 2.91 (t, J = 5.5 Hz, 2H), δ 2.79 (t, J = 5.0 Hz, 2H), δ 2.43 (t, J = 6.0 Hz, 1H) 19 F NMR (376 MHz, CDCl3) δ -74.4 (m, 2F), δ -86.7 (s, 3F), δ -88.1 - -88.6 (m, 10F)

[0124] (6) Amidite formation In the diethanolamine containing the C7-PFPE group obtained in (5) above, the hydroxyl group not protected with a DMTr group was converted into an amidite.

[0125] [ka]

[0126] 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (1.76 g, 5.9 mmol) was dissolved in dry acetonitrile. 5-(Ethylthiotetrazole) (ETT) (7.61 g, 5.9 mmol) was added to the reaction mixture under argon, followed by the addition in portions of a solution of DMTr-protected diethanolamine (3.26 g, 3.9 mmol) containing the C7-PFPE group obtained in (5) above dissolved in THF (5 mL) and acetonitrile (5 mL). The reaction mixture was then stirred at room temperature under argon for 4 hours. The solvent was evaporated under reduced pressure. The resulting crude product was purified by column chromatography under argon using degassed hexane / ethyl acetate (1:4 volume ratio) as the mobile phase, and the target compound (N[C7-PFPE]amidite) was isolated as a colorless oil (78% yield).

[0127] 1 H NMR (400 MHz, Acetone-d6) δ 8 7.41 (d, J = 8.7 Hz, 2H), δ 7.32-7.14(m, 7H), δ 6.83-6.79 (m, 4H), δ 3.80-3.51 (m, 6H) δ 3.77 (s, 6H) δ 3.50 (t, J = 11.4 Hz, 2H), δ 3.25 (t, 11.4 Hz, 2H), δ 3.15 (t, J = 6.0 Hz, 2H), δ 2.94-2.89 (m, 4H), 2.54 (t, J = 6.4 Hz, 2H), δ 1.16 (d, J = 6.9 Hz, 6H), δ 1.12 (d, J = 6.9 Hz, 6H) 19 F NMR (376 MHz, Acetone-d6) δ -75.0 (m, 2F), δ -86.6 (s, 3F), δ -88.3 - -88.8 (m, 10F) 31 P NMR (162 MHz, Acetone-d6), δ -148.4 (s)

[0128] A 5'-N[C7-PFPE] modified DNA was synthesized by binding an N[C7-PFPE] group to the 5' end of an oligonucleotide consisting of SEQ ID NO: 1. 5'-N[C7-PFPE]-modified DNA, in which an N[C7-PFPE] group was attached to the 5' end of an oligonucleotide consisting of SEQ ID NO: 1, was synthesized as follows. First, DNA synthesis (trityl-off) was performed on a 1000 Å CPG solid support column (1 μmole scale). Next, under a nitrogen atmosphere, the N[C7-PFPE] amidite synthesized in (6) above (0.1 M acetonitrile solution, 300 μL) and activator solution (300 μL) were mixed using a syringe in the presence of CPG. Five minutes after mixing, the solution was removed from the column, and the strands were capped, oxidized, and deblocked using a DNA synthesizer. The nucleic acids were deprotected and purified by HPLC in the same manner as described above. The synthesized nucleic acids are shown in Table 6.

[0129] [Table 6]

[0130] Figure 5 shows the results of flow cytometry of cells that had been loaded with fluorescein-modified nucleic acids (cont-rDNA, FE(2)1, FE(2)2, and FE(2)5). The single-stranded nucleic acids modified with N[C7-PFPE] at the 5' end were all loaded into cells at higher amounts than unmodified nucleic acids (cont-rDNA).

[0131] [Example 4] A phosphoramidite containing a perfluoroalkyl group having four carbon atoms (-C4F9: hereinafter referred to as C4-PFC group) that does not have an ether-bonded oxygen atom was synthesized, and nucleic acids containing C4-PFC groups were produced using this by the amidite method.

[0132] (1) Synthesis of trifluoromethanesulfonate Trifluoromethanesulfonates containing C4-PFC groups were synthesized.

[0133] [ka]

[0134] Fluorinated alcohol (CFCHOH) (500 mg, 2.0 mmol) was dissolved in dry dichloromethane (1 mL), followed by the addition of dry triethylamine (1 mL) and cooling to -78 °C. Trifluoromethanesulfonic anhydride (8.46 g, 3 mmol) was added dropwise to the reaction mixture over 30 minutes, causing the reaction mixture to turn dark. The reaction mixture was stirred at 0 °C for 1 hour, then warmed to room temperature and stirred for an additional 18 hours. The reaction was then quenched by the addition of saturated NaHCO (10 mL) and extracted three times with dichloromethane (50 mL). All organic phases were combined, washed with brine, dried over MgSO, and the solvent was removed under vacuum to give the desired compound as a thick black oil. The resulting crude product was 1 It was analyzed by H NMR and used in the next step without further purification (yield 61%).

[0135] 1 H NMR (400 MHz, CDCl3) δ 4.91 (t, J = 12.3 Hz, 2H) 19 F NMR (376 MHz, 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)

[0136] (2) Diethanolamine The trifluoromethanesulfonate containing a C4-PFC group obtained in (1) above was converted to diethanolamine.

[0137] [ka]

[0138] A solution of the C4-PFC group-containing trifluoromethanesulfonate (459 mg, 1.2 mmol) obtained in (1) above and diethanolamine (254 mg, 2.4 mmol) was added to dry DMF (5 mL). The resulting reaction mixture was placed in an oil bath at 100°C and stirred for 18 hours. The reaction mixture was then cooled and dissolved in water (10 mL), and the product was extracted three times with dichloromethane (20 mL). All organic phases were combined, washed with brine, dried over MgSO4, and the solvent was removed under vacuum to obtain the target compound. The resulting crude product was 1 Analyzed by 1 H NMR and used in the next step without further purification.

[0139] 1 H NMR (400 MHz, CDCl3) δ 3.61 (t, J = 5.0 Hz, 4H), δ 3.21 (t, J = 17.4 Hz, 2H), δ 2.89 (t, J = 5.5 Hz, 4H) 19 F NMR (376 MHz, CDCl3) δ -81.2 (s, 3F), δ -117.0 (s, 2F), δ -124.7 (s, 2F), δ -126.2 (s, 2F)

[0140] (3)DMTr protection One of the two hydroxyl groups of the diethanolamine containing a C4-PFC group obtained in (2) above was protected with DMTr (4,4'-dimethoxytriphenylmethyl group).

[0141] [ka]

[0142] The C4-PFC group-containing diethanolamine (3.20 g, 9.5 mmol) obtained in (2) above was dissolved in dry dichloromethane (20 mL) and triethylamine (5 mL). DMTrCl (3.21 g, 9.5 mmol) was added portionwise to the reaction mixture, which was then stirred at room temperature for 2.5 hours. The solvent was then removed under vacuum to give a dark brown oily substance. This oily substance was purified by column chromatography using a silica column pretreated with triethylamine and a mixture of EtOAc and hexane (volume ratio 1:4) to give the target compound as a yellow oily substance (yield 54%).

[0143] 1 H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.3 Hz, 2H), δ 7.33-7.18 (m, 7H), δ 6.82 (d, J = 9.2 Hz, 4H), δ 3.78 (s, 6H), δ 3.52 (dd, J = 5.5 Hz, 10.5 Hz, 2H), δ 3.31 (t, J = 16.9 Hz, 2H), δ 3.23 (t, J = 5.5 Hz, 2H) δ 2.94 (t, J = 5.5 Hz, 2H), δ 2.81 (t, J = 5.0 Hz, 2H), δ 2.44 (t, J = 6.2 Hz, 1H) 19 F NMR (376 MHz, CDCl3) δ -79.9 (s, 3F), δ -117.1 (s, 2F), δ -125.0 (s, 2F), δ -125.9 (s, 2F)

[0144] (4) Amidite formation In the diethanolamine containing a C4-PFC group obtained in (3) above, the hydroxyl group not protected with a DMTr group was converted into an amidite.

[0145] [ka]

[0146] 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (1.84 g, 6.1 mmol) was dissolved in dry acetonitrile. 5-(Ethylthiotetrazole) (ETT) (1.05 g, 7.7 mmol) was added to the reaction mixture under argon, followed by the addition in portions of a solution of the DMTr-protected diethanolamine (3.26 g, 5.1 mmol) containing the C4-PFC group obtained in (3) above dissolved in THF (5 mL) and acetonitrile (5 mL). The reaction mixture was stirred at room temperature under argon for 4 hours, and the solvent was evaporated under reduced pressure. The resulting crude product was purified by column chromatography under argon using degassed hexane / ethyl acetate (1:4 volume ratio) as the mobile phase, and the target compound (N[C4-PFC] amidite) was isolated as a colorless oil (45% yield).

[0147] 1 H NMR (400 MHz, Acetone-d6) δ 7.41 (t, J = 8.7 Hz, 2H), δ 7.33-7.17 (m, 7H), δ 6.85 (td, J = 2.3 Hz, 8.7 Hz, 4H), δ 3.84-3.56 (m, 6H) δ 3.76 (s, 6H) δ 3.37 (t, J = 16.5 Hz, 2H), δ 3.20 (t, 6.0 Hz, 2H), δ 2.94 (t, J = 5.5 Hz, 2H), δ 2.90 (t, J = 6.4 Hz, 2H), δ 2.56 (t, J = 6.4 Hz, 2H), δ 1.16 (dd, J = 6.9 Hz, 6H), δ 1.12 (d, J = 6.9 Hz, 6H) 19 F NMR (376 MHz, Acetone-d6) δ -80.9 (m, 2F), δ -117.7 (s, 2F), δ -124.3 (s, 2F), δ -126.0 (s, 2F) 31 P NMR (162 MHz, Acetone-d6), δ -148.4 (s) [Industrial Applicability]

[0148] The present invention provides a nucleic acid containing a perfluoroalkyl group and a method for producing the same. The nucleic acid according to the present invention has excellent cell membrane permeability and is therefore expected to be used in the pharmaceutical field as a physiologically active substance, for example, as a carrier for introducing medicinal ingredients into target cells.

[0149] The entire contents of the specification, claims, drawings and abstract of Japanese Patent Application No. 2019-177622, ​​filed on September 27, 2019, are hereby incorporated by reference as the disclosure of the specification of the present invention.

Claims

1. A nucleic acid having a structure represented by the following general formula (A1): 【Chemistry 1】 In the formula, the groups have the following meanings. R ＦＥ 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 circle represents a bond.

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

3. The nucleic acid of claim 1, wherein the perfluoroalkyl group is indirectly introduced between two nucleotides.

4. The nucleic acid according to claim 1, wherein the number of the perfluoroalkyl groups is 2 to 10.

5. The nucleic acid according to claim 4, wherein the number of perfluoroalkyl groups is 5 to 10.

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

7. A method for producing a nucleic acid containing a perfluoroalkyl group, comprising synthesizing the nucleic acid containing a perfluoroalkyl group according to any one of claims 1 to 5 by a phosphoramidite method using a compound represented by the following general formula (A2) as a raw material: 【Chemistry 2】 In the formula, the groups have the following meanings. R ＦＥ 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 a 4,4'-dimethoxytriphenylmethyl group, and i-Pr is an isopropyl group.

8. A nucleic acid drug containing a nucleic acid as an active ingredient, The nucleic acid drug, wherein the nucleic acid has a structure represented by the following general formula (A1): 【Transformation 3】 In the formula, the groups have the following meanings. R ＦＥ 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 circle represents a bond.

9. A method for improving the cell membrane permeability of a nucleic acid, comprising introducing a structure represented by the following general formula (A1) into a nucleic acid to improve the cell membrane permeability: 【Chemistry 4】 In the formula, the groups have the following meanings. R ＦＥ 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 circle represents a bond.

10. The method for improving cell membrane permeability of nucleic acid according to claim 9, wherein the perfluoroalkyl group is indirectly bound to the 5'-end or 3'-end of the nucleic acid.

11. A compound represented by the following general formula (A2): 【Transformation 5】 In the formula, the groups have the following meanings. R ＦＥ 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 a 4,4'-dimethoxytriphenylmethyl group, and i-Pr is an isopropyl group.

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