Modified Polynucleotides

Abstract

To provide a technique for delivering a polynucleotide into a cell in a simplified, less cytotoxic manner.SOLUTION: This problem is solved by a polynucleotide modified by a molecule that includes a structure including a disulfide bond and / or a thiol group, represented by the formula (2). [DMTr is a dimethyl trityl group; R1 is a single bond or at least one bivalent group selected from -S-S- and -SH; Rx is -O-R31 (R31 is at least one monovalent group selected from -S-S- and -SH) or -O-(CH2)2-CN; Z1 is -O- or -NH-, where at least one of R1 and Rx includes at least one group selected from the group consisting of -S-S- and -SH].SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to modified polynucleotides, etc. [Background technology]

[0002] In the medical field, etc., technology is being developed to introduce polynucleotides such as siRNA into cells to express their functions. Polynucleotides are negatively charged due to the presence of phosphate groups, and therefore have low permeability through the cell membrane, which is also negatively charged. For this reason, when introducing polynucleotides into cells, the introduction efficiency is increased by complexing the polynucleotide with a cationic lipid and introducing the complex into the cell (Patent Document 1).

[0003] However, cationic lipids require complexation with polynucleotides prior to intracellular delivery, and cationic lipids are known to be cytotoxic. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2002-529439 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a technique for introducing a polynucleotide into a cell more easily and with reduced cytotoxicity, and preferably, to provide a technique for introducing a polynucleotide into a cell more efficiently. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted intensive research and have found that a polynucleotide modified with a molecule containing a structure containing a disulfide bond and / or a thiol group can be introduced into cells more efficiently by simply adding the modified polynucleotide to a cell culture medium, without the need for complicated procedures such as complex formation with a cationic lipid. The present inventors have also found that the modified polynucleotide has lower cytotoxicity. Based on these findings, the present inventors have conducted further research and completed the present invention.

[0007] That is, the present invention includes the following aspects.

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

[0009] [ka]

[0010] [In the formula, R 1 and R 2 R may be the same or different and represents a single bond or a divalent group which may contain at least one group selected from the group consisting of -SS- and -SH. 3 -O - -OR 31 (In the formula, R 31 represents a monovalent group which may contain at least one group selected from the group consisting of -SS- and -SH. 1 and Z 2 are the same or different and represent -O- or -NH-. 1 , R 2 , and R 3 At least one of the groups contains at least one group selected from the group consisting of -SS- and -SH, and n represents an arbitrary integer. A modified polynucleotide comprising a molecule having a structure represented by the formula:

[0011] Section 2. Said R 1 is an alkylene group or a group derived from a ring having -SS- in the ring structure, 2is a single bond, and the R 3 -O - -OR 31a -SSR 31b (In the formula, R 31a is an alkylene group or -R 311a -NH-COO-R 312a -(R 311a and R 312a R is the same or different and represents an alkylene group. 31b represents a protecting group.

[0012] Section 3. (A) Above R 1 is an alkylene group, and the R 2 is a single bond, and the R 3 -OR 31a -SSR 31b (In the formula, R 31a is an alkylene group or -R 311a -NH-COO-R 312a -(R 311a and R 312a R is the same or different and represents an alkylene group. 31b represents a protecting group; or (B) The above R 1 is a group derived from a ring having a group represented by -SS- in the ring structure, 2 is a single bond, and the R 3 -O - That is, Item 3. A modified polynucleotide according to item 1 or 2.

[0013] Section 4. (A1) Above R 1 is an alkylene group having 3 to 6 carbon atoms, and the R 2 is a single bond, and the R 3 -OR 31a -SSR 31b (In the formula, R 31a is an alkylene group having 3 to 6 carbon atoms or -R 311a -NH-COO-R 312a -(R 311a and R 312aR is the same or different and represents an alkylene group having 2 to 6 carbon atoms. 31b represents an alkyl group or an aryl group; (B1) R 1 is a group derived from a 4- to 8-membered ring having a group represented by -SS- in the ring structure, 2 is a single bond, and the R 3 -O - That is, Item 4. The modified polynucleotide according to any one of Items 1 to 3.

[0014] Item 5. The modified polynucleotide according to any one of Items 1 to 4, wherein the n is 1 to 30.

[0015] Item 6. The above Z 1 and Z 2 and both are -O-. The modified polynucleotide according to any one of Items 1 to 5.

[0016] Item 7. The modified polynucleotide according to any one of Items 1 to 6, wherein the polynucleotide is a polynucleotide for introduction into a cell.

[0017] Item 8. The modified polynucleotide according to any one of Items 1 to 7, wherein the polynucleotide is at least one selected from the group consisting of antisense polynucleotides, siRNAs, miRNAs, miRNA precursors, aptamers, guide RNAs, mRNAs, non-coding RNAs, DNAs, non-naturally occurring nucleic acids (LNAs, PNAs, and morpholino nucleic acids).

[0018] Item 9. The modified polynucleotide according to any one of Items 1 to 8, wherein the base length of the polynucleotide is 200 bases or less.

[0019] Item 10. The modified polynucleotide according to any one of Items 1 to 9, wherein the structure represented by the general formula (1) is linked to an end of the polynucleotide.

[0020] Item 11. The modified polynucleotide according to any one of Items 1 to 10, wherein the structure represented by the general formula (1) is linked to the 5'-end of the polynucleotide.

[0021] Item 12. An agent for introducing a modified polynucleotide into a cell, comprising the modified polynucleotide according to any one of items 1 to 11.

[0022] Item 13. A pharmaceutical comprising the modified polynucleotide according to any one of items 1 to 11.

[0023] Item 14. A reagent comprising the modified polynucleotide according to any one of items 1 to 11.

[0024] Section 15. General formula (1):

[0025] [ka]

[0026] [In the formula, R 1 and R 2 R may be the same or different and represents a single bond or a divalent group which may contain at least one group selected from the group consisting of -SS- and -SH. 3 -O - -OR 31 (In the formula, R 31 represents a monovalent group which may contain at least one group selected from the group consisting of -SS- and -SH. 1 and Z 2 are the same or different and represent -O- or -NH-. 1 , R 2 , and R 3 At least one of the groups contains at least one group selected from the group consisting of -SS- and -SH, and n represents an arbitrary integer. A molecule for modifying a polynucleotide comprising a structure represented by the formula: Effect of the Invention

[0027] According to the present invention, by using a polynucleotide modified with a molecule having a structure containing a disulfide bond and / or a thiol group, a technique for introducing a polynucleotide into a cell more easily and with reduced cytotoxicity can be provided. In a preferred embodiment of the present invention, a polynucleotide can be introduced into a cell more efficiently than the conventional technique using a cationic lipid. [Brief description of the drawings]

[0028] [Figure 1] The results of the cell membrane permeability test in Test Example 1 are shown. On the horizontal axis, "NA" indicates the case where no polynucleotide was added to the medium, and the others indicate the cases where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (DNA (Comparative Example 1), PS-DNA (Comparative Example 3), 5 DTT (Example 1), 10 DTT (Example 2), 5 ThioL (Example 3), 10 ThioL (Example 4)), or the case where DNA (Comparative Example 1) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (DNA / Lipo3000). The vertical axis indicates the relative value of the fluorescence intensity (average value) derived from FAM added to the polynucleotide. The bars on each column indicate the standard error. On each column, ** indicates that the P value for "NA" is less than 0.01, *** indicates that the P value for "NA" is less than 0.001, # indicates that the P value for "DNA / Lipo3000" is less than 0.05, ## indicates that the P value for "DNA / Lipo3000" is less than 0.01, and ### indicates that the P value for "DNA / Lipo3000" is less than 0.001. [Diagram 2]The results of the expression suppression test of Test Example 2 are shown. On the horizontal axis, "NA" indicates the case where no polynucleotide was added to the medium, and the others indicate the cases where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (DNA (Comparative Example 1), PS-DNA (Comparative Example 3), 10ThioL (Example 4)), or where DNA (Comparative Example 1), R-DNA (Comparative Example 2), or 10ThioL (Example 4) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (~ / Lipo3000). The vertical axis indicates the relative value of the luciferase activity ratio (P. pyralis luciferase activity value / Renilla luciferase activity value) (average value). The bar on each column indicates the standard error. On each column, * indicates that the P value for "NA" is less than 0.05. [Diagram 3] The results of the cytotoxicity test of Test Example 3 are shown. On the horizontal axis, "NA" indicates the case where no polynucleotide was added to the medium, and the others indicate the cases where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (DNA (Comparative Example 1), PS-DNA (Comparative Example 3), 5 x DTT (Example 1), 10 x DTT (Example 2), 5 x ThioL (Example 3), 10 x ThioL (Example 4)), or the case where DNA (Comparative Example 1) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (DNA / Lipo3000). The vertical axis indicates the relative value of absorbance (average value). The black column indicates the case where the polynucleotide concentration in the added polynucleotide solution was 1 μM, and the gray column indicates the case where the same concentration was 5 μM. The bar on each column indicates the standard error. In each column, * indicates that the P value for "NA" is less than 0.05, ** indicates that the P value for "NA" is less than 0.01, and *** indicates that the P value for "NA" is less than 0.001. [Figure 4]The results of the cell membrane permeability test in Test Example 4 are shown. The horizontal axis shows the cases where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (PS-DNA (Comparative Example 4), 5×tBu (Example 5), 10×tBu (Example 6)), or where PS-DNA (Comparative Example 4) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (PS-DNA / Lipo). The vertical axis shows the fluorescence intensity (average value) derived from FAM added to the polynucleotide. The bars above each column show the standard error. [Diagram 5] The results of the expression inhibition test in Test Example 5 are shown. On the horizontal axis, "NA" indicates the case where no polynucleotide was added to the medium, and the others indicate the case where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (PS-DNA (Comparative Example 4), 5×tBu (Example 5)), or the case where PS-DNA (Comparative Example 4) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (PS-DNA / Lipo). The vertical axis indicates the relative value of the luciferase activity ratio (P. pyralis luciferase activity value / Renilla luciferase activity value) (average value). The bars on each column indicate the standard error. [Figure 6] The results of the cytotoxicity test of Test Example 6 are shown. On the horizontal axis, "NA" indicates the case where no polynucleotide was added to the medium, and the others indicate the cases where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (PS-DNA (Comparative Example 4), 5×tBu (Example 5)), or where PS-DNA (Comparative Example 4) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (PS-DNA / Lipo). The vertical axis indicates the relative value of absorbance (average value). The bars above each column indicate the standard error. [Figure 7]The results of the expression suppression test of Test Example 7 are shown. On the horizontal axis, "NA" indicates the case where no polynucleotide was added to the medium, and the others indicate the cases where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (siRNA (Comparative Example 5), 5×ThioL (3' & 3') (Example 7), 5×ThioL (3' & 5') (Example 8)), or the case where siRNA (Comparative Example 5) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (siRNA / Lipo). The vertical axis indicates the relative value of the luciferase activity ratio (P. pyralis luciferase activity value / Renilla luciferase activity value) (average value). The bars on each column indicate the standard error. *** indicates that the P value for "NA" is less than 0.001. ## indicates that the P value for "siRNA / Lipo" is less than 0.01. [Figure 8] The results of the cell membrane permeability test in Test Example 8 are shown. The horizontal axis shows the cases where the polynucleotides of the Examples or Comparative Examples were added directly to the medium (PSDNA (Comparative Example 6), 5× linker (Example 9)), or where PSDNA (Comparative Example 6) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (PSDNA / Lipo). The vertical axis shows the fluorescence intensity (average value) derived from FAM added to the polynucleotide. The bars above each column show the standard error. *** indicates that the P value is less than 0.001. [Figure 9] The results of the cytotoxicity test of Test Example 9 are shown. On the horizontal axis, "control" indicates the case where no polynucleotide was added to the medium, and the others indicate the cases where the polynucleotide of the Examples or Comparative Examples was added directly to the medium (PSDNA (Comparative Example 6), 5× linker (Example 9)), or where PSDNA (Comparative Example 6) was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000) (PSDNA / Lipo). The vertical axis indicates the relative value of absorbance (average value). The bars above each column indicate the standard error. * indicates a P value of less than 0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0030] 1. Modified polynucleotides and modifying molecules In one aspect, the present invention provides a compound represented by general formula (1):

[0031] [ka]

[0032] The present invention relates to a modified polynucleotide (sometimes referred to as the "modified polynucleotide of the present invention" herein) that has been modified with a molecule containing a structure represented by the following formula: and a molecule for modifying a polynucleotide that contains said structure (sometimes referred to as the "molecule for modification of the present invention" herein). These will be explained below.

[0033] R 1 and R 2 are the same or different and each represents a single bond, or a divalent group which may contain at least one group selected from the group consisting of -SS- and -SH.

[0034] R 1 or R 2 Examples of the divalent group represented by the formula (I) include a group that can function as a linker (hereinafter, sometimes simply referred to as a "linker"), a group derived from a ring, and a group formed by linking two or more of these.

[0035] Examples of the linker include an alkylene group, an alkenylene group, and a heteroalkylene group (for example, a heteroalkylene group containing -SS- in the main chain, a heteroalkylene group containing -NH-COO- in the main chain, etc.). The linker may be either linear or branched, and is preferably linear. The number of atoms constituting the main chain of the linker (the number of carbon atoms in the case of an alkylene group and an alkenylene group) is not particularly limited, and is, for example, 3 to 10, preferably 3 to 6, and more preferably 3 to 4. Examples of the linker include preferably an alkylene group, more preferably an alkylene group having 3 to 6 carbon atoms, further preferably an alkylene group having 3 to 4 carbon atoms, and even more preferably a linear alkylene group having 3 to 4 carbon atoms. Specific examples of such alkylene groups include an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, and the like.

[0036] The ring-derived group is a divalent group obtained by removing two hydrogen atoms from a ring, and is not particularly limited thereto. The ring is not particularly limited, and is preferably a monocyclic or bicyclic ring, and more preferably a monocyclic ring. The ring-constituting atoms are not particularly limited, and may be, for example, a carbon atom only, or a carbon atom and a heteroatom (for example, a nitrogen atom, a sulfur atom, an oxygen atom, etc.). The number of ring-constituting atoms is not particularly limited, and may be, for example, 3 to 20, preferably 3 to 12, more preferably 4 to 8, and even more preferably 5 to 7. Examples of such rings include rings having -SS- in the ring structure, cycloalkanes, cycloalkenes, benzene, naphthalene, etc., and preferably rings having -SS- in the ring structure. Specific examples of rings having -SS- in the ring structure include rings in which one -CC- in a cycloalkane or cycloalkene (preferably a cycloalkane) is replaced with -SS-.

[0037] R 1Preferably, the group is a divalent group which may contain at least one group selected from the group consisting of -SS- and -SH, more preferably an alkylene group, a group derived from a ring having -SS- in the ring structure, and more preferably an alkylene group.

[0038] R 2 A preferred example of the group is a single bond.

[0039] R 3 -O - -OR 31 Shows.

[0040] R 31 R represents a monovalent group which may contain at least one group selected from the group consisting of -SS- and -SH. 31 Examples of the monovalent group represented by the formula (I) include an alkyl group, an alkenyl group, a heteroalkyl group (e.g., a heteroalkyl group containing -SS- in the main chain, a heteroalkyl group containing -NH-COO- in the main chain, etc.), groups derived from the above rings, and groups formed by linking two or more of these groups. 31 The monovalent group represented by the formula R may be either linear or branched. 31 The number of atoms constituting the main chain of the monovalent group represented by the following formula is not particularly limited and is, for example, 1 to 20, preferably 3 to 12, and more preferably 5 to 9.

[0041] -OR 31 As the aryl group, preferably -OR 31a -SSR 31b Examples of the groups include those represented by the following formula:

[0042] R 31a is an alkylene group or -R 311a -NH-COO-R 312a -(R 311a and R 312a R is the same or different and represents an alkylene group. 31a is preferably an alkylene group.

[0043] R 31aThe alkylene group represented by the formula (I) includes both linear and branched alkylene groups, and is preferably linear. The number of carbon atoms in the alkylene group is not particularly limited, and is, for example, 3 to 10, preferably 3 to 6, and more preferably 3 to 4. Specific examples of such alkylene groups include an n-propylene group, an isopropylene group, an n-butylene group, and an isobutylene group.

[0044] R 311a or R 312a The alkylene group represented by the formula (I) includes both linear and branched alkylene groups, and is preferably linear. The number of carbon atoms in the alkylene group is not particularly limited, and is, for example, 2 to 10, preferably 2 to 6, and more preferably 2 to 3. Specific examples of such alkylene groups include an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, and an isobutylene group.

[0045] R 31b represents a protecting group. The protecting group is not particularly limited as long as it has the function of protecting the disulfide bond, and examples thereof include an alkyl group, an aryl group, and the like.

[0046] R 31b The alkyl group represented by the formula (I) includes both linear and branched alkyl groups. The alkyl group is preferably a branched alkyl group. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1 to 6, preferably 2 to 5, more preferably 3 to 5, and further preferably 4. 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. Among these, a tert-butyl group is preferable.

[0047] R 31bThe aryl group represented by is not particularly limited, but 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 either monocyclic or polycyclic (e.g., bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include a phenyl group, a naphthyl group, a biphenyl group, a pentalenyl group, an indenyl group, an anthranyl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perylenyl group, a fluorenyl group, and a phenanthryl group. Among these, a phenyl group is preferable.

[0048] R 3 As the aryl group, preferably -OR 31 and more preferably -OR 31a -SSR 31b Examples include:

[0049] In a preferred embodiment of the present invention, R 1 , R 2 , R 3 As a combination of Preferably (A) The above R 1 is an alkylene group, and the R 2 is a single bond, and the R 3 -OR 31a -SSR 31b (In the formula, R 31a is an alkylene group or -R 311a -NH-COO-R 312a -(R 311a and R 312a R may be the same or different and represents an alkylene group. 31b represents a protecting group; More preferably (A1) R 1 is an alkylene group having 3 to 6 carbon atoms, and the R 2 is a single bond, and the R 3 -OR 31a -SSR 31b (In the formula, R 31a is an alkylene group having 3 to 6 carbon atoms or -R311a -NH-COO-R 312a -(R 311a and R 312a R is the same or different and represents an alkylene group having 2 to 6 carbon atoms. 31b represents a t-butyl group.

[0050] In another preferred embodiment of the present invention, R 1 , R 2 , R 3 As a combination of Preferably (B) The above R 1 is a group derived from a ring having a group represented by -SS- in the ring structure, 2 is a single bond, and the R 3 -O - The combinations are: More preferably (B1) R 1 is a group derived from a 4- to 8-membered ring having a group represented by -SS- in the ring structure, 2 is a single bond, and the R 3 -O - The combination of R 1 , R 2 , R 3 Among these combinations, the combinations (A) and (A1) are preferred, and the combination (A1) is more preferred.

[0051] Above R 1 , R 2 , and R 3 At least one of the R 1 , R 2 , and R 3 When none of the above simultaneously contains at least one group selected from the group consisting of -SS- and -SH, they are excluded from the structure represented by general formula (1).

[0052] Z 1 and Z 2are the same or different and each represents -O- or -NH-. 1 and Z 2 At least one of is -O-, and more preferably Z 1 and Z 2 Both are -O-.

[0053] n is an arbitrary integer, for example, 1 to 50, preferably 1 to 30, more preferably 3 to 30, even more preferably 5 to 20, still more preferably 7 to 20, and particularly preferably 8 to 15.

[0054] The phrase "modified with a molecule containing a structure represented by general formula (1)" means that a polynucleotide is modified with the molecule.

[0055] The mode of modification is not particularly limited. The modified polynucleotide of the present invention is preferably a modified polynucleotide having a structure represented by general formula (1) linked to the terminal of the polynucleotide, more preferably a modified polynucleotide having a structure represented by general formula (1) linked to the 5' terminal of the polynucleotide. In addition, the structure represented by general formula (1) may be linked to both terminals of the polynucleotide.

[0056] The orientation of the structure represented by general formula (1) is not particularly limited. For example, Z 1 is on the 5' side of the modified polynucleotide and R 2 may be oriented in the 3' direction of the modified polynucleotide, or vice versa, i.e., Z 1 is on the 3' side of the modified polynucleotide and R 2 is on the 5' side of the modified polynucleotide.

[0057] In the modified polynucleotide of the present invention, the terminal Z 1 or R 2 may or may not have another molecule (e.g., a fluorescent label) linked to it. In the latter case, a hydrogen atom is usually linked to it. 1 or R 2may be linked to the polynucleotide to be modified (preferably at the terminal nucleotide).

[0058] The polynucleotide to be modified is not particularly limited, and may be DNA, RNA, or other known chemical modifications, as exemplified below. In order to prevent degradation by hydrolases such as nucleases, the phosphoric acid residue (phosphate) of each nucleotide may be replaced with a chemically modified phosphoric acid residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, such as by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. In addition, BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety, can also be preferably used.

[0059] The polynucleotide to be modified may be linked to another molecule, such as a fluorescent label.

[0060] The base length of the polynucleotide to be modified is not particularly limited, and is, for example, 500 bases or less, preferably 200 bases or less, more preferably 100 bases or less, even more preferably 50 bases or less, and even more preferably 30 bases or less. The lower limit is not particularly limited, and is, for example, 5 bases, 10 bases, or 15 bases.

[0061] The polynucleotide to be modified is preferably one that is primarily intended to be introduced into cells for use. Examples of such polynucleotides include antisense polynucleotides, siRNAs, miRNAs, miRNA precursors, aptamers, guide RNAs, and mRNAs.

[0062] The modified polynucleotide of the present invention and the modifying molecule of the present invention can be synthesized according to or in accordance with a known method. For example, they can be obtained by synthesizing nucleic acid by the phosphoramidite method using a phosphoramidite monomer capable of forming a structure represented by general formula (1). 1 is a divalent group which may contain at least one group selected from the group consisting of -SS- and -SH, and R 2 is a single bond, the phosphoramidite monomer may be, for example, a compound represented by the general formula (2):

[0063] [ka]

[0064] [In the formula, DMTr represents a dimethyltrityl group. x -OR 31 or -O-(CH2)2-CN. The rest is the same as above.] The compound represented by the following formula (I) is used to synthesize nucleic acid by phosphoramidite method according to a standard method or a similar method.

[0065] After synthesis, the product can be isolated and purified by conventional methods such as chromatography. The structure of the product can also be determined by elemental analysis, MS (FD-MS) analysis, IR analysis, 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0066] 2.Applications The modified polynucleotide of the present invention can efficiently penetrate cell membranes by itself without forming a complex with a cationic lipid or the like, and can therefore be efficiently introduced into cells. Furthermore, the modified polynucleotide of the present invention has relatively low cytotoxicity. For this reason, the modified polynucleotide of the present invention can be used in various applications aimed at introducing the modified polynucleotide into cells, such as an agent for introducing the modified polynucleotide into cells, a medicine, a reagent, etc. (hereinafter, these may be collectively referred to as the "agent of the present invention").

[0067] The agent of the present invention is not particularly limited as long as it contains the modified 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, flavors, chelating agents, etc.

[0068] 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).

[0069] 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.

[0070] 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.

[0071] The dosage form of the agent of the present invention is not particularly limited, and can be suitable according to its mode of use.For example, when administered to animals, it can be oral preparations such as tablets, capsules, granules, powders, fine granules, syrups, enteric-coated drugs, sustained release capsules, chewable tablets, drops, pills, liquids for internal use, confectionery tablets, sustained release agents, sustained release granules, etc.; external preparations such as nasal drops, inhalants, rectal suppositories, inserts, enemas, and jellies.The agent of the present invention can also be any of solid preparations, semisolid preparations, and liquid preparations.

[0072] The content of the modified 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, but can be, for example, 0.0001 to 95% by weight, preferably 0.001 to 50% by weight.

[0073] 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 modified polynucleotide of the present invention, which is an active ingredient. The 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. EXAMPLES

[0074] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0075] Reference Example 1. Synthesis of DTT-type phosphoramidite monomer According to the following synthesis scheme, a DTT-type phosphoramidite monomer (compound 5) was synthesized.

[0076] [ka]

[0077] <Synthesis of compound 3> Dimethyl sulfoxide (0.78 mL) was added to dithiothreitol (DTT) (1.54 g, 10.0 mmol) and the mixture was stirred at 110° C. for 4 hours. After cooling to room temperature, the resulting solid was suspended in dichloromethane and collected by suction filtration. The collected solid was washed with a small amount of ethyl acetate and dried under vacuum to obtain the target compound 3 as a white solid (1.26 g, 82%). 1 H NMR (400 MHz, DMSO-d6)δ: 5.24 (2H, d, J = 3.2 Hz), 3.33 (2H, br. s), 3.03 (2H, d, J = 13.2 Hz), 2.74 (1H, d, J = 13.2 Hz), 2.72 (1H, d, J = 13.6 Hz).

[0078] <Synthesis of compound 4> Compound 3 (1 g, 6.57 mmol) was azeotroped twice with methanol and twice with anhydrous pyridine. The residue was dissolved in anhydrous pyridine (15 mL), and dimethoxytrityl chloride (3.0 g, 8.85 mmol) was added and stirred at room temperature. After 4 hours, dimethoxytrityl chloride (2.0 g, 5.90 mmol) was added and stirred at room temperature. After 4 hours, methanol was added to the reaction solution, and the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with water and then with saturated saline. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by neutral flash silica gel column chromatography (hexane / ethyl acetate = 90:10 + 1% triethylamine → 80:20 + 1% triethylamine) to obtain the target compound 4 as a white foamy solid (1.67 g, 56%). ESI-MS: calcd. for C 25 H 26 NaO4S2477.1170[M+Na] + , found: 477.1262 [M+Na] + .

[0079] <Synthesis of compound 5> Compound 4 (300 mg, 0.66 mmol) was azeotroped twice with anhydrous acetonitrile. The residue was dissolved in dichloromethane (2 mL), and N,N-diisopropylethylamine (350 μL, 1.98 mmol) was added. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (217 μL, 0.99 mmol) was added dropwise at 0°C and stirred. After 20 minutes, the reaction solution was diluted with ethyl acetate and washed with water and then with saturated saline. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by neutral flash silica gel column chromatography (hexane / ethyl acetate = 20 : 1, + 1% triethylamine → 8 : 1, + 1% triethylamine) to obtain the target compound 5 as a white foamy solid (280 mg, 65%). 31 P NMR (121 MHz, CDCl3)δ: 148.4, 148.1 ESI-MS: calculation for C 34 H 43 N2NaO5PS2677.2249[M+Na] + , found: 677.2134 [M+Na] + .

[0080] Reference Example 2. Synthesis of ThioL-type phosphoramidite monomer ThioL-type phosphoramidite monomer (compound 4) was synthesized according to the following synthesis scheme. Using 3-mercaptopropanol as a starting material, the thiol group was converted to a disulfide structure to obtain compound 1. ThioL-type phosphoramidite monomer 4 was obtained by coupling with a separately synthesized C3 linker derivative 3.

[0081] [ka]

[0082] <Synthesis of Compound 1> Under an argon atmosphere, 3-mercaptopropanol (808 μL, 9.36 mmol) was added dropwise to a stirred solution (36 mL) of di-tert-butyl 1-(tert-butylthio)-1,2-hydrazinedicarboxylate (3.0 g, 9.36 mmol) and triethylamine (1.09 mL, 7.80 mmol). After stirring overnight at room temperature, the reaction solution was concentrated under reduced pressure. Hexane (10 mL) was added to the resulting residue, and the resulting precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by neutral flash silica gel column chromatography (toluene / ethyl acetate = 15 : 1) to obtain the target compound 1 as a pale yellow oily substance (731 mg, 85%). 1 H NMR (400 MHz, CDCl3) δ : 3.75 (2H, t, J = 6.0 Hz), 2.82 (2H, t, J = 7.2 Hz), 1.93 (2H, m, J = 6.8 Hz), 1.33 (s, 9H).

[0083] <Synthesis of compound 2> A pyridine solution (20 mL) of dimethoxytrityl chloride (3.0 g, 9.10 mmol) was added to a pyridine solution (52 mL) of propanediol (13 mL, 181.0 mmol) and stirred at room temperature. After 26 hours, the reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate (20 mL), washed twice with water (80 mL), and then with saturated saline. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by neutral flash silica gel column chromatography (hexane / ethyl acetate = 3:1 → 1:1) to obtain the target compound 2 as a white solid (2.50 g, 83%). 1H NMR (400 MHz, CDCl3) δ:7.43-7.38 (2H, m), 7.33-7.26 (5H, m), 7.24-7.16 (2H, m), 6.79-6.85 (4H, m), 3.87 (1H, t, J = 5.6 Hz), 3.79 (3H, s), 3.78 (3H, s), 3.28 (1H, t, J = 5.6 Hz), 1.91-1.80 (2H, m), 1.60 (1H, br. s) ESI-MS: calcd. for C 24 H 26 NaO4401.1723 [M+Na] + , found: 401.176 [M+Na] + .

[0084] <Synthesis of compound 4> A solution of bis(N,N-diisopropylamino)chlorophosphine (240 mg, 0.79 mmol) and triethylamine (120 μL) in anhydrous tetrahydrofuran (5 mL) was added dropwise to a solution of bis(N,N-diisopropylamino)chlorophosphine (240 mg, 0.79 mmol) and anhydrous tetrahydrofuran (3 mL) at 0°C. The reaction solution was stirred at room temperature, and the completion of the reaction was confirmed by 31P NMR after 100 minutes (135.5 ppm to 121.8 ppm, C6D6). Compound 1 (142 mg, 0.79 mmol) was added to the reaction solution, followed by a solution of 5-benzylthio-1H-tetrazole in acetonitrile (0.25M, 750 μL). The reaction solution was stirred at room temperature, and the completion of the reaction was confirmed by 31P NMR after 80 minutes (121.8 ppm to 147.0 ppm, C6D6). The reaction solution was diluted with ethyl acetate (70 mL) and washed twice with saturated sodium bicarbonate water (70 mL), water (70 mL), and then saturated saline (70 mL). The organic layer was concentrated under reduced pressure, and the resulting residue was purified by neutral flash silica gel column chromatography (hexane / ethyl acetate = 15 : 1, +1% triethylamine) to give the target compound 4 as a pale yellow oil (189 mg, 63%). 31 P NMR (121 MHz, C6D6) δ: 146.97 1 H NMR (400 MHz, C6D6) δ:7.68 (d, 2H, J = 11.0 Hz), 7.51 (4H, d, J = 11.2 Hz), 7.19 (3H, d, J = 11.4 Hz), 6.79 (4H, d, J = 11 Hz), 3.31(14H, m), 2.11(2H, m), 1.92 (2H, m), 1.19 (9H, s, 3CH3; 12H, d, 3CH3) 13 C NMR (100 MHz, C6D6) δ:158.7, 145.9, 136.7, 130.2, 128.4, 127.7, 126.5, 113.1, 86.0, 61.6, 61.4, 60.4, 54.4, 47.1, 42.8, 37.2, 32.2, 31.1, 29.6, 24.5, ESI-MS : calcd. for C 37 H 54 NO5PS2, 598.276 [M-C4H9S] + ; found : 598.277[M-C4H9S] + .

[0085] Example 1. Synthesis of DTT-modified polynucleotide (5DTT-DNA) 5´-AACCGCTTCCCGACTTCC (coordination number 1) is the 5´ end of DNA, and the following formula (A):

[0086]

change

[0087] A DTT-type modified polynucleotide (5DTT-DNA) having a structure represented by the following formula (I) linked thereto and fluorescently labeled (FAM-labeled) at the 3' end was synthesized by a standard method using β-cyanoethyl phosphoramidite (Glen Research), a DTT-type phosphoramidite monomer (Reference Example 1), and 6-FAM, using a DNA / RNA solid-phase synthesizer (NTS M-2-MX, Nippon Techno Service Co., Ltd.). SEQ ID NO: 1 is the antisense sequence of the luciferase gene derived from P. pyralis. The resulting modified polynucleotide was de-resinized and deprotected by a standard method, and purified by high performance liquid chromatography. Column: C18Hydrosphere 10 x 250 mm (YMC), column temperature: room temperature, measurement wavelength: 260 nm, mobile phase: 50 mM triethylammonium acetate buffer (pH 7.0) (+5% acetonitrile) / acetonitrile = 100:0 → 70:30 (20 min, linear gradient). Fractions containing modified polynucleotides were collected and concentrated using a centrifugal evaporator. Structure determination was performed using mass spectrometry (MALDI-TOF MS). The obtained modified polynucleotides were dissolved in ultrapure water and appropriately diluted, and the UV absorption spectrum was measured to determine the concentration. MALDI-MS: calcd. 7361.628 [M+H] + ; found :7362.888 [M+H] + .

[0088] Example 2. Synthesis of DTT-modified polynucleotide (10DTT-DNA) The 5' end of the DNA having the base sequence shown in SEQ ID NO:1 is a DNA having the following formula (B):

[0089] [ka]

[0090] A DTT-modified polynucleotide (10DTT-DNA) having a structure represented by the following formula (I) linked thereto and fluorescently labeled (FAM-labeled) at the 3' end was synthesized in the same manner as in Example 1. MALDI-MS: calcd.8434.762 [M+H] + ; found : 8434.22 [M+H] + .

[0091] Example 3. Synthesis of ThioL-type modified polynucleotide (5ThioL-DNA) The 5' end of the DNA having the base sequence shown in SEQ ID NO:1 is a DNA having the following formula (C):

[0092] [ka]

[0093] A ThioL-type modified polynucleotide (5ThioL-DNA) having a structure represented by the following formula (I) linked thereto and fluorescently labeled (FAM-labeled) at the 3' end was synthesized in the same manner as in Example 1, except that a ThioL-type phosphoramidite monomer (Reference Example 2) was used instead of a DTT-type phosphoramidite monomer (Reference Example 1). MALDI-MS: calcd. 7789.695 [M+H] + ; found : 7789.289 [M+H] + .

[0094] Example 4. Synthesis of ThioL-type modified polynucleotide (10ThioL-DNA) The 5' end of the DNA having the base sequence shown in SEQ ID NO:1 is represented by the following formula (D):

[0095] [ka]

[0096] A ThioL-type modified polynucleotide (10ThioL-DNA) having a structure represented by the following formula (I) linked thereto and fluorescently labeled (FAM labeled) at the 3' end was synthesized in the same manner as in Example 3. MALDI-MS: calcd. 9287.426 [M+H] + ; found : 9288.527 [M+H] + .

[0097] Comparative Example 1. Synthesis of Unmodified Polynucleotide (DNA) A DNA (DNA) consisting of the base sequence shown in SEQ ID NO: 1 and having a fluorescently labeled (FAM-labeled) 3' end was synthesized by a standard method. MALDI-MS: calcd. 6212.113 [M+H] + ; found : 6212.705 [M+H] + .

[0098] Comparative Example 2. Synthesis of Unmodified Polynucleotide (R-DNA) A DNA (R-DNA) consisting of the base sequence represented by 5'-ACACGTCCTCTCAGCCCTC (SEQ ID NO: 2) and having a fluorescently labeled (FAM-labeled) 3' end was synthesized by a standard method.

[0099] Comparative Example 3. Synthesis of phosphorothioate DNA (PS-DNA) Phosphorothioate DNA (PS-DNA) consisting of the base sequence shown in SEQ ID NO: 1 and having a fluorescently labeled (FAM-labeled) 3' end was synthesized by a standard method. MALDI-MS: calcd. 6499.02 [M+H] + ; found : 6502.773[M+H] + .

[0100] Test Example 1. Cell membrane permeability test 1 The polynucleotides modified with FAM in the Examples or Comparative Examples were added to the culture medium, and the fluorescence intensity of the cells (= the amount of polynucleotide taken up into the cells) was measured to examine the cell membrane permeability of the polynucleotides.

[0101] HeLa cells were transfected with pGL3 (Promega) and pTK-Green Renilla Luc (Thermo Fisher Scientific), which are expression plasmids for luciferase derived from Photinus pyralis and Renilla reniformis. The cells were cultured in the presence of G418 and puromycin to establish a cell line, HeLa-LucRluc, which stably expresses luciferase. The HeLa cells were cultured in DMEM medium (Wako) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture conditions were 37°C, 5% CO2 concentration in the atmosphere, and 95% humidity. 2 × 10 cells were cultured per well of a 12-well plate. 4 HeLa p13 cells were seeded and cultured for 1 day. Each of the polynucleotides of the examples and comparative examples was added to 0.3 mL of serum-free DMEM medium to a concentration of 1 μM and mixed. The resulting mixture was added to the medium and incubated for 3 hours. On the other hand, as a positive control, the unmodified polynucleotide of Comparative Example 1 was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000, manufactured by Invitrogen) according to the instructions in the reagent manual and incubated. After the incubation was completed, the cells were rinsed with the medium and then detached by trypsin treatment. The fluorescence intensity derived from FAM of the obtained cells was measured with a flow cytometer (BD FACSCanto II, manufactured by Becton Dickinson). The higher the fluorescence intensity, the more polynucleotides (modified with FAM) were introduced into the cells by penetrating the cell membrane.

[0102] The results are shown in Figure 1. DNA (Comparative Example 1) showed almost no membrane permeability. On the other hand, it was found that DTT (Examples 1 and 2) and ThioL (Examples 3 and 4) were each taken up into the cells. In particular, it was found that 10ThioL (Example 4) was taken up into the cells at a rate more than twice as high as that in the case where commercially available Lipoacetamine 3000 was used.

[0103] Test Example 2. Expression inhibition test using antisense sequences 1 Polynucleotides of the Examples and Comparative Examples having an antisense sequence of the luciferase gene derived from P. pyralis were added to the culture medium, and the expression level of luciferase was measured. This was used to examine whether the polynucleotides could permeate the cell membrane and exert their function (the effect of suppressing gene expression as an antisense nucleic acid). Specifically, the procedure was as follows.

[0104] HeLa-LucRluc cells (HeLa cells stably expressing P. pyralis luciferase and Renilla luciferase) were cultured in DMEM medium (Wako) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture conditions were 37°C, 5% CO2 concentration, and 95% humidity. 2 × 10 4 HeLa-LucRluc cells were seeded and cultured for 1 day. Each of the polynucleotides of the examples and comparative examples was added to 0.3 mL of serum-free DMEM medium to a concentration of 1 μM and mixed. The resulting mixture was added to the medium and incubated. Meanwhile, each of the polynucleotides of Example 4, Comparative Examples 1 and 3 was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000, Invitrogen) according to the instructions in the reagent manual and incubated. After the incubation, the cells were lysed with reporter lysis buffer (Promega). The luciferase activity (P. pyralis luciferase activity and Renilla luciferase activity) of the obtained cell lysate was measured using a luminometer (uminescencer-PSN, ATTO) and a luciferase assay reagent (Promega).

[0105] The results are shown in Figure 2. DNA / Lipo3000 and PS-DNA (Comparative Example 3) showed no gene expression inhibitory effect, but 10ThioL (Example 4) did. This indicates that this method may be more useful than liposomes as a method for introducing polynucleotides into cells.

[0106] Test Example 3. Cytotoxicity Test 1 The cytotoxicity of the polynucleotides of the Examples and Comparative Examples was examined by MTT assay. Specifically, the procedure was as follows.

[0107] HeLa-LucRluc cells (HeLa cells stably expressing P. pyralis luciferase and Renilla luciferase) were cultured in DMEM medium (Wako) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture conditions were 37°C, 5% CO2 concentration, and 95% humidity. 2 × 10 4 HeLa-LucRluc cells were seeded and cultured for 1 day. Each of the polynucleotides of the examples and comparative examples was added to 0.3 mL of serum-free DMEM medium to a concentration of 1 μM or 5 μM and mixed. The resulting mixture was added to the medium and incubated. On the other hand, as a positive control, the unmodified polynucleotide of Comparative Example 1 was added to the medium using a nucleic acid transfer reagent (Lipofectamine 3000, Invitrogen) according to the instructions in the reagent manual and incubated. After 24 hours of incubation, 20 μL of CellTiter 96 (registered trademark) AQueous One Solution Reagent (Promega) was added and incubated for 2.5 hours. The absorbance at 490 nm was measured using a 96-well plate reader. The higher the absorbance, the lower the cytotoxicity of the added polynucleotide.

[0108] The results are shown in Figure 3. DNA / Lipo3000 and PS-DNA (Comparative Example 3) showed toxicity, but the polynucleotides of the Examples (5 x DTT (Example 1), 10 x DTT (Example 2), 5 x ThioL (Example 3), 10 x ThioL (Example 4)) did not show toxicity. From this, it can be said that the polynucleotides of the Examples have low toxicity.

[0109] Example 5. Synthesis of ThioL-type modified polynucleotide (5×tBu-PS-DNA) The 5' end of the PS-DNA consisting of 5'-CGGTATCCAGATCCACAAC (SEQ ID NO: 3) is represented by the following formula (C):

[0110] [ka]

[0111] A ThioL type modified polynucleotide (5×tBu-PS-DNA) having a structure represented by the following formula (I) linked thereto and fluorescently labeled (FAM labeled) at the 3′ end was synthesized in the same manner as in Example 3.

[0112] Example 6. Synthesis of ThioL-type modified polynucleotide (10xtBu-PS-DNA) The 5' end of the PS-DNA consisting of the base sequence shown in SEQ ID NO: 3 is represented by the following formula (D):

[0113] [ka]

[0114] A ThioL type modified polynucleotide (10×tBu-PS-DNA) having a structure represented by the following formula (I) linked thereto and fluorescently labeled (FAM labeled) at the 3′ end was synthesized in the same manner as in Example 3.

[0115] Comparative Example 4. Synthesis of phosphorothioate DNA (PS-DNA) Phosphorothioate DNA (PS-DNA) consisting of the base sequence shown in SEQ ID NO: 3 and fluorescently labeled (FAM labeled) at the 3' end was synthesized by a standard method.

[0116] Example 7. Synthesis of ThioL-type siRNA (5×ThioL siRNA (3′ & 3′)) The 3' end of the RNA consisting of 5'-UUUCGAAGUACUCAGCGUAAGUU (SEQ ID NO: 4) is represented by the following formula (C):

[0117] [ka]

[0118] A ThioL-type modified single-stranded RNA (5×ThioL guide strand (3′)) having structures represented by the following formula (I) linked thereto was synthesized in the same manner as in Example 3.

[0119] On the other hand, the 3' end of the RNA consisting of 5'-CUUACGCUGAGUACUUCGAAAUU (SEQ ID NO: 5) is represented by the following formula (C):

[0120] [ka]

[0121] A ThioL-type modified single-stranded RNA (5×ThioL passenger strand (3′)) in which the structures represented by the following formula (I) were linked was synthesized in the same manner as in Example 3.

[0122] The 5×ThioL guide strand (3') and the 5×ThioL passenger strand (3') were mixed and then adjusted to the desired concentration (0.1-1 μM) in 1×annealing buffer (60 mM KCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl2), and incubated at 90°C for 3 minutes, then allowed to cool to room temperature for 3 hours to form double-stranded RNA (ThioL-type siRNA (5×ThioL siRNA (3' & 3'))).

[0123] Example 8. Synthesis of ThioL-type siRNA (5×ThioL siRNA (3′ & 5′)) The 5' end of the RNA having the base sequence shown in SEQ ID NO:5 is represented by the following formula (C):

[0124] [ka]

[0125] A ThioL-type modified single-stranded RNA (5×ThioL passenger strand (5′)) in which the structures represented by the following formula (I) were linked was synthesized in the same manner as in Example 3.

[0126] The 5×ThioL guide strand (3') and the 5×ThioL passenger strand (5') were mixed and then adjusted to the desired concentration (0.1-1 μM) in 1×annealing buffer (60 mM KCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl2), incubated at 90°C for 3 minutes, and then allowed to cool to room temperature for 3 hours to form double-stranded RNA (ThioL-type siRNA (5×ThioL siRNA (3' & 5'))).

[0127] Comparative Example 5. Synthesis of unmodified siRNA RNA consisting of the base sequence shown in SEQ ID NO: 4 and RNA consisting of the base sequence shown in SEQ ID NO: 5 were synthesized by standard methods, and double-stranded (unmodified siRNA) was formed in the same manner as in Example 7.

[0128] Test Example 4. Cell membrane permeability test 2 The same procedure as in Test Example 1 was carried out, except that the antisense polynucleotides of Examples 5, 6, and Comparative Example 4 were used as polynucleotides. The polynucleotide concentration was 100 nM.

[0129] The results are shown in Figure 4. In the case of ThioL modification, cell membrane permeability was equal to or greater than that in the case of using commercially available Lipoacetamine3000.

[0130] Test Example 5. Expression inhibition test using antisense sequences 2 The same procedure as in Test Example 2 was repeated, except that the antisense polynucleotides of Example 5 and Comparative Example 4 were used as polynucleotides. The polynucleotide concentration was 1 μM.

[0131] The results are shown in Figure 5. In the case of ThioL modification, the gene expression suppression effect was equivalent to that in the case of using commercially available Lipofectamine3000.

[0132] Test Example 6. Cytotoxicity Test 2 The same procedure as in Test Example 3 was carried out, except that the antisense polynucleotides of Example 5 and Comparative Example 4 were used as polynucleotides. The polynucleotide concentration was 5 μM.

[0133] The results are shown in Figure 6. The polynucleotide of the present example (5xtBu-PS-DNA) showed no toxicity.

[0134] Test Example 7. siRNA-based expression suppression test 1 The same procedure as in Test Example 2 was carried out, except that the siRNAs of Examples 7, 8, and Comparative Example 5 were used as polynucleotides. The polynucleotide concentration was 1 μM or 100 nM.

[0135] The results are shown in Figure 7. In the case of ThioL modification, the gene expression suppression effect was equal to or greater than that in the case of using commercially available Lipoacetamine3000.

[0136] Reference Example 3. Synthesis of procationic phosphoramidite monomers A procationic phosphoramidite monomer (compound 4) was synthesized according to the following synthesis scheme.

[0137] [ka]

[0138] [ka]

[0139] <Synthesis of compound b: 2-(tert-Butyldisulfanyl)ethanol> 2-Methyl-2-propanethiol (12 mL, 230 mmol, 10 eq.) was added to 100% ethanol (22 mL), 2-mercaptoethanol (0.77 mL, 23 mmol, 1.0 eq.), and the mixture was stirred in an ice bath. I2 (3.0 g, 36.8 mmol, 1.1 eq.) was dissolved in 100% ethanol (17 mL) and added dropwise to the reaction solution until the color changed from colorless to red, and the reaction was allowed to proceed overnight. NaHCO3 (saturated, 23 mL) was added until the pH reached 7 or higher, and the solution was evaporated under reduced pressure. AcOEt was added, and the mixture was separated using 10% Na2S2O5 and brine, and the organic layer was evaporated under reduced pressure. The residue was purified by column chromatography (neutral flash, Hexane / AcOEt = 2 / 1) to give compound b (1.62 g, 9.72 mmol, 91%) as an oil. 1 H-NMR (400 MHz, CDCl3): δ 3.842 (2H, t, J = 6.0 Hz), 2.827 (2H, t, J = 6.0 Hz), 1.317 (9H, s) and other spectral data were in good agreement with the data reported in the literature (Tetrahedron, 2005, 61, 6138.).

[0140] <Synthesis of compound c: 2-(tert-Butyldisulfanyl)ethyl 4-Nitrophenyl carbonate> Compound b (5.45 g, 32.8 mmol, 1.0 eq.) was added with 4-nitrophenyl chloroformate (9.89 g, 49.1 mmol, 1.5 eq.) and THF (10 mL) and reacted in an ice bath under an argon atmosphere. TEA (6.8 mL) and dehydrated THF (25 mL) were then added dropwise and reacted in an ice bath for 10 minutes, then at room temperature for 18 hours. The reaction solution was filtered, washed (Hexane / AcOEt = 5 / 1), and evaporated under reduced pressure. Column chromatography (neutral flash, Hexane / AcOEt = 4 / 1) was performed to obtain compound c (9.69 g, 29.2 mmol, 89%). 1 H-NMR (400 MHz, CDCl3): δ 8.27 (2H, m), 7.37 (2H, m), 4.51 (3H, t, J = 6.8 Hz), 2.99 (3H, t, J = 6.8 Hz), 1.34 (9H, s) and other spectral data were in good agreement with the data reported in the literature (Angew. Chem. Int. Ed., 2012, 51, 10347.).

[0141] <Synthesis of compound d: 2-(tert-butyldisulfanyel)ethyl (3-hydroxypropyl)carbamate> 3-amino-1-propanol (3.2 mL, 41.4 mmol, 1.0 eq.) in CHCl(25 mL) was added with TEA (6.4 mL) and compound c (9.15 g, 27.6 mmol, 1.0 eq.) in CHCl(15 mL) and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, the solution was dissolved in AcOEt (15 mL) and washed with water, saturated NaHCO3, 0.1M NaOH, and brine. The solution was dried over NaSO4 and removed under reduced pressure. Oily compound 4 (7.49 g, 27.0 mmol, 97%) was obtained. 1H-NMR (400 MHz, CDCl3): δ 4.28 (2H, t, J = 6.4 Hz), 3.67 (2H, t, J = 5.6 Hz), 3.13 (2H, t, J = 6.0 Hz), 2.88 (2H, t, J = 6.4 Hz), 1.69 (2H, t, J = 6.0 Hz), 1.31 (9H, s); 13 C-NMR (100 MHz, CDCl3):δ 157.22, 63.24, 59.60, 48.04, 39.19, 37.77, 32.55, 29.92; HRMS (ESI) calcd for C 10 H 21 NO3S2[M+Na] + : 290.10, found for 290.0913.

[0142] <Synthesis of Compound 2: 3-(4,4'-dimethoxytrityl)propan-1-ol> 4,4'-dimethoxytrityl chloride (3.09 mg, 9.12 mmol) was dissolved in pyridine (72 mL), and propan-1,3-diol (13 mL, 181 mmol) was added under argon atmosphere and stirred for 5.5 hours. The solvent was removed under reduced pressure, and the residue was extracted with Et2O / H2O. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (Hexane / AcOEt = 3 / 1 → 1 / 1, 1% TEA) to obtain compound 2 (1.58 g, 3.28 mmol, 65%) as a yellow oily substance. 1 H NMR (400 MHz, CDCl3): δ 7.43-7.40 (2H, m), 7.33-7.20 (7H, m), 6.88-6.78 (4H, m), 3.82-3.74 (8H, m), 3.27 (2H, q, J = 5.6 Hz), 2.24 (1H, t, J = 5.6 Hz), 1.85 (2H, q, J = 5.6). Other spectral data were in good agreement with those reported in the literature.

[0143] <Compound 4: Synthesis of 2-(tert-butyldisulfaneyl)ethyl(3-(((3-(bis(4-methoxyphenyl)(phenyl)ethoxy)propoxy) (diisopropylamino)phosphaneyl)propyl)carbamate> THF (dehydrated, 8 mL) and TEA (dehydrated, 0.5 mL, 3.96 mmol, 1.4 eq.) were added to N,N-bis(diisopropylamino)chlorophosphine (776 mg, 2.91 mmol, 1.0 eq.) azeotroped with pyridine. THF (dehydrated, 3 mL) was added to compound 2 (1.06 mg, 2.8 mmol, 1.0 eq.) and dropped at 0 °C under argon atmosphere, and the mixture was allowed to react at room temperature for 2 hours. THF (dehydrated, 4 mL) was added to compound 4 (530 mg, 1.99 mmol, 0.7 eq.) azeotroped with pyridine, and the mixture was added dropwise to the reaction solution at 0 °C under argon atmosphere. THF (anhydrous, 3 mL) was added to 1H-tetrazole (95.5 mg, 1.36 mmol, 0.5 eq.), and the mixture was allowed to react at room temperature for 2 hours. CH2Cl2 and NaHCO3 were cooled to 0 °C in advance and separated. The organic layer was distilled under reduced pressure to obtain a yellow oily crude (2.13 g). The product was purified by column chromatography (neutral flash, Hexane / AcOEt = 15 / 1 → 10 / 1, 1% TEA) to obtain colorless oily compound 4 (284 mg, 0.37 mmol, 19%). 1 H-NMR (400 MHz, CDCl3):δ 7.24 (9H, m), 6.80 (4H, d), 4.23 (2H, t, J = 7.2 Hz), 3.78 (6H, s), 3.59 (6H, m), 3.23 (2H, t, J = 6.0 Hz), 3.14 (2H, t, J = 6.4 Hz), 2.85 (t, 2H, J = 6.8 Hz) , 1.91 (t, 2H, J = 6.8 Hz), 1.73 (t, 2H, J = 6.4 Hz), 1.31 (s, 9H), 1.12 (d, 12H, J = 7.2 Hz);13 C-NMR (100 MHz, CDCl3):δ 158.39, 145.36, 136.62, 130.12, 128.27, 127.73, 126. 67, 113.04, 85.85, 62.96, 60.49, 60.36, 47.93, 42.95, 42.82, 39.28, 38.84, 32.04, 31.96, 29.94, 24.72; 31 P-NMR (162 MHz, CDCl3): δ 146.21; HRMS (ESI) calcd for C 40 H 59 N2O7PS2[M+H] + : 775.35, found for 776.3627.

[0144] Example 9. Synthesis of procationically modified polynucleotide (5xcation-PS-DNA) The 5' end of the PS-DNA consisting of the base sequence shown in SEQ ID NO:1 is a PS-DNA having the following formula (E):

[0145] [ka]

[0146] A procationic modified polynucleotide (5xcation-PS-DNA) having a structure represented by the following formula (I) linked thereto and fluorescently labeled (FAM-labeled) at the 3' end was synthesized in the same manner as in Example 1 using a procationic phosphoramidite monomer (Reference Example 3).

[0147] Comparative Example 6. Synthesis of phosphorothioate DNA (PS-DNA) Phosphorothioate DNA (PS-DNA) consisting of the base sequence shown in SEQ ID NO: 1 and having a fluorescently labeled (FAM-labeled) 3' end was synthesized by a standard method.

[0148] Example 10. Synthesis of procationic siRNA (5×ThioL siRNA (3′ & 3′)) The 3' end of the RNA having the base sequence shown in SEQ ID NO:4 is represented by the following formula (E):

[0149] [ka]

[0150] A procationically modified single-stranded RNA (5×cagtion guide strand (3′)) having a structure represented by the following formula (I) linked thereto was synthesized in the same manner as in Example 1 using a procationic phosphoramidite monomer (Reference Example 3).

[0151] On the other hand, the 3' end of the RNA having the base sequence shown in SEQ ID NO:5 is represented by the following formula (E):

[0152] [ka]

[0153] A procationically modified single-stranded RNA (5×cation passenger strand (3′)) having a structure represented by the following formula (I) linked thereto was synthesized in the same manner as in Example 1 using a procationic phosphoramidite monomer (Reference Example 3).

[0154] The 5×cation guide strand (3') and 5×cation passenger strand (3') were mixed and then adjusted to the desired concentration (0.1-1 μM) in 1×annealing buffer (60 mM KCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl2), and incubated at 90°C for 3 minutes, then allowed to cool to room temperature for 3 hours to form double-stranded RNA (procationic siRNA (5×cation siRNA (3' & 3'))).

[0155] Example 11. Synthesis of procationic siRNA (5xcation siRNA (3' & 5')) The 5' end of the RNA having the base sequence shown in SEQ ID NO:5 is represented by the following formula (E):

[0156] [ka]

[0157] A procationically modified single-stranded RNA (5×cation passenger strand (5′)) having a structure represented by the following formula (I) linked thereto was synthesized in the same manner as in Example 1 using a procationic phosphoramidite monomer (Reference Example 3).

[0158] The 5×cation guide strand (3') and 5×cation passenger strand (5') were mixed and then adjusted to the desired concentration (0.1-1 μM) in 1×annealing buffer (60 mM KCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl2), and incubated at 90°C for 3 minutes, then allowed to cool to room temperature for 3 hours to form double-stranded RNA (procationic siRNA (5×cation siRNA (3' & 5'))).

[0159] Test Example 8. Cell membrane permeability test 3 The same procedure as in Test Example 1 was repeated, except that the antisense polynucleotides of Example 9 and Comparative Example 6 were used as polynucleotides. The polynucleotide concentration was 100 nM.

[0160] The results are shown in Figure 8. In the case of procation modification, cell membrane permeability was equivalent to that in the case of using commercially available Lipofetamine3000.

[0161] Test Example 9. Cytotoxicity Test 3 The same procedure as in Test Example 3 was repeated except that the antisense polynucleotides of Example 9 and Comparative Example 6 were used as polynucleotides. The polynucleotide concentration was 1 μM.

[0162] The results are shown in Figure 9. The polynucleotide of the present example (5xcation-PS-DNA) showed no toxicity.

Claims

1. General formula (2): 【Chemistry 1】 [In the formula, DMTr represents a dimethyltrityl group. 1 R represents an alkylene group having 3 to 6 carbon atoms. x represents -O-R 31a -S-S-R 31b (wherein R 31a represents an alkylene group having 3 to 6 carbon atoms or -R 311a -NH-COO-R 312a - (R 311a and R 312a are the same or different and represent alkylene groups having 2 to 6 carbon atoms), and R 31b represents an alkyl group or an aryl group). Z 1 represents -O- or -NH-.] A compound represented by the formula:

2. R 31a is an alkylene group having 3 to 6 carbon atoms, and R 31b The compound of claim 1 , wherein is an alkyl group.

3. A method for producing a modified polynucleotide, comprising synthesizing nucleic acid by a phosphoramidite method using the compound according to claim 1 or 2 as a phosphoramidite monomer.

4. The method according to claim 3, which comprises modifying the end of a polynucleotide by using the compound according to claim 1 or 2 as a phosphoramidite monomer.

5. The method according to claim 3, wherein the polynucleotide is at least one selected from the group consisting of an antisense polynucleotide, an siRNA, an miRNA, an miRNA precursor, an aptamer, a guide RNA, an mRNA, a non-coding RNA, a DNA, an LNA, a PNA, and a morpholino nucleic acid.

6. The modified polynucleotide has the general formula (1): 【Chemistry 2】 [In the formula, R 1 R represents an alkylene group having 3 to 6 carbon atoms. 2 R indicates a single bond. 3 represents -O-R 31a -S-S-R 31b (wherein R 31a represents an alkylene group having 3 to 6 carbon atoms or -R 311a -NH-COO-R 312a - (R 311a and R 312a are the same or different and represent alkylene groups having 2 to 6 carbon atoms), and R 31b represents an alkyl group or an aryl group). Z 1 represents -O- or -NH-; and n represents any integer. The method according to claim 3, wherein the modified polynucleotide is modified with a molecule having a structure represented by the following formula:

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