Nucleic Acid Monomers

Abstract

To provide techniques for reducing stability of formed polynucleotide double strands.SOLUTION: The disclosed method comprises introducing into a polynucleotide at least one kind of nucleoside unit selected from a group consisting of: nucleoside unit 1 in which a group of a formula (1): -NH-C(=O)-R1 (in the formula, R1 is a hydrogen atom or an alkyl group) is linked to 2' carbon atom of a sugar: nucleoside unit 2 in which the amino group in cytosine is replaced by a hydrocarbon group; and a nucleoside unit 3 in which a group of a formula 3: -(O-CH2-)n-(S-)m-R4 (in the formula: R4 is a hydrocarbon group, n and m are respectively an integer of 1-3) is linked to 2' carbon atom of a sugar.SELECTED DRAWING: None

Description

Technical field

[0001] The present invention relates to nucleic acid monomers and the like. [Background technology]

[0002] In RNAi, siRNA (small interfering RNA), which is a double-stranded RNA homologous to a specific region of a gene, is incorporated into Argonaute (AGO) protein, becomes a single strand on AGO, and becomes mRNA with a complementary sequence. This is a phenomenon in which AGO cleaves mRNA upon pairing. It is known that in RNAi, genes other than the target gene may be suppressed, even though siRNA with a sequence that is completely complementary to the target gene is used. The present inventors revealed that genes with a complementary sequence of only the 7 bases 2-8 bases (called the seed region) of the siRNA guide strand from the 5' end of the siRNA guide strand are suppressed by the off-target effect. (Non-patent Document 1). In other words, in the case of siRNA, genes that have completely complementary sequences over their entire length (target genes) are suppressed by the RNAi effect, but not only that, genes that have complementary sequences only in their seed regions (non-target genes) may also be suppressed by an originally unintended effect called an off-target effect. [Prior art documents] [Non-patent literature]

[0003] [Non-patent document 1] Ui-Tei K, Naito Y, Nishi K, Juni A, Saigo K. (2008) Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect. Nucleic Acids Res.36, 7100-7109. [Summary of the invention] [Problem to be solved by the invention]

[0004] While conducting research against the above background, the present inventor focused on suppressing off-target effects by reducing the stability of the double strands in the seed region of siRNA.

[0005] Therefore, an object of the present invention is to provide a technique for reducing stability during polynucleotide duplex formation. [Means to solve the problem]

[0006] As a result of intensive research in view of the above problems, the present inventor discovered that the 2'-position carbon of sugar has the general formula (1): -NH-C(=O)-R. 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group. ), the nucleoside unit 2 is formed by replacing the amino group in cytosine with a hydrocarbon group, and the 2' carbon of the sugar has the general formula (3): -(O -CH 2 -) n -(S-) m -R 4 (In the formula: R 4 indicates a hydrocarbon group. n and m each represent an integer from 1 to 3. ) is introduced into a polynucleotide at least one type of nucleoside unit selected from the group consisting of 3 nucleoside units formed by linking the groups represented by the formula (3) to reduce the stability of the polynucleotide during double strand formation. I found out that it can be done. The present inventor conducted further research based on this knowledge and completed the present invention. That is, the present invention includes the following aspects.

[0007] Item 1. General formula (1): -NH-C(=O)-R at the 2'-position carbon of the sugar 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group. ) A nucleic acid monomer formed by linking groups represented by

[0008] Section 2. General formula (1A):

[0009] [ka] [In the formula: Base represents a monovalent group obtained by removing one hydrogen atom from a nucleobase. R 1 represents a hydrogen atom or an alkyl group. ] Item 1. The nucleic acid monomer according to Item 1, comprising a partial structure represented by:

[0010] Item 3. The nucleic acid monomer according to item 1 or 2, which is a nucleoside, nucleotide, or a derivative thereof.

[0011] Item 4. The nucleic acid monomer according to any one of Items 1 to 3, which is a phosphoramidite monomer.

[0012] Section 5. Said R 1 5. The nucleic acid monomer according to any one of Items 1 to 4, wherein is a hydrogen atom.

[0013] Item 6. General formula (3):-(O-CH 2 -) n -(S-) m -R 4 (In the formula: R 4 indicates a hydrocarbon group. n and m each represent an integer from 1 to 3. ) is a nucleic acid monomer formed by linking groups.

[0014] Section 7. Said R 4 7. The nucleic acid monomer according to item 6, wherein is an alkyl group.

[0015] Item 8. General formula (1): -NH-C(=O)-R at the 2' position of the sugar 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group. ) is connected to the nucleoside unit 1, and the 2' carbon of the sugar has the general formula (3): -(O-CH 2 -) n -(S-) m -R 4 (In the formula: R 4 indicates a hydrocarbon group. n and m each represent an integer from 1 to 3. ) A polynucleotide comprising at least one nucleoside unit selected from the group consisting of 3 nucleoside units connected to each other.

[0016] Item 9. The polynucleotide according to Item 8, which is siRNA.

[0017] Item 10. According to item 9, at least one nucleoside unit selected from the group consisting of the nucleoside unit 1 and the nucleoside unit 3 is contained in the 2nd to 5th nucleoside sequences from the 5' end of the siRNA guide strand. polynucleotide.

[0018] Item 11. Nucleic acid base has general formula (2)

[0019] [ka] [In the formula: R 2 indicates a hydrocarbon group. ] A reagent for producing polynucleotides containing a nucleic acid monomer having a group represented by:

[0020] Item 12. The reagent according to item 11, for use in producing a polynucleotide with reduced stability during double-strand formation, or for use in producing siRNA.

[0021] Item 13. Nucleic acid base has general formula (2)

[0022] [ka] [In the formula: R 2 indicates a hydrocarbon group. ] siRNA containing nucleoside unit 2, which is a group represented by.

[0023] Item 14. The siRNA according to Item 13, wherein the nucleoside unit 2 is included in the 2nd to 5th nucleoside sequence from the 5' end of the siRNA guide strand.

[0024] Item 15. A reagent or medicament containing at least one selected from the group consisting of the polynucleotide according to Item 9 or 10 and the siRNA according to Item 13 or 14.

Effect of the invention

[0025] According to the present invention, it is possible to provide a technique for reducing stability during polynucleotide duplex formation. [Brief explanation of the drawing]

[0026]

Figure 1

Figure 2

[0027] In this specification, the expressions "contain" and "comprising" include the concepts of "containing", "containing", "consisting essentially" and "consisting only".

[0028] 1.Nucleic acid monomer In one embodiment of the present invention, the 2' carbon of the sugar has the general formula (1): -NH-C(=O)-R 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group. ) A nucleic acid monomer (herein sometimes referred to as "nucleic acid monomer 1 of the present invention") formed by linking groups represented by the following. Furthermore, in one embodiment of the present invention, the nucleobase is represented by the general formula (2).

[0029] [ka] [In the formula: R 2 indicates a hydrocarbon group. ] It relates to a nucleic acid monomer that is a group represented by (herein sometimes referred to as "nucleic acid monomer 2 of the present invention"). Furthermore, in one embodiment of the present invention, the 2' carbon of the sugar has the general formula (3):-(O-CH 2 -) n -(S-) m -R 4 (In the formula: R 4 indicates a hydrocarbon group. n and m each represent an integer from 1 to 3. ) is linked to a nucleic acid monomer (herein sometimes referred to as "nucleic acid monomer 3 of the present invention"). In this specification, the nucleic acid monomers 1 to 3 of the present invention may be collectively referred to as "the nucleic acid monomers of the present invention." These will be explained below.

[0030] <1-1. Nucleic acid monomer> As used herein, a nucleic acid monomer is a compound that can serve as a constituent unit of a polynucleotide with some atoms and / or groups removed, and includes a partial structure in which a nucleobase and a sugar are linked. There are no particular restrictions. This section explains nucleic acid monomers.

[0031] As the nucleic acid base, any base constituting a nucleic acid can be used without particular limitation. The bases that make up nucleic acids include only typical bases found in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.). However, bases other than these, such as hypoxanthine (I) and modified bases, are also included. Examples of modified bases include 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-carboxymethyl Aminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxy Aminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxy Examples include acetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, and the like.

[0032] The sugar is not particularly limited as long as it can constitute a polynucleotide, but typical examples include pentose sugars such as ribose and deoxyribose. In addition, sugars also include those that have been modified. Such sugars include, for example, a hydroxyl group at the 2' position of ribose, -OR (R is a hydroxyl protecting group (e.g., TBDMS group (tert-butyldimethylsilyl group), TOM group (triisopropylsiloxymethyl group) , alkyl group (e.g. methyl group, ethyl group, etc.), alkoxyalkyl group (e.g. methoxyethyl group, etc.), -CH 2 CH 2 NHC(NH)NH 2 ,-CH 2 CONHCH 3 ,-CH 2 CH 2 CN, etc.), the 2' and 4' carbons of the sugar are cross-linked through other atoms (e.g., oxygen atoms, carbon atoms) or directly, thereby increasing the composition of the sugar moiety. Examples include sugars with a fixed N-type formation, sugars with a halogen atom (eg, fluorine atom) linked to the 2′ carbon of the sugar, and the like.

[0033] Nucleic acid monomers typically have the general formula (X):

[0034] [ka] [In the formula: Base represents a monovalent group obtained by removing one hydrogen atom from a nucleobase. R a is a hydrogen atom, a halogen atom, or -OR group, etc.), alkoxyalkyl group (e.g., methoxyethyl group, etc.), -CH 2 CH 2 NHC(NH)NH 2 ,-CH 2 CONHCH 3 ,-CH 2 CH 2 CN etc.). ) shows. ] Contains the substructure represented by .

[0035] Nucleic acid monomers include nucleosides, nucleotides, derivatives thereof, and the like. Derivatives include, but are not particularly limited to, derivatives for use in producing polynucleotides (eg, phosphoramidite monomers, etc.).

[0036] A nucleoside is a compound formed by linking a nucleobase and a sugar. The nucleoside typically includes a compound in which a hydrogen atom is linked to the oxygen atom linked to the 3' and 5' carbons of a sugar in the partial structure represented by the above general formula (X).

[0037] A nucleotide is typically a compound in which a phosphate group is bonded to a nucleoside. Examples of the nucleotide include monophosphate, diphosphate, triphosphate, etc. depending on the number of bonded phosphate groups. Furthermore, compounds with modified phosphate groups are also included in nucleotides.

[0038] The phosphoramidite monomer is a monomer used in solid phase synthesis of polynucleotides, and is not particularly limited as far as this is concerned. As a phosphoramidite monomer, for example, general formula (X1):

[0039] [ka] [In the formula: Base and R a is the same as above. R b and R c are the same or different and represent a hydroxyl-protecting group. R d and R e are the same or different and represent an alkyl group. ] Examples include compounds represented by:

[0040] R b Any group can be used without particular limitation as long as it can function as a protecting group for a hydroxyl group, and a wide range of known protecting groups used in amidite monomers can be used. R b is preferably the general formula (X2):

[0041] [ka] [In the formula: R 31 ,R 32 and R 33 are the same or different and represent a hydrogen atom or an alkoxy group. ] Examples include groups represented by:

[0042] R 31 ,R 32 and R 33 It is preferable that one of the groups is hydrogen and the remaining two are alkoxy groups, and the alkoxy group is particularly preferably a methoxy group.

[0043] R c Any protective group that can function as a protecting group for a hydroxyl group can be used without particular limitation, and a wide variety of known protecting groups used in amidite monomers can be used. R cExamples include alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, haloalkyl groups, aryl groups, heteroaryl groups, arylalkyl groups, cycloalkenyl groups, cycloalkylalkyl groups, cyclylalkyl groups, hydroxyalkyl groups, Aminoalkyl group, alkoxyalkyl group, heterocyclylalkenyl group, heterocyclylalkyl group, heteroarylalkyl group, silyl group, silyloxyalkyl group, mono-, di- or trialkylsilyl group, mono-, di- or trialkylsilyloxyalkyl group, etc. and these may be substituted with electron-withdrawing groups.

[0044] R c 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, an arylsulfonyl group, a trihalomethyl group, a trialkylamino group, and preferably a cyano group. R c is particularly preferably -(CH 2 ) 2 -CN.

[0045] R d and R e The alkyl group represented by may be linear or branched, preferably an alkyl group having 1 to 12 carbon atoms, and more preferably an alkyl group having 1 to 6 carbon atoms. Alkyl groups include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl. The alkyl group herein also includes alkyl moieties such as an alkoxy group. R 5 and R 6 may be bonded to each other to form a cyclic structure.

[0046] R d and R e are particularly preferably both isopropyl groups.

[0047] Nucleic acid monomers also include salts. Salts are not particularly limited, and include, for example, 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; lysine; Examples include salts with basic amino acids such as ornithine and arginine, and ammonium salts. The salt may be an acid addition salt, and examples of such salt include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; formic acid, acetic acid, Organic acids such as propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid; aspartic acid, glutamic acid, etc. and acid addition salts with acidic amino acids. Nucleic acid monomers also include hydrates, solvates, crystal polymorphs, and the like.

[0048] <1-2. Nucleic acid monomer of the present invention> Nucleic acid monomer 1 of the present invention has the general formula (1): -NH-C(=O)-R at the 2'-position carbon of the sugar. 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group. ) is formed by connecting groups.

[0049] R 1 The alkyl group represented by may be linear or branched, preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, even more preferably 1 to 4 carbon atoms, even more preferably Examples include alkyl groups having 1 to 2 carbon atoms. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, and the like.

[0050] R 1 is particularly preferably a hydrogen atom.

[0051] Nucleic acid monomer 1 of the present invention preferably has the general formula (1A):

[0052] [ka] [In the formula: Base and R 1 is the same as above. ] Contains the substructure represented by .

[0053] Nucleic acid monomer 1 of the present invention can be produced by various methods.

[0054] Nucleic acid monomer 1 of the present invention includes, for example, a nucleic acid monomer (compound 1) in which an amino group is linked to the 2' carbon of a sugar, and a general formula (1B): HO-C(=O)-R 1 (In the formula: R 1 is the same as above. ) (Compound 2).

[0055] As Compound 1 and Compound 2, commercially available compounds can be used as they are, or if necessary, compounds synthesized according to or according to known methods and / or according to or according to the methods described in Examples are used. You can also do that.

[0056] The amount of Compound 2 to be used is usually preferably 0.5 to 3 mol, more preferably 1 to 2 mol, per 1 mol of Compound 1, from the viewpoint of yield, ease of synthesis, and the like.

[0057] This reaction is usually carried out in the presence of a reaction solvent. Examples of the reaction solvent include, but are not limited to, dichloromethane, dimethylformamide, acetonitrile, tetrahydrofuran, acetone, toluene, and the like, with dichloromethane being preferred. A single solvent may be used, or a plurality of solvents may be used in combination.

[0058] This reaction is usually carried out in the presence of a base. The base is not particularly limited, and for example, a wide variety of known bases can be used. Specific examples of the base include diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP). Further, this reaction is preferably carried out in the presence of a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC).

[0059] In this reaction, in addition to the above-mentioned components, additives can also be used as appropriate within a range that does not significantly impair the progress of the reaction.

[0060] The reaction temperature can be any of heating, room temperature, and cooling, and is usually preferably carried out at 10 to 80°C. The reaction time is not particularly limited and can usually be from 30 minutes to 30 hours.

[0061] The progress of the reaction can be followed by conventional methods such as chromatography. After the reaction is completed, the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. In addition, the structure of the product was determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 It can be identified by C-NMR etc.

[0062] The nucleic acid monomer 2 of the present invention has a nucleic acid base represented by the general formula (2)

[0063] [ka] [In the formula: R 2 indicates a hydrocarbon group. ] It is a group represented by

[0064] R 2 The hydrocarbon group represented by is not particularly limited, and includes, for example, an alkyl group, an aryl group, and a group formed by any combination of these groups (for example, an aralkyl group, an alkylaryl group, an alkylaralkyl group), etc. It will be done.

[0065] R 2 The alkyl group represented by may be linear or branched, preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, even more preferably 1 to 4 carbon atoms, and even more preferably Examples include alkyl groups having 1 to 2 carbon atoms. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, and the like.

[0066] R 2 The aryl group represented by is not particularly limited, but it preferably has 6 to 12 carbon atoms, more preferably 6 to 12 carbon atoms, and even more preferably 6 to 8 carbon atoms. The aryl group may be monocyclic or polycyclic (eg, bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include phenyl group, naphthyl group, biphenyl group, pentalenyl group, indenyl group, anthranyl group, tetracenyl group, pentacenyl group, pyrenyl group, perylenyl group, fluorenyl group, phenanthryl group, etc. and preferably phenyl group.

[0067] R 2 Although the aralkyl group represented by Examples include aralkyl groups in which a hydrogen atom) is substituted with the above aryl group. Specific examples of the aralkyl group include benzyl group and phenethyl group.

[0068] R 2 The alkylaryl group represented by is not particularly limited, but for example, the hydrogen atom (for example, 1 to 3, preferably 1 hydrogen atom) of the above aryl group is a straight or branched chain having 1 to 6 carbon atoms ( Preferred examples include alkylaryl groups substituted with the alkyl groups 1 to 2). Specific examples of the alkylaryl group include tolyl group and xylyl group.

[0069] R 2 The alkylaralkyl group represented by Examples include alkylaralkyl groups substituted with 1 to 6 (preferably 1 to 2) alkyl groups.

[0070] R 2 is particularly preferably an alkyl group.

[0071] Nucleic acid monomer 2 of the present invention preferably has general formula (2A):

[0072] [ka] [In the formula: R 2 and R a is the same as above. ] Contains the substructure represented by .

[0073] Nucleic acid monomer 2 of the present invention can be produced according to or analogously to a known method. For example, it can be produced according to the method described in a known document (NUCLEOSIDES & NUCLEOTIDES, 14(3-5), 1129-1 132 (1995) Jane A. Grasby, Mohinder Singh, Jonathan Karn & Michael J. Gait).

[0074] Nucleic acid monomer 3 of the present invention has the general formula (3):-(O-CH 2 -) n -(S-) m -R 4 (In the formula: R 4 indicates a hydrocarbon group. n and m each represent an integer from 1 to 3. ) is formed by connecting groups.

[0075] R 3 The hydrocarbon group represented by is not particularly limited, and includes, for example, an alkyl group, an aryl group, and a group formed by any combination of these groups (for example, an aralkyl group, an alkylaryl group, an alkylaralkyl group), etc. It will be done.

[0076] R 2 The alkyl group represented by may be linear or branched, preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms. It will be done. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, and the like.

[0077] R 3 For the aryl group, aralkyl group, alkylaryl group, and alkylaralkyl group represented by R 2 It is similar to

[0078] R 3 is particularly preferably an alkyl group.

[0079] n is preferably 2 to 3 or 1 to 2, particularly preferably 2.

[0080] m is preferably 1-2.

[0081] Nucleic acid monomer 3 of the present invention preferably has the general formula (3A):

[0082] [ka] [In the formula: Base, R 4 , n, and m are the same as above. ] Nucleic acid monomer 3 of the present invention can be produced by various methods. Nucleic acid monomer 3 of the present invention can be produced, for example, according to or in accordance with the method described in the Examples below.

[0083] Nucleic acid monomers of the invention are preferably nucleosides, nucleotides, or derivatives thereof. Among these, the nucleic acid monomer of the present invention is particularly preferably a phosphoramidite monomer.

[0084] Regarding the nucleic acid monomer of the present invention, matters not explained above are as described in "1-1. Nucleic acid monomer" above.

[0085] <2. Polynucleotide> In one embodiment of the present invention, the carbon at the 2' position of the sugar has the general formula (1): -NH-C(=O)-R 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group. ) The nucleoside unit 1 is formed by linking the groups represented by

[0086] [ka] [In the formula: R 2 is the same as above. ] The nucleoside unit 2 is a group represented by the general formula (3):-(O-CH 2 -) n -(S-) m -R 4 (In the formula: R 4 indicates a hydrocarbon group. n and m each represent an integer from 1 to 3. ) containing at least one nucleoside unit selected from the group consisting of 3 nucleoside units connected to each other (herein sometimes referred to as "polynucleotide of the present invention") ). This will be explained below.

[0087] In this specification, the nucleoside unit is a repeating unit that constitutes a polynucleotide, and is not particularly limited as long as it includes a partial structure formed by linking a nucleobase and a sugar. The nucleoside unit typically has the general formula (X):

[0088] [ka] [In the formula: Base and R a is the same as above. ] Contains the substructure represented by .

[0089] Nucleoside unit 1 has the general formula (1): -NH-C(=O)-R on the carbon at the 2' position of the sugar. 1 (In the formula: R 1 is the same as above. ) is formed by connecting groups.

[0090] The nucleoside unit 1 preferably has the general formula (1A):

[0091] [ka] [In the formula: Base and R 1 is the same as above. ] Contains the substructure represented by .

[0092] Nucleoside unit 2 has a nucleobase of general formula (2)

[0093] [ka] [In the formula: R 2 is the same as above. ] It is a group represented by

[0094] Nucleoside unit 2 preferably has the general formula (2A):

[0095] [ka] [In the formula: R 2 and R a is the same as above. ] Contains the substructure represented by .

[0096] Nucleoside unit 3 has the general formula (3):-(O-CH 2 -) n -(S-) m -R 4 (In the formula: R 4 indicates a hydrocarbon group. n and m each represent an integer from 1 to 3. ) is formed by connecting groups.

[0097] The nucleoside unit 3 preferably has the general formula (3A):

[0098] [ka] [In the formula: Base, R 4 , n, and m are the same as above. ] Contains the substructure represented by .

[0099] The number of nucleoside units 1, the number of nucleoside units 2, the number of nucleoside units 3, or the total number thereof contained in the polynucleotide of the present invention is not particularly limited, but for example, 1 to 20, 1 to 10, 1 to 5 , 1~3, 1~2, or 1.

[0100] The ratio of the number of nucleoside units 1, the number of nucleoside units 2, the number of nucleoside units 3, or their total number contained in the polynucleotide of the present invention to the total number of nucleoside units constituting the polynucleotide of the present invention is particularly For example, but not limited to, 80% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 2% or less, or 1% or less.

[0101] In the polynucleotide of the present invention, the constituent units other than nucleoside unit 1, nucleoside unit 2, and nucleoside unit 3 are not particularly limited, and constituent units of various artificial nucleic acids including natural nucleic acids, aTNA, and SNA can be employed. Specifically, the nucleic acids that can be employed include, in addition to DNA, RNA, etc., those that have been subjected to known chemical modifications, as exemplified below. The hydroxyl group at the 2' position of the sugar (ribose) of each ribonucleotide is replaced by -OR (R is, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3 , CH2CH2CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introducing a methyl group or cationic functional group to the 5-position of the pyrimidine base, or replacing the carbonyl group at the 2-position with thiocarbonyl. can be mentioned. Further examples include, but are not limited to, those in which the phosphoric acid moiety or hydroxyl moiety is modified with biotin, an amino group, a lower alkylamine group, an acetyl group, or the like. In addition, BNA (LNA), etc., in which the conformation of the sugar part of the nucleotide is fixed to N-type by cross-linking the 2' oxygen and 4' carbon of the sugar part of the nucleotide, can also be preferably used.

[0102] In the polynucleotide of the present invention, the bonding mode between nucleoside units is not particularly limited, and examples include phosphodiester bonds, phosphorothioate bonds, methylphosphonate bonds, hydroxymethylphosphonate bonds, phosphorodithionate bonds, and the like.

[0103] The base length of the polynucleotide of the present invention is not particularly limited, and is, for example, 6 to 10,000. The base length is preferably 6 to 1000, more preferably 6 to 500, even more preferably 6 to 200, even more preferably 6 to 100, particularly preferably 6 to 50, from the viewpoint of ease of synthesis.

[0104] The polynucleotides of the present invention also include those to which other molecules are linked. Other molecules are not particularly limited and include, for example, fluorescent labels, biotin, azide, ethynyl groups, and the like. Fluorescent labels include fluorescein, rhodamine, Texas red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM (trademark), Cy (registered trademark) 3, Cy (registered trademark) 5, Alexa Fluor (registered trademark) ) series, etc.

[0105] The polynucleotide of the present invention can be single-stranded or double-stranded. Moreover, the polynucleotide of the present invention can be chain-like or circular.

[0106] The polynucleotide of the present invention preferably exhibits various functions by binding to a target polynucleotide. Specific examples of the polynucleotide of the present invention include siRNA, miRNA, antisense nucleic acids, and the like. Among these, siRNA is particularly preferred. The siRNA of the present invention can also target genes that are difficult to target with existing siRNAs (for example, genes whose seed region contains a large amount of CG). Furthermore, the siRNA of the present invention can suppress the target gene while suppressing off-target effects.

[0107] The positions of nucleoside unit 1, nucleoside unit 2, and nucleoside unit 3 in the polynucleotide of the present invention are not particularly limited, but these nucleoside units are located at the 2nd to 8th (preferably 2nd to 5th) nucleosides from the 5' end. Preferably, it is included in the sequence. When the polynucleotide of the present invention is siRNA, off-target activity can be suppressed more efficiently by including nucleoside unit 1, nucleoside unit 2, and nucleoside unit 3 at the above positions in the guide strand. This position is also preferable from the viewpoint of suppressing the influence on the RNAi activity of the siRNA toward the target gene.

[0108] siRNA is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of a target gene. In one embodiment, the siRNA preferably has a length of, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more. Further, the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. It is envisaged that the upper and lower limits of the length of siRNA described herein may be combined arbitrarily. For example, lengths where the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases. a certain length; the lower limit is 20 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 21 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; A combination of lengths that are bases is envisaged.

[0109] siRNA may be shRNA (small hairpin RNA). shRNA can be designed so that a portion thereof forms a stem-loop structure. For example, in shRNA, if the sequence of a certain region is sequence a, and the complementary strand to sequence a is sequence b, then these sequences exist in one RNA strand in the order of sequence a, spacer, and sequence b. It can be designed to have a total length of 45 to 60 bases. Sequence a is a partial region of the base sequence encoding the target Fchsd1, and the target region is not particularly limited, and any region can be used as a candidate. The length of sequence a is 19 to 25 bases, preferably 19 to 21 bases.

[0110] Fchsd1-specific siRNA may have additional bases at the 5' or 3' end. The length of the additional base is usually about 2 to 4 bases. The additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Sequences of such additional bases include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3. Examples include, but are not limited to, sequences such as ', uuuuu-3'.

[0111] The siRNA may have a protrusion sequence (overhang) at the 3' end, and specific examples include those to which dTdT (dT represents deoxythymidine) is added. Alternatively, it may have a blunt end without any terminal addition. The sense strand and the antisense strand of siRNA may have different numbers of bases, for example, "asymmetrical interfering RNA (asymmetrical interfering RNA)" in which the antisense strand has overhangs at the 3' and 5' ends. aiRNA). A typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and an overhang structure of 3 bases at each end of the antisense strand.

[0112] The location of the target sequence of siRNA is not particularly limited, but in one embodiment, the target sequence may be selected from the 5'-UTR and up to about 50 bases from the start codon, and from a region other than the 3'-UTR. desirable. For the selected target sequence candidate group, we performed BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) to confirm the specificity of the selected target sequence. For the target sequence for which specificity has been confirmed, a sense strand with a 3'-terminal overhang of TT or UU at bases 19-21 after AA (or NA), a sequence complementary to the 19-21 bases, and a TT or A double-stranded RNA consisting of a UU strand and an antisense strand having a 3'-terminal overhang may be designed as siRNA. In addition, for shRNA, which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5 to 25 bases) that can form a loop structure is appropriately selected, and the sense strand and antisense strand are connected via the linker sequence. It can be designed by connecting them.

[0113] The sequences of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include the following: siRNA Target Finder provided by Ambion (http: / / www.ambion.com / jp / techlib / misc / siRNA_finder.html) pSilencer(R) Expression Vector insert design tool (http: / / www.ambion.com / jp / techlib / misc / psilencer_converter.html) GeneSeer provided by RNAi Codex (http: / / codex.cshl.edu / scripts / newsearchhairpin.cgi).

[0114] The polynucleotide of the present invention can be easily produced according to known genetic engineering techniques, artificial nucleic acid production techniques, and the like. For example, it can be produced using PCR, restriction enzyme cleavage, nucleic acid ligation techniques, etc. Moreover, it can be suitably produced according to the production method of the present invention described below.

[0115] The polynucleotide of the present invention can be used in medicines, reagents, etc. (hereinafter, these may be collectively referred to as "the agent of the present invention").

[0116] 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. Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, and binders. , disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.

[0117] 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 thereof. The agent of the present invention can be used, for example, in vitro (eg, added to the culture medium of cultured cells) or in vivo (eg, administered to animals).

[0118] The subject to which the agent of the present invention can be applied is not particularly limited, and includes, for example, various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; animal cells; and the like. The type of cells is not particularly limited, and examples include blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, and muscle cells. , epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, etc.

[0119] When the agent of the present invention is used as an anticancer agent, the type of cancer cells used is not particularly limited, and examples include kidney cancer cells, leukemia cells, esophageal cancer cells, gastric cancer cells, colon cancer cells, and liver cancer cells. Examples include cells, pancreatic cancer cells, lung cancer cells, prostate cancer cells, skin cancer cells, breast cancer cells, and cervical cancer cells.

[0120] The agent of the present invention can be in any dosage form, such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, Oral preparations such as hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jelly preparations; ), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (e.g. ointments, poultices, lotions), suppository inhalants, eye preparations, eye ointments, nasal drops. Parenteral preparations such as tablets, ear drops, and liposomes can be used.

[0121] The route of administration of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and includes oral administration, tube feeding, enteral administration such as enema administration; transvenous administration, transarterial administration, intramuscular administration; Examples include parenteral administration such as 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 to which it is applied, the condition of the subject, etc., and is not limited to, for example, 0.0001 to 100% by weight, preferably 0.001% by weight. ~50% by weight.

[0122] The dosage when administering the agent of the present invention to animals is not particularly limited as long as it is an effective amount that exhibits the medicinal effect, and is generally administered orally as the weight of the polynucleotide of the present invention, which is the active ingredient. 0.1-1000 mg / kg body weight per day, preferably 0.5-50 mg / kg body weight per day, and 0.01-100 mg / kg body weight per day, preferably 0.1-100 mg / kg body weight per day for parenteral administration. 10 mg / kg body weight. The above dosage is preferably administered once a day or divided into 2 to 3 times a day, and can be increased or decreased as appropriate depending on age, pathological condition, and symptoms.

[0123] Regarding the polynucleotide of the present invention, matters not explained above are as described in "1. Nucleic acid monomer" above.

[0124] <3.Reagent> In one aspect, the present invention provides a reagent (herein referred to as , may also be referred to as "the reagent of the present invention". This will be explained below.

[0125] The reagent of the present invention is not particularly limited as long as it contains the nucleic acid monomer of the present invention, and may further contain other components as necessary. Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients. Other ingredients include ingredients with pharmacological effects as well as additives. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, Examples include chelating agents.

[0126] The content of the nucleic acid monomer of the present invention in the reagent of the present invention is, for example, 0.001 to 100% by mass.

[0127] The mode of use of the reagent of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type thereof. The reagent of the present invention can be used, for example, as a reagent for producing polynucleotides (preferably for producing polynucleotides with reduced stability during double-strand formation) or for producing siRNA (siRNA with suppressed off-target activity). It can be used as a reagent for use in In addition, it can be used, for example, in vitro (eg, added to the culture medium of cultured cells) or in vivo.

[0128] The reagents of the invention can also be in the form of a kit. The kit may contain other reagents and instruments (eg, reagents and instruments used for producing polynucleotides) as necessary.

[0129] Regarding the reagent of the present invention, matters not explained above are as described in "1. Nucleic acid monomer" and "2. Polynucleotide" above.

Example

[0130] The present invention will be described in detail below based on Examples, but the present invention is not limited to these Examples.

[0131] Synthesis Example 1. Synthesis of phosphoramidite of 2´-formamide uridine The following phosphoramidite of 2'-formamide uridine was synthesized.

[0132] [ka] The synthesis scheme is as follows.

[0133] [ka]

[0134] <Spectrum measurement> NMR spectra were measured using JEOL ECS-400 (400 MHz). 1 The chemical shift value of the H-NMR spectrum is determined by the residual protons of the solvent (CHCl 3 : 7.26 ppm, CD 3 SOCD 2 H: 2.50 ppm) was used as an internal standard. 13 The chemical shift value of the C-NMR spectrum is the signal derived from the measurement solvent (CDCl 3 : 77.16 ppm, CD 3 SOCD 3 : 33.52 ppm) was used as an internal standard. 31 For chemical shift values ​​of P-NMR spectra, 85% phosphoric acid (0 ppm) was used as an internal standard. The signal multiplicity was expressed using the abbreviations s: singlet, d: doublet, t: triplet, q: quartet, and m: multiplet, respectively.

[0135] MS spectra were measured using Bruker Daltonics micrOTOF-Q II (ESI-MS).

[0136] <Synthesis of compound 2> Dissolve 2,2´-O-cyclouridine (compound 1) (5.00 g, 22.1 mmol, 1 eq.) in pyridine (50 mL) and add DMTrCl (8.24 g, 24.3 mmol, 1.1 eq.), DMAP with stirring. (0.0540 g, 0.442 mmol, 0.02 eq.) was added and stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate and washed with aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered through a cotton plug, and the solvent was distilled off. The crude product was purified by neutral flash column chromatography (ethyl acetate / methanol=10 / 1) to obtain compound 2 (10.4 g, 19.8 mmol, 90%). 1 H-NMR (400 MHz, DMSO-d 6 ): δ 7.906 (d, J = 7.6 Hz, 1H), 7.239-7.085 (m, 9H), 6.822-6.777 (m, 4H), 6.287 (d, J = 5.6 Hz, 1H), 5.938 (br, s , 1H), 5.842 (d, J = 7.6 Hz, 1H), 5.169 (d, J = 5.6 Hz, 1H), 4.262 (s, 1H), 4.177 (m, 1H), 3.692 (s, 6H), 2.901 (dd, J = 10.4, 4.4 Hz, 1H), 2.773 (dd, J = 10.4, 7.4 Hz, 1H); 13 C-NMR (100 MHz, DMSO-d 6 ): δ 171.493, 159.800, 158.564, 145.181, 137.287, 135.715, 130.063, 128.386, 127.965, 127.210, 113.757, 109.417, 90.295, 88 .963, 87.383, 85.907, 75.254, 63.346, 55.499; HRMS (ESI + ) calc. m / z529.1969 (M + H + ), 551.1789 (M + Na + ), 567.1528 (M + K + ), found m / z 529.1970 (M + H + ), 551.1792 (M + Na + ), 567.1529 (M + K + )..

[0137] <Synthesis of compound 3> Dissolve compound 2 (10.0 g, 18.9 mmol, 1eq.) in DMF (62.5 mL), add sodium azide (6.5 g, 100 mmol, 5 eq.), 15-crown-5 (37.5 mL, 189.5 mmol, 10 eq. .) and stirred at 120°C for 42 hours. The reaction solution was diluted with a 2 / 1 mixture of hexane / ethyl acetate, and washed with aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered through a cotton plug, and the solvent was distilled off. The crude product was purified by neutral flash column chromatography (hexane / ethyl acetate=1 / 2) to obtain compound 3 (7.01 g, 12.3 mmol, 65%). 1 H-NMR (400 MHz, CDCl 3 ): δ 8.934 (br, s, 1H), 7.894 (d, J = 8.4 Hz, 1H), 7.364-7.233 (m, 9H), 6.835 (d, J = 4.4 Hz, 4H), 5.964 (d, J = 2.8 Hz, 1H), 5.365 (d, J = 8.0 Hz, 1H), 4.475 (q, J = 6.4 Hz, 1H), 4.159 (q, J = 2.8 Hz, 1H), 4.023 (m, 1H), 3.782 (s, 6H), 3.589 (dd, J = 11.4, 2.2 Hz, 1H), 3.474 (dd, J = 11.4, 2.6 Hz, 1H), 2.524 (d, J=6.8 Hz, 1H); 13 C-NMR (100 MHz, CDCl 3 ): δ 163.062, 158.865, 150.129, 144.254, 139.609, 135.203, 135.060, 130.167, 128.164, 127.363, 113.448, 102.527, 87.811, 87 .382, 83.290,69.823, 67.267, 61.421, 55.365; HRMS (ESI + ) calc. m / z 594.1959 (M + Na + ), 610.1699 (M + K + ), found m / z 594.1940 (M + Na + ), 610.1708 (M + K + )..

[0138] <Synthesis of compound 4> Compound 3 (6.50 g, 11.4 mmol) was dissolved in methanol (115 mL), palladium / carbon (10% palladium, approximately 55% water wet product) (0.95 g) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. . The reaction solution was filtered through Celite to remove the palladium catalyst, and the solvent was distilled off to obtain Compound 4 (5.66 g, 10.4 mmol, 91%). 1 H-NMR (400 MHz, CDCl 3 ): δ 7.778 (d, J = 8.4 Hz, 1H), 7.199-7.363 (m, 9H), 6.842-6.812 (m, 4H), 5.879 (d, J = 6.4 Hz, 1H), 5.401 (d, J = 8.4 Hz, 1H), 4.212-4.180 (m, 2H), 3.775 (s, 6H), 3.580 (t, J = 6.0 Hz, 1H), 3.472-3.380 (m, 2H); 13 C-NMR (100 MHz, CDCl 3 ): δ 163.157, 158.789, 151.188, 144.311, 140.010, 135.346, 135.193, 130.129, 128.126, 127.249, 113.400, 102.508, 89.928, 87 .172, 85.474, 71.683, 63.462, 59.628, 55.346; HRMS (ESI + ) calc. m / z 546.2235 (M + H + ), 568.2054 (M + Na + ), found m / z 546.2276 (M + H + ), 568.2074 (M + Na + )..

[0139] <Synthesis of compound 5> Compound 4 (5.50 g, 10.1 mmol, 1 eq.) was dissolved in dichloromethane (75 mL), DIPEA (26.4 mL, 152 mmol, 15 eq.), EDC·HCl (2.90 g, 15.1 mmol, 1.5 eq.), DMAP (0.0620 g, 0.507 mmol, 0.05 eq.) and formic acid (0.571 mL, 15.1 mmol, 1.5 eq.) were added, and the mixture was stirred at room temperature for 19 hours. The reaction solution was washed with aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered through a cotton plug, and the solvent was distilled off. The crude product was purified by neutral silica gel column chromatography (dichloromethane / methanol=10 / 1) to obtain compound 5 (3.30 g, 5.75 mmol, 57%). 1 H-NMR (400 MHz, CDCl 3 ): δ 8.185 (s, 1H), 7.608 (d, J= 8.4 Hz, 1H), 7.543 (d, J = 7.2 Hz, 1H), 7.367-7.165 (m, 9H), 6.818-6.796 (m, 4H) ), 6.103 (d, J = 8.8 Hz, 1H), 5.393 (d, J = 8.0 Hz, 1H), 4.760 (dd, J = 8.4 Hz, J = 14 Hz, 1H), 4.375 (d, J= 5.6 Hz, 1H), 4.162 (m, 1H), 3.717 (s, 6H), 3.364 (m, 2H); 13 C-NMR (100 MHz, CDCl 3 ): δ 163.594, 163.153, 158.727, 151.935, 144.146, 140.007, 135.342, 130.197, 128.262, 128.157, 127.237, 113.432, 103.478, 8 7.201, 85.591, 85.524, 71.786, 63.815, 55.327, 54.992; HRMS (ESI + ) calc. m / z596.2003 (M + Na + ), 612.1743 (M + K + ), found m / z596.2026 (M + Na + ), 612.1764 (M + K + )..

[0140] <Synthesis of compound 6> Compound 5 (3.00 g, 5.23 mmol, 1 eq.) was azeotroped with toluene, dissolved in dichloromethane (55 mL) in an ice bath, DIPEA (5.50 mL, 31.6 mmol, 6 eq.), 2-cyanoethyl-N ,N-diisopropylchlorophosphoramidite (2.90 mL, 13.0 mmol, 2.5 eq.) was added, stirred in an ice bath for 3 hours, and quenched with methanol. It was diluted with ethyl acetate and washed with aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered through a cotton plug, and the solvent was distilled off. The crude product was purified by neutral silica gel column chromatography (hexane / ethyl acetate / triethylamine=9 / 90 / 1→0 / 99 / 1) to obtain compound 6 (1.16 g, 1.50 mmol, 29%). 1 H-NMR (400 MHz, CDCl 3 ): δ 8.268 (s, 1H), 7.635 (d, J = 8.8 Hz, 1H), 7.404-7.209 (m, 9H), 6.857-6.818 (m, 4H), 6.603 (d, 8.8 Hz, 1H), 6.104 (d, J = 8.8 Hz, 1H), 5.449, (d, J = 8.4 Hz, 1H), 4.911-4.853 (m, 1H), 4.575 (q, J = 5.6 Hz, 1H), 4.215 (m, 1H), 3.895-3.847 (m, 1H), 3.784 (s, 1H), 3.766-3.707 (m, 1H), 3.631-3.569 (m, 2H), 3.392 (d, J = 3.2 Hz, 2H), 2.638 (t, J = 6.0 Hz, 2H), 1.184-1.109 (m, 12H); 13 C-NMR (100 MHz, CDCl 3 ): δ 162.661, 161.936, 158.837, 150.892, 144.015, 140.000, 135.212, 135.041, 130.177, 128.202, 127.296, 117.740, 113.467, 1 03.090, 87.439, 86.161, 84.940, 75.060, 63.519, 58.207, 55.374, 53.772, 43.405, 24.597 , 20.582; 31 P-NMR (162 MHz, CDCl 3 ): δ 151.932; HRMS (ESI + ) calc. m / z796.3082 (M + Na + ), 812.2821 (M + K + ), found m / z796.3089 (M + Na + ), 812.2828 (M + K + )..

[0141] Test example 1. Melting temperature measurement 1 Using the phosphoramidite form of 2'-formamidouridine (Synthesis Example 1), polynucleotides (DNA / RNA, containing 2'-formamidouridine) (Table 1: SEQ ID NO: 1 and 2) were produced using an automatic nucleic acid synthesizer. was synthesized. A corresponding polynucleotide that does not contain 2'-formamide uridine was also synthesized. The 50% melting temperature (T m value) was measured. The measurement method is as follows. A JASCO V-650 Spectrophotometer was used for absorbance measurement. Prepare the sample so that the final concentration of single-stranded oligonucleic acid is 3 μM in 1 M NaCl, 10 mM phosphate buffer (pH 7.0), heat the prepared sample at 90 °C for 5 minutes, and let it stand. Annealing was performed by returning the temperature to room temperature. The sample was transferred to a cell, and the absorbance at 260 nm was measured while changing the temperature from 15°C to 90°C at a rate of 0.5°C / min. Draw the midline of the obtained sigmoid curve, the straight line for the double-stranded state and the straight line for the single-stranded state, and calculate the temperature at which the midline intersects with the sigmoid curve as T. m And so.

[0142] The results are shown in Table 1. It was revealed that the introduction of 2'-formamide uridine decreased the stability of the double strands.

[0143]

table 1

[0144] Test example 2. Thermodynamic parameter measurement The thermodynamic parameters of polynucleotide duplex formation shown in Table 2 (SEQ ID NO: 1 and 2) were measured. The measurement method is as follows. A JASCO V-650 Spectrophotometer was used for absorbance measurement. Samples were adjusted to final concentrations of single-stranded oligonucleotides of 3, 6, 9, and 12 μM in 1 M NaCl, 10 mM phosphate buffer (pH 7.0), and incubated at 90 °C for 5 minutes. Annealing was performed by heating, leaving to stand, and returning to room temperature. The sample was transferred to a cell, and the absorbance at 260 nm was measured while changing the temperature from 15°C to 90°C at a rate of 0.5°C / min. Draw the midline of the obtained sigmoid curve, the straight line for the double-stranded state and the straight line for the single-stranded state, and calculate the temperature at which the midline intersects with the sigmoid curve as T. m And so. The obtained T m Each thermodynamic parameter was calculated by applying the value and the concentration at that time to the following two equations.

[0145]

number

[0146]

number

[0147] The results are shown in Table 2. It was revealed that the introduction of 2'-formamide uridine gave an entropic advantage during duplex formation, but destabilized the duplex formation in an enthalpy-dominant manner.

[0148] [Table 2]

[0149] Test example 3. Base discrimination ability evaluation 1 Polynucleotides (DNA / RNA, containing 2'-formamidouridine) (Table 3: SEQ ID NOs: 3 to 5) were produced using an automatic nucleic acid synthesizer using the phosphoramidite form of 2'-formamidouridine (Synthesis Example 1). was synthesized. A corresponding polynucleotide that does not contain 2'-formamide uridine was also synthesized. Using the polynucleotides shown in Table 3 (SEQ ID NOS: 1-5), 50% melting temperature (T m value) was measured in the same manner as in Test Example 1 to evaluate the base discrimination ability of the oligonucleic acid incorporating 2'-formamide uridine.

[0150] The results are shown in Table 3. It was revealed that there was no significant change in base discrimination ability even when 2'-formamide uridine was introduced.

[0151] [Table 3]

[0152] Test example 4. Evaluation of off-target effects 1 Using the phosphoramidite form of 2´-formamidouridine (Synthesis Example 1), a polynucleotide (siRNA passenger strand / guide strand, containing 2´-formamidouridine) (Table 4: SEQ ID NO: 6) was synthesized using an automatic nucleic acid synthesizer. ~11) was synthesized. A corresponding polynucleotide that does not contain 2'-formamide uridine was also synthesized. In Table 4, the notation 2 (or 3, 4, 6, or 7) U>U* refers to the second (or 3, 4 , 6, or 7) is an siRNA in which 2'-formamide uridine is introduced into the nucleoside. Those without this notation are siRNAs in which 2'-formamide uridine has not been introduced.

[0153] [Table 4]

[0154] For each siRNA, RNAi activity toward the target gene (CLTC, VIM, or MC4R) and off-target activity were measured. The measurement method is as follows. 24 hours before transfection, HeLa cells were added at 1.0X10 per well of a 24-well cell culture plate. 5It was seeded with cells. siRNA, pGL3-Control (100ng / μL), a firefly luciferase gene expression vector, and a target vector (psiCheck1_gCM or psiCheck1_gSM) in which the siRNA target sequence is inserted into the 3'UTR of the Renilla luciferase gene, were added to HeLa cells at a concentration of 10ng / μL. were simultaneously transfected using Lipofectamine2000. After 24 hours, the cells were lysed and collected, and the activities of Renilla luciferase and firefly luciferase were measured using the Dual Luciferase Reporter Assay System. The relative value of (Renilla luciferase activity / Fireflu luciferase activity) was calculated, and the relative value when using the control siRNA was set as 100%, and the relative value of each siRNA was calculated as %. psiCheck1_gCM is a target vector with a sequence completely complementary to siRNA, and was used to measure the RNAi effect. psiCheck1_gSM is a targeting vector that has three sequences complementary to only the seed portion of siRNA, and off-target effects were measured using this vector.

[0155] The results are shown in Figure 1. When 2´-formamide uridine was introduced at the 2nd, 3rd, and 4th positions from the 5´ end, the off-target effect was suppressed, but when it was introduced at the 6th and 7th positions, the degree of suppression of the off-target effect was reduced. ing.

[0156] Synthesis Example 2. Synthesis of phosphoramidite of 4-methylcytidine The following phosphoramidite of 4-methylcytidine was synthesized.

[0157] [ka]

[0158] The synthesis was performed according to NUCLEOSIDES & NUCLEOTIDES, 14(3-5), 1129-1 132 (1995) Jane A. Grasby, Mohinder Singh, Jonathan Karn & Michael J. Gait.

[0159] Test example 5. Melting temperature measurement 2 Polynucleotides (RNA, containing 4-methylcytidine) (Table 5: SEQ ID NOs: 12 and 13) were synthesized with an automatic nucleic acid synthesizer using a 4-methylcytidine phosphoramidite (Synthesis Example 2). A corresponding polynucleotide that does not contain 4-methylcytidine was also synthesized. 50% melting temperature (T m value) was measured in the same manner as in Test Example 1.

[0160] The results are shown in Table 5. It was revealed that the introduction of 4-methylcytidine decreased the stability of the double strands.

[0161] [Table 5]

[0162] Test example 6. Base discrimination ability evaluation 2 Synthesize polynucleotides (DNA / RNA, with 4-methylcytidine) (Table 6: SEQ ID NOs: 14 to 16) using a 4-methylcytidine phosphoramidite (synthesis example 2) using an automatic nucleic acid synthesizer. did. A corresponding polynucleotide that does not contain 4-methylcytidine was also synthesized. Using the polynucleotides shown in Table 6 (SEQ ID NOS: 12-16), 50% melting temperature (T m value) was measured in the same manner as in Test Example 1 to evaluate the base discrimination ability of the oligonucleic acid incorporating 4-methylcytidine.

[0163] The results are shown in Tables 6 and 7. In Table 7, it was revealed that there was no significant change in the base discrimination ability even when 4-methylcytidine was introduced.

[0164] [Table 6]

[0165] [Table 7]

[0166] Test example 7. Evaluation of off-target effects 2 Using a phosphoramidite form of 4-methylcytidine (Synthesis Example 2), a polynucleotide (siRNA passenger strand / guide strand, containing 4-methylcytidine) (Table 8: SEQ ID NOS: 6 to 9) was produced using an automatic nucleic acid synthesizer. ) was synthesized. A corresponding polynucleotide that does not contain 4-methylcytidine was also synthesized. In Table 8, the notation 5 (or 6 or 8)C>C* refers to the 5th (or 6th or 8th) nucleoside from the 5´ end of the guide strand (the strand displayed at the bottom of each siRNA). Indicates that the siRNA has 4-methylcytidine introduced. Those without this description are siRNAs in which 4-methylcytidine has not been introduced.

[0167] [Table 8]

[0168] For each siRNA, RNAi activity toward the target gene (CLTC or VIM) and off-target activity were measured in the same manner as in Test Example 4.

[0169] The result is shown in figure 2. When 4-methylcytidine was introduced at the 5th position from the 5′ end, off-target effects were suppressed, but when 4-methylcytidine was introduced at the 6th and 8th positions, almost no suppression of off-target effects was observed.

[0170] Synthesis Example 3.Synthesis of S-modified phosphoramidite Two types of S-modified phosphoramidites shown below were synthesized.

[0171] [ka]

[0172] <Synthesis Example 3-1.Synthesis of S-modified phosphoramidite A> The synthesis scheme of S-modified phosphoramidite A is as follows.

[0173] [ka]

[0174] Step 1: 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine (3) Starting material uridine (15.00 g, 61.4 mmol, 1 eq.) was suspended in perhydrated pyridine (approximately 250 mL). Next, the protecting reagent 1,3-dichloro-1,1,3,3,3-tetraisopropyldisiloxane (21.1 mL, 66 mmol, 1.1 eq.) was added dropwise. After the addition was complete, the reaction mixture was kept stirring at room temperature for about 6 hours until the reaction was complete as monitored by TLC [hexane / ethyl acetate, 1:1 (v / v)]. The reaction mixture was first evaporated to remove as much of the solvent pyridine as possible and then redissolved in an appropriate amount of ethyl acetate. The reaction mixture was then washed twice with saturated aqueous NaHCO3 solution and once with brine. The organic layer was then separated using a funnel and dried over Na2SO4 to remove the water content. After filtering through cotton and concentrating to a syrup by evaporation, the crude mixture was applied to a neutral silica gel column with an eluent gradient of hexane / ethyl acetate [3:1 (v / v) to 1:1 (v / v)]. Purification was performed using chromatography to obtain the target compound. Finally, the fractions containing the desired product were collected, evaporated and dried under vacuum. The desired product 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine was obtained in 21.8 g with a yield of 73%. 1 H-NMR (400 MHz, CDCl 3 ) δ: 7.65 (1H, m); 5.72 (1H, s); 5.70-5.67 (1H, m); 4.40-4.38 (1H, m); 4.16-4.02 (4H, m); 1.09-1.03 (28H, m). ESI-MS: calc for C 21 H 38 N 2 O 7 Si 2, m / z: 486.22 found [M+Na] + 509.20.

[0175] Step 2: 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2'-O-[(methylthiomethoxy)methyl]uridine (4) 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine(4) (2.44 g, 5.02 mmol, 1.0 eq.) obtained in the previous synthesis. Bis(methylthiomethyl)ether (2.08 g, 15.05 mmol, 3.0 eq.) was dissolved in perhydrated THF (10.0 mL) and cooled to -45°C. Trifluoromethanesulfonic acid (1.50 g / 0.90 mL, 9.99 mmol, 2.0 eq.) was carefully added to the reaction solution, stirred for about 15 minutes, and then further stirred for 30 minutes. Then, an anhydrous THF solution containing N-iodosuccinimide (2.26 g, 10.04 mmol, 2.0 eq.) was carefully injected into the reaction mixture. The reaction was kept stirring under Ar for more than 24 hours until most of the starting material was transferred to the product as monitored by TLC [hexane / ethyl acetate, 1:1 (v / v)]. The reaction was finally stopped by adding triethylamine (2.54 g, 25.10 mmol, 5.0 eq.) and stirring for 15 minutes. After these steps, the reaction mixture was allowed to return to room temperature and evaporated to a syrupy state as much as possible. The remaining material was rediluted with ethyl acetate and sonicated to complete dissolution, then washed three times with saturated aqueous sodium thiosulfate and once with brine. The resulting organic layer was separated in a funnel. The washed sodium thiosulfate aqueous solution was extracted again with ethyl acetate. The organic phases were combined and dried over Na2SO4 to remove the contained water. After evaporating again to a residue, the remaining material was transferred onto a neutral silica gel column using an eluent gradient of hexane / ethyl acetate [5:1 (v / v) to 1:1 (v / v)]. It was purified by chromatography. Fractions containing the target compound were pooled together, evaporated and foamed. The residue was completely dried in vacuo to obtain the desired product. 2.89 g of the compound 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2'-O-[(methylthiomethoxy)methyl]uridine (4) It was obtained in a yield of 80%. 1 H-NMR (400 MHz, CDCl 3 ) δ: 7.88 (1H, d); 5.73 (1H, s); 5.68-5.66 (1H, d); 5.14-4.71 (5H, m); 4.27-4.11 (4H, m); 3.97-3.94 (1H, d); 2.18-2.14 (3H, m); 1.10-0.91 (28H, m). ESI-MS calc for C 24 H 44 N 2 O 8 SSi 2 , m / z=576.24, found [M+Na] + 599.23, [M+K] + 615.20. ([2M+Na] + , [3M+Na] + also found).

[0176] Steps 3~5: 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2'-O-[(tert-butyldithiomethoxy)methyl]uridine (6) Starting material 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2'-O [(methylthiomethoxy)methyl]uridine (4) (7.24 g, 12.56 mmol, 1 eq.) was dissolved in super dry dichloromethane (125 mL) containing DIPEA (1.62 g / 2.07 mL, 12.53 mmol, 1 eq.). The reaction mixture was stirred at room temperature for about 30 minutes under argon protection and then cooled to 0° C. in an ice bath. A solution of sulfuryl chloride (1.70 g / 1.02 mL, 12.59 mmol, 1 eq.) in dry dichloromethane (5 mL) was carefully added dropwise to the reaction mixture. Thereafter, the mixture was stirred for an additional 1.5 hours at room temperature. A solution of potassium thiotosylate (5.69 g, 25.13 mmol, 2 eq.) in dry DMF (5 mL) was then added and the reaction mixture was stirred at room temperature for about 16 hours.

[0177] After transferring all starting materials to the product monitored by TLC [hexane / ethyl acetate, 1:1 (v / v)], the next step was to transfer tert-butyl mercaptan (2.26 g / 2.83 mL, 25.06 mmol, 2 eq.) was added directly and allowed to react at room temperature with stirring for over 8 hours. After the reaction was completed as monitored by TLC [hexane / ethyl acetate, 1:1 (v / v)], stirring was stopped and the reaction mixture was passed through a Celite layer to remove insoluble matter, and then diluted with saturated aqueous sodium bicarbonate solution. Washed twice, once with brine, and the organic layer was separated in a funnel. The organic layer was separated using a funnel and then dried over Na2SO4 to remove the water content. This solution was filtered through cotton wool and finally evaporated to obtain a solid crude product, 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)- 2'-O-[(tert-butyldithiomethoxy)methyl]uridine (6) was obtained. The resulting crude material was obtained as a foam and was used directly in the next step without further purification.

[0178] Step 6: 2'-O-[(tert-butyldithiomethoxy)methyl]uridine (7) The starting material crude compound obtained in the last step 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2'-O-[(tert-butyl Dithiomethoxy)methyl]uridine (6) was suspended in dry methanol (125 mL). Ammonium hydrogen fluoride (3.58 g, 67.76 mmol, 5 eq.) was added to the reaction mixture, stirred at room temperature overnight (approximately 20 hours) under Ar until the disiloxyl protecting group was completely cleaved, and then analyzed by TLC [dichloromethane / Methanol, 10:1 (v / v)] and [hexane / ethyl acetate, 1:1 (v / v)] were simultaneously monitored. After the end of the reaction, the mixture was first evaporated to as much syrup as possible, then rediluted with ethyl acetate and sonicated to ensure complete dissolution. The solution was then washed with two portions of saturated NaHCO3 solution and one portion of brine was added. The organic phase was separated using a funnel, dried over Na2SO4 to remove the water content, and filtered through cotton. These steps were followed by concentration by evaporation to obtain the crude product. This crude compound was obtained as a foam in an amount of 1.07 g and a yield of 21% by combining the above four steps. This crude product was then used directly in the next step without further purification.

[0179] A portion of this material was purified by silica gel column chromatography on a neutral silica gel column using an eluent gradient of dichloromethane / methanol (90:1 (v / v) to 10:1 (v / v)). After obtaining the desired fraction, it was evaporated and dried under vacuum. The resulting pale pink foam was analyzed by MS. ESI-MS:C 15 H 24 N 2 O 7 S 2 , m / z: 408.10, found [M+H] + 409.10, [M+Na] + 431.09.

[0180] Step 7: 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(tert-butyldithiomethoxy)methyl]uridine (8) The starting material crude 2'-O-(tert-butyldithiomethyl)uridine (7) (0.77 g, 1.88 mmol, 1 eq.) obtained in the last step was dissolved in perhydrated pyridine (20 mL) and diluted with 4 ,4'-dimethoxytrityl chloride (0.77 g, 2.26 mmol, 1.2 eq.) was added directly to the solution. The reaction mixture was stirred at room temperature under Ar for more than 8 hours until complete conversion of starting material was achieved, which was monitored by TLC [hexane / ethyl acetate, 1:1 (v / v)]. The reaction mixture was then evaporated to remove as much of the pyridine solvent as possible to a residue. The residue was redissolved in ethyl acetate and washed twice with saturated aqueous sodium bicarbonate solution and once with brine. The organic layer was separated in a funnel and then dried over Na2SO4. After filtration through cotton, the solution was finally concentrated by evaporation to give the crude product. The resulting crude mixture was purified by chromatography on a neutral silica gel column using an eluent gradient of hexane / ethyl acetate (5:1 (v / v) to 1:1 (v / v)); After obtaining the desired fractions, they were evaporated and dried under vacuum. The target compound 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(tert-butyldithiomethoxy)methyl]uridine (8) (880 mg, 1.24 mmol) was purified in 66% yield. Isolated as a red foam. 1 H-NMR (400 MHz, CDCl 3 ) δ: 7.54 (d, 1H); 7.41-7.13 (m, 9H); 6.87-6.82 (m, 4H); 5.76 (d, 1H); 5.50 (s, 2H); 5.01-4.67 (m, 6H) ; 4.36 (dd, 1H); 3.79 (s, 6H); 3.51-3.39 (d, 2H); 1.33 (s, 9H). ESI-MS: calc for C 36 H 42 N 2 O 9 S 2 , m / z: 710.23, found [M+Na] + 733.21.

[0181] Step 8: 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(tert-butyldithiomethoxy)methyl]uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphorami Daito)(9) 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(tert-butyldithiomethoxy)methyl]uridine (8) obtained in the previous step (192 mg, 0.27 mmol, 1 eq. ) was dissolved in super dry dichloromethane (3.0 mL) containing DIPEA (235 μL, 1.35 mmol, 5 eq.). After stirring for several minutes, the amidite reagent 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (150 μL, 0.68 mmol, 2.5 eq.) was added. The reaction mixture was stirred at room temperature under argon until complete conversion of starting material to product (over 4 hours) and analyzed by TLC [hexane / ethyl acetate, 1:1 (v / v) with 5% triethylamine addition]. I monitored it. After the reaction was completed, the mixture was diluted with dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution and once with brine. The organic layer was separated using a funnel and dried over Na2SO4 to remove water. After filtering through cotton, the solution was evaporated and concentrated to a residue to obtain the crude product. The resulting crude mixture was purified on neutral silica gel using an eluent gradient of hexane / ethyl acetate (10:1 (v / v) to 1:1 (v / v)) with 5% triethylamine in the mobile phase. Purification was performed by chromatography on a column. Fractions containing the target compound were collected, concentrated to a residue (red foam-like), and then redissolved in around 3.0 mL of super-dry dichloromethane. The flask was filled with dehydrated hexane (15.0 mL) and placed in a cold bath at approximately -78°C. The product solution in dry dichloromethane was added dropwise to the cold hexane solution with stirring. The amidite product immediately precipitated out as a white solid suspended in the solution mixture. The mixture was then filtered under reduced pressure and the resulting solid was washed several times with cold hexane (-78°C). The resulting residue was redissolved in perhydrated dichloromethane, evaporated again, and dried in vacuo to give the target compound 0.20 g 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(tert-butyl Dithiomethoxy)methyl]uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (9) was obtained in a white foamy state with a yield of 81%. 31 P-NMR (400 MHz, CD 3 CN) 150.79, 149.74 ppm. ESI-MS: calc for C 45 H 59 N 4 O 10 P.S. 2 , m / z: 910.34, found [M+Na] + 933.22, [M+K] + 949.20.

[0182] <Synthesis Example 3-2.Synthesis of S-modified phosphoramidite B> The synthesis scheme of S-modified phosphoramidite B is as follows.

[0183] [ka]

[0184] Step 3: 2'-O-[(methylthiomethoxy)methyl]uridine (7b) 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2'-O-[(methylthiomethoxy)methyl]uridine(4) (2.00 g, 3.47 mmol, 1 eq.) was dissolved in methanol (40 mL), ammonium hydrogen fluoride (1.00 g, 17.53 mmol, 5 eq.) was added, and the reaction mixture was stirred at room temperature overnight (about 16 hours). Ammonium hydrogen fluoride (1.00 g, 17.53 mmol, 5 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature overnight (about 16 hours) until the disiloxyl protecting group was completely cleaved. After the end of the reaction, the mixture was first evaporated to as much syrup as possible, then rediluted with ethyl acetate and, if necessary, sonicated for complete dissolution. The dissolved solution was then washed twice with saturated NaHCO3 solution and once with brine. The organic phase was separated using a funnel, dried over Na2SO4 to remove the water content, and filtered through cotton. After these processes, it was concentrated by evaporation to afford the crude product, which was isolated as a red foam in an amount of 976 mg with a yield of 84%. This crude compound was used in the next step without further purification. A small portion of this material was analyzed by ESI-MS. ESI-MS: calc for C 12 H 18 N 2 O 7 S, m / z: 334.08, found [M+Na] + 357.07, [M+K] + 373.09.

[0185] Step 4: 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(methylthiomethoxy)methyl]uridine (8b) The starting material 2'-O-[(methylthiomethoxy)methyl]uridine (7b) (976 mg, 2.91 mmol, 1 eq.) obtained in the last step was dissolved in perhydrated pyridine (15.0 mL) and diluted with 4, 4'-dimethoxytrityl chloride (1.18 g, 3.50 mmol, 1.2 eq.) was carefully added to the solution. The reaction mixture was stirred at room temperature under Ar for more than 6 h until complete conversion of starting material was achieved, which was monitored by TLC [hexane / ethyl acetate, 1:1 (v / v)]. .

[0186] The reaction mixture was then evaporated to a residue to remove as much of the pyridine solvent as possible. The residue was redissolved in ethyl acetate, treated with ultrasonic waves for complete dissolution, and washed twice with saturated aqueous sodium bicarbonate solution and once with brine. The organic layer was separated in a funnel and then dried over Na2SO4. After filtration through cotton, the solution was finally concentrated by evaporation to give the crude product. The resulting crude mixture was purified by chromatography on a neutral silica gel column using an eluent gradient of hexane / ethyl acetate to obtain fractions containing the target compound. The solutions were then collected, evaporated and dried under vacuum. The target compound 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(methylthiomethoxy)methyl]uridine (8b) (1.42 g, 2.23 mmol) as a light red foam with a yield of 77%. isolated. 1 H-NMR (400 MHz, CDCl 3 ) δ: 7.65 (d, 1H); 7.28-7.15 (m, 9H); 6.85-6.81 (m, 4H); 5.76 (d, 1H); 5.51 (s, 2H); 5.02-4.70 (m, 6H) ; 4.37 (dd, 1H); 3.80 (s, 6H); 3.52-3.38 (d, 2H); 2.16 (s, 3H). ESI-MS: calc for C 33 H 36 N 2 O 9 S, m / z: 636.21, found [M+Na] + 659.18.

[0187] Step 5: 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(methylthiomethoxy)methyl]uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)( 9b) 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(methylthiomethoxy)methyl]uridine (8b) (164 mg, 0.26 mmol, 1 eq.) obtained in the previous step, Dissolved in super dry dichloromethane (2.15 mL) containing DIPEA (225 μL, 1.30 mmol, 5 eq.). After stirring for several minutes, the amidite reagent 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (152 mg, 0.64 mmol, 2.5 eq.) was added directly. The reaction mixture was stirred at room temperature under argon until complete conversion of starting material to product (over 6 h) and was subjected to TLC [hexane / ethyl acetate, 1:3 (v / v) with addition of 5% triethylamine; on NH2 silica plates]. After the reaction was completed, the mixture was diluted with an appropriate amount of dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution and once with brine. The organic layer was separated using a funnel and dried over Na2SO4 to remove water. After filtration through cotton, the solution was concentrated to a residue by evaporation to give the crude product. The crude mixture was purified by chromatography on a NH2 silica gel column using an eluent gradient of hexane / ethyl acetate [1:3 (v / v)] with 5% triethylamine in the mobile phase. Fractions containing the target compound were collected and concentrated to a residue. The residue was then redissolved in dry dichloromethane, evaporated again and dried in vacuo to yield the target compound 5'-O-(4,4'-dimethoxytrityl)-2'-O-[(methylthiomethoxy)methyl]uridine. 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (9b) 166 mg, yield 77% (red foamy solid) was obtained. 31 P-NMR (400 MHz, CD 3 CN) 150.73, 149.56 ppm. ESI-MS: calc for C 42 H 53 N 4 O 10 PS, m / z: 836.32, found [M+Na] + 859.28.

[0188] Test example 8. Evaluation of off-target effects 3 Using S-modified phosphoramidite B (Synthesis Example 3-2), polynucleotides (siRNA passenger strand / guide strand, with S-modified nucleoside) (Table 9: SEQ ID NO: 8~ 11 and 17-18) were synthesized. We also synthesized a corresponding polynucleotide that does not contain S-modified nucleosides. In Table 9, the notation 2 (or 3, 4, 5, 6, or 7)U>2 refers to the second (or 3, This indicates that the siRNA has an S-modified nucleoside introduced into the 4th, 5th, 6th, or 7th nucleoside. Those without this notation are siRNAs in which no S-modified nucleoside has been introduced.

[0189] [Table 9]

[0190] For each siRNA, RNAi activity toward the target gene (VIM, MC4R, or KIF23) and off-target activity were measured in the same manner as in Test Example 4.

[0191] The results are shown in Figure 3. Results with the same tendency as Test Examples 4 and 7 were obtained.

Claims

1. The 2'-carbon of the sugar is linked to the general formula (1): -NH-C(=O)-R 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group.) A nucleic acid monomer comprising groups represented by the following formula:

2. General formula (1A): 【Chemistry 1】 [wherein Base represents a monovalent group obtained by removing one hydrogen atom from a nucleic acid base. R 1 represents a hydrogen atom or an alkyl group. The nucleic acid monomer according to claim 1, comprising a partial structure represented by:

3. The nucleic acid monomer according to claim 1 or 2, which is a nucleoside, a nucleotide, or a derivative thereof.

4. The nucleic acid monomer according to any one of claims 1 to 3, which is a phosphoramidite monomer.

5. R 1 The nucleic acid monomer according to any one of claims 1 to 4, wherein is a hydrogen atom.

6. The 2'-carbon of the sugar is substituted with a compound of the general formula (3): -(O-CH 2 -) n - (S-) m -R 4 (In the formula: R 4 represents a hydrocarbon group; n and m each represent an integer of 1 to 3.

7. R 4 The nucleic acid monomer of claim 6, wherein is an alkyl group.

8. The 2'-position of the sugar is attached to the carbon atom of the general formula (1): -NH-C(=O)-R 1 (In the formula: R 1 represents a hydrogen atom or an alkyl group.) and a nucleoside unit 1 having a group represented by the general formula (3): -(O-CH 2 -) n - (S-) m -R 4 (In the formula: R 4 represents a hydrocarbon group; n and m each represent an integer of 1 to 3. A polynucleotide comprising at least one nucleoside unit selected from the group consisting of 3 nucleoside units formed by linking groups represented by the following formula:

9. The polynucleotide of claim 8, which is an siRNA.

10. The polynucleotide of claim 9, wherein at least one nucleoside unit selected from the group consisting of nucleoside unit 1 and nucleoside unit 3 is contained in the second to fifth nucleoside sequence from the 5' end of the siRNA guide strand.

11. The nucleic acid base is represented by the general formula (2) 【Chemistry 2】 [In the formula: R 2 represents a hydrocarbon group.] A reagent for producing a polynucleotide, comprising a nucleic acid monomer represented by the following group:

12. The reagent according to claim 11, for use in producing a polynucleotide having reduced stability when formed into a double strand, or for use in producing siRNA.

13. The nucleic acid base is represented by the general formula (2) 【Transformation 3】 [In the formula: R 2 represents a hydrocarbon group.] An siRNA comprising 2 nucleoside units each of which is a group represented by:

14. The siRNA of claim 13, wherein the nucleoside unit 2 is contained in the second to fifth nucleoside sequence from the 5' end of the siRNA guide strand.

15. A reagent or pharmaceutical comprising at least one selected from the group consisting of the polynucleotide of claim 9 or 10 and the siRNA of claim 13 or 14.

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