Single-stranded polynucleotide

Abstract

The present invention addresses the problem of providing a technique by which self-duplex formation of a single-stranded polynucleotide, said single-stranded polynucleotide comprising a palindromic structure comprising an acyclic polynucleotide constitutional unit, is suppressed whereas the binding properties thereof to a target polynucleotide such as miRNA can be enhanced. This problem is solved by a single-stranded polynucleotide which comprises a palindromic structure comprising an acyclic polynucleotide constitutional unit, wherein adenine in the palindromic structure is substituted by diaminopurine and thymine located at the position complementary to the aforesaid adenine is substituted by a thiouracil derivative.

Description

[Technical field]

[0001] The present invention relates to single-stranded polynucleotides and the like. [Background technology]

[0002] It has been reported that artificial polynucleotides such as SNA (serinol nucleic acid) and aTNA (acyclic threoninol nucleic acid) recognize DNA and RNA in a sequence-specific manner (Non-Patent Documents 1 and 2). These are non-cyclic polynucleotides that do not have a sugar backbone, and therefore are highly resistant to degradation by enzymes in the body. They are also said to be easy to synthesize. For this reason, non-cyclic polynucleotides are expected to be used as anti-miRNA, anti-mRNA, siRNA, and even molecular beacons. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] H. Kashida, K. Murayama, T. Toda, H. Asanuma, Angew. Chem. Int. Ed. 2011, 50, 1285-1288. [Non-Patent Document 2] K. Murayama, H. Kashida, H. Asanuma, Chem.Commun.,2015,51,6500-6503. Summary of the Invention [Problem to be solved by the invention]

[0004] In the course of research, the present inventors have discovered that a non-circular polynucleotide is represented by the general formula (2): A 1 -BA 2 (In the formula: A 1 and A 2indicate mutually complementary base sequences. B indicates an arbitrary base sequence.) is found to have the problem that even a small mismatch will result in the formation of a self-duplex and the inability to bind to a target polynucleotide (such as miRNA or mRNA in a living organism).

[0005] Therefore, an object of the present invention is to provide a technique that can suppress the formation of a self-duplex of a single-stranded polynucleotide containing a palindrome containing a non-cyclic polynucleotide building block, while enhancing the binding ability to a target polynucleotide such as miRNA. In another aspect, an object of the present invention is to provide a technique that can improve the antisense activity of a single-stranded polynucleotide containing a palindrome containing a non-cyclic polynucleotide building block. [Means for solving the problem]

[0006] As a result of intensive research, the present inventors have found that the above-mentioned problems can be solved by a single-stranded polynucleotide comprising a palindrome containing a non-cyclic polynucleotide building block, in which adenine in the palindrome is replaced with diaminopurine and thymine at the complementary position of the adenine is replaced with a thiouracil derivative.

[0007] Furthermore, when synthesizing acyclic single-stranded polynucleotides containing diaminopurine and / or thiouracil derivatives by the phosphoramidite method, if conventional amidite monomers of diaminopurine and thiouracil derivatives are used, various impurities remain. As a result of further research, the present inventors have found that this problem can be solved by adopting protecting groups removable with acid as protecting groups for the bases in the acyclic amidite monomers of diaminopurine and thiouracil derivatives.

[0008] The present inventors have conducted further research based on these findings and have completed the present invention. That is, the present invention includes the following aspects.

[0009] Section 1. A single-stranded polynucleotide comprising a palindrome containing a non-cyclic polynucleotide building block, in which adenine and / or its analogue in the palindrome is replaced with diaminopurine and thymine and / or its analogue at the complementary position of the adenine is replaced with a thiouracil derivative.

[0010] Section 2. The non-cyclic polynucleotide building block has the general formula (1):

[0011] [ka] [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups. Base refers to a nucleic acid base.) Item 2. The single-stranded polynucleotide according to Item 1, wherein the structural unit is represented by the following formula:

[0012] Section 3. R 1 is a hydrogen atom or a chain hydrocarbon group, and the R 2 Item 3. The single-stranded polynucleotide according to Item 2, wherein is a hydrogen atom.

[0013] Section 4. The palindrome is represented by the general formula (2): A 1 -BA 2 (In the formula: A 1 and A 2 The length of A is 3 or more. 1 and A 2 The base length of B is 0 to (A 1 Base length + A 2 4. The single-stranded polynucleotide according to any one of items 1 to 3, which is a single-stranded polynucleotide consisting of a base sequence represented by the following formula:

[0014] Section 5. Item 5. The single-stranded polynucleotide according to any one of Items 1 to 4, wherein the palindrome is composed of the non-cyclic polynucleotide building block.

[0015] Section 6. Item 6. The single-stranded polynucleotide according to any one of Items 1 to 5, wherein the number of each of the diaminopurine derivatives and the thiouracil derivatives is 2 or more.

[0016] Section 7. Item 7. The single-stranded polynucleotide according to any one of Items 1 to 6, which has a base length of 6 to 500.

[0017] Section 8. 8. The single-stranded polynucleotide according to any one of Items 1 to 7, which is complementary to a cell-resident polynucleotide.

[0018] Section 9. Item 9. The single-stranded polynucleotide according to any one of Items 1 to 8, which is an anti-miRNA or anti-mRNA polynucleotide.

[0019] Section 10. A double-stranded polynucleotide comprising the single-stranded polynucleotide according to any one of Items 1 to 9.

[0020] Section 11. General formula (3):

[0021] [ka] [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups.) 3 and R 4 R may be the same or different and represent a protecting group for a hydroxyl group. 5 and R 6are the same or different and each represents an alkyl group. Base' represents a nucleic acid base in which the amino group of diaminopurine is protected with a protecting group that can be removed by acid, or a nucleic acid base in which the thiocarbonyl group of a thiouracil derivative is protected with a protecting group that can be removed by acid.] or a salt thereof, or a solvate thereof.

[0022] Section 12. The compound has the general formula (3A) or (3B):

[0023] [ka] [In the formula: R 1 , R 2 / R 3 , R 4 , R 5 and R 6 is the same as above. 7 , R 8 , R 9 , R 10 and R 11 R may be the same or different and represents a protecting group removable with an acid. 12 represents a hydrogen atom or an alkyl group.], or a salt thereof, or a solvate thereof according to item 11.

[0024] Section 13. R 7 , R 8 , R 9 and the above R 10 is a Boc group, and the R 9 Item 13. The compound or salt thereof, or a solvate thereof according to item 12, wherein is an MMPM group.

[0025] Section 14. R 3 is represented by the general formula (4):

[0026] [ka] [In the formula: R 31 , R 32 and R33 are the same or different and each represents a hydrogen atom or an alkoxy group. 4 is -(CH2)2-CN, and R 5 and R 6 Item 14. The compound or salt thereof, or a solvate of the compound or salt according to any one of items 11 to 13, wherein is an isopropyl group.

[0027] Section 15. Item 15. A method for producing a single-stranded polynucleotide by a phosphoramidite method, using the compound or a salt thereof, or a solvate of the compound or salt according to any one of Items 11 to 14, as an amidite monomer. Effect of the Invention

[0028] According to the present invention, it is possible to provide a single-stranded polynucleotide containing a palindrome containing a non-cyclic polynucleotide building block, which exhibits higher binding affinity to a target polynucleotide such as miRNA while suppressing the formation of a self-duplex. Also, according to the present invention, it is possible to improve the antisense activity of a single-stranded polynucleotide containing a palindrome containing a non-cyclic polynucleotide building block. Furthermore, according to the present invention, it is possible to provide an amidite monomer capable of efficiently synthesizing a non-cyclic single-stranded polynucleotide containing a diaminopurine and / or thiouracil derivative, and a method for synthesizing the polynucleotide. [Brief description of the drawings]

[0029] [Figure 1] 3 shows the HPLC profile of the SNA polynucleotide synthesized in Synthesis Example 3. [Diagram 2] 1 shows the melting curve obtained in Test Example 1. The vertical axis indicates the relative value of absorbance at 260 nm, and the horizontal axis indicates the temperature. [Diagram 3] 1 shows the melting curve obtained in Test Example 2. The vertical axis shows the relative value of absorbance at 260 nm, and the horizontal axis shows the temperature. [Figure 4] 1 shows the native MS profile obtained in Test Example 2. [Diagram 5]1 shows a polyacrylamide gel electrophoresis diagram obtained in Test Example 2. [Figure 6] 1 shows the sequence of the single-stranded polynucleotide of Synthesis Example 4 and the results of Test Example 3. The grey areas indicate self-complementary regions. [Figure 7] The results of Test Example 4 are shown. The vertical axis indicates the relative value of luciferase activity. The leftmost horizontal axis indicates the case where a control reporter plasmid (without miR21 binding site) was introduced, and the others indicate the cases where a reporter plasmid containing a miR21 binding site was introduced. In addition, the horizontal axis indicates the cases where AMO was introduced, and its name is shown. [Figure 8] 1 shows the sequence of the single-stranded polynucleotide of Synthesis Example 5 and the results of Test Example 5. The grey areas indicate self-complementary regions. [Figure 9] The sequence of the single-stranded polynucleotide of Synthesis Example 6 and the results of Test Example 6 are shown. The grey portion indicates the self-complementary region. [Figure 10] 4 shows the results of Test Example 7. The vertical and horizontal axes are the same as in FIG. [Figure 11] The sequence of the single-stranded polynucleotide of Synthesis Example 7 and the results of Test Example 8 are shown. [Figure 12] 8 shows the results of Test Example 9. The vertical and horizontal axes are the same as those in FIG. [Figure 13] 1 shows the sequence of the single-stranded polynucleotide of Synthesis Example 8 and the results of Test Example 10. The grey areas indicate self-complementary regions. [Figure 14] 7 shows the results of Test Example 11. The vertical and horizontal axes are the same as those in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0031] 1. Single-stranded polynucleotide In one aspect, the present invention relates to a single-stranded polynucleotide (sometimes referred to as the "single-stranded polynucleotide of the present invention" in this specification) that includes a palindrome containing a non-cyclic polynucleotide building block, in which adenine in the palindrome is replaced with diaminopurine and thymine at the complementary position of the adenine is replaced with a thiouracil derivative. This will be described below.

[0032] The non-cyclic polynucleotide building block is not particularly limited as long as it is a building block corresponding to a nucleotide constituting a polynucleotide and does not contain a sugar backbone. Representative examples of the non-cyclic polynucleotide building block include those represented by the general formula (1):

[0033] [ka] [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups. Base refers to a nucleic acid base.) Examples of the structural unit include those represented by the following formula:

[0034] The organic group is not particularly limited, and examples thereof include hydrocarbon groups.

[0035] The hydrocarbon group is preferably a chain-like hydrocarbon group. Examples of the chain-like hydrocarbon group include an alkyl group, an alkenyl group, and an alkynyl group, and among these, an alkyl group is preferred. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group. The number of carbon atoms in the hydrocarbon group is not particularly limited. The number of carbon atoms is preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 4, still more preferably 1 to 2, and particularly preferably 1. In addition, an alkyl group containing an alkynyl group (-C≡C-, -C≡CH) at the end or inside thereof, which allows the introduction of various functional groups by click reaction or the like, is more preferred.

[0036] In addition to the above, the organic group may be a monovalent group obtained by removing one hydrogen atom or functional group from various molecules, for example, a molecule used for modifying a polynucleotide. Examples of such molecules include polyethylene glycol chains, dye molecules, polycations (spermine), groove binders, amino groups, hydroxyl groups, thiol groups, metal ligands, photocleavable functional groups, and sugar chains. These can be linked directly or indirectly to the skeleton of the above-mentioned structural units. For example, they can be linked by using a click reaction (for example, the above-mentioned reaction between an alkyne and an azide).

[0037] Molecular modeling of non-circular polynucleotides such as SNA and LaTNA 1 and R 2 It is believed that even if a relatively large organic group is used, the double-stranded chain forming ability and its structure are not affected.

[0038] In a preferred embodiment of the present invention, R 1 is a hydrogen atom or a chain hydrocarbon group, and R 2 is preferably a hydrogen atom.

[0039] As the nucleic acid base, any base constituting a nucleic acid can be used without any particular limitation. The base constituting a nucleic acid includes not only typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also other bases such as hypoxanthine (I) and modified bases. Modified bases include, for example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypo Examples of such amino acids include xanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine.

[0040] In general formula (1), *1 and *2 indicate the direction in constructing a polynucleotide. 1 is an organic group and R 2 When R is a hydrogen atom, *1 is the 3' side and *2 is the 1' side. 1 is a hydrogen atom and R 2 When R is an organic group, *1 is the 1' side and *2 is the 3' side. 1 is a hydrogen atom and R 2 is a hydrogen atom, *1 is the (S) side and *2 is the (R) side.

[0041] The palindrome is not particularly limited as long as it is a single-stranded polynucleotide consisting of a base sequence capable of forming a self-duplex (intramolecular (hairpin) or intermolecular (dimer)).

[0042] The palindrome contains the above-mentioned non-cyclic polynucleotide building blocks. The ratio of the non-cyclic polynucleotide building blocks to 100% of the polynucleotide building blocks (number of nucleotides) constituting the palindrome is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 100% (i.e., the palindrome is composed of non-cyclic polynucleotide building blocks). The ratio of the non-cyclic polynucleotide building blocks to 100% of the polynucleotide building blocks (number of nucleotides) constituting the single-stranded polynucleotide of the present invention is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 100% (i.e., the single-stranded polynucleotide of the present invention is composed of non-cyclic polynucleotide building blocks). When at least a part of the above ratios are satisfied, the problem of self-duplex formation becomes more prominent, and therefore the effect of applying the technology of the present invention becomes more prominent. In addition, by satisfying at least a part of the above ratios, the resistance to degradation by enzymes in the living body can be further increased.

[0043] A palindrome is typically represented by the general formula (2): 1 -BA 2 It is a single-stranded polynucleotide consisting of the base sequence represented by the formula:

[0044] A 1 and A 2 indicate complementary base sequences. 1 is the base sequence *1-ATGCCG, 2is a base sequence complementary to the base sequence, typically a base sequence *1-CGGCAT (perfectly complementary base sequence). In this specification, "complementary" includes not only a perfect complementary relationship of bases (perfectly complementary: for example, A and T or U, and G and C), but also a complementary relationship to the extent that hybridization can occur under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). For example, washing conditions after hybridization can usually be about "1xSSC, 0.1% SDS, 37°C". It is preferable that the hybridization state is maintained even when washed under such conditions. Although not particularly limited, examples of more stringent hybridization conditions include approximately "0.5×SSC, 0.1% SDS, 42° C.", and examples of even more stringent hybridization conditions include washing conditions of approximately "0.1×SSC, 0.1% SDS, 65° C." 2 A 1 A base sequence that has, for example, 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity, even more preferably 98% or more identity, still more preferably 99% or more identity, and particularly preferably 100% identity, to a base sequence that is completely complementary to the base sequence.

[0045] A 1 and A 2 The base length of A is preferably 3 or more. The base length is preferably 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. The upper limit of the base length is not particularly limited, and is, for example, 100, 50, 20, or 10. The base length of B is preferably 0 to (A 1 Base length + A 2The base length of the palindrome is an integer of (base length of the palindrome) / 4. The base length of the palindrome is preferably 6 or more, more preferably 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, or 20 or more. There is no particular upper limit to the base length of the palindrome, and it is, for example, 200, 100, 50, or 20. When at least a part of these ranges is satisfied, the problem of self-duplex formation becomes more pronounced, and therefore the effect of applying the technology of the present invention becomes more pronounced.

[0046] The proportion of palindrome in the single-stranded polynucleotide of the present invention (the proportion of polynucleotide constituent units that form a palindrome to 100% of the polynucleotide constituent units (number of nucleotides) that constitute the single-stranded polynucleotide of the present invention) is not particularly limited as long as it can form a self-duplex. The proportion is, for example, 10% or more, preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and even more preferably 50% or more. When these ranges are satisfied, the problem of self-duplex formation becomes even more pronounced, and therefore the effect of applying the technology of the present invention becomes even more pronounced.

[0047] In the single-stranded polynucleotide of the present invention, the constituent units other than the non-cyclic polynucleotide constituent units are not particularly limited, and various artificial nucleic acid constituent units including natural nucleic acid, aTNA and SNA can be adopted. Specific examples of nucleic acids that can be adopted include DNA, RNA, and the like, as well as those that have been subjected to known chemical modifications, as exemplified below. In order to prevent decomposition by hydrolases such as nucleases, the phosphate residues of each nucleotide can be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. In addition, the hydroxyl group at the 2' position of the sugar (ribose) of each ribonucleotide can be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a carbonyl group at the 2-position with a thiocarbonyl group. Further examples include those in which the phosphate portion or the hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc., but are not limited thereto. In addition, BNA (LNA), which is a nucleotide in which the conformation of the sugar portion is fixed to N-type by bridging the 2' oxygen and the 4' carbon of the sugar portion of the nucleotide, may also be preferably used.

[0048] In the single-stranded polynucleotide of the present invention, adenine and / or its analogs in a palindrome (not including diaminopurine and thiouracil derivatives) are replaced with diaminopurine (2,6-diaminopurine) and thymine and / or its analogs at the complementary position of the adenine are replaced with thiouracil derivatives (2-thiouracil, 2-thiothymine, etc.). The complementary position is the position that faces when the palindrome is arranged so that it is complementary to the other palindrome. For example, when the base sequence of the palindrome is *1-TAACGTTA, the complementary position of the second A from the *1 side is the position of the seventh T from the *1 side.

[0049] It is particularly preferred that the number of each of these diaminopurine and thiouracil derivatives is 2 or more. This can suppress the formation of a self-duplex of a single-stranded polynucleotide containing a palindrome containing an acyclic polynucleotide building block, while further enhancing the binding to a target polynucleotide such as miRNA. The upper limit of the number is not particularly limited, and is, for example, 50, 20, 10, or 5. It is also preferred that the diaminopurine and thiouracil derivatives are present separately and / or adjacent to each other.

[0050] The ratio of the total number of diaminopurine and thiouracil derivatives to the number of bases in the palindrome (100%) is, for example, 10 to 90%, preferably 20 to 80%, more preferably 30 to 70%, and further preferably 40 to 60%.

[0051] In the single-stranded polynucleotide of the present invention, thymine and / or its analogs other than those in the palindrome are preferably not replaced with thiouracil derivatives.

[0052] The base length of the single-stranded polynucleotide of the present invention is not particularly limited and is, for example, 6 to 10000. From the viewpoint of ease of synthesis, etc., the base length is preferably 6 to 1000, more preferably 6 to 500, even more preferably 6 to 200, still more preferably 6 to 100, and particularly preferably 6 to 50.

[0053] The single-stranded polynucleotide of the present invention also includes those to which other molecules are linked. The other molecules are not particularly limited, and examples thereof include fluorescent labels, biotin, azide, and ethynyl groups. Fluorescent labels include fluorescein, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM (trademark), Cy (registered trademark) 3, Cy (registered trademark) 5, and Alexa Fluor (registered trademark) series.

[0054] The single-stranded polynucleotide of the present invention can exert various functions by binding to a target polynucleotide. Therefore, the single-stranded polynucleotide of the present invention (preferably a palindrome) is complementary to all or a part of the target polynucleotide. Examples of the target polynucleotide include miRNA, mRNA, and DNA. The target polynucleotide is preferably an endogenous polynucleotide in a cell. The single-stranded polynucleotide of the present invention can be suitably used as an anti-miRNA polynucleotide.

[0055] In one aspect, the present invention can provide a double-stranded polynucleotide comprising the single-stranded polynucleotide of the present invention.

[0056] The single-stranded polynucleotide of the present invention can be easily prepared according to known genetic engineering techniques, techniques for producing artificial nucleic acids, etc. For example, it can be prepared by using PCR, restriction enzyme digestion, nucleic acid ligation techniques, etc. In addition, it can be preferably prepared according to the production method of the present invention described below.

[0057] The single-stranded polynucleotide of the present invention and the double-stranded polynucleotide (collectively referred to as "polynucleotide of the present invention") can be used in medicines, reagents, etc. (hereinafter, collectively referred to as "agents of the present invention").

[0058] The agent of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention, and may further contain other components as necessary. The other components are not particularly limited as long as they are pharma- ceutically acceptable, and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0059] The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type. For example, the agent of the present invention can be used in vitro (for example, added to a culture medium for cultured cells) or in vivo (for example, administered to an animal).

[0060] The subject of application of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, etc.; animal cells, etc. The types of cells are also not particularly limited, and examples thereof include blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, etc.

[0061] When the agent of the present invention is used as an anticancer agent, the type of cancer cells is not particularly limited, and examples include kidney cancer cells, leukemia cells, esophageal cancer cells, gastric cancer cells, colon cancer cells, liver cancer cells, pancreatic cancer cells, lung cancer cells, prostate cancer cells, skin cancer cells, breast cancer cells, cervical cancer cells, etc.

[0062] The agent of the present invention may take any dosage form, for example, oral preparation forms such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; and parenteral preparation forms such as injection preparations (for example, drip injections (for example, intravenous drip preparations, etc.), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), external preparations (for example, ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

[0063] The route of administration of the agent of the present invention is not particularly limited as long as the desired effect can be obtained, and examples thereof include enteral administration such as oral administration, tube feeding, and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration. The content of the polynucleotide of the present invention in the agent of the present invention depends on the mode of use, the subject of application, the condition of the subject of application, etc., and is not limited, and can be, for example, 0.0001 to 95% by weight, preferably 0.001 to 50% by weight.

[0064] The dosage of the agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is usually 0.1 to 1000 mg / kg body weight, preferably 0.5 to 50 mg / kg body weight, per day in the case of oral administration, and 0.01 to 100 mg / kg body weight, preferably 0.1 to 10 mg / kg body weight, per day in the case of parenteral administration, in terms of the weight of the polynucleotide of the present invention as an active ingredient. The above dosage is preferably administered once a day or in 2 to 3 divided doses, and may be appropriately increased or decreased depending on the age, pathological condition, and symptoms.

[0065] 2. Amidite Monomers In one aspect, the present invention provides a compound represented by general formula (3):

[0066] [ka] [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups.) 3 and R 4 R may be the same or different and represent a protecting group for a hydroxyl group. 5 and R 6 are the same or different and each represents an alkyl group. Base' represents a nucleic acid base in which the amino group of diaminopurine is protected with a protecting group that can be removed by acid, or a nucleic acid base in which the thiocarbonyl group of a thiouracil derivative is protected with a protecting group that can be removed by acid.] The present invention relates to a compound represented by the following formula (1): or a salt thereof, or a solvate thereof (also referred to as the "amidite monomer of the present invention" in this specification). This will be explained below.

[0067] R 1 , R 2 The diaminopurine and thiouracil derivatives are as explained above in the section "1. Single-stranded polynucleotides."

[0068] R 3 As the protecting group for the hydroxyl group, any protecting group that can function as a protecting group for a hydroxyl group can be used without any particular limitation, and a wide variety of known protecting groups that are used in amidite monomers can be used. 3 is preferably represented by the general formula (4):

[0069] [ka] [In the formula: R 31 , R 32 and R 33 and are the same or different and each represents a hydrogen atom or an alkoxy group.

[0070] R 31 , R 32 and R 33 It is preferred that one of the groups is hydrogen and the remaining two are alkoxy groups, and the alkoxy group is particularly preferably a methoxy group.

[0071] R 4 As the protecting group for the hydroxyl group, any protecting group that can function as a protecting group for a hydroxyl group can be used without any particular limitation, and a wide variety of known protecting groups that are used in amidite monomers can be used. 4Examples of the alkyl group include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a haloalkyl group, an aryl group, a heteroaryl group, an arylalkyl group, a cycloalkenyl group, a cycloalkylalkyl group, a cyclylalkyl group, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a heterocyclylalkenyl group, a heterocyclylalkyl group, a heteroarylalkyl group, a silyl group, a silyloxyalkyl group, a mono-, di- or trialkylsilyl group, or a mono-, di- or trialkylsilyloxyalkyl group, which may be substituted with an electron-withdrawing group.

[0072] R 4 R is preferably an alkyl group substituted with an electron-withdrawing group. Examples of the electron-withdrawing group include a cyano group, a nitro group, an alkylsulfonyl group, a halogen atom, an arylsulfonyl group, a trihalomethyl group, and a trialkylamino group, and is preferably a cyano group. 4 is particularly preferably -(CH2)2-CN.

[0073] R 5 and R 6 The alkyl group represented by may be either linear or branched, and is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl. The alkyl group here also includes an alkyl moiety such as an alkoxy group. R 5 and R 6 may be bonded to each other to form a cyclic structure.

[0074] R 5 and R 6 and are particularly preferably both an isopropyl group.

[0075] The protecting group that can be removed with an acid is not particularly limited, and examples thereof include a Boc group (tert-butoxycarbonyl group) and an MMPM group (4-methoxy-2-methylbenzyl group).

[0076] A preferred embodiment of the compound represented by general formula (3) is a compound represented by general formula (3A) or (3B):

[0077] [ka] [In the formula: R 1 , R 2 / R 3 , R 4 , R 5 and R 6 is the same as above. 7 , R 8 , R 9 , R 10 and R 11 R may be the same or different and represents a protecting group removable with an acid. 12 represents a hydrogen atom or an alkyl group (preferably a methyl group). Examples of the compound include compounds represented by the following formula:

[0078] The salt of the amidite monomer of the present invention is not particularly limited, and examples thereof include salts with inorganic bases such as sodium salts, magnesium salts, potassium salts, calcium salts, and aluminum salts; salts with organic bases such as methylamine, ethylamine, and ethanolamine; salts with basic amino acids such as lysine, ornithine, and arginine, and ammonium salts. The salt may be an acid addition salt, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. The amidite monomer of the present invention also includes hydrates, solvates, and crystal polymorphs.

[0079] The amidite monomer of the present invention can be produced according to the methods described in the Examples below or by methods with appropriate modifications of these methods as necessary.

[0080] 3. Method for producing single-stranded polynucleotides In one aspect, the present invention relates to a method for producing a single-stranded polynucleotide by the phosphoramidite method using the amidite monomer of the present invention (also referred to as the "production method of the present invention" in this specification). This will be described below.

[0081] The phosphoramidite method can be carried out according to a known method, for example, using a commercially available automated nucleic acid synthesizer. Specifically, the method includes the steps of (A) deprotecting the hydroxyl group at the 5' position (or a position corresponding thereto), (B) condensing the amidite monomer of the present invention, (C) capping the hydroxyl group at the 5' position (or a position corresponding thereto) of the unreacted compound, (D) converting the phosphorous group to a phosphate group or a thiophosphate group, (E) cleaving the obtained compound from the solid phase support and deprotecting the hydroxyl groups at the 2' position and the nucleic acid base, and (F) deprotecting the hydroxyl group at the 5' position (or a position corresponding thereto). By repeating the steps (A) to (D), a compound containing a polynucleotide backbone of a desired chain length can be produced.

[0082] In step (D), it is preferable to use tert-butyl hydroperoxide as the oxidizing agent.

[0083] In the deprotection in step (E), the protecting group of the nucleic acid base derived from the amidite monomer of the present invention is usually removed first with an acid such as TFA, and then deprotection is performed with a base such as aqueous ammonia. The deprotection time with an acid such as TFA is preferably 2 to 6 hours. In the deprotection with a base, the thiouracil derivative is destroyed if the treatment time is long, so in order to prevent this, the treatment time is preferably about 4 to 6 hours.

[0084] The obtained single-stranded polynucleotide may be isolated and purified as necessary. Usually, it can be isolated by using a method for precipitating, extracting and purifying RNA. Specifically, a method of precipitating RNA by adding a solvent with low solubility for RNA, such as ethanol or isopropyl alcohol, to the reaction solution, or a method of adding a phenol / chloroform / isoamyl alcohol solution to the reaction solution to extract RNA into the aqueous layer is adopted. Then, it can be isolated and purified by known high performance liquid chromatography (HPLC) techniques such as reverse phase column chromatography, anion exchange column chromatography and affinity column chromatography.

[0085] The single-stranded polynucleotide of the present invention can be preferably produced by the production method of the present invention. EXAMPLES

[0086] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0087] Synthesis Example 1: Synthesis of Boc-protected 2,6-diaminopurine SNA

[0088] [ka] The reagents and conditions were as follows: a) Boc2O, DMAP, THF, rt, 18 h; b) NaHCO3, MeOH-H2O (2:1), 50°C, 1 h, then rt, 18 h; c) BrCH2CO2Et, K2CO3, DMF, rt, 20 h; d) NaOH, MeOH, 1,4-dioxane, H2O, 0°C, 10 min; e) DMT-MM, DMF, triethylamine, rt, 1 h; f) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, dry CH2Cl2, Et2N, 0°C, 30 min.

[0089] Synthesis Example 1-1: Synthesis of Compound 2 (Step a) 2,6-Diaminopurine (compound 1) (5.0 g, 33.3 mmol) and DMAP (0.61 g, 5.0 mmol) were dissolved in THF (100-200 mL) under nitrogen atmosphere. Boc2O (61.2 g, 64.4 mL) was added to this solution at 0 °C. After stirring at 0 °C for 5 min, the mixture was stirred at room temperature for 18 h. After the reaction was completed, the solvent was removed by evaporation. The residue was dissolved in CHCl3 and then mixed with H2O. The organic layer was collected and dried with MgSO4, and the solvent was removed by evaporation. The residue was purified by silica gel column chromatography (hexane / AcOEt, 10:3) to obtain compound 2 (yield 82%). 1 H-NMR [CDCl3, 500 MHz] δ=8.53 (s, 1H, 8-purine), 1.69 (s, 9H), 1.45 (s, 18H), 1.43 (s, 18H) 13 C-NMR [CDCl3, 125 MHz] δ=153.7, 152.9, 151.6, 150.6, 149.7, 145.5, 143.8, 127.8, 87.3, 83.9, 83.2, 41.9, 27.8, 27.7, 27.6 HRMS (FAB) Calcd for compound 2 (M+H+) 651.3348. Found 651.3371.

[0090] Synthesis Example 1-2: Synthesis of Compound 3 (Step b) Compound 2 (17.6 g, 27 mmol) was dissolved in AcOEt (400 mL) and washed with 1 M HCl (30 mL) and a saturated solution of NaCl. The organic layer was dried with MgSO4, the solvent was removed by evaporation, and dried in vacuum. The mixture was dissolved in MeOH (200 mL), NaHCO3 solution (90 mL) was added, and the mixture was placed in an oil bath at 50 °C and stirred for 1 h. After the reaction was completed, MeOH was removed by evaporation, and H2O (100 mL) was added. Extraction was performed three times with CHCl3 (50 mL), the organic layers were combined, dried with MgSO4, and the solvent was removed by evaporation. The residue was purified by silica gel column chromatography (hexane / AcOEt, 2:1 to AcOEt 100%) to give 14.3 g of compound 3 (yield 96%). 1 H-NMR [CDCl3, 500 MHz] δ=8.38 (s, 1H), 1.49 (s, 18H), 1.44 (s, 18H) 13 C-NMR [CDCl3, 125 MHz] δ=151.66, 150.95, 150.06, 144.99, 83.58, 27.99 HRMS (FAB) Calcd for compound 3 (M+H+) 551.2824. Found 551.2829.

[0091] Synthesis Example 1-3: Synthesis of Compound 4 (Step c) Compound 3 (14.3 g, 26.0 mmol) and K2CO3 (1.15 equiv., 4.0 g, 29.9 mmol) were suspended in dry DMF (60 mL) under nitrogen atmosphere. Ethyl bromoacetate (1.1 equiv., 28.6 mmol) was added dropwise to the mixture at 0°C and then stirred for 10 min. The mixture was then stirred at room temperature for 20 h. The reaction solution was removed by azeotropic distillation with toluene, followed by addition of H2O (150 mL). Extraction was performed three times with CHCl3 (100 mL), and the organic layers were combined and washed with H2O. The organic layers were dried over MgSO4, and the solvent was removed by evaporation. The residue was purified by silica gel column chromatography (CHCl3 / MeOH, 40:1) to give 14.6 g of compound 4 (88% yield). 1 H-NMR [CDCl3, 500 MHz] δ=8.18 (s, 1H), 5.03 (s, 2H,), 4.27-4.26 (d, 2H), 1.42 (s, 18H), 1.41 (s, 18H), 1.30 (m, 3H) 13 C-NMR [CDCl3, 125 MHz] δ=166.69, 154.38, 152.35, 151.18, 150.85, 145.92, 83.84, 83.36, 62.49, 44.47, 27.88, 27.75, 14.16 HRMS (FAB) Calcd for compound 4 (M+H+) 637.3192 Found 637.3197.

[0092] Synthesis Example 1-4: Synthesis of Compound 5 (Step d) Compound 4 (14.6 g, 23 mmol) was dissolved in MeOH (120 mL) and 1,4-dioxane (40 mL). Then, 2 M NaOH solution (40 mL) was added on ice and stirred for 10 minutes. After the reaction was completed, 2 M HCl was added to adjust the pH of the reaction solution to 3, and MeOH and 1,4-dioxane were removed by evaporation. Extraction was performed three times with AcOEt, and the organic layers were combined and washed with H2O. The organic layer was dried with MgSO4, and the solvent was removed by evaporation and dried in vacuum. The crude product (compound 5) was used in the next reaction step without further purification (quantitative yield). 1 H-NMR [CDCl3, 500 MHz] δ=8.46 (s, 1H), 5.03 (s, 2H), 1.41 (s, 18H), 1.40 (s, 18H) 13 C-NMR [CDCl3, 125 MHz] δ=168.9, 154.31, 152.28, 150.94, 150.59, 149.97, 147.2, 126.28, 84.06, 83.61, 27.85, 27.74 HRMS (FAB) Calcd for compound 5 (M+H+) 609.2879. Found 609.2900.

[0093] Synthesis Example 1-5: Synthesis of Compound 7 (Step e) Compound 5 (5.4 g, 8.8 mmol) was dissolved in DMF and condensed with 1.96 g (1.0 equiv, 8.0 mmol) of compound 6 in the presence of Et3N (3.0 equiv) and DMT-MM (1.2 equiv, 5 mmol). After the reaction was completed, CHCl3 was added and the organic layer was washed with saturated NaHCO3. The mixture was dried over MgSO4. The solvent was removed by evaporation and the residue was purified by silica gel column chromatography (CHCl3 / MeOH, 30:1 to 10:1, containing 3% Et3N) to give 7.44 g (7.6 mmol) of compound 7 (yield 94%). 1 H-NMR [CDCl3, 500 MHz] δ=8.19 (s, 1H), 7.45-7.20 (m, 9H), 6.88-6.80 (m, 4H), 6.59-6.55 (d, 1H), 4.91-4.75 (dd, 2H), 4.18-4.10 (m, 1H), 3.85-3.80 (m, 1H), 3.79 (s, 6H), 3.70-3.65 (m, 1H), 3.35- 3.20 (m, 2H), 1.40 (s, 18H), 1.39 (s, 18H) 13 C-NMR [CDCl3, 125 MHz] δ=165.28, 162.69, 158.69, 154.38, 152.11, 151.13, 151.09, 146.44, 135.79, 130.08, 130.03, 128.12, 128.05, 127.07, 113.35, 86.58, 83.98, 83.67, 62.53, 62.41, 55.31, 51.55, 46.16, 36.58, 31.55; 28.31, 28.21, 27.91, 27.77 HRMS (FAB) Calcd for compound 7 (M+H+) 984.4713. Found 984.4686.

[0094] Synthesis Example 1-6: Synthesis of Compound 8 (Step f) Compound 7 (6.46 g, 6.56 mmol) was dissolved in anhydrous CHCl (7 mL) and EtN (4.55 mL), and 2.2 mL (7.84 mmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added dropwise under nitrogen atmosphere at 0 °C. After stirring for 20 min at room temperature, the reaction mixture was purified by silica gel column chromatography (hexane / AcOEt, 4:1 to 1:1, containing 3% EtN) to give compound 8 (4.81 g (4.06 mmol)) (yield 62%). 1 H-NMR [CDCl3, 500 MHz] δ=8.23-8.19 (1H), 7.43-7.20 (m, 9H), 6.85-6.82 (m, 4H), 6.37, (m, 1H), 4.93-4.73 (m, 2H), 4.40-4.20 (m, 1H), 4.00-3.80 (m, 2H), 3.79(s, 6H), 3.70-3.60 (m, 1H), 3.61-3.52 (m, 2H), 3.42-3.35 (m, 1H), 3.21-3.15 (m, 1H), 2.70-2.40 (m, 2H), 1.41 (s, 18H), 1.39 (s, 18H), 1.20-1.10 (m, 12H) 1313C-NMR [CDCl3, 125 MHz] δ=164.77, 158.58, 154.41, 152.05, 150.84, 150.03, 146.58, 146.48, 144.73, 144.63, 135.91, 135.83, 135.75, 130.13, 130.09, 130.07, 130.04, 128.16, 128.11, 127.93, 126.94, 126.80, 118.59, 118.48, 113.23, 113.20, 86.29, 86.27, 83.73, 83.72, 83.28, 83.24, 65.86, 61.45, 58.54, 58.44, 58.40, 58.29, 55.26, 50.43, 50.37, 45.55, 45.46, 43.18, 43.08, 30.95, 27.82, 27.71, 24.68, 24.62, 20.62, 20.60, 20.60, 20.56, 15.31 31 31P-NMR [CDCl3, 202 MHz] δ=148.15, 147.71 HRMS (FAB) Calcd for compound 8 (M+H+) 1185.5792. Found 1184.5794.。

[0095] Synthesis Example 2: Synthesis of MMPM-protected thiouracil SNA

[0096]

Chem.

[0097] Synthesis Example 2-1: Synthesis of Compound 10 (Step g) Compound 9 (2.03 g, 12.8 mmol) was dissolved in EtOH (32 mL), and then NaBH4 was added. The mixture was stirred at room temperature for 5 minutes. After the reaction was completed, H2O (240 mL) was mixed and 4 M HCl was added to adjust the pH of the reaction solution to 4. Extraction was performed three times with Et2O, and the organic layers were combined and dried over MgSO4. The solvent was removed by evaporation, and the obtained crude product (compound 10) was used in the next step without further purification (quantitative yield). 1 H-NMR [CDCl3, 500 MHz] δ=7.22 (d, 1H), 6.74-6.71 (m, 2H), 4.61 (s, 2H), 3.79 (s, 3H), 2.36 (s, 3H), 1.63 (s, 1H) 13 C-NMR [CDCl3, 125 MHz] δ=158.82, 137.70, 131.14, 129.17, 115.91, 129.17, 115.91, 110.47, 62.41, 54.94, 18.66 HRMS (FAB) Calcd for compound 10 (M+H + ) 152.0837. Found 152.0837.

[0098] Synthesis Example 2-2: Synthesis of Compound 12 (Steps h and i) Compound 10 (1.93 g, 12.7 mmol) was dissolved in dry CH2Cl2 under nitrogen atmosphere. PBr3 was added dropwise over 2.5 h at 0 °C, and after stirring for 15 min, NaHCO3 mixed with crushed ice was mixed with the reaction solution. Extracted three times with Et2O, the combined organic layers were dried over MgSO4, and the solvent was removed by evaporation. The obtained crude product (compound 11) was used in the next step without further purification (quantitative yield). 2-Thiouracil (833 mg, 6.50 mmol) was suspended in EtOH (8 mL), 1 M KOH solution (8 mL) was added, and the mixture was heated to 45°C. After cooling to room temperature, compound 11 dissolved in EtOH (5 mL) was added dropwise and stirred at room temperature overnight. The reaction solution was mixed with saturated NaHCO3 solution (15 mL). The precipitate was collected by filtration, washed with H2O, EtOH, AcOEt, and Et2O, and then dried in vacuum to give compound 12 (72% yield). 1 H-NMR [CDCl3, 500 MHz] δ=7.91 (s, 1H), 7.29-7.28 (d, 1H), 6.78-6.78 (d, 1H), 6.72-6.70 (dd, 1H), 6.12 (s, 1H), 4.35 (s, 2H), 3.71 (s, 3H), 2.31 (s, 3H) 13 C-NMR [CDCl3, 125 MHz] δ=163.98, 143.39, 136.45, 131.13, 121.05, 116.53, 37.10, 24.57 HRMS (FAB) Calcd for compound 12 (M+H + )263.0849. Found 263.0844.

[0099] Synthesis Example 2-3: Synthesis of Compound 13 (Step j) Compound 12 (1.74 g, 4.71 mmol) was dissolved in dry EtOH, then in NaOEt (2.0 equiv., 20% in EtOH). The solution was refluxed at 90 °C, BrCH2CO2Et (2.0 equiv.) was mixed, and stirred for 2 h. Then, after cooling to room temperature, saturated NaHCO3 solution (30 mL) was mixed and EtOH was removed by evaporation. Extraction was performed three times with a mixture of CH2Cl2:MeOH (3:1). The organic layers were combined, dried over MgSO4, and removed by evaporation. The residue was purified by silica gel column chromatography (hexane / AcOEt, 5:1 to 1:1, AcOEt, AcoEt / MeOH, 9:1) to obtain 0.63 g (1.81 mmol) of compound 13 (yield 38%). 1 H-NMR [CDCl3, 500 MHz] δ=7.26-7.24 (d, 1H), 6.73-6.71 (d, 1H), 6.70-6.67 (dd, 1H), 6.08-6.05 (d, 1H), 4.56 (s, 2H), 4.46 (s, 2H), 4.26-4.21 (q, 2H), 3.77 (s, 3H), 2.34 (s, 3H), 1.27-1.24 (t, 3H) 13 C-NMR [CDCl3, 125 MHz] δ=167.91, 166.04, 163.40, 159.54, 144.06, 138.89, 131.83, 124.28, 116.24, 111.44, 109.88, 62.63, 55.21, 52.83, 34.87, 19.51, 14.01 HRMS (FAB) Calcd for compound 13 (M+H + ) 349.1217. Found 349.1233.

[0100] Synthesis Example 2-4: Synthesis of Compound 14 (Step k) Compound 13 (0.63 g, 1.81 mmol) was dissolved in MeOH (4.7 mL), THF (11.1 mL), and 2 M LiOH (1.6 mL) and stirred at room temperature for 10 min. The solvent was removed by evaporation, and the precipitate was filtered, washed with AcOEt, Et2O, and dried in vacuum. The obtained crude product (compound 14) was used in the next step without further purification (quantitative yield). 1 H-NMR [CDCl3, 500 MHz] δ=7.52-7.51 (d, 1H), 7.45-7.43 (d, 1H), 7.31-7.28 (d, 1H), 6.78-6.76 (d ,1H), 6.72-6.68 (dd, 1H), 5.82- 5.80 (d,1H), 5.72-5.69 (d, 1H), 4.28 (s, 1H), 4.10 (s, 1H), 4.05 (s, 1H), 3.79 (s 1H), 3.71 (s, 1H), 2.31 (s, 3H), 1.66 (s, 3H) 13 C-NMR [CDCl3, 125 MHz] δ=175.82, 170.59, 168.50, 167.28, 167.90, 162.26, 158.81, 156.59, 146.39, 145.05, 138.36, 131.43, 125.71, 115.82, 111.34, 107.70, 106.63, 55.78, 55.05, 54.78, 53.26, 32.85, 25.22, 19.13. HRMS (FAB) Calcd for compound 14 (M+H + ) 327.0985. Found 327.0988.

[0101] Synthesis Example 2-5: Synthesis of Compound 15 (Step I) Compound 14 (0.57 g, 1.67 mmol) was dissolved in DMF and coupled with 0.64 g (1.6 equiv, 2.59 mmol) of compound 6 in the presence of Et3N (3.0 equiv) and DMT-MM (1.8 equiv). After the reaction, the mixture was diluted with CHCl3 and the organic layer was washed with saturated NaHCO3. The mixture was dried over MgSO4. The solvent was removed by evaporation and the residue was purified by silica gel column chromatography (CHCl3 / MeOH, 20:1 to 10:1, containing 2% Et3N) to give 1.32 g (0.93 mmol) of compound 7 (yield 56%). 1 H-NMR [CDCl3, 500 MHz] δ=7.37-7.35 (m, 2H), 7.27-7.00 (m, 10H), 6.79-6.76 (m 4H), 6.66-6.64 (d, 1H), 6.58-6.55 (m, 1H), 5.98-5.96 (d, H), 4.47-4.40 (dd, 2H), 4.35-4.20 (dd, 2H), 4.14 (m, 1H), 3.78-3.73 (m, 10H), 3.69-3.65 (m, 1H), 3.29-3.19 (m, 2H), 2.20 (s, 3H). 13 C-NMR [CDCl3, 125 MHz] δ=168.53, 164.87, 163.95, 159.59, 158.69, 158.67, 144.76, 144.68, 138.97, 135.83, 135.72, 131.92, 130.14, 130.05, 128.12, 128.03, 127.04, 124.32, 116.35, 113.34, 111.51, 109.71, 86.51, 62.71, 55.34, 54.44, 52.05, 46.13, 34.99, 19.55. HRMS (FAB) Calcd for compound 15 (M+H + ) 696.2738. Found 696.2768.

[0102] Synthesis Example 2-6: Synthesis of Compound 16 (Step m) Compound 15 (0.92 g, 1.32 mmol) was dissolved in dry CHCl (5 mL) and EtN (0.9 mL) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.44 mL, 1.57 mmol) was added dropwise under nitrogen at 0 °C. After stirring for 20 min at room temperature, the reaction solution was purified by silica gel column chromatography (CHCl / acetone, 4:1 to 2:1 with 3% EtN) to give 0.78 g (0.87 mmol) of compound 8 (yield 66%). 1 H-NMR [CDCl3, 500 MHz] δ=7.40-7.38 (m, 2H), 7.30-7.18 (m, 9H), 7.11-7.07 (d, 1H), 6.81-6.78 (m, 4H), 6.69-6.66 (m, 1H), 6.65-6.59 (m, 1H),6.43-6.38 (m, 1H), 6.03-6.036(m, 1H), 4.51-4.29 (m, 5H), 3.93-3.85 (m, 1H), 3.77 (m, 10H), 3.56-3.45 (m, 3H), 3.35-3.25 (m, 1H), 3.18-3.12 (m, 1H), 2.52-2.35 (m, 2H), 2.26-2.25 (m, 3H), 1.15-1.05 (m, 12H) 13C-NMR [CDCl3, 125 MHz] δ=164.77, 158.58, 154.41, 152.05, 150.84, 150.03, 146.58, 146.48, 144.73, 144.63, 135.91, 135.83, 135.75, 130.13, 130.09, 130.07, 130.04, 128.16, 128.11, 127.93,126.94, 126.80, 118.59, 118.48, 113.23, 113.20, 86.29, 86.27, 83.73, 83.72, 83.28, 83.24, 65.86, 61.45, 58.54, 58.44, 58.40, 58.29, 50.43, 50.37, 45.55, 45.46, 43.18, 43.08, 30.95, 27.82, 27.71, 24.68, 24.62, 20.62, 20.60, 20.56, 15.31 31 P-NMR [CDCl3, 202 MHz] δ=148.03, 147.76 HRMS (FAB) Calcd for compound 16 (M+H + ) 896.3817. Found 896.3824.

[0103] Synthesis Example 3: Synthesis of single-stranded polynucleotide 1 Four single-stranded polynucleotides consisting of the palindrome shown below (D represents 2,6-diaminopurine, and sU represents 2-thiouracil) were prepared. SNAa, D2, and sU2 are SNA polynucleotides. RNAb is an RNA polynucleotide. · SNAa: (S)-ATTGCAAT-(R) RNAb: 5'-AUUGCAAT-3' SNAa-D2: (S)-ATTGCDDT-(R) ·SNAa-sU2: (S)-AsUsUGCDDT-(R).

[0104] SNAa and RNAb were obtained from Fasmac and Hokkaido System Science Co., Ltd.

[0105] SNAa-D2 (hereinafter referred to as D2) and SNAa-sU2 (hereinafter referred to as sU2) were synthesized using an amidite SNA monomer of a natural nucleic acid base, an amidite SNA monomer of 2,6-diaminopurine (compound 8 in Synthesis Example 1), and an amidite SNA monomer of 2-thiouracil, using an automatic DNA synthesizer (ABI-3400 DNA synthesizer, Applied Biosystems). When using an amidite SNA monomer of 2-thiouracil, a decomposition reaction occurs when a normal oxidizing agent (iodine) is used in the phosphorus oxidation step of the phosphoramidite method, so tert-butyl hydroperoxide was used as an oxidizing agent. The CPG-linked SNA polynucleotide was incubated with TFA (13.9% m-cresol and 2.8% H2O) at room temperature for 3 hours to deprotect the acid-removable protecting group on the amidite SNA monomer. The CPG-linked SNA polynucleotide was then washed. After drying, cleavage and deprotection were carried out under 28% NH4OH solution for 4-6 h at 55 °C. The SNAs were then purified by reversed-phase HPLC (Kanto Chemical, Mightysil RP 18 GPII column).

[0106] The results of MALDI-TOF MS analysis of the synthesized SNA polynucleotide are shown in Table 1. Furthermore, no peaks due to insufficient deprotection were observed.

[0107] [Table 1] The HPLC profile of the synthesized SNA polynucleotide is shown in FIG.

[0108] Test Example 1: Melting Temperature Measurement 1 The melting temperature was measured for each of the SNA polynucleotides (SNAa, D2, and sU2). Specifically, the melting temperature was measured as follows. Each SNA polynucleotide was dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl (final concentration 2 μM). The solution was analyzed with a Shimadzu UV-1800, and a melting curve was obtained by measuring the change in absorbance at 260 nm versus temperature. The melting curve was measured at 1.0°C min -1 The melting curves were measured using a temperature ramp of 100 s. Tm values ​​were determined from the maximum of the first derivative of the melting curve.

[0109] The melting curves obtained are shown in Figure 2. The Tm values ​​are shown in Table 2.

[0110] [Table 2] Comparison of D2 and sU2 revealed that the melting temperature of Homo Dimer could be significantly lowered by introducing sU into the complementary site of D.

[0111] Test Example 2: Melting Temperature Measurement 2 The melting temperature of each of the SNA polynucleotides (SNAa, D2, and sU2) and its complementary RNA polynucleotide (RNAb) was measured in the same manner as in Test Example 1, except that each SNA polynucleotide and RNA polynucleotide were dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl (final concentration of SNA polynucleotide: 2 μM, final concentration of RNA polynucleotide: 2 μM).

[0112] The melting curves obtained are shown in Figure 3. The Tm values ​​are shown in Table 3.

[0113] [Table 3] In the case of D2 / RNAb, two sigmoid curves were observed, and the D2 strands and the RNAb strands formed a duplex, respectively, whereas in the case of sU2 / RNAb, the sigmoid curves merged into one, and the Tm was higher than that of the sU2 / sU2 duplex (30.6°C), suggesting that sU2 / RNAb formed a duplex.

[0114] To confirm this, sU2 / RNAb samples were analyzed by native MS. Specifically, this was done as follows: sU2 / RNAb samples were buffer exchanged into 100 mM triethylammonium acetate, pH 7.0, by passing them through a Bio-Rad Micro Bio-Spin 6 column. Buffer-exchanged samples were immediately analyzed by nanoflow electrospray ionization mass spectrometry using gold-coated glass capillaries (approximately 2–5 μL of sample was loaded per analysis). Spectra were recorded on a Waters SYNAPT G2-Si HDMS mass spectrometer in negative ionization mode at 1.33 kV, with sampling cone and source offset voltages of 150 V, 4 V trap and 2 V transfer collision energies, and a trap gas flow rate of 5 mL / min. Spectra were calibrated using 1 mg / mL cesium iodide and analyzed using MassLynx software (Waters).

[0115] The obtained native MS profile is shown in Figure 4. It was confirmed that sU2 / RNAb formed a double strand.

[0116] To quantitatively demonstrate that sU2 / RNAb formed a double strand, polyacrylamide gel electrophoresis (PAGE) was used to analyze the results. Specifically, the procedure was as follows: 25 μM RNAb, SNAa-sU2, and a mixture of RNAb and SNAa-sU2 in 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl were heated to 80 °C and then cooled to 4 °C. Loading buffer without bromophenol blue was mixed with the samples, and the mixtures were analyzed by 20% native-PAGE containing 10% glycerol at 750 CV, 2 h, and 4 °C.

[0117] The PAGE obtained was stained with Fast Blast DNA stain (Biorad) and then imaged using an imaging analyzer (FLA9500 (GE Healthcare)). The results are shown in Figure 5. The band obtained by mixing sU2 and RNAb was located at a position clearly different from the bands of sU2 and RNAb alone, confirming that sU2 and RNAb formed a double strand. Furthermore, a comparison of the band intensities of sU2 and sU2 / RNAb revealed that more than 90% of sU2 formed a double strand with RNAb.

[0118] Synthesis Example 4: Synthesis of single-stranded polynucleotide 2 Four single-stranded polynucleotides (AMO (Anti miRNA oligonucleotide): SEQ ID NO: 1) containing the palindrome (D represents 2,6-diaminopurine, and sU represents 2-thiouracil) shown in Figure 6 were prepared in the same manner as in Synthesis Example 3. All of these are SNA polynucleotides targeting miRNA-21 (miR-21).

[0119] Test Example 3: Melting Temperature Measurement 3 For each of the polynucleotides of Synthesis Example 4, the Tm value was determined in the same manner as in Test Example 1. The Tm values ​​are shown in Figure 6. Introduction of the D-sU pair suppressed the formation of the Homo double strand of SNA-AMO, improving the RNA recognition ability.

[0120] Test Example 4: Measurement of antisense activity 1 Antisense activity was measured for each of the polynucleotides (AMO) of Synthesis Example 4. Specifically, 40 nM of nucleic acid to be introduced (a reporter plasmid containing a luciferase gene (control) or a reporter plasmid containing a miR21 binding site downstream of the luciferase gene (expression of luciferase is suppressed by the miR21 binding site), and AMO) was introduced into HeLa cells using Lipofectamine 2000, and luciferase activity was measured after 24 hours of incubation. Improvement of luciferase activity by introduction of AMO indicates the expression of antisense activity of AMO.

[0121] The results are shown in Figure 7. Introduction of the D-sU pair significantly improved the antisense activity.

[0122] Synthesis Example 5: Synthesis of single-stranded polynucleotide 3 Five single-stranded polynucleotides (AMO: SEQ ID NO: 1) containing the palindrome (D represents 2,6-diaminopurine, and sU represents 2-thiouracil) shown in Figure 8 were prepared in the same manner as in Synthesis Example 3. All of these are SNA polynucleotides targeting miRNA-21 (miR-21).

[0123] Test Example 5: Melting Temperature Measurement 4 For each of the polynucleotides of Synthesis Example 5, the Tm value was determined in the same manner as in Test Example 1. The Tm values ​​are shown in Figure 8. Introduction of the D-sU pair suppressed the formation of the Homo double strand of SNA-AMO, improving the RNA recognition ability.

[0124] Synthesis Example 6: Synthesis of single-stranded polynucleotide 4 Six SNA single-stranded polynucleotides (AMO: sequence number 1) and two LNA single-stranded polynucleotides (AMO: sequence number 1 or a short sequence thereof) containing the palindrome (D represents 2,6-diaminopurine, and sU represents 2-thiouracil) shown in FIG. 9 were prepared in the same manner as in Synthesis Example 3.

[0125] Test Example 6: Melting Temperature Measurement 5 For each of the polynucleotides of Synthesis Example 6, the Tm value was determined in the same manner as in Test Example 1. The Tm values ​​are shown in Figure 9. Introduction of the D-sU pair suppressed the formation of the Homo double strand of SNA-AMO, improving the RNA recognition ability.

[0126] Test Example 7: Measurement of antisense activity 2 The antisense activity of each of the polynucleotides (AMO) of Synthesis Example 6 was measured in the same manner as in Test Example 5. The results are shown in Figure 10. A D-sU-introduced SNA-AMO with extremely high activity was successfully developed.

[0127] Synthesis Example 7: Synthesis of single-stranded polynucleotide 5 Nine SNA single-stranded polynucleotides (AMO: sequence number 1) containing a palindrome (D represents 2,6-diaminopurine, and sU represents 2-thiouracil) shown in FIG. 11, two LNA single-stranded polynucleotides (AMO: sequence number 1 or a short sequence thereof), and one DNA single-stranded polynucleotide (AMO: sequence number 1) were prepared in the same manner as in Synthesis Example 3.

[0128] Test Example 8: Melting Temperature Measurement 6 For each of the polynucleotides of Synthesis Example 7, the Tm value was determined in the same manner as in Test Example 1. The Tm values ​​are shown in Figure 11. Introduction of the D-sU pair suppressed the formation of the Homo double strand of SNA-AMO, improving the RNA recognition ability.

[0129] Test Example 9: Measurement of antisense activity 3 The antisense activity of each of the polynucleotides (AMO) of Synthesis Example 7 was measured in the same manner as in Test Example 5. The results are shown in Figure 12. A D-sU-introduced SNA-AMO with extremely high activity was successfully developed.

[0130] Synthesis Example 8: Synthesis of single-stranded polynucleotide 6 Three SNA single-stranded polynucleotides (AMO: SEQ ID NO: 1) containing a palindrome (D represents 2,6-diaminopurine, and sU represents 2-thiouracil) shown in FIG.

[0131] Test Example 10: Melting Temperature Measurement 7 For each of the polynucleotides of Synthesis Example 8, the Tm value was determined in the same manner as in Test Example 1. The Tm values ​​are shown in Figure 13. The introduction of sU alone does not affect the melting temperature.

[0132] Test Example 11: Measurement of antisense activity 4 The antisense activity of each of the polynucleotides (AMO) of Synthesis Example 8 was measured in the same manner as in Test Example 5. The results are shown in Figure 14. The introduction of sU alone does not affect the antisense activity.

Claims

1. A single-stranded polynucleotide comprising a palindrome containing a non-cyclic polynucleotide building block, in which adenine and / or its analogue in the palindrome is replaced with diaminopurine and thymine and / or its analogue at the complementary position of the adenine is replaced with a thiouracil derivative.

2. The non-cyclic polynucleotide building block has the general formula (1): 【Chemistry 1】 [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups.) Base represents a nucleic acid base.) The single-stranded polynucleotide according to claim 1, which is a structural unit represented by:

3. R 1 is a hydrogen atom or a chain hydrocarbon group, and the R 2 The single-stranded polynucleotide of claim 2, wherein is a hydrogen atom.

4. The palindrome is represented by the general formula (2): A 1 -B-A 2 (In the formula: A 1 and A 2 The length of A is 3 or more. 1 and A 2 The base length of B is 0 to (A 1 Base length + A 2 4. The single-stranded polynucleotide according to claim 1, which is a single-stranded polynucleotide consisting of a base sequence represented by the formula:

5. The single-stranded polynucleotide according to any one of claims 1 to 4, wherein the palindrome is composed of the non-circular polynucleotide building blocks.

6. The single-stranded polynucleotide according to any one of claims 1 to 5, wherein the number of each of the diaminopurine derivatives and the thiouracil derivatives is 2 or more.

7. The single-stranded polynucleotide according to any one of claims 1 to 6, which has a base length of 6 to 500.

8. The single-stranded polynucleotide of any one of claims 1 to 7, which is complementary to a cell-resident polynucleotide.

9. The single-stranded polynucleotide according to any one of claims 1 to 8, which is an anti-miRNA or anti-mRNA polynucleotide.

10. A double-stranded polynucleotide comprising the single-stranded polynucleotide according to any one of claims 1 to 9.

11. General formula (3): 【Chemistry 2】 [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups.) R 3 and R 4 R may be the same or different and represent a protecting group for a hydroxyl group. 5 and R 6 are the same or different and each represents an alkyl group. Base' represents a nucleic acid base in which the amino group of diaminopurine is protected with a protecting group that can be removed by acid, or a nucleic acid base in which the thiocarbonyl group of a thiouracil derivative is protected with a protecting group that can be removed by acid.] or a salt thereof, or a solvate thereof.

12. The compound has the general formula (3A) or (3B): 【Chemistry 3】 [In the formula: R 1 , R 2 / R 3 , R 4 , R 5 and R 6 is the same as above. 7 , R 8 , R 9 , R 10 and R 11 R may be the same or different and represents a protecting group removable with an acid. 12 represents a hydrogen atom or an alkyl group.], or a salt thereof, or a solvate thereof according to claim 11.

13. R 7 , R 8 , R 9 and the above R 10 is a Boc group, and the R 9 The compound according to claim 12, or a salt thereof, or a solvate thereof, wherein is an MMPM group.

14. R 3 is represented by the general formula (4): 【Chemistry 4】 [In the formula: R 31 , R 32 and R 33 are the same or different and each represents a hydrogen atom or an alkoxy group. 4 - (CH 2 ) 2 -CN, and said R 5 and R 6 The compound or a salt thereof, or a solvate thereof according to any one of claims 11 to 13, wherein is an isopropyl group.

15. A method for producing a single-stranded polynucleotide by a phosphoramidite method, comprising using the compound according to any one of claims 11 to 14, or a salt thereof, or a solvate of either of them as an amidite monomer.

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