JPWO2006025154A1 - Modified oligonucleotide - Google Patents

Abstract

It is possible to provide a modified oligonucleotide having a modified 3'end, which is excellent in three properties such as permeability to cell membrane, nuclease resistance and double-strand forming ability, and which is difficult to produce. The 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide is solved by a modified oligonucleotide in which the bond of formula (I-1) is replaced. -X1-C(=Y1)-Z1- (I) In the above formula, X1 is O, NH or S, Y1 is O or S, and Z1 is O, NH or S. This modified oligonucleotide can be produced by solid phase synthesis using the unit compound for solid phase synthesis represented by formula (X-1) as a starting material. In the formula (X-1), R2, W11, W21, B1, B2, A, X2, X1, Y1, Z1 and M are as defined in the specification.

Description

  The present invention relates to modified oligonucleotides.

  In recent years, a methodology of treating genetic information itself as a target of treatment is becoming widespread. One of the methodologies is an antisense method using an oligonucleotide. In the antisense method, a chemically synthesized oligonucleotide (for example, single-stranded DNA consisting of 15 to 20 base pairs) is added to cells to form a target messenger RNA (mRNA) and DNA/mRNA double-stranded nucleic acid. By this method, the expression of the target gene is specifically suppressed in the base sequence, and the translation process from mRNA to protein is inhibited. In the antisense method, when the base sequence of the virus or gene that causes the disease is known, it is possible to theoretically design the antisense molecule and synthesize it, and thus it has been considered difficult to cure. It is expected to be one of the effective treatment methods for diseases caused by various viruses and genetic diseases.

  Also, very recently, attention has been focused on a method using RNAi (RNA interference) as a gene expression suppressing method using an oligonucleotide. This RNAi refers to a phenomenon in which, when a double-stranded RNA is introduced into a cell, RNA derived from a cell chromosome having the same base sequence is decomposed and cleaved. The mechanism of this RNAi is currently considered as follows. First, a long double-stranded RNA is a double-stranded RNA (called siRNA (short interfering RNA)) having a length of about 21 bases having a 3'-UU type dangling end structure by an enzyme (called Dicer). Is hydrolyzed to. The siRNA forms an RNA/mRNA double-stranded nucleic acid with a target mRNA, and an intracellular protein that recognizes this double-stranded nucleic acid (called RISC (RNA-induced Silencing Complex)) and this double-stranded nucleic acid The target mRNA is bound by this binding and the bound body is cleaved. According to this method utilizing RNAi, in many cases, an equivalent effect is obtained by using RNA at a concentration of about 1/100 as compared with the antisense method. Therefore, the method utilizing RNAi is also expected to be one of the effective therapeutic methods for diseases and genetic diseases caused by various viruses, which have been considered difficult to cure.

  In the method using an oligonucleotide such as the antisense method or the method utilizing RNAi, it is necessary to introduce the oligonucleotide into cells. In addition, although it is necessary to make the oligonucleotide once introduced into the cell stably exist, a nucleic acid hydrolase (nuclease) exists inside and outside the cell, and the introduced oligonucleotide, especially the natural oligonucleotide is There was a problem that it was easily disassembled.

  Various modified oligonucleotides have been developed for the purpose of improving permeability to cell membranes and improving nuclease resistance. For example, a DNA oligonucleotide in which a modified TT dimer in which a 5′,3′-phosphodiester bond between thymidine-thymidine dimers is replaced with a carbamate bond is introduced is known (for example, see Non-Patent Document 1). .. It has been confirmed that a DNA oligonucleotide containing such a modified TT dimer has a slightly improved nuclease resistance. However, this modified TT dimer can be introduced into a site other than the 3'end of a DNA oligonucleotide due to the nature of its production method, but it has not been realized into the 3'end.

  In addition, nuclease is known to decompose an oligonucleotide from its 3'end. For example, a DNA oligonucleotide containing a modified TT dimer as described above is considered to be usually degraded by a nuclease between the 3'end of the DNA oligonucleotide and the modified TT dimer. That is, although it shows some nuclease resistance, the base sequence of the DNA oligonucleotide containing the modified TT dimer is partially decomposed, the sequence is not preserved, and the action of the DNA oligonucleotide is reduced. I was afraid. Considering the reaction characteristics of the nuclease as described above, a modified oligo showing resistance to nuclease by modifying the 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide. You can expect nucleotides. However, in oligonucleotide synthesis using solid-phase synthesis technology, modification of the site is difficult, and such modified oligonucleotide has not been realized.

Furthermore, in the method using an oligonucleotide such as the antisense method and the method utilizing RNAi, the oligonucleotide is required to have a double-strand forming ability with a natural oligonucleotide and the stability of the formed double strand. However, a non-natural oligonucleotide having a modified chemical structure usually has a problem that its double-stranded ability is reduced.
Adrian Waldner, Alain De Mesmaeker and Jacques Lebreton, "Synthesis of Oligodeoxyribonucleotides containing Dimers with Carbamate Moieties as Replacement of the Natural Phosphodiester linkage", Bioorganic & Medicinal Chemistry Letters, 1994 years, Vol. 4, p. 405

  Therefore, an object of the present invention is to provide an oligonucleotide having a modified 3'end, which is excellent in the three properties of permeability to cell membrane, nuclease resistance and double-strand forming ability.

The present invention provides a modified oligo in which the 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide is replaced with a bond represented by the following formula (I-1). It is a nucleotide.
^{-X 1 -C (= Y 1)} -Z 1 - (I-1)
In the formula (I-1), X ¹ is O, NH or S, Y ¹ is O or S, and Z ¹ is O, NH or S.

  The present invention is based on the successful production of an oligonucleotide having a modified 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end, which has been difficult to produce by conventional methods. It has been completed. In the present invention, as the modification, replacement with a bond represented by the formula (I-1) was selected, and improvement of nuclease resistance of the modified oligonucleotide, improvement of cell membrane permeability and improvement of double-strand forming ability were realized. .. Furthermore, the improved nuclease resistance of the modified oligonucleotides of the present invention is far superior to that expected due to the modified 3'end.

FIG. 1 is a diagram showing an example of a modified double-stranded oligonucleotide. FIG. 2 is a graph showing RNase L expression levels of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide. FIG. 3 is a graph showing RNase L expression levels of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide. FIG. 4 is a diagram showing cell membrane permeability of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide. FIG. 5 is a diagram showing intracellular RNase L expression levels of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide. FIG. 6 is a graph showing exonuclease resistance of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide. FIG. 7 is a diagram showing an example of a modified double-stranded oligonucleotide. FIG. 8 is a graph showing endonuclease resistance of an example of the modified double-stranded oligonucleotide.

  In the present invention, the “oligonucleotide” refers to, for example, a polymer of nucleoside subunits, and the number of subunits is not particularly limited, but is, for example, 4 to 100. Among them, when the modified oligonucleotide of the present invention is DNA, the number of subunits thereof is preferably 4 to 100, more preferably 4 to 30, and if it is RNA, 4 to 50. Preferably, 4 to 30 are more preferable. The "oligonucleotide" in the present invention is not particularly limited, except that the 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end is modified. For example, in the case of a nucleoside that is a subunit of an oligonucleotide, the sugar moiety (eg, 2′-substituted form) and the base may be modified forms known to those skilled in the art.

Bond ^-X 1 -C represented by formula (I-1) in the modified oligonucleotides of the present invention ⁽⁼ Y ¹⁾ -Z 1 - is conveniently, the 5 'carbon atom of the sugar moiety of the nucleoside The bond to be bonded is shown on the left and the bond to the 3'-carbon atom is shown on the right. For example, when the 3'end of the modified oligonucleotide is TT and the 5',3'-phosphodiester bond between the TTs is replaced with the bond represented by the formula (I-1), expressed.

In the above formula, W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
Here, R ¹¹ and R ²¹ are each independently a protecting group.

  The bond represented by the formula (I-1) is, for example, -OC(=O)-NH-, -NH-C(=O)-O-, -NH-C(=O)-NH. -, -NH-C(=O)-S-, -SC-(=O)-NH-, -OC(=S)-NH-, -NH-C(=S)-O-, -NH-C(=S)-NH-, -NH-C(=S)-S-, -SC-(=S)-NH-, etc. are mentioned. Among them, the bond represented by the above formula (I-1) is -OC(=O)-NH-, -NH-C(=O)-O- or -NH-C(=O)-. NH- is preferred.

The modified oligonucleotide of the present invention preferably satisfies at least one of the following (i) and (ii). In addition, regarding the bonds represented by the following formula (I-2) and the following formula (I-3), similarly, for the sake of convenience, to the left, a bond that bonds to the carbon atom at the 5′-position of the sugar moiety of the nucleoside is placed on the left. The bond attached to the 3′ carbon atom is shown on the right.
(I) The 5',3'-phosphodiester bond between the 2nd and 3rd nucleosides from the 3'end of the oligonucleotide is replaced with a bond represented by the following formula (I-2).
^{-X 2 -C (= Y 2)} -Z 2 - (I-2)
In the formula (I-2), X ² is O, NH or S, Y ² is O or S, and Z ² is O, NH or S.
(Ii) The 5',3'-phosphodiester bond between the 3rd and 4th nucleosides from the 3'end of the oligonucleotide is replaced with a bond represented by the following formula (I-3).
^{-X 3 -C (= Y 3)} -Z 3 - (I-3)
In the formula (I-3), X ³ is O, NH or S, Y ³ is O or S, and Z ³ is O, NH or S.

  The bond represented by the formula (I-2) and the bond represented by the formula (I-3) are each independently, for example, -OC(=O)-NH, -NH-C(. =O)-O-, -NH-C(=O)-NH-, -NH-C(=O)-S-, -S-C(=O)-NH-, -OC(=S). )-NH-, -NH-C(=S)-O-, -NH-C(=S)-NH-, -NH-C(=S)-S-, -SC-(=S)-. It is preferably selected from NH- and the like. Among them, the bond represented by the formula (I-2) is -OC(=O)-NH-, -NH-C(=O)-O- or -NH-C(=O)-NH. -Is more preferable. In addition, among them, the bond represented by the formula (I-3) is -OC(=O)-NH-, -NH-C(=O)-O- or -NH-C(=O). -NH- is more preferable.

  When the above (i) is satisfied, the combination of the bond represented by the formula (I-1) and the bond represented by the formula (I-2) is not particularly limited, but is, for example, -OC(=O). -NH- and -OC(=O)-NH-, -NH-C(=O)-O- and -NH-C(=O)-O-, -NH-C(=O)-NH And -NH-C(=O)-NH- and the like. The bond represented by formula (I-1) and the bond represented by formula (I-2) may be the same or different. Further, when the above (i) is satisfied, the combination of the bond represented by the formula (I-1) and the bond represented by the formula (I-3) is not particularly limited, but, for example, -OC(= O)-NH- and -O-C(=O)-NH-, -NH-C(=O)-O- and -NH-C(=O)-O-, -NH-C(=O). -NH- and -NH-C(=O)-NH- and the like. The bond represented by formula (I-1) and the bond represented by formula (I-3) may be the same or different. Furthermore, when both (i) and (ii) are satisfied, the bond represented by formula (I-1), the bond represented by formula (I-2) and the formula (I-3) are represented. The combination of bonds is not particularly limited, but for example, -OC(=O)-NH- and -NH-C(=O)-O- and -OC(=O)-NH-, -NH. -C(=O)-O-, -OC(=O)-NH-, -NH-C(=O)-O- and the like. The bond represented by formula (I-1), the bond represented by formula (I-2) and the bond represented by formula (I-3) may be the same or different.

  The modified oligonucleotide of the present invention may be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or the like. The modified oligonucleotide of the present invention is excellent in double-strand forming ability, and thus is preferable for use as antisense, siRNA and the like. When the modified oligonucleotide is double-stranded, 5',3'-phosphate between the first and second nucleosides from the 3'end of the single-stranded oligonucleotide of one or both of the double-stranded oligonucleotides The diester bond is preferably replaced with the bond represented by the formula (I-1). The 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end of both single-stranded oligonucleotides of the double-stranded oligonucleotide is represented by the above formula (I-1). When the bond is replaced with another bond, the bonds represented by formula (I-1) may be the same or different.

  When the modified oligonucleotide is double-stranded, it is an oligonucleotide that causes RNAi (RNA interference), and the modified oligonucleotide is a modified oligonucleotide having the same base sequence as a part of mRNA encoding an endonuclease or the like. preferable. This is because such modified oligonucleotides are useful for RNAi research and the like. Examples of the endonuclease include RNaseL and the like.

  When the modified oligonucleotide is double-stranded, the modified oligonucleotide is siRNA (short interfering RNA), and a part of the mRNA is located at least 75 bases upstream from the start codon and follows the first AA sequence. It is preferable that a part of the mRNA is a base sequence and is specific to the mRNA, and the modified oligonucleotide is a combination of a sense strand and an antisense strand of the 19 base sequence. Such a sense strand can be obtained, for example, as follows. First, NCBI (National Center for Biotechnology Information), EMBL-EBI (European Molecular Biological Laboratory-European mRNA Incorporation, etc.) is a publicly known gene using a sequence of a known gene such as a database for obtaining nucleases. In the gene sequence, find the first AA sequence 75 bases or more downstream from the start codon. It is confirmed that the 19-base sequence following the AA sequence, that is, a total of 21-base sequence is specific to the target gene again using a known gene database. It is also confirmed that this 19-base sequence has a GC content of about 50%. After confirming these two, the obtained 19-base sequence is the sense strand as described above.

  When the modified oligonucleotide is a siRNA as described above, it is preferred that one or both of the sense strand and the antisense strand have a nucleotide further containing two thymidines at their 3'ends. This is because having such a sequence at the 3'end facilitates RNAi. The modified oligonucleotide has a double-stranded structure since the thymidine-thymidine sequence, which is the first and second nucleosides from the 3'end, is replaced by the bond represented by the formula (I-1). It is preferable in terms of excellent forming ability and exhibiting siRNA function.

  In addition, when the modified oligonucleotide is single-stranded, the modified oligonucleotide is antisense DNA or antisense RNA, and the modified oligonucleotide is complementary to a part or all of mRNA encoding an endonuclease or the like. It is preferable to have a specific sequence. This is because the modified oligonucleotide can form a double strand with such mRNA and suppress the expression of endonuclease and the like.

  The modified oligonucleotides of the invention are preferably nuclease resistant. When the modified oligonucleotide of the present invention is taken up into cells, it can be prevented from being decomposed by nuclease, and as a result, the intracellular activity of the modified oligonucleotide can be sustained. ..

  The gene expression inhibitor of the present invention contains the modified oligonucleotide of the present invention. In such a gene expression inhibitor, the modified oligonucleotide can, for example, cleave the mRNA of the target gene or form a double strand with the mRNA of the target gene, and as a result, suppress the gene expression. ..

  The pharmaceutical composition of the present invention is for treating a disease associated with gene expression and contains the gene expression inhibitor. When a disease associated with gene expression, for example, the expression of a protein causes the disease, this pharmaceutical composition can be used to suppress the gene expression and treat the disease associated with the gene expression. Is.

  Such a pharmaceutical composition preferably further contains an excipient for cell introduction. This is because it becomes possible to facilitate introduction into cells when treating a disease associated with gene expression. Examples of the cell-introducing excipient include transfection reagents and the like. The transfection reagent is, for example, a DNA molecule (the above-mentioned pharmaceutical composition) wrapped in an artificial lipid vesicle (liposome) composed of phospholipid, and the artificial lipid vesicle is added to a cell suspension, It is a reagent for forming an artificial lipid vesicle, which is used in a lipofection method in which a DNA molecule in an artificial lipid vesicle is incorporated into a cell by being attached to the cell surface and being fused with a cell membrane. Examples of the transfection reagent include Lipofectamine (manufactured by Invitrogen), Lipofectamine 2000 (manufactured by Invitrogen), Oligofectamine (manufactured by Invitrogen), Transmessenger (TransMessenger (QIAGEN)). )), siRNA transfection kit Jet SI (manufactured by Ambion), GeneSlicer siRNA transfection reagent (manufactured by Gene Therapy Systems), and the like.

  As described above, the pharmaceutical composition of the present invention can be introduced into cells by using an electroporation method, a particle gun method, or the like, in addition to the inclusion of the cell introduction excipient. The electroporation method is, for example, a method in which an electric pulse is applied to a cell to make a hole in the cell wall, and the pharmaceutical composition of the present invention is introduced into the cell through the hole. In the particle gun method, molecules such as DNA molecules (the above-mentioned pharmaceutical composition) are attached to gold fine particles, and compressed gold helium gas is used with a particle gun (particle gun) to shoot the gold fine particles into a cell membrane. And the DNA molecule (the above-mentioned pharmaceutical composition) is introduced into cells.

  The RNAi kit of the present invention includes a modified oligonucleotide that is siRNA. Other examples of such a kit include plates on which wells and modified oligonucleotides and the like are fixed, fibers, immobilized carriers such as biochips, and the like. Such a kit may contain, in addition to the modified oligonucleotide and the like, for example, a drug, a coloring reagent that develops a color by reacting, a detection reagent that facilitates detection, and the like.

  The RNAi research reagent of the present invention includes a modified oligonucleotide that is siRNA. When dsRNA of 30 bp or more is introduced into a cell during RNAi research, a cell-specific defense reaction is activated and a reaction of randomly degrading mRNA occurs, and it is determined whether RNAi occurs in the cell. Sometimes you can't. The random degradation of this mRNA is considered to occur by the following mechanism. First, 2-5 oligoadenylate synthase (2-5AS) is activated by dsRNA, and 2-5A generated thereby activates RNAaseL. The RNAase L decomposes mRNA at random. Since the modified oligonucleotide, which is siRNA, can suppress the expression of this RNAaseL, it can suppress the random degradation of mRNA, and as a result, for example, it becomes easy to determine whether or not RNAi occurs in the cell.

  The gene expression suppressing method of the present invention is a method of suppressing gene expression using a modified oligonucleotide. In this method, the modified oligonucleotide can, for example, cleave the mRNA of the target gene or form a double strand with the mRNA of the target gene, resulting in suppression of gene expression.

  The method of causing RNAi of the present invention is a method of causing RNAi using a modified oligonucleotide that is siRNA. In this way, RNAi can be triggered because the modified oligonucleotide is siRNA.

  Next, a production method for producing the modified oligonucleotide of the present invention will be described with reference to examples. By such a production method, it becomes possible to produce the modified oligonucleotide of the present invention which could not be produced conventionally.

First, the 3',5'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide is represented by the following formula (I-1) (wherein X ¹ is O, NH or S). , Y ¹ is O or S, and Z ¹ is O, NH, or S).

^{-X 1 -C (= Y 1)} -Z 1 - (I-1)
For example, using a unit compound for solid phase synthesis represented by the following formula (X-1) as a starting material, R ^{2 of the} unit compound for solid phase synthesis represented by the formula (X-1) is removed, The modified oligonucleotide can be obtained by extending a nucleotide at the 5'end of the unit compound for solid phase synthesis represented by (X-1) and then cutting out from the solid phase carrier.

In the formula (X-1), R ² is a protecting group,
B ¹ and B ² are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
Here, R ¹¹ and R ²¹ are each independently a protecting group,
X ¹ and X ² , independently of each other, are O, NH or S,
Y ¹ is O or S,
Z ¹ is O, NH or S,
M is a solid phase carrier.

In addition, the 3',5'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide is represented by the above formula (I-1) (wherein X ¹ is O, NH or S). , Y ¹ is O or S, Z ¹ is O, NH or S), and the 5′,3′-phosphodiester bond between the second and third nucleosides from the 3′ end of the oligonucleotide is Production Example of Modified Oligonucleotide Substituted by a Bond represented by Formula (I-2) (wherein X ² is O, NH or S, Y ² is O or S, Z ² is O, NH or S) Will be explained.

^{-X 2 -C (= Y 2)} -Z 2 - (I-2)
For example, a unit compound for solid phase synthesis represented by the following formula (XI-1) is used as a starting material, R ^{3 of the} unit compound for solid phase synthesis represented by the formula (XI-1) is removed, and then the compound represented by the formula (XI The modified oligonucleotide can be obtained by extending a nucleotide at the 5'end of the unit compound for solid phase synthesis represented by XI-1) and then cutting out from the solid phase carrier.

In the above formula, R ³ is a protecting group,
B ¹ , B ² and B ³ are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
Here, R ¹¹ , R ²¹ and R ³¹ are each independently a protecting group,
X ¹ , X ² and X ³ , independently of each other, are O, NH or S,
Y ¹ and Y ² , independently of each other, are O or S,
Z ¹ and Z ² , independently of each other, are O, NH or S,
M is a solid phase carrier.

In addition, the 3',5'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide is represented by the formula (I-1) (wherein X ¹ is O, NH or S). , Y ¹ is O or S, Z ¹ is O, NH or S), and the 5′,3′-phosphodiester bond between the 3rd and 4th nucleosides from the 3′-end of the oligonucleotide is Production Example of Modified Oligonucleotide Substituted by Bond Represented by Formula (I-3) (wherein X ³ is O, NH or S, Y ³ is O or S, Z ³ is O, NH or S) Will be explained.

^{-X 3 -C (= Y 3)} -Z 3 - (I-3)
For example, using a solid-phase synthesis unit compound represented by the following formula (XII-1) as a starting material, R ⁴ of the solid-phase synthesis unit compound represented by the formula (XII-1) is removed, and then the formula (XII-1) is used. The modified oligonucleotide can be obtained by extending a nucleotide at the 5′ end of the unit compound for solid phase synthesis represented by XII-1) and then cutting out from the solid phase carrier.

In the above formula, R ⁴ and R are, independently of each other, a protecting group,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ and f are, independently of each other, O or S,
Z ¹ and Z ³ , independently of each other, are O, NH or S,
M is a solid phase carrier.

In addition, the 3',5'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide is represented by the above formula (I-1) (wherein X ¹ is O, NH or S). , Y ¹ is O or S, Z ¹ is O, NH or S), and the 5′,3′-phosphodiester bond between the second and third nucleosides from the 3′-end of the oligonucleotide is A bond represented by the formula (I-2) (in the above formula, X ² is O, NH or S, Y ² is O or S, Z ² is O, NH or S), and is from the 3′ end of the oligonucleotide. The 5′,3′-phosphodiester bond between the 3rd and 4th nucleosides has the formula (I-3) (wherein X ³ is O, NH or S, Y ³ is O or S, Z). ³ represents O, NH, or S), and describes an example of producing a modified oligonucleotide in which the bond is replaced.

For example, using a unit compound for solid phase synthesis represented by the following formula (XIII-1) as a starting material, R ^{4 of the} unit compound for solid phase synthesis represented by the formula (XIII-1) is removed, and then R The modified oligonucleotide can be obtained by extending a nucleotide at the 5′ end of the unit compound for solid phase synthesis represented by XIII-1) and then cutting out from the solid phase carrier.

In the above formula, R ^4's independently of each other are protective groups,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ² , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ , Y ² and Y ³ , independently of each other, are O or S,
Z ¹ , Z ² and Z ³ , independently of one another, are O, NH or S,
M is a solid phase carrier.

  The solid phase carrier is not limited as long as it is a solid phase carrier suitable for synthesizing DNA, RNA and the like with the solid phase carrier, and examples thereof include CPG (control pore glass) and HCP (highly cross-linked polystyrene). Etc.

The protecting group for R ² , R ³ and R ⁴ is selected from conventionally known protecting groups according to the reactivity of —X ² —H, —X ³ —H and —X ⁴ —H. For ^example, when X 2, ^{X 3} or ^{X 4} is O, and protecting groups known primary alcohol, for example, known in the field of nucleoside chemistry, 4,4'-dimethoxytrityl (DMTr), 4-mono- Methoxytrityl (MMTr), (9-phenyl)xanthen-9-yl [pixyl] and the like can be used as the protecting group.

The protecting group for R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ is selected from conventionally known protecting groups depending on the reactivity of the 2′-position hydroxyl group. For example, tert-butyldimethylsilyl (TBDMS), [(triisopropylsilyl)oxy]methyl (Tom), tetrahydropyranyl (THP) and the like can be used as the protecting group.

  The protecting group for R is selected from conventionally known protecting groups according to the reactivity of phosphoric acid. For example, cyanoethyl (CE), allyl (Allyl), trimethylsilylethyl (TMSE), p-nitrophenethyl (NPE) and the like can be used as the protecting group.

In addition, with regard to B ¹ , B ² , B ³ and B ⁴ , the protective group whose functional group is protected is selected from the protective groups known in nucleic acid chemistry. For example, benzoyl (Bz), isobutyryl (iBu), phenoxyacetyl (Pac), allyloxycarbonyl (AOC) and the like can be used as the protective group.

In order to extend the nucleotide to the 5'end of the unit compound for solid phase synthesis, a technique known in the art of oligonucleotide synthesis can be used to sequentially couple nucleosides according to the sequence of the modified oligonucleotide. ..
As the nucleoside, the coupling reagent, the deprotection reagent, the washing reagent and the like, those usually used for nucleic acid solid phase synthesis are used. The modified oligonucleotide thus obtained on the solid phase carrier is deprotected, if necessary, from the oligonucleotide side chain and then cut out from the solid phase carrier to obtain a crude modified oligonucleotide. The reagent used for excision can be appropriately selected from conventionally known reagents according to the structure of the solid phase carrier and the linker (portion connecting the solid phase carrier and the modified oligonucleotide), and the like. This crude modified oligonucleotide may be purified by HPLC or the like, if necessary.

  Next, the production when the modified oligonucleotide is double-stranded will be described with an example. For example, a single-stranded modified oligonucleotide is first produced according to the method described above. A single-stranded natural oligonucleotide having a sequence complementary to the modified oligonucleotide is also separately produced according to a conventionally known method. Next, the obtained single-stranded modified oligonucleotide is dissolved in an annealing buffer (for example, a buffer containing 100 mM KOAc aqueous solution, 2 mM MgOAc solution, and 30 mM HEPES-KOH (pH 7.4)). And a single-stranded natural oligonucleotide dissolved in an annealing buffer are mixed, treated at 95° C. for 5 minutes, and then gradually cooled to 25° C. Chain modified oligonucleotides can be obtained. This double-stranded modified oligonucleotide can be isolated and purified by further performing phenol/chloroform extraction, ethanol precipitation and the like, if necessary.

  A unit compound for solid phase synthesis of the formula (X-1), a unit compound for solid phase synthesis of the formula (XI-1), a unit compound for solid phase synthesis of the formula (XII-1) and a formula (XIII) used in the production method. The unit compound for solid phase synthesis of -1) can be produced, for example, by the following method.

  First, the production of the unit compound (X-1) will be described with reference to Scheme 1. A nucleoside derivative of the formula (IV-1) and a nucleoside derivative of the formula (III) are combined with a diimidazole derivative of the formula (V-1) and optionally a base (eg pyridine etc.), a catalyst (eg 4-dimethylamino). The reaction is performed in the presence of pyridine (DMAP) etc.) to obtain a dimer (VI). The nucleosides of the formula (IV-1) and the formula (III) may be commercially available or may be produced in-house using known literatures.

  Next, in the obtained dimer (VI), an anhydride (VII), optionally a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine (DMAP), etc.) and the like are present. React below to give the dimer (VIII).

  Next, the obtained dimer (VIII) and the solid support (IX) having an amino group are coupled with each other by a coupling reagent (for example, WSC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Hydrochloric acid salt)) to condense to obtain a unit compound (X-1).

In the above formula,
R ² is a protecting group,
B ¹ and B ² are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
Here, R ¹¹ and R ²¹ are each independently a protecting group, and X ¹ and X ² are independently O, NH or S,
Y ¹ is O or S,
Z ¹ is O, NH or S,
M is a solid phase carrier.

Next, a production example of the unit compound (XI-1) will be described with reference to Scheme 2. For example, R ² of the dimer of formula (VI) is replaced with H by a method depending on the property of R ² to obtain a dimer of formula (VI-1) in the free form at the 5′-position. Then, the dimer of formula (VI-1) and the nucleoside derivative of formula (IV-2) are combined with the diimidazole derivative of formula (V-2) and optionally a base (eg, pyridine), a catalyst ( For example, it is reacted in the presence of 4-dimethylaminopyridine (DMAP) etc. to obtain a trimer (XIV-1). The nucleoside of formula (IV-2) may be commercially available or may be produced in-house using known literature.

  Next, the obtained trimer (XIV-1) is optionally mixed with an anhydride (VII), a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine (DMAP), etc.) and the like. To give a trimer (XV-1).

  Next, the obtained trimer (XV-1) and the solid support (IX) having an amino group are coupled with each other by a coupling reagent (for example, WSC(1-ethyl-3-(3-dimethylaminopropyl)-). Carbodiimide/hydrochloride)) is condensed in the presence of unit compound (XI-1).

In the above formula, R ² and R ³ are each independently a protecting group,
B ¹ , B ² and B ³ are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
Here, R ¹¹ , R ²¹ and R ³¹ are each independently a protecting group, and X ¹ , X ² and X ³ are each independently O, NH or S,
Y ¹ and Y ² , independently of each other, are O or S,
Z ¹ and Z ² , independently of each other, are O, NH or S,
M is a solid phase carrier.

Next, a production example of the unit compound (XII-1) will be described with reference to Scheme 3. For example, R ² of the dimer of the formula (VI-2) is replaced with H by a method depending on the property of R ² to obtain a dimer of the formula (VI-3) in the free form at the 5′-position. Then, the dimer of formula (VI-3) and the nucleoside derivative of formula (IV-3) are added in the presence of a phosphate binding reagent (eg, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite). Condensation gives the trimer of formula (XIV-2). The nucleoside of the formula (IV-3) may be commercially available or may be produced in-house using known literature.

  Next, the obtained trimer (XIV-2) is optionally mixed with an anhydride (VII), a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine (DMAP), etc.) and the like. To give a trimer (XV-2).

  Next, the obtained trimer (XV-2) and the solid phase carrier (IX) having an amino group are coupled with each other by a coupling reagent (for example, WSC(1-ethyl-3-(3-dimethylaminopropyl)-). Carbodiimide/hydrochloride)) is condensed in the presence of unit compound (XII-1).

In the above formula, R ² , R ³ and R independently of one another are protective groups,
B ¹ , B ² and B ³ are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
Here, R ¹¹ , R ²¹ and R ³¹ are each independently a protecting group, and X ¹ and X ³ are each independently O, NH or S,
Y ¹ is, independently of each other, O or S,
Z ¹ is, independently of each other, O, NH or S,
M is a solid phase carrier.

Next, a production example of the unit compound (XIII-1) will be described with reference to Scheme 4. For example, R ³ of the trimer of formula (XIV-1) is replaced with H by a method depending on the property of R ³ to give a trimer of formula (XIV-3) in the 5′-position in a free form. Then, the trimer of formula (XIV-3) and the nucleoside derivative of formula (IV-4) are combined with the diimidazole derivative of formula (V-3) and optionally a base (for example, pyridine), a catalyst ( For example, it is reacted in the presence of 4-dimethylaminopyridine (DMAP) etc. to obtain a tetramer (XVI-1). The nucleoside of the formula (IV-4) may be commercially available or may be produced in-house using known literature.

  Next, the obtained tetramer (XVI-1) is optionally mixed with an anhydride (VII), a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine (DMAP), etc.) and the like. To give tetramer (XVII-1).

  Next, the obtained tetramer (XVII-1) and the solid support (IX) having an amino group are coupled with a coupling reagent (for example, WSC(1-ethyl-3-(3-dimethylaminopropyl)- Carbodiimide/hydrochloride)) is condensed in the presence of unit compound (XIII-1).

In the above formula,
R ³ and R ⁴ are protecting groups,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ² , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ , Y ² and Y ³ , independently of each other, are O or S,
Z ¹ , Z ² and Z ³ , independently of one another, are O, NH or S,
M is a solid phase carrier.

  In each step of Schemes 1 to 4, if necessary, a protective group may be introduced into each functional group, the protective group may be deprotected, or the protective group may be changed. The selection of a protecting group according to the type of functional group, the introduction of the protecting group, and the removal of the protecting group can be performed according to a method known in the art. in Organic Synthesis"), T. Greene et al., John Wiley & Sons, Inc. Publishing" and the like.

In the description of the present specification, the following abbreviations are used.
Ar: Argon APS: Ammonium peroxodisulfate (ammonium peroxodisulfate)
CPG: controlled pore glass
DIAD: diisopropyazocarboxylate
DMAP: 4-dimethylaminopyridine (4-dimethylaminopyridine)
DMTrCl: 4,4'-dimethoxytrityl chloride (4,4'-dimethyoxytrityl chloride)
DMF: dimethylformamide
EDTA: ethylenediamine-N,N,N',N'-tetraacetic acid-disodium salt-dehydrate (ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dehydrate)
FAB: fast atom bombardment
HRMS: high-resolution mass spectrometry
Im ₂ CO: 1-1′-Carbonylbis-1H-imidazole (1-1′-Carbonylbis-1H-imidazole)
mRNA: messenger ribonucleic acid (messenger ribonucleic acid)
NBA: 3-nitrobenzyl alcohol (3-nitrobenzyl alcohol)
PAGE: Polyacrylamide gel electrophoresis
TBAF: Tributylammonium Fluoride
TBE: Tris-boric acid-EDTA (Tris-boric acid-EDTA)
TEAA: triethylamine-acetic acid
TBDMSCl: tert-butyldimethylsilyl chloride (tert-butyldimethylsilyl chloride)
TEMED: N,N,N',N'-tetramethyl-ethylenediamine (N,N,N',N'-tetramethyl-ethylenediamine)
THF: Tetrahydrofuran
TRIS: tris(hydroxymethyl)aminomethane (tris(hydroxymethyl)aminomethane)
WSC: 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide,hydrochloride)
The activity of the compound on CPG was calculated as follows.

6 mg of dried CPG was placed on a glass filter, and a mixture of HClO ₄ and ethanol (HClO ₄ :EtOH=3:2) was poured therein. The absorbance (wavelength 498 nm (absorption wavelength of DMTr group)) of the obtained filtrate was measured, and the value was calculated by substituting it in the following formula.

In the above formula, Abs. Is the absorbance of CPG at a wavelength of 498 nm,
Vol. Is the measured filtrate volume,
weight is the measured weight of CPG.

  1. Monomer production for the production of modified single-stranded oligonucleotides or modified double-stranded oligonucleotides

(1) Production of 5'-O-(4,4'-dimethoxytrityl)thymidine (IV-11) (5'-O-(4,4'-dimethyoxytrityl)thymidine) (S. Agrawal et al., Methods in Molecular). Produced according to Biology Vol. 20, 360-366.)
A solution of thymidine (2.03 g, 8.43 mmol) in pyridine (16 mL) was stirred at room temperature under Ar atmosphere. DMTrCl (3.08 g, 9.11 mmol, 1.1 equivalent) was added thereto, and the mixture was further stirred at room temperature. After 12 hours, 1 equivalent (0.28 g, 0.83 mmol) of DMTrCl was added to the reaction mixture, and the reaction mixture was further stirred. After TLC analysis confirmed that thymidine was not consumed any more, methanol was added to the reaction mixture under ice cooling. The solvent was distilled off from the reaction mixture under reduced pressure, and the obtained residue was diluted with chloroform. The chloroform solution was washed successively with saturated aqueous NaHCO ₃ solution (2 times) and saturated aqueous NaCl solution (1 time), then dried over sodium sulfate. The solvent was distilled off from the chloroform solution under reduced pressure, and the obtained residue was azeotropically distilled with toluene. The obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =0-4%) to give the title compound as white foamy crystals (3.55 g, 6.51 mmol, yield 77%). ..

¹ H NMR (400 MHz, CDCl ₃ ) δ: 1.47 (3H,s,5-CH ₃ ), 2.32-2.41 (2H,m,2′-H), 3.35-3.49. (2H, m, 5'-H ), 3.79 (6H, s, -OCH 3), 4.06 (1H, m, 4'-H), 4.54 (1H, m, 3'-H ), 6.44 (1H, t, J=6.8Hz, 1'-H), 6.83-7.40 (13H, m, DMTr), 7.59 (1H, s, 6-H), 8.66 (1H, s, 1-NH).

  (2) Production of 3'-O-tert-butyldimethylsilylthymidine (III-12) (produced according to S. Agrawal et al., Methods in Molecular Biology vol. 20, 360-366).

(I) 3'-O-tert-butyldimethylsilyl-5'-O-(4,4'-dimethoxytrityl)thymidine (3'-O-tert-butyldimethylsilyl-5'-O-(4,4'- Preparation of dimethyoxytrityl)thymidine 5′-O-(4,4′-dimethoxytrityl)thymidine (1.04 g, 1.91 mmol) and imidazole (0.32 g, 4.74 mmol, 2.5 eq) in Ar atmosphere. It was then dissolved in DMF (8.0 mL) and the resulting solution was stirred under Ar atmosphere. TBDMSCl (0.36 g, 2.37 mmol, 1. eq) was added to the above solution and the reaction mixture was stirred under M atmosphere for 12 h.
After confirming by TLC analysis that 5′-O-(4,4′-dimethoxytrityl)thymidine was not consumed any more, methanol (4 mL) was added to the reaction mixture under ice cooling. The solvent was distilled off from the reaction mixture under reduced pressure, and the obtained residue was diluted with ethyl acetate. The ethyl acetate solution was washed successively with saturated aqueous NaHCO ₃ solution (2 times) and saturated aqueous NaCl solution (1 time), then dried over sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =0 to 5%) to obtain the title compound as white foamy crystals. (1.18 g, 1.79 mmol, 94% yield).

¹ H NMR (400 MHz, CDCl ₃ ) δ: 0.09 (6H,s, TBDMS-CH ₃ ), 0.81 (9H,s,t-butyl), 1.47 (3H,s,5-CH _{3 ).} ), 2.16-2.33 (2H, m, 2'-H), 3.23-3.46 (2H, m, 5'-H), 3.77 (6H, s, OCH 3), 3.94-3.95 (1H, m, 4'-H), 4.49-4.50 (1H, m, 3'-H), 6.32 (1H, t, J=6.6Hz, 1'-H), 6.80-7.39 (13H, m, DMTr), 7.63 (1H, s, 6-H), 8.15 (1H, s, NH).

(Ii) Production of 3'-O-tert-butyldimethylsilylthymidine (III-12) (3'-O-tert-butyldimethylsilylthymidine) 3'-O-tert-butyldimethylsilyl-5'-O-(4. 80% aqueous acetic acid solution (26 mL) was added to 4'-dimethoxytrityl)thymidine (1.18 g, 1.79 mmol), and the reaction mixture was stirred. After 1 hour, the disappearance of 3'-O-tert-butyldimethylsilyl-5'-O-(4,4'-dimethoxytrityl)thymidine was confirmed by TLC analysis, and the solvent was distilled off from the reaction mixture under reduced pressure. Left. The obtained residue was diluted with ethyl acetate. The ethyl acetate solution was washed successively with saturated aqueous NaHCO ₃ solution (2 times) and saturated aqueous NaCl solution (1 time), and then dried over sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =0 to 3%) to obtain the title compound as white crystals (0. 60 g, 1.68 mmol, yield 94%).

¹ H NMR (400 MHz, CDCl ₃ ) δ: 0.03 (6H,s, TBDMS-CH ₃ ), 0.81 (9H,s,t-butyl), 1.83 (3H,s,5-CH _{3 ).} ), 2.10-2.41 (2H, m, 2'-H), 3.64-3.69 (1H, m, 4'-H), 3.81-3.86 (2H, m, 5'-H), 4.39-4.42 (1H, m, 3'-H), 6.02-6.05 (1H, t, J=6.8Hz, 1'-H), 7. 26(1H,s,6-H), 8.34(1H,s,NH).
(3) Production of 5′-aminothymidine (III-11) (produced according to T. Hata et al., J. Chem. Soc., Perkin trans. 1, 1980, 306-310).

(I) 5'azidothymidine (5'-azidothymidine) manufacturing thymidine _{(2.00g, 8.26mmol), Ph 3} P (2.00g, 8.26mmol, 1.0 eq) and _NaN 3 (2.60 g , 41.3 mmol, 5.0 equivalent) was dissolved in DMF (40 mL), and CBr ₄ (2.80 g, 8.40 mmol, 1.0 equivalent) was added to the solution. The resulting reaction mixture was stirred at 85°C under Ar atmosphere. After 25 hours, the disappearance of thymidine was confirmed by TLC analysis, and then the reaction mixture was diluted with ethyl acetate and washed successively with water (3 times), saturated aqueous NaHCO ₃ solution (1 time), and saturated NaCl aqueous solution (1 time). And then dried over sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =1:49 to 7:93) to obtain the title compound as white crystals. (Yield 2.01 g, 7.52 mmol, 91% yield).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 1.78 (3H,s,5-CH ₃ ), 2.06-2.28 (2H,m,2′-H), 3.55 (2H , D, J=4.8 Hz, 5′-H), 3.83 (1H, m, 4′-H), 4.08 (1H, m, 3′-H), 3.40 (1H, d). , J=3.6 Hz, 3-OH), 6.19 (1 H, t, J=6.8 Hz, 1′-H), 7.55 (1 H, s, 6-H), 11.32 (1 H , S, NH).

(Ii) Production of 5'-aminothymidine (III-11) (5'-aminothymidine) 5'-azidothymidine (0.23 g, 0.94 mmol) and 5% Pd catalyst (0.065 g) were added to ethanol and methylene chloride. In a mixed solvent (EtOH:CH ₂ Cl ₂ =1:1) (3.1 mL) under hydrogen atmosphere for 24 hours. After confirming the disappearance of 5'-azidothymidine by TLC analysis, the reaction mixture was filtered through Celite. The solvent was distilled off from the obtained filtrate under reduced pressure to give the title compound as white crystals (yield 0.22 g, 0.89 nmol, yield 95%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 1.74 (3H,s,5-CH ₃ ), 2.00-2.16 (2H,m,2′-H),2.49-2 .77 (2H, m, 5'-H), 3.20-3.45 (1H, br, 3'-OH), 3.63-3.66. (1H, m, 4'-H), 4.16-4.20 (1H, m, 3'-H), 6.13 (1H, t, J=6.8Hz, 1'-H), 7 .63 (1H, s, 6-H).

  (4) Production of 3'-amino-5'-O-tert-butyldimethylsilylthymidine (IV-12) (EW Harkins et al., Current Protocols in Nucleic Acid Chemistry, 4.7.15-4.7). .17.)

(I) Production of 5'-O-tert-butyldimethylsilylthymidine (5'-O-tert-butyldimethylsilylthymidine) Thymidine (2.00 g, 8.26 mmol) was azeotroped with DMF to give DMF (16.5 mL). Melted To the solution was Et ₃ N (1.36 mL, 9.91 mmol, 1.2 eq), DMAP (0.05 g, 0.41 mmol, 0.05 eq) and TBDMSCl (1.36 g, 9.02 mmol, 1. 1 eq.) were added sequentially and the resulting reaction mixture was stirred at room temperature under Ar atmosphere for 2.5 hours. Further TBDMSCl (0.25g, 1.66mmol, 0.2eq) was added to the reaction mixture and stirred for a further 30 minutes. After TLC analysis confirmed the disappearance of thymidine, the reaction mixture was diluted with ethyl acetate. The resulting ethyl acetate solution was washed successively with water (3 times), saturated aqueous NaHCO ₃ solution (1 time), saturated aqueous NaCl solution (1 time), and then dried over sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =0:1 to 1:20) to obtain the title compound as white crystals. (Yield 2.44 g, 6.84 mmol, yield 83%).

¹ H NMR (400 MHz, CDCl ₃ ) δ: 0.12 (6H, m, TBDMS-CH ₃ ), 0.92 (9H, s, t-butyl), 1.89 (3H, s, 5-CH _{3 ).} ), 2.08-2.39 (3H, m, 2'-H and 3'-OH), 3.82-3.92 (2H, m, 5'-H), 4.03 (1H, m. , 4'-H), 4.47 (1H, m, 3'-H), 6.37 (1H, m, 1'-H), 7.50 (1H, s, 6-H), 8. 44 (1H, br, NH).

(Ii) Preparation of 2,3'-anhydro-5'-O-tert-butyldimethylsilylthymidine (2,3'-anhydro-5'-O-tert-butyldimethylsilthymidine) 5'-O-tert-butyldimethyl A solution of silylthymidine (2.19 g, 6.14 mmol) and Ph ₃ P (2.60 g, 9.91 mmol, 1.6 eq) in DMF (7.0 mL) was added to DIAD (3.2 mL, 9.00 mmol, 1). A solution of 0.6 equiv., 40% toluene solution in DMF (3.1 mL) was added and the resulting reaction mixture was stirred at room temperature under Ar atmosphere. After 3 and a half hours, TLC analysis confirmed the disappearance of 5′-O-tert-butyldimethylsilylthymidine, and the reaction mixture was diluted with ethyl acetate, water (3 times), saturated aqueous NaHCO ₃ solution (1 time), saturated. It was washed successively with aqueous NaCl solution (once) and then dried over sodium sulphate. The solvent was distilled off from the obtained ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane:EtOAc:MeOH=1:1:0 to 0:10:1) to give a white residue. The title compound as crystals was obtained (amount 1.63 g, 4.82 mmol, yield 79%).

¹ H NMR (400 MHz, CDCl ₃ ) δ: 0.07 (6H,s, TBDMS-CH ₃ ), 0.88 (9H,s,t-butyl), 1.95 (3H,s,5-CH _{3 ).} ), 2.43-2.67 (2H, m, 2'-H), 3.73-3.83 (2H, m, 5'-H), 4.28 (1H, t, J=5. 6 Hz, 4'-H), 5.19 (1H, s, 3'-H), 5.47 (1H, s, 1'-H), 6.95 (1H, s, 6-H).

(Iii) Production of 3'-azido-5'-O-tert-butyldimethylsilylthymidine (3'-azido-5'-O-tert-butyldimethylsilthymidine) 2,3'-anhydro-5'-O-tert - butyldimethylsilyl thymidine (0.48 g, 1.41 mmol) in DMF (4.2 mL) solution _{of, NaN 3 (0.14g, 2.21mmol,} 1.5 eq) was added and the reaction mixture stirred under M atmosphere And stirred at 120°C. After 48 hours, TLC analysis confirmed the disappearance of 2,3′-anhydro-5′-O-tert-butyldimethylsilylthymidine. The reaction mixture was diluted with ethyl acetate, washed sequentially with water (3 times), saturated aqueous NaHCO ₃ solution (1 time), saturated aqueous NaCl solution (1 time), and then dried over sodium sulfate. The solvent was distilled off from the obtained ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane:EtOAc=0:1) to obtain the title compound as an oil (yield: 0. 48 g, 1.25 mmol, yield 88%).

¹ H NMR (400 MHz, CDCl ₃ ) δ: 0.01 (6H, m, TBDMS-CH ₃ ), 0.80 (9H, m, t-butyl), 1.80 (3H, s, 5-CH _{3 ).} ), 2.07-2.34 (2H, m, 2'-H), 3.66-3.70 (1H, m, 4'-H), 3.81-3.86 (2H, m, 5'-H), 4.12 (1H, m, 3'-H), 6.12 (1H, s, 1'-H), 7.32 (1H, s, 6-H), 10.00. (1H, br, NH).

(Iv) Production of 3'-amino-5'-O-tert-butyldimethylsilylthymidine (IV-12) (3'-amino-5'-O-tert-butyldimethylsilylthymidine) 3'-azido-5'-O -Tert-Butyldimethylsilylthymidine (1.15 g, 3.02 mmol) and 5% Pd catalyst (0.15 g) were stirred in methanol (10 mL) under Ar atmosphere at room temperature for 19 hours. After confirming the disappearance of 3′-azido-5′-O-tert-butyldimethylsilylthymidine by TLC analysis, the reaction mixture was filtered through Celite. The solvent was distilled off from the obtained filtrate under reduced pressure to obtain the title compound as white crystals (yield 1.03 g, 2.89 nmol, yield 95%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 0.05 (6H, m, TBDMS-CH ₃ ), 0.86 (9H, s, t-butyl), 1.75 (3H, s, 5′). _{-CH 3), 2.01-2.09 (2H,} m, 2'-H), 3.30 (2H, s, NH 2), 3.41-3.46 (1H, m, 4'- H), 3.64-3.77 (2H, m, 5'-H), 3.80-3.84 (1H, m, 3'-H), 6.11-6.16 (1H, t. , 9.4 Hz, 1'-H), 7.45 (1 H, s, 6-H).

  2. Production of a unit compound bound to a solid phase carrier for producing a modified single-stranded oligonucleotide or a modified double-stranded oligonucleotide

(Example 1-01)
(1) Production of unit compound represented by (X-11)

(I) 5'-O-(4,4'-dimethoxytrityl)-3'-(5''-aminothymidylyl)carbamoylthymidine (5'-O-(4,4'-dimethyoxytrityl)-3'-(5 ''-Aminothymidylyl)carbamoylthymidine) Preparation of 5'-O-(4,4'-dimethoxytrityl)thymidine (IV-11) (0.76 g, 1.4 mmol) in pyridine (8.0 mL) was added to DMAP (8.0 mL). Small amount) and Im ₂ CO (0.23 g, 1.4 mmol, 1.0 eq) were added and the reaction mixture was stirred at room temperature under Ar atmosphere. After 24 hours, a solution of 5′-aminothymidine (III-11) (0.50 g, 2.07 mmol, 1.5 eq) in pyridine (6.0 mL) was added dropwise to the reaction mixture. After 48 hours, the reaction mixture was diluted with ethyl acetate. The obtained ethyl acetate solution was washed successively with water (once), saturated aqueous NaHCO ₃ solution (once) and saturated aqueous NaCl solution (once), and then dried over sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =1:20 to 1:10) to obtain the title compound as white crystals (yield: 0.71 g, 0.87 mmol, yield 62%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 1.38 (3H,s,5-CH ₃ 5′T), 1.75 (3H,s,5-CH ₃ 3′T), 1.98. -2.48 (4H, m, 2'- H), 3.14-3.33 (4H, m, 5'-H), 3.73 (6H, s, OCH 3), 3.99-4 .04 (2H, m, 4'-H), 4.12 (1H, s, 3'-H 5'T), 5.22 (1H, br, OH), 5.28-5.29 (1H , M, 3'-H 3'T), 6.10-6.14 (1H, m, 1'-H 5'T), 6.20-6.24 (1H, m, 1'-H 3 'T), 6.28-7.38 (13H, m, DMTr), 7.48 (1H, s, 6-H 5'T), 7.51 (1H, s, 6-H 3'T) , 7.60 (1H, t, J=6.0 Hz, NH), 11.28 (1H, s, NH 5′T), 11.37 (1H, s, NH 3′T).
¹³ C-NMR (100 MHz, CDCl ₃ ) δ: 11.44, 12.34, 37.97, 38.90, 55.22, 63.94, 71.23, 76.33, 76.68, 77. 32, 84.00, 84.36, 84.87, 87.19, 110.75, 111.96, 113.28, 127.19, 128.01, 128.11, 130.11, 135.04. 135.21, 135.56, 144.16, 151.14, 156.38, 158.70, 164.07, 164.41.
_{FAB-MS (NBA) calcd for} C 42 H 45 N 2 O 12 (MH -), 810.29867; found, 810.29930.

Production of Unit Compound (X-11) 5′-O-(4,4′-dimethoxytrityl)-3′-(5″-aminothymidylyl)carbamoylthymidine (0.71 g, 0.87 mmol) pyridine (8. 7 mL) solution was added DMAP (minor amount) and succinic anhydride (0.29 g, 2.87 mmol, 3.3 eq) and the reaction mixture was stirred at room temperature under Ar atmosphere for 16 h. After confirming the disappearance of 5′-O-(4,4′-dimethoxytrityl)-3′-(5″-aminothymidylyl)carbamoylthymidine by TLC analysis, the reaction mixture was diluted with chloroform. The chloroform solution was washed successively with water (once) and saturated aqueous NaCl solution (once) and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the chloroform solution. A part of the obtained residue (0.80 g, 0.87 mmol, 4 equivalents) was dissolved in DMF (22 mL). CPG (136 μmol/g, 1.61 g, 0.22 mol) was added to the solution to swell the CPG, and then WSC (0.17 g, 0.87 mmol, 4 eq) was added to the reaction mixture. The reaction mixture was shaken at room temperature for 2 days. After the reaction mixture was filtered under reduced pressure, the obtained filtered product was washed with pyridine and then dried. A 0.1 M pyridine solution of DMAP and acetic anhydride mixture solution (pyridine:acetic anhydride=9:1) (15 mL) was added to the filtered material, and the mixture was shaken at room temperature for 15 hours. The reaction mixture was filtered under reduced pressure, and the obtained filtered product was washed with methanol and acetone successively and then dried to obtain the title unit compound (CPG compound activity=26.7 μmol/g) (yield 1 0.53 g).

(Example 1-02)
(2) Production of unit compound represented by (X-12)

(I) 5′-O-tert-butyldimethylsilyl-3′-amino-(3″-O-tert-butyldimethylsilylthymidylyl)carbamoylthymidine (5′-O-tert-butyldimethylsilyl-3′- Preparation of amino-(3''-O-tert-butyldimethylsilylthymidine)carbamoylthymidine) 3'-O-tert-butyldimethylsilylthymidine (III-12) (2.27 g, 6.37 mmol) in pyridine (35 mL) solution, DMAP (minor amount) and Im ₂ CO (0.76 g, 4.70 mmol, 1 eq) were added and the reaction mixture was stirred for 1 day. A solution of 3'-amino-5'-O-tert-butyldimethylsilylthymidine (IV-12) (1.70 g, 4.79 mmol, 1 eq) in pyridine (12 mL) was added dropwise, and the reaction mixture was then added. Was stirred for 1 day. The reaction mixture was diluted with ethyl acetate. The ethyl acetate solution was washed successively with water (once), saturated aqueous NaHCO ₃ solution (once) and saturated aqueous NaCl solution (once), then dried over sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =0:1 to 1:10) to obtain the title compound as white crystals (yield 2.46 g, 3.33 mmol, yield 70%).

¹ H NMR (400 MHz, CDCl ₃ ) δ: 0.08-0.13 (12H, m, TBDMS-CH ₃ ), 0.89 and 0.93 (18H, s, t-butyl), 1.90 ( _{3H, s, 5-CH 3} 5'T), 1.93 (3H, s, 5-CH 3 3'T), 2.15-2.35 (4H, m, 2'-H), 3. 85-3.94 (2H, m, 5'-H 5'T), 4.01-4.03 (2H, m, 5'-H 3'T), 4.24-4.34 (2H, m, 4'-H), 6.04-6.08 (1H, m, 3'-H 5'T), 6.30-6.37 (1H, m, 3'-H 3'T), 7.17 (1H, s, 6-H 5'T), 7.56 (1H, s, 6-H 3'T), 8.83 (1H, s, NH 5'T), 8.95 ( 1H,s,NH3'T).
¹³ C-NMR (100 MHz, CDCl ₃ ) δ: −5.98, −4.91, 12.42, 18.30, 25.88, 40.46, 52.51, 55.21, 61.70, 63.97, 66.45, 71.52, 83.67, 84.82, 86.96, 87.56, 110.83, 110.91, 111.42, 130.02, 130.69, 135. 05, 137.03, 150.23, 155.72, 163.80.
_{FAB-HRMS (NBA) calcd for} C 33 H 55 N 5 O 10 Si 2 (MH +), 738.3566; found, 738.3579.

(Ii) Production of 3'-amino-thymidylylcarbamoylthymidine (3'-amino-thymidylylcarbamoylthymidine) 5'-O-tert-butyldimethylsilyl-3'-amino-(3''-O-tert-butyldimethylsilyl) To a solution of thymidylyl)carbamoylthymidine (0.90 g, 1.44 mmol) in THF (14 mL) was added TBAF (2 mL, 2 mmol, 2 eq) and the reaction mixture was stirred for 1 day. The solvent was distilled off from the reaction mixture under reduced pressure, and the obtained residue was purified by silica gel column chromatography (MeOH:CHCl ₃ =1:20 to 1:5) to obtain the title compound as white crystals (yield 0 .72 g, 1.43 mmol, yield 99%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 1.77 (6H,s,5-CH ₃ ), 2.00-2.24 (4H,m,2′-H),3.44-3 .64 (2H, m, 5'-H 5'T), 3.76-3.91 (2H, m, 5'-H 3'T), 4.01-4. 21 (2H, m, 4). '-H), 5.00-5.22 (1H, m, 3'-H 5'T), 5.37-5.43 (1H, m, 3'-H 3'T), 6.13 -6.21 (2H, m, 1'-H), 7.44-79 (2H, m, 6-H), 11.28 (1H, s, NH5'T), 11.31 (1H, s, NH 3'T).
¹³ C-NMR (100 MHz, DMSO-d ₆ )δ: 12.23, 12.29, 37.17, 50.80, 61.24, 64.53, 70.74, 83.46, 83.86, 84.11, 84.83, 109.40, 109.72, 135.00, 136.12, 150.41, 150.50, 155.58, 163.71, 163.76.
_{FAB-HRMS (NBA) calcd for} C 21 H 27 N 5 O 10 (MH +), 510.18406; found, 510.18357.

(Iii) Preparation of 5'-O-(4,4'-dimethoxytrityl)-3'-aminothymidylylcarbamoylthymidine (5'-O-(4,4'-dimethyoxytrityl)-3'-aminothymidylcarbamoylthymidine) 3' To a solution of -amino-thymidylylcarbamoylthymidine (0.21 g, 0.41 mmol) in pyridine (1.4 mL) was added DMAP (minor amount) and DMTrCl (0.21 g, 0.62 mmol, 1.5 eq), The reaction mixture was stirred for 1 day. The reaction mixture was diluted with ethyl acetate. The ethyl acetate solution was washed successively with water (once), saturated aqueous NaHCO ₃ solution (once) and saturated aqueous NaCl solution (once), then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the obtained ethyl acetate solution, and the obtained residue was purified by silica gel column chromatography (n-hexane:AcOEt:MeOH=9:1:0 to 0:1:20) to give white. The title compound was obtained as crystals (yield 0.19 g, 0.22 mmol, 54%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 1.46 (3H,s,5-CH ₃ 5′T), 1.68 (3H,s,5-CH ₃ 3′T), 1.90. -2.36 (4H, m, 2'- H), 3.05-3.32 (4H, m, 5'-H), 3.71 (6H, s, OCH 3), 3.89-3 .92 (2H, m, 4'-H), 4.02-4.31 (4H, m, 3'-H), 5.38 (1H, d, J = 4.4Hz, OH), 6. 15 (1H, t, J=8.0 Hz, 1'-H 5'T), 6.21 (1H, t, J=8.0 Hz, 1'-H 3'T), 6.84-7. 42 (13H, m, DMTr), 7.55 (1H, s, 6-H 5'T), 7.75-7.55 (1H, m, 6-H 3'T), 7.76 (1H , D, J=7.6 Hz, NH), 11.30 (1H, s, NH 5′T), 11.33 (1H, s, NH 3′T).
¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 11.78, 12.14, 37.01, 38.60, 50.65, 54.98, 55.00, 63.11, 64.63, 70.68, 82.68, 83.56, 83.77, 84.06, 85.76, 109.50, 109.67, 113.17, 126.73, 127.65, 127.84, 129. 67, 129.71, 135.24, 135.31, 135.78, 135.91, 144.68, 150.28, 150.47, 155.49, 158.09, 158.12, 163.63, 163.68.
_{FAB-HRMS (NBA) calcd for} C 42 H 45 N 5 O 12 (MH +), 812.3143; found, 812.3137.

(Iv) Production of Unit Compound (X-12) 5′-O-(4,4′-dimethoxytrityl)-3′-aminothymidylylcarbamoylthymidine (0.50 g, 0.61 mmol) in pyridine (6.1 mL) ) To the solution was added DMAP (minor amount) and succinic anhydride (0.18 g, 1.83 mmol, 3.0 eq) and the reaction mixture was stirred for 2 days. The reaction mixture was diluted with chloroform. The chloroform solution was washed with water (3 times) and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the chloroform solution. Part of the obtained residue (0.55 g, 0.61 mmol, 4.0 eq) was dried under vacuum overnight. CPG (1.65 g, 0.15 mmol, 90.4 μmol/g) was added to a DMF (15 mL) solution of the residue to swell the CPG. After 30 minutes, more WSC (0.12 g, 0.60 mmol, 1.0 eq) was added to the reaction mixture and shaken for 3 days. After the reaction mixture was filtered under reduced pressure, the obtained filtered product was washed with pyridine and then dried. A solution (20 mL) of a mixture of DMAP 0.1 M pyridine and acetic anhydride (pyridine:acetic anhydride=9:1) was added to the filtered material, and the mixture was shaken at room temperature for 1 day. The reaction mixture was filtered under reduced pressure, and the obtained filtered product was washed with methanol and acetone successively and dried to obtain the title unit compound (CPG compound activity=44.4 μmol/g) (yield 0.93 g).

(Example 1-03)
(3) Production of unit compound represented by (X-13)

(I) 5′-O-tert-butyldimethylsilyl-3′-amino-(5″-aminothymidylyl)ureathymidine (5′-O-tert-butyldimethylsilyl-3′-amino-(5″-aminothymidylyl) Preparation of 3'-amino-5'-O-tert-butyldimethylsilylthymidine (IV-12) (0.26 g, 0.74 mmol) in pyridine (5 mL) was added to DMAP (0.018 g, 0. 15 mmol, 0.2 eq) and Im ₂ CO (0.12 g, 0.74 mmol, 1 eq) were added and the reaction mixture was stirred for 3.5 h. A solution of 5′-aminothymidine (III-11) (0.22 g, 0.89 mmol, 1.2 eq) in pyridine (2 mL) was added dropwise to the reaction mixture, which was then stirred for 12 hours. did. The reaction mixture was extracted with ethyl acetate, and the solvent was distilled off under reduced pressure from the ethyl acetate solution. The obtained residue was purified by silica gel column chromatography (CHCl ₃ :CH ₃ OH=100:3 to 10:1) to give the title compound as white crystals (yield 0.26 g, 0.42 mmol, yield). 57%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 0.06 (6H,s, TBDMS-CH ₃ ), 0.87 (9H,s,t-butyl), 1.77 (3H,s,5-). _{CH 3 5'T), 1.79 (3H} , s, 5-CH 3 3'T), 2.05-2.17 (4H, m, 2'-H), 3.13-3.16 ( 4H, m, 5'-H), 3.70-3.84 (2H, m, 4'-H), 4.10-4.12 (1H, m, 3'-H 5'T), 5 .26-5.27 (1H, m, -OH), 5.98 (1H, t, 1'-H 5'T), 6.12-6.13 (1H, m, 3'-H 3'. T), 6.45-6.47 (1H, m, 1'-H 3'T), 7.48-7.50 (2H, s, 6-H), 11.29-11.32 (2H , S, NH).
¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: −5.47, −5.43, 12.10, 12.26, 18.08, 25.81, 37.67, 38.45, 41. 78, 49.99, 63.46, 71.13, 79.19, 83.58, 83.76, 85.24, 85.51, 109.41, 109.74, 135.49, 136.10, 150.34, 150.47, 157.53, 163.69, 163.74.
_{FAB-HRMS (NBA) calcd for} C 27 H 43 N 6 O 9 Si (MH +), 623.28707; found, 623.28606.

(Ii) Preparation of 3′-amino-(5″-aminothymidylyl)ureathymidine (3′-amino-(5″-aminothymidylyl)ureathymidine) 5′-O-tert-butyldimethylsilyl-3′-amino- TBAF (2 mL, 2 mmol, 1.4 eq) was added to a solution of (5″-aminothymidylyl)ureathymidine (0.90 g, 1.44 mmol) in THF (14 mL), and the reaction mixture was stirred for 10 hours. The solvent was distilled off from the reaction mixture under reduced pressure, and the obtained residue was purified by silica gel column chromatography (CHCl ₃ :CH ₃ OH=20:1 to 5:1) to obtain the title compound as white crystals ( Yield 0.72 g, 1.43 mmol, yield 99%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 1.76 (3H,s,5-CH ₃ 5′T), 1.78 (3H,s,5-CH ₃ 3′T), 1.94. -2.20 (4H, m, 2'-H), 3.42-3.69 (4H, m, 5'-H), 4.10-4.24 (2H, m, 4'-H) , 5.04-5.07 (1H, m, OH 5'T), 5.27-5.28 (1H, m, OH 3'T), 6.02 (1H, m, 3'-H 5 'T), 6.09-6.15 (2H, m, 1'-H 5'T and 3'-H 3'T), 6.43-6.45 (1H, m, 1'-H 3 'T), 7.48 (1H, s, 6H 5'T), 7.73 (1H, s, 6H 3'T), 11.28 (2H, s, NH).
¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 12.07, 12.25, 37.66, 38.35, 49.78, 61.23, 71.08, 83.33, 83.69, 85.43, 109.27, 109.74, 136.05, 150.39, 150.45, 157.63, 163.71, 163.73.
_{FAB-HRMS (NBA) calcd for} C 21 H 28 N 6 O 9 (MH +), 509.1996; found, 509.1996.

(Iii) 5'-O-(4,4'-dimethoxytrityl)-3'-amino-(5''-aminothymidylyl)urea thymidine (5'-O-(4,4'-dimethyoxytrityl)-3'- Production of amino-(5″-aminothymidylyl)ureathymidine) 3′-amino-(5″-aminothymidylyl)ureathymidine (0.80 g, 1.57 mmol) in pyridine (15.7 mL) solution, DMAP (small amount). And DMTrCl (0.64 g, 1.90 mmol, 1.2 eq) were added and the reaction mixture was stirred for 1 day. The reaction mixture was diluted with ethyl acetate. The ethyl acetate solution was washed successively with water (once), saturated aqueous NaHCO ₃ solution (once) and saturated aqueous NaCl solution (once), then dried over sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane:AcOEt:MeOH=9:1:0 to 0:1:20) to give white crystals. The title compound was obtained (yield 0.30 g, 0.37 mmol, yield 24%).

¹ H NMR (400 MHz, DMSO-d ₆ )δ: 1.43 (3H,s,5-CH ₃ 5′T), 1.73 (3H,s,5-CH ₃ 3′T), 1.98. -2.32 (4H, m, 2'- H), 3.14-3.28 (4H, m, 5'-H), 3.72 (6H, s, OCH 3), 3.86-4 .04 (2H, m, 4'-H), 4.07-4.36 (1H, m, 3'-H 5'T), 5.27 (1H, m, OH), 6.03 (1H). , T, J=6 Hz, 1'-H 5'T), 6.03-6.16 (1H, m, 3'-H 3'T), 6.38-6.40 (1H, m, 1). '-H 3'T), 6.80-7.30 (13H, m, DMTr), 7.49 (1H, s, 6-H 5'T), 7.53 (1H, s, 6-H) 3'T), 11.29 (1H, s, NH 5'T), 11.34 (1H, s, NH 3'T).
¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 11.77, 12.08, 14.10, 20.78, 39.92, 40.13, 41.83, 49.75, 55.03. 59.77, 63.31, 71.13, 83.30, 83.56, 83.75, 85.51, 85.78, 109.46, 109.76, 113.22, 123.93, 126. 76, 127.72, 127.89, 129.77, 135.29, 135.45, 135.71, 136.08, 144.77, 149.63, 150.34, 150.48, 157.44, 158.13, 163.72.
_{FAB-MS (NBA) calcd for} C 42 H 46 N 6 O 11 (MH -), 809.31468; found, 809.31376.

(Iv) Production of Unit Compound (X-13) 5′-O-(4,4′-dimethoxytrityl)-3′-amino-(5″-aminothymidylyl)ureathymidine (0.72 g, 0.88 mmol) To pyridine (8.8 mL) solution of was added DMAP (small amount) and succinic anhydride (0.29 g, 2.87 mmol, 3.0 eq), and the reaction mixture was stirred for 2 days. The reaction mixture was diluted with chloroform. The chloroform solution was washed with water (3 times) and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the chloroform solution. A part of the obtained residue (0.55 g, 0.61 mmol, 4.0 eq) was dried under vacuum overnight. CPG (1.10 g, 0.15 mmol, 136 μmol/g) was added to DMF (15 mL) of the residue to swell. After 30 minutes, WSC (0.12 g, 0.60 mmol, 1.0 equivalent) was further added to the reaction mixture, and the mixture was shaken for 3 days. After the reaction mixture was filtered under reduced pressure, the obtained filter cake was washed with pyridine and then dried. A 0.1 M pyridine solution of DMAP and an acetic anhydride mixture (pyridine:acetic anhydride=9:1) (20 mL) was added to the filtered material, and the mixture was shaken at room temperature for 1 day. The reaction mixture was filtered under reduced pressure, and the obtained filtered product was washed with methanol and acetone successively and dried to give the title unit compound (CPG compound activity=38.4 μmol/g) (yield 1.09 g).

The 3',5'-phosphodiester bond between the 1st and 2nd nucleosides from the 3'end of the single-stranded oligonucleotide is -OC(=O)-NH-, -NH-C(= O)-O- or -NH-C(=O)-NH- is substituted to produce a modified single-stranded oligonucleotide. The target protein is Homo sapiens ribonuclease L (Homo sapiens ribonuclease L) (2', 5'-. oligoisoadenylate synthetase-dependent, RNase L) [gi: 30795246], and the target sequence was the base sequence consisting of SEQ ID NO: 1 from the 92nd position to the 114th position from the start codon. As a modified single-stranded oligonucleotide, a modified RNA sense strand (SEQ ID NOS: 2, 3 and 4) of the designed siRNA sequence and a modified RNA antisense strand (SEQ ID NO: 5, 6 and 7) were produced. For comparison, an RNA sense strand (SEQ ID NO: 8) and an RNA antisense strand (SEQ ID NO: 9) of the designed siRNA sequence were produced for the base sequence consisting of SEQ ID NO: 1.

(Reference example 1-1s)
For the RNA sense strand consisting of SEQ ID NO: 8, a commercially available dT resin (bound to CPG resin) (manufactured by Glen Research) was used in order to synthesize the TT dimer sequence at the 3′ end. Otherwise, according to SEQ ID NO: 8, the oligonucleotide of SEQ ID NO: 8 bound to CPG resin was produced by the phosphoramidite method using an automatic nucleic acid synthesizer. 1 μmol of the dT resin was used and each condensation time was 15 minutes.

  Synthesis of the oligonucleotide of SEQ ID NO: 8 bound to the CPG resin was completed by the automatic nucleic acid synthesizer in the state where the DMTr group was removed.

The oligonucleotides bound to the CPG resin were reacted in ethanol and ammonia mixed (EtOH:NH ₃ =1:3) solution (2 mL) at 55° C. for 12 hours, respectively. The reaction mixture was filtered under reduced pressure, the obtained filtrate was transferred to an Eppendorf tube, and then concentrated under reduced pressure. To each of the obtained concentrates, 1M TBAF in THF (1 mL) was added, and the mixture was shaken for 12 hours. The reaction solution was diluted to 30 mL with 0.1 M TEAA buffer. The diluted solution was passed through a C-18 reverse phase column (Sep-Pak) (CH ₃ CN (10 mL) and 0.1 M TEAA buffer (10 mL) were sequentially equilibrated in advance) to pass through the column. Adsorbed. The salt was removed by washing the column with sterile water and then eluted respectively with a mixture of acetonitrile and water (50% _CH 3 CN water (3 mL)). The obtained eluates were concentrated under reduced pressure. The resulting concentrates were each dissolved in a loading solution (1×TBE solution in 90% formamide) (100 μL). The solution was separated using 20% PAGE (20 A, 6 hours) (1×TBE buffer was used as the migration buffer), and the band of the desired oligonucleotide was cut out. A 0.1 M EDTA aqueous solution (20 mL) was added to each of the cut bands, and the band was left overnight. The liquid portion of the EDTA aqueous solution, C-18 reverse phase column chromatography (Sep-Pak): to give respectively by (eluant _{water, 50% CH 3 CN (3mL} )), to obtain an oligonucleotide of interest ..

  The method for preparing the following solution used in the Reference Example will be described.

  The 0.1 M TEAA buffer was prepared as follows. First, water was added to a mixture of 2N acetic acid (114.38 mL) and triethylamine (277.6 mL) to make 1 L. It was prepared by adding acetic acid to the solution to adjust the pH to 7.0 and then diluting the solution 20 times.

  20% PAGE was prepared as follows. First, a 40% acrylamide (acrylamide:N,N′-methylenebisacrylamide=19:1) solution (45 mL), urea (37.8 g) and 10×TBE buffer (9 mL) were mixed and dissolved, and then water was added. To 90 mL. APS (62 mg) was added to and dissolved in the solution, and TEMED (45 μL) was added and shaken. The solution was poured between two glass plates fixed with a 1.5 mm spacer in between and allowed to stand for 1 hour or more to solidify to obtain 20% PAGE.

  A 0.1 M EDTA aqueous solution was prepared by dissolving EDTA.4Na (1.80 g) in water (40 mL).

  A 40% acrylamide (acrylamide:N,N′-methylenebisacrylamide=19:1) solution was prepared by dissolving acrylamide (190 g) and N,N′-bisacrylamide (10 g) in water to make 500 mL. did.

  The 10×TBE buffer was prepared by dissolving Tris (109 g), boric acid (55 g) and EDTA.2Na (7.43 g) in water to make 1 L.

  The obtained oligonucleotide was dissolved in water (1 mL) and diluted 100 times with water to prepare a diluted solution. The absorbance (260 nm) of the diluted solution was measured, and the yield was calculated. The ε value of the absorbance, the absorbance at 260 nm and the yield are shown in Table 1. The molecular weight of the obtained oligonucleotide was confirmed by MALDI-TOF/MS. The results are also shown in Table 1.

(Reference example 1-1a)
An RNA antisense strand consisting of SEQ ID NO: 9 was produced in the same manner as in Reference Example 1-1s except that SEQ ID NO: 9 was used instead of SEQ ID NO: 8.

(Reference example 1-2)
Single-stranded oligonucleotides consisting of SEQ ID NO: 9 were produced in the same manner as in Reference Example 1-1a, except that the ³² P label was attached to the 5′ end of the modified single-stranded oligonucleotide consisting of SEQ ID NO: 9.

(Reference Example 1-3a)
Similarly, the modified single-stranded oligonucleotide consisting of SEQ ID NO: 9 modified at the 5'end with fluorescein has, as the 5'end, fluorescein phosphoramidite (manufactured by Glen Research). In the same manner as in Reference Example 1-1s, except that was added, a single-stranded oligonucleotide of SEQ ID NO: 9 modified at the 5′ end with fluorescein was produced.

(Reference example 1-4)
An RNA single strand consisting of SEQ ID NO: 12 was produced in the same manner as in Reference Example 1-1s except that SEQ ID NO: 12 was used instead of SEQ ID NO: 8.

(Example 1-1s)
Further, the modified single-stranded oligonucleotide consisting of SEQ ID NO: 2 was the same as Reference Example 1-1s, except that the unit compound represented by (X-11) was used instead of the dT resin, A modified single-stranded oligonucleotide consisting of SEQ ID NO:2 was produced.

(Example 1-1a)
The modified single-stranded oligonucleotide consisting of SEQ ID NO: 5 was the same as Reference Example 1-1s, except that the unit compound represented by (X-11) was used instead of the dT resin. A modified single-stranded oligonucleotide consisting of SEQ ID NO:5 was produced.

(Example 1-2s)
The modified single-stranded oligonucleotide consisting of SEQ ID NO: 3 was the same as Reference Example 1-1s except that the unit compound represented by (X-12) was used instead of the dT resin. A modified single stranded oligonucleotide consisting of SEQ ID NO:3 was produced.

(Example 1-2a)
The modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 was the same as Reference Example 1-1s, except that the unit compound represented by (X-12) was used instead of the dT resin. A modified single-stranded oligonucleotide consisting of SEQ ID NO:6 was produced.

(Examples 1-3s)
The modified single-stranded oligonucleotide consisting of SEQ ID NO: 4 was prepared in the same manner as in Reference Example 1-1s, except that the unit compound represented by (X-13) was used instead of the dT resin. A modified single-stranded oligonucleotide consisting of SEQ ID NO:4 was produced.

(Example 1-3a)
The modified single-stranded oligonucleotide consisting of SEQ ID NO: 7 was the same as Reference Example 1-1s except that the unit compound represented by (X-13) was used instead of the dT resin. A modified single-stranded oligonucleotide consisting of SEQ ID NO:7 was produced.

(Example 1-4a)
Similarly, the modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 modified at the 5'end with fluorescein has 5'end as fluorescein phosphoramidite (manufactured by Glen Research). A modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 modified at the 5′ end with fluorescein was produced in the same manner as in Example 1-2a except that was added.

(Example 1-5a)
'Except subjected to ^{32 P} labeled at the end, in the same manner as Example 1-2a, 5 consisting of SEQ ID NO: 6 5' modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 ^{32 P} labeled modified one at the end A chain oligonucleotide was prepared.

(Example 1-6)
The modified single-stranded oligonucleotide consisting of SEQ ID NO: 10 was the same as Reference Example 1-1s except that the unit compound represented by (X-12) was used instead of the dT resin. A modified single-stranded oligonucleotide consisting of SEQ ID NO: 10 was produced.

(Example 1-7)
The modified single-stranded oligonucleotide consisting of SEQ ID NO: 11 was the same as Reference Example 1-1s, except that the unit compound represented by (X-13) was used instead of the dT resin. A modified single stranded oligonucleotide consisting of SEQ ID NO: 11 was prepared.

  The oligonucleotides of SEQ ID NOS: 8 and 9 obtained in Reference Examples 1-1s and 1-1a are hereinafter referred to as (dT-s) and (dT-a), respectively. The oligonucleotide consisting of SEQ ID NO: 9 obtained in Reference Example 1-2 is hereinafter referred to as (PdT-a). The oligonucleotide consisting of SEQ ID NO: 9 modified at the 5'end with fluorescein obtained in Reference Example 1-3a is hereinafter referred to as (FdT-a). The oligonucleotide of SEQ ID NO: 12 obtained in Reference Example 1-4 is hereinafter referred to as (d-sa).

  The modified single-stranded oligonucleotides consisting of SEQ ID NOs: 2 and 5 obtained in Examples 1-1s and 1-1a are hereinafter referred to as (11-s) and (11-a), respectively. The modified single-stranded oligonucleotides consisting of SEQ ID NOS: 3 and 6 obtained in Examples 1-2s and 1-2a are hereinafter referred to as (12-s) and (12-a), respectively. The modified single-stranded oligonucleotides consisting of SEQ ID NOs: 4 and 7 obtained in Examples 1-3s and 1-3a are hereinafter referred to as (13-s) and (13-a), respectively. The modified single-stranded oligonucleotide having the 5'end modified with fluorescein and consisting of SEQ ID NO: 6 obtained in Example 1-4a is hereinafter referred to as (F12-a). The modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 obtained in Example 1-5a is hereinafter referred to as (P12-a). The modified single-stranded oligonucleotides consisting of SEQ ID NOs: 10 and 11 obtained in Examples 1-6 and 1-7 are hereinafter referred to as (16-sa) and (17-sa), respectively.

  The absorbance and yield of the oligonucleotide were measured as follows.

The oligonucleotide was dissolved in water to prepare an aqueous solution, and the aqueous solution was diluted so that the absorbance (Abs ₂₆₀ ) at a wavelength of 260 nm was within the effective range of the absorptiometer. Abs ₂₆₀ was measured at room temperature using a quartz cell for absorbance measurement with an optical path length (l) of 1 cm. The OD ₂₆₀ value was calculated using the following formula (1).
OD ₂₆₀ (ε·M·mL ⁻¹ )=Abs ₂₆₀ (ε·cm·M)×V ⁻¹ (mL)×1 ⁻¹ (cm) (1)
In the formula (1), Abs ₂₆₀ represents the absorbance of the oligonucleotide solution at a wavelength of 260 nm, V represents the total amount of the solution, l represents the optical path length, and M represents the molar concentration.

In the formula (1), the molar extinction coefficient ε260 of the oligonucleotide was calculated using the following formula (2).
ε=2{ε(N _1p N ₂ )+ε(N _2p N ₃ )+...+ε(N _n−1p N _n )}−{ε(N ₂ )+ε(N ₃ )+...+ε( N _n-1 )} (2)
In the formula _{(2), ε (N n} ) represents the epsilon ₂₆₀ nucleic acid _{N n located, ε (N n-1p N} n) represents a certain nucleic acid dimeric _{N n-1p N n} of epsilon _260. In addition, (11-s), (11-a), (12-s), (12-a), (13-s), (13-a), (F12-s), (F12-a), For (P-12-a) and (P13-a), ε ₂₆₀ was calculated using the TT sequence of the modified 3′ end portion as the UU sequence.

The concentration C (mol/L) was calculated using the following formula (3).
C=Abs ₂₆₀ x ε ₂₆₀ ^-1 x 1 ^-1 (3)

In the formula (3), Abs ₂₆₀ , ε ₂₆₀ , and 1 are as described above.
Production of modified double-stranded oligonucleotides

(Example 2-1)
(11-s) (35 nmol) prepared in Example 1-1s and (11-a) (35 nmol) prepared in Example 1-1a were treated with annealing buffer (10 mM Tris-HCl (pH 7.5) and It was dissolved in 100 mM NaCl). The solution was incubated at 90° C. for 1 minute and then 37° C. for 1 hour to obtain a modified double-stranded oligonucleotide consisting of SEQ ID NO:2 and SEQ ID NO:5 as shown in FIG.

(Example 2-2)
As in Example 2-1, except that (12-s) and (12-a) were used instead of (11-s) and (11-a), the sequence shown in FIG. A modified double-stranded oligonucleotide consisting of SEQ ID NO:3 and SEQ ID NO:6 was obtained.

(Example 2-3)
As in Example 2-1, except that (13-s) and (13-a) were used instead of (11-s) and (11-a), the sequence shown in FIG. A modified double-stranded oligonucleotide consisting of No. 4 and SEQ ID No. 7 was obtained.

(Example 2-4)
An array as shown in FIG. 1 was prepared in the same manner as in Example 2-1, except that (12-s) and (F12-a) were used instead of (11-s) and (11-a). A modified double-stranded oligonucleotide consisting of SEQ ID NO:3 and SEQ ID NO:6 further modified at the 5'end with fluorescein was obtained.

(Example 2-5)
(12-s) (80 pmmol) produced in Example 1-2s and (P12-a) (80 pmmol) produced in Example 1-5a were treated with annealing buffer (10 mM Tris-HCl (pH 7.5) and It was dissolved in 100 mM NaCl). The solution was heated at 95° C. for 3 minutes and then incubated at room temperature for 1 hour to obtain a modified dinucleotide consisting of SEQ ID NO:3 and SEQ ID NO:6 with a ³² P label at the 5′ end, as shown in FIG. A single-stranded oligonucleotide was obtained.

(Example 2-6)
As in Example 2-1, except that (16-sa) and (16-sa) were used instead of (11-s) and (11-a), the sequence as shown in FIG. A modified double-stranded oligonucleotide consisting of No. 10 and SEQ ID No. 10 was obtained.

(Example 2-7)
As in Example 2-1, except that (17-sa) and (17-sa) were used instead of (11-s) and (11-a), the sequence shown in FIG. A modified double-stranded oligonucleotide consisting of SEQ ID NO: 11 and SEQ ID NO: 11 was obtained.

(Reference example 2-1)
Sequences as shown in FIG. 1 were obtained in the same manner as in Example 2-1, except that (dT-s) and (dT-a) were used instead of (11-s) and (11-a). A double-stranded oligonucleotide consisting of No. 8 and SEQ ID No. 9 was obtained.

(Reference example 2-2)
Sequences as shown in FIG. 1 were obtained in the same manner as in Example 2-1, except that (dT-s) and (PdT-a) were used instead of (11-s) and (11-a). A double-stranded oligonucleotide consisting of No. 8 and SEQ ID No. 9 was obtained.

(Reference example 2-3)
As in Example 2-1, except that (d-sa) and (d-sa) were used instead of (11-s) and (11-a), the sequence as shown in FIG. A double-stranded oligonucleotide consisting of No. 12 and SEQ ID No. 12 was obtained.

(Evaluation of modified double-stranded oligonucleotide)
1. Evaluation of RNase L expression in cells (1) Cell culture Proliferate HT1080 cells at 37°C in RPMI1640 medium supplemented with 10% FBS, penicillin (100 units/ml) and streptomycin (0.1 mg/ml). Let Cells were passaged regularly to maintain exponential growth. Twenty-four hours prior to transfection at approximately 25% confluency, HT1080 cells were trypsinized, diluted with fresh media without antibiotics and transferred to 35 millidish (2 ml/dish).

(2) Transfection of modified double-stranded oligonucleotide into cells Using Lipofectamine 2000 reagent (manufactured by Invitrogen), the modified double-stranded oligonucleotide was transferred to adherent cell lines by the method described below. First, double-stranded oligonucleotide (50, 100 nM or 200 nM per 250 μl of the medium) was added to RPMI1640 medium (containing no antibiotic and FBS) (the double-stranded oligonucleotide obtained in Example 2-2). Were dissolved and incubated for 5 minutes at room temperature. An equal volume of 50 times diluted Lipofectamine 2000 reagent was added to the solution and then incubated for 20 minutes at room temperature. From the obtained solution, 500 μl was collected and transferred to the 35 millidish prepared in (1) above for transfection. The cells in the 35 millidishes were incubated 24 hours and 48 hours after transfection. In addition, thereafter, no toxicity to the transfected cells was confirmed with any of the double-stranded oligonucleotides obtained in Example 2-3 or Reference Example 2-1.

(3) Monitoring of RNase L expression by Western blotting HT1080 cells obtained after 24 hours incubation and 48 hours incubation from transfection were trypsinized and washed twice with cold PBS(-). Then, the obtained cell pellet was mixed with 2 volumes of hypotonic buffer A (0.5% (v/v) Nonidet P-40, 20 mM Hepes (pH 7.5), 10 mM CH ₃ ). It was suspended in CO ₂ K, 15 mM (CH ₃ CO ₂ ) ₂ Mg, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 10 μg/ml aprotinin).

  The obtained cell suspension was incubated on ice for 10 minutes, and then homogenized by stroke up and down 30 times on ice using a tight-fitting glass dounce homogenizer. The obtained homogenizing solution was centrifuged at 4° C. for 10 minutes using an ultracentrifuge (10000×g). Then, the centrifuged supernatant liquid is collected, and an equal amount of 2× sample buffer solution (0.14 M Tris-HCl pH 6.8, 0.2% (v/v) glycerol, 2.8% (v/v) SDS, an appropriate amount of bromophenol blue) was added and incubated in boiling water for 5 minutes. The incubated supernatant was separated by electrophoresis using 7.5% polyacrylamide gel (ATTO) at 20 mA for 70 minutes, and then the protein was transferred to a PVDF membrane (manufactured by Millipore).

  The PVDF membrane was incubated with 5% bovine serum albumin for 1 hour at 25°C. Then, the PVDF membrane was rinsed twice with TBST (200 mM Tris pH 7.6, 1.37 M sodium chloride, 0.1% Tween-20). The PVDF membrane was incubated with 0.5 μg/ml anti-RNase L monoclonal antibody at 4° C. for 16 hours. Then, the PVDF membrane was sequentially washed with TBST once for 15 minutes and then for 5 minutes three times. The PVDF membrane was then incubated with horseradish peroxidase-labeled anti-mouse IgG (500 μg/mL diluted 100,000 times) for 1 hour at 25°C. Then, the PVDF membrane was sequentially washed with TBST once for 15 minutes and then for 5 minutes three times.

  The detection of the horseradish peroxidase label on the PVDF membrane was performed using an ECL plus western blotting detection kit (manufactured by Amersham). The quantification of horseradish peroxidase labeling on the PVDF membrane was performed using a densitometer (Kodak Digital Science DC290 Zoom Digital Camera). The relative ratio of the RNase L intensities of the cells treated with the double-stranded oligonucleotide was determined by setting the RNase L intensity in the cells not treated with the double-stranded oligonucleotide to 1. The calculated values were standardized by setting the GAPDH intensity to 1. The obtained results are shown in FIG.

  (4) (1) except that the double-stranded oligonucleotides obtained in Example 2-3 and Reference Example 2-1 are used instead of the double-stranded oligonucleotide obtained in Example 2-2. In the same manner as in (3) to (3), the relative ratio of the RNase L intensity of the cells treated with the double-stranded oligonucleotide was determined. The results are shown in FIGS. 2(b) and 2(c), respectively.

  As shown in FIGS. 2(a) to 2(c), the modified double-stranded oligonucleotide of the present invention has a significantly improved effect of suppressing the expression of RNaseL as compared with the unmodified double-stranded oligonucleotide. I was able to confirm that It is considered that this is because the modified double-stranded oligonucleotide of the present invention has excellent cell membrane permeability and improved stability or retention in cells.

2. Evaluation of cytotoxicity (1) 1. The cells transfected with the modified double-stranded oligonucleotide obtained in Example 2-2 as described in (2) were counted under a phase contrast microscope using a hemocytometer. The viability of the transfected cells was determined by the trypan blue dye exclusion assay. At that time, the dye could not be eliminated due to the decrease in the cell membrane function, and as a result, the cells stained with trypan blue were regarded as dead cells. The survival rate was defined as the percentage (%) of the number of viable cells to the total number of cells. As a result, no cytotoxicity was observed at any concentration (50 nM, 100 nM, and 200 nM) of the modified double-stranded oligonucleotide.

  (2) Other than using the double-stranded oligonucleotides obtained in Examples 2-1, 2-3 and Reference Example 2-1, instead of the double-stranded oligonucleotide obtained in Example 2-2 2. Cytotoxicity was evaluated in the same manner as (1). As a result, no cytotoxicity was observed at any concentration of the double-stranded oligonucleotide (50 nM, 100 nM, and 200 nM).

3. Measurement of IC ₅₀ for RNase L in cells (1) 1. The concentration of the double-stranded oligonucleotide used in (2) is 1 nM, 5 nM or 10 nM instead of 50, 100 nM or 200 nM per 250 μl of the above medium, except that the concentration of the double-stranded oligonucleotide is 1. Similar to (1) to (4), the relative ratio of the RNase L intensity of the cells treated with the double-stranded oligonucleotide was determined. The result is shown in FIG.

  (2) 3 except that the double-stranded oligonucleotides obtained in Example 2-3 and Reference Example 2-1 are used instead of the double-stranded oligonucleotides obtained in Example 2-2. ． In the same manner as in (1), the relative ratio of the RNase L intensity of the cells treated with the double-stranded oligonucleotide was determined. The result is shown in FIG.

(3) IC ₅₀ was calculated from the graph of FIG. 3 and is shown in Table 2.

  As shown in Table 2 above, it was confirmed that the modified double-stranded oligonucleotide of the present invention markedly improved the effect of suppressing the expression of RNaseL as compared with the unmodified double-stranded oligonucleotide. This remarkable improvement is an excellent effect more than expected from the fact that the modified double-stranded oligonucleotide of the present invention has excellent cell membrane permeability and improved nuclease resistance.

4. Evaluation of effect of introducing modified double-stranded oligonucleotide into cells (1) Observation of intracellular RNase L by immunostaining Method 24 hours before transfection, poly-L-lysine (manufactured by Sigma) was used. The coated cover glass was placed in a 35 millidish. HT1080 cells were pre-fixed on the cover glass by adding a volume of 2 mL of the HT1080 cell solution onto the cover glass in the 35 millidish and then culturing.

  The double-stranded oligonucleotide obtained in Example 2-2 was diluted with RPMI1640 medium (GIBCO) containing no antibiotics and FBS, and then incubated at room temperature for 5 minutes (dilution concentration=two per 250 μl of medium). Strand oligonucleotide 10 nM or 50 nM). Separately, Lipofectamine 2000 reagent (Invitrogen) (5 μg) was diluted with RPMI1640 medium (GIBCO) (250 μl) containing no antibiotics and FBS, and then incubated at room temperature for 5 minutes. After mixing 250 μl of the diluted double-stranded oligonucleotide diluted solution and 250 μl of the incubated Lipofectamine 2000 reagent solution, the mixed solution was incubated at room temperature for 20 minutes. 500 μl of the mixed solution was added to a cover glass on which the HT1080 cells prepared above were fixed in advance for transfection.

  After incubating for 24 hours after transfection, the HT1080 cells on the coverslips were washed twice with PBS(-). The cells on the coverslips were then incubated with 4% paraformaldehyde for 15 minutes at 4°C. After washing 3 times with PBS(−), the cells on the cover glass were incubated with a 0.2% Triton-X 100 solution at room temperature for 5 minutes. After washing 3 times with PBS(−), the cells on the coverslips were incubated with 0.5% bovine serum albumin for 30 minutes at room temperature. The cells on the cover glass were washed 3 times with PBS(-) and then incubated with 1 µg/ml of anti-RNase L monoclonal antibody for 60 minutes at room temperature. The cells on the coverslips were washed 3 times with PBS(-). 10 μg/ml fluorescent (Alexa 488) labeled mouse IgG was added to the cells on the coverslips and incubated for 30 minutes at room temperature. Then, after washing with PBS(-) three times, the cells on the cover glass were immediately observed with a fluorescence microscope. The result is shown in FIG. As a control, the results obtained in the same manner as above are also shown, except that the double-stranded oligonucleotide obtained in Reference Example 2-1 was used in place of the double-stranded oligonucleotide obtained in Example 2-2. ..

  As shown in FIG. 5, the modified double-stranded oligonucleotide of the present invention showed a marked decrease in RNaseL fluorescence intensity, that is, a strong inhibitory effect on the expression of RNaseL, as compared with the unmodified double-stranded oligonucleotide. I was able to confirm that

(2) Method for calculating intracellular introduction effect of modified double-stranded oligonucleotide by fluorescence microscope The concentration of the double-stranded oligonucleotide (10 nM or 50 nM) obtained in Example 2-2 was 50 nM or 25 nM instead of 10 nM. And using the fluoroscein-labeled double-stranded oligonucleotide obtained in Example 2-4 instead of the double-stranded oligonucleotide obtained in Example 2-2. Double-stranded oligonucleotides were transfected into HT1080 cells on coverslips as described in (1).

  After incubating for 24 hours after transfection, the HT1080 cells on the cover glass were washed twice with PBS(-). The cells on the coverslips were then incubated with 4% paraformaldehyde for 15 minutes at 4°C. After washing three times with PBS(-), the cells on the cover glass were immediately observed with a fluorescence microscope. The result is shown in FIG. From the obtained results, the ratio of the number of fluorescein-labeled double-stranded oligonucleotide positive cells to the total number of cells was calculated. The results are shown in Table 3. Further, as a control, instead of the mixed solution, only 500 μl of the lipofectamine 2000 reagent solution after incubation was added to the cover glass pre-fixed with HT1080 cells previously prepared, and the results obtained with HT1080 cells transfected were obtained. Is also shown in FIG.

  As shown in FIG. 4 and Table 3 above, it was confirmed that the modified double-stranded oligonucleotide of the present invention significantly reduced the fluorescence intensity of RNAaseL, that is, suppressed the RNAaseL.

5. Modified single-stranded and double-stranded oligonucleotide consisting of SEQ ID NO: 6 obtained in Evaluation Example 1-5a exonuclease-resistant-modified single-stranded oligonucleotides (5 'that labeled end at ^{32 P),} Reference examples 1-2 (which was labeled at the 5 'end with ^{32 P)} unmodified single-stranded oligonucleotide consisting of SEQ ID NO: 9 obtained in the resulting modified double-stranded oligonucleotide in example 2-5 (5 the 'end what was labeled with ^{32 P)} and reference examples 2-2 obtained in the unmodified double-stranded oligonucleotide (5' that labeled end at ^{32 P),} to evaluate the exonuclease-resistant. Snake venom phosphorodiesterase (SVP) was used as the exonuclease. SVP selectively cleaves the phosphodiester bond and degrades the oligonucleotide into 5'-monophosphate nucleotides.

The reaction solution (5 μl) was added to the filling solution (7 M urea XC BPB) (5 μl) that had been dispensed into each Eppendorf tube after 1, 3, 5, 10, 30, 60 minutes from the reaction solution having the composition shown below. ) Was sampled to stop the reaction. The obtained sample at each time was separated by electrophoresis using a 20% polyacrylamide gel (containing 7 M urea) at 20 mA for 180 minutes, and the residual rate (%) of the complete chain of the modified oligonucleotide was expressed as log _10. 6(a) and 6(b) were prepared with the vertical axis representing the results and the horizontal axis representing the reaction time. Then, from the graphs of FIGS. 6(a) and 6(b), the time (t ₅₀ ) when the residual rate of the complete chain was 50% was calculated. The results are shown in Table 4.

Composition of reaction solution of modified single-stranded oligonucleotide (Example 1-5a) 20 μM Modified single-stranded oligonucleotide aqueous solution 4 μL
6 μL of buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
2u/mL SVP aqueous solution 4μL
Water 26 μL
40 μL in total

Composition of reaction solution of unmodified single-stranded oligonucleotide (Reference Example 1-2) 20 μM Unmodified single-stranded oligonucleotide aqueous solution 4 μL
6 μL of buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
2u/mL SVP aqueous solution 4μL
Water 26 μL
40 μL in total

Composition of reaction solution of modified double-stranded oligonucleotide (Example 2-5) 20 μM Modified double-stranded oligonucleotide aqueous solution 4 μL
6 μL of buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
2u/mL SVP aqueous solution 4μL
Water 26 μL
40 μL in total

Composition of reaction solution of unmodified double-stranded oligonucleotide (Reference Example 2-2) 20 μM Unmodified double-stranded oligonucleotide aqueous solution 4 μL
6 μL of buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
2u/mL SVP aqueous solution 4μL
Water 26 μL
40 μL in total

  From FIG. 6 and Table 4, it was confirmed that the modified single-stranded oligonucleotide has improved exonuclease resistance as compared with the unmodified single-stranded oligonucleotide. It was also confirmed that the modified double-stranded oligonucleotide has significantly improved exonuclease resistance as compared with the unmodified double-stranded oligonucleotide. In particular, the double-stranded modified oligonucleotide was confirmed to have a markedly improved nuclease resistance as compared with the result of the single-stranded modified oligonucleotide, and this result is beyond the expected range.

6. Evaluation of Thermodynamic Stability of Modified Double-stranded Oligonucleotide Modified double-stranded oligonucleotide consisting of SEQ ID NO: 10 obtained in Example 2-6 and modified consisting of SEQ ID NO: 11 obtained in Example 2-7 Thermodynamic stability was evaluated for the double-stranded oligonucleotide and the double-stranded oligonucleotide consisting of SEQ ID NO: 12 obtained in Reference Example 2-3.

For the above oligonucleotides, the van't Hoff transition enthalpy (ΔH°), entropy (ΔS°) and free energy (ΔG°) at 298K were calculated according to the method of Marky and Bleslaur. ΔG° indicates how much the thermodynamic energy is inclined from the equilibrium state of the reaction at a certain temperature when a 1-mol single strand becomes a 1-mol double strand in the standard state. The thermodynamic stability of the chains can be evaluated. The minus sign of this value indicates that the double-stranded state is more stable than the single-stranded state, and the larger the absolute value, the more stable the double-stranded state. ΔH° represents the change in the thermal energy balance when 1 mol of single strand becomes 1 mol of double strand in the standard state, and the − sign represents an exothermic reaction, and the larger this absolute value is, the larger the double strand is. Is stable in terms of thermal energy. ΔS° represents a change in disorder when a 1-mol single strand becomes a 1-mol double strand in the standard state, and the − sign indicates a more disordered state. Indicates that the smaller is the more advantageous for double-strand formation. The relationship between ΔG° and ΔH° and ΔS° is expressed by the following equation (4).
ΔG°=ΔH°-TΔS° (4)
That is, the thermodynamic stability of the duplex is determined by the difference between the thermal energy balance of the reaction and the change in randomness.

The Tm was measured by dividing the concentration of the oligonucleotides into several steps. The Tm value was measured with a Beckman Coulter DU 640 spectrophotometer. Measurement, using _{10mM NaH 2 PO 4 -Na 2 HPO} 4, 1M NaCl (pH7.0) as a buffer. Using the obtained Tm value, draw a graph of 1/Tm vs. In Ct/4 (Ct is the total concentration of oligonucleotide) based on the equation (5), and calculate ΔH° and ΔS° from the slope and intercept. did. Then, ΔG° was calculated from the equation (4). The results obtained are shown in Table 5.
1/Tm=(R/ΔH°)lnCt/4+(ΔS°/ΔH°) (5)

As shown in Table 5, the modified double-stranded oligonucleotide of the present invention was confirmed to have an increased absolute value of -ΔG ⁰ ₂₉₈ and an increased absolute value of _ΔH as compared with the unmodified oligonucleotide. It was confirmed that the stability as a double strand was improved. In addition, the modified double-stranded oligonucleotide of the present invention has an increased absolute value of ΔS as compared with the unmodified oligonucleotide, which confirms that it forms a more ordered double-stranded chain. ..

7. Evaluation of Endonuclease Resistance of Modified Double-stranded Oligonucleotide Modified single-stranded oligonucleotide consisting of SEQ ID NO: 5 obtained in Example 1-5a (5′ end labeled with ³² P), Reference Example 1-2 Unmodified single-stranded oligonucleotide consisting of SEQ ID NO: 9 (labeled at the 5′ end with ³² P) obtained in Example 2, modified double-stranded oligonucleotide obtained in Example 2-5 ( ^{32 at the} 5′ end) The endonuclease resistance of the unmodified double-stranded oligonucleotide (labeled with P) and the unmodified double-stranded oligonucleotide obtained in Reference Example 2-2 (5′ end labeled with ³² P) was evaluated. RNase A was used as the endonuclease.

  The reaction solution (10 μl) was sampled into the filling solution (8 M urea XC BPB) (10 μl) that had been dispensed into each Eppendorf tube after 30 minutes from the reaction solution having the composition shown below to stop the reaction. .. The obtained samples at each time were separated by electrophoresis using a 20% polyacrylamide gel (containing 7 M urea) at 20 mA for 180 minutes, and the residual ratio of the complete strand of the modified or unmodified single-stranded oligonucleotide ( %) was measured. The results are shown in Table 6.

Composition of reaction solution of modified single-stranded oligonucleotide (Example 1-5a) 20 μM Modified single-stranded oligonucleotide aqueous solution 2 μL
Buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
1.5 μL
10 μg/mL RNase aqueous solution 0.5 μL
Water 6 μL
10 μL in total

Composition of reaction solution of unmodified single-stranded oligonucleotide (Reference Example 1-2) 20 μM Unmodified single-stranded oligonucleotide aqueous solution 2 μL
Buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
1.5 μL
10 μg/mL RNase aqueous solution 0.5 μL
Water 6 μL
10 μL in total

  In addition, the reaction solution was added to the filling solution (8 M urea XC BPB) (10 μl) that had been dispensed into each Eppendorf tube after 5, 10, 20, 30, 60, and 90 minutes from the reaction solution having the composition shown below. (10 μl) was sampled to stop the reaction. The obtained samples at each time were separated by electrophoresis using a 20% polyacrylamide gel (containing 7M urea) at 20 mA for 180 minutes, and the residual ratio of the complete strand of the modified or unmodified double-stranded oligonucleotide ( %) on the vertical axis and the reaction time on the horizontal axis to prepare FIG.

Composition of reaction solution of modified double-stranded oligonucleotide (Example 2-5) 20 μM Modified double-stranded oligonucleotide aqueous solution 2 μL
Buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
1.5 μL
10 μg/mL RNase aqueous solution 0.5 μL
Water 6 μL
10 μL in total

Composition of reaction solution of unmodified double-stranded oligonucleotide (Reference Example 2-2) 20 μM Unmodified double-stranded oligonucleotide aqueous solution 2 μL
Buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 7.0))
1.5 μL
10 μg/mL RNase aqueous solution 0.5 μL
Water 6 μL
10 μL in total

  From Table 6, it was confirmed that the modified double-stranded oligonucleotide has significantly improved endonuclease resistance as compared with the unmodified double-stranded oligonucleotide. Moreover, from FIG. 8, it was confirmed that the modified double-stranded oligonucleotide had significantly improved endonuclease resistance as compared with the unmodified double-stranded oligonucleotide.

  The modified oligonucleotide of the present invention is useful, for example, as a therapeutic agent for diseases.

SEQ ID NO:1 Target sequence of RNaseL SEQ ID NO:2 Modified sense strand of siRNA sequence for target sequence of SEQ ID NO:1 SEQ ID NO:3 Modified sense strand of siRNA sequence for target sequence of SEQ ID NO:1 SEQ ID NO:4 siRNA for target sequence of SEQ ID NO:1 Sequence of modified sense strand SEQ ID NO:5 Modified antisense strand of siRNA sequence for the target sequence of SEQ ID NO:1 SEQ ID NO:6 Modified antisense strand of siRNA sequence for target sequence of SEQ ID NO:1 SEQ ID NO:7 siRNA for the target sequence of SEQ ID NO:1 Sequence modification Antisense strand SEQ ID NO: 8 Sense strand of siRNA sequence for the target sequence of SEQ ID NO: SEQ ID NO: 9 Antisense strand of siRNA sequence for the target sequence of SEQ ID NO: 10 SEQ ID NO: 10 of the siRNA sequence for the target sequence of SEQ ID NO: 12 Modified strand SEQ ID NO: 11 Another modified strand of siRNA sequence to the target sequence of SEQ ID NO: 12 SEQ ID NO: 12 target sequence

Claims (33)

A modified oligonucleotide in which the 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end of the oligonucleotide is replaced with a bond represented by the following formula (I-1).
^{-X 1 -C (= Y 1)} -Z 1 - (I-1)
In the formula (I-1), X ¹ is O, NH or S, Y ¹ is O or S, and Z ¹ is O, NH or S. The bond represented by the formula (I-1) is -OC(=O)-NH-, -NH-C(=O)-O-, -NH-C(=O)-NH-, -NH-C(=O)-S-, -S-C(=O)-NH-, -O-C(=S)-NH-, -NH-C(=S)-O-, -NH The modified oligonucleotide according to claim 1, which is selected from the group consisting of -C(=S)-NH-, -NH-C(=S)-S-, and -SC-(=S)-NH-. The bond represented by the formula (I-1) is from -OC(=O)-NH-, -NH-C(=O)-O- and -NH-C(=O)-NH-. The modified oligonucleotide according to claim 2, which is selected from the group consisting of: The modified oligonucleotide according to claim 1, which satisfies at least one of the following (i) and (ii).
(I) The 5',3'-phosphodiester bond between the 2nd and 3rd nucleosides from the 3'end of the oligonucleotide is replaced with a bond represented by the following formula (I-2).
^{-X 2 -C (= Y 2)} -Z 2 - (I-2)
In the formula (I-2), X ² is O, NH or S, Y ² is O or S, and Z ² is O, NH or S.
(Ii) The 5',3'-phosphodiester bond between the 3rd and 4th nucleosides from the 3'end of the oligonucleotide is replaced with a bond represented by the following formula (I-3).
^{-X 3 -C (= Y 3)} -Z 3 - (I-3)
In the formula (I-3), X ³ is O, NH or S, Y ³ is O or S, and Z ³ is O, NH or S. The bond represented by the formula (I-2) and the bond represented by the formula (I-3) are, independently of each other, -OC(=O)-NH-, -NH-C(=. O)-O-, -NH-C(=O)-NH-, -NH-C(=O)-S-, -S-C(=O)-NH-, -O-C(=S). -NH-, -NH-C(=S)-O-, -NH-C(=S)-NH-, -NH-C(=S)-S- and -S-C(=S)-NH. The modified oligonucleotide according to claim 4, which is selected from the group consisting of: The modified oligonucleotide according to claim 1, wherein the modified oligonucleotide is a double-stranded oligonucleotide. The 5',3'-phosphodiester bond between the first and second nucleosides from the 3'end of at least one single-stranded oligonucleotide of the double-stranded oligonucleotide is represented by the formula (I-1). 7. The modified oligonucleotide according to claim 6, which is replaced by a bond that is The modified oligonucleotide according to claim 6, wherein the modified oligonucleotide is an oligonucleotide that causes RNAi (RNA interference), and the modified oligonucleotide has the same base sequence as a part of mRNA encoding an endonuclease. The modified oligonucleotide is siRNA (short interfering RNA),
A part of the mRNA is
A 19-base sequence 75 bases or more upstream from the start codon, which follows the first AA sequence,
A portion of said mRNA is specific for said mRNA,
The modified oligonucleotide according to claim 8, wherein the modified oligonucleotide is a combination of a sense strand and an antisense strand having the 19-base sequence. The modified oligonucleotide according to claim 9, wherein at least one of the sense strand and the antisense strand has a nucleotide further containing two thymidines at the 3'end thereof. The modified oligonucleotide according to claim 1, wherein the modified oligonucleotide is a single-stranded oligonucleotide. The modified oligonucleotide according to claim 11, wherein the modified oligonucleotide is antisense DNA or antisense RNA, and the modified oligonucleotide has a sequence complementary to at least a part of mRNA encoding an endonuclease. The modified oligonucleotide according to claim 1, wherein the oligonucleotide is nuclease resistant. A gene expression inhibitor containing an oligonucleotide, wherein the oligonucleotide is the modified oligonucleotide according to claim 1. A pharmaceutical composition for treating a disease associated with gene expression, comprising:
A pharmaceutical composition, which comprises the gene expression inhibitor according to claim 14. The pharmaceutical composition according to claim 15, further comprising an excipient for introducing cells. The pharmaceutical composition according to claim 16, wherein the cell-introducing excipient is a transfection reagent. An RNAi kit, wherein the kit comprises the modified oligonucleotide of claim 8. A reagent for RNAi research, wherein the reagent comprises the modified oligonucleotide according to claim 8. A method for suppressing gene expression using an oligonucleotide, wherein the oligonucleotide is the modified oligonucleotide according to claim 1. A method of using an oligonucleotide to induce RNAi, wherein the oligonucleotide is the modified oligonucleotide of claim 8. A method for producing the modified oligonucleotide according to claim 1, wherein the unit compound for solid phase synthesis represented by the following formula (X-1) is used as a starting material.
R ^{2 of the} unit compound for solid phase synthesis represented by the formula (X-1) is removed,
A method for producing a modified oligonucleotide by extending a nucleotide at the 5′ end of a unit compound for solid phase synthesis represented by formula (X-1) and then cleaving the nucleotide from the solid phase carrier.

In the formula (X-1), R ² is a protecting group,
B ¹ and B ² are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
Here, R ¹¹ and R ²¹ are each independently a protecting group,
X ¹ and X ² , independently of each other, are O, NH or S,
Y ¹ is O or S,
Z ¹ is O, NH or S,
M is a solid phase carrier. A method for producing the modified oligonucleotide according to claim 4, wherein (i) is satisfied using a unit compound for solid phase synthesis represented by the following formula (XI-1) as a starting material,
R ^{3 of the} unit compound for solid phase synthesis represented by the formula (XI-1) is removed,
A method for producing a modified oligonucleotide by extending a nucleotide at the 5′ end of a unit compound for solid phase synthesis represented by formula (XI-1) and then cleaving the nucleotide from the solid phase carrier.

In the above formula, R ³ is a protecting group,
B ¹ , B ² and B ³ are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
Here, R ¹¹ , R ²¹ and R ³¹ are each independently a protecting group,
X ¹ , X ² and X ³ , independently of each other, are O, NH or S,
Y ¹ and Y ² , independently of each other, are O or S,
Z ¹ and Z ² , independently of each other, are O, NH or S,
M is a solid phase carrier. A method for producing the modified oligonucleotide according to claim 4, wherein the unit compound for solid phase synthesis represented by the following formula (XII-1) is used as a starting material to satisfy (ii),
R ^{4 of the} unit compound for solid phase synthesis represented by the formula (XII-1) is removed,
A process for producing a modified oligonucleotide by extending a nucleotide at the 5'end of a unit compound for solid phase synthesis represented by the formula (XII-1) and then cleaving it from a solid phase carrier.

In the above formula, R ⁴ and R are, independently of each other, a protecting group,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ and Y ³ , independently of each other, are O or S,
Z ¹ and Z ³ , independently of each other, are O, NH or S,
M is a solid phase carrier. A method for producing a modified oligonucleotide according to claim 4, which comprises using a unit compound for solid phase synthesis represented by the following formula (XIII-1) as a starting material and satisfying both (i) and (ii):
R ^{4 of the} unit compound for solid phase synthesis represented by the formula (XIII-1) is removed,
A process for producing a modified oligonucleotide by extending a nucleotide at the 5′ end of a unit compound for solid phase synthesis represented by formula (XIII-1) and then cleaving the nucleotide from the solid phase carrier.

In the above formula, R ⁴ is a protecting group,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ² , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ , Y ² and Y ³ , independently of each other, are O or S,
Z ¹ , Z ² and Z ³ , independently of one another, are O, NH or S,
M is a solid phase carrier. A unit compound for solid phase synthesis represented by the following formula (X-1), for producing the modified oligonucleotide according to claim 1.

In the formula (X-1), R ² is a protecting group,
B ¹ and B ² are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
Here, R ¹¹ and R ²¹ are each independently a protecting group,
X ¹ and X ² , independently of each other, are O, NH or S,
Y ¹ is O or S,
Z ¹ is O, NH or S,
M is a solid phase carrier. A method for producing a unit compound for solid phase synthesis represented by formula (X-1) according to claim 26,
A nucleoside derivative represented by the following formula (IV-1) and a nucleoside derivative represented by the following formula (III) are condensed with a diimidazole derivative represented by the following formula (V-1) to give the following formula ( VI) to obtain a dimer (VI)

The dimer represented by the above formula (VI) and the anhydride represented by the following formula (VII) are condensed to obtain a dimer represented by the following formula (VIII),

A unit compound for solid phase synthesis represented by formula (X-1) by condensing the dimer represented by formula (VIII) with a solid support having an amino group represented by formula (IX) below. Manufacturing method for obtaining.

In the above formula,
R ² is a protecting group,
B ¹ and B ² are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
Here, R ¹¹ and R ²¹ are each independently a protecting group,
X ¹ and X ² , independently of each other, are O, NH or S,
Y ¹ is O or S,
Z ¹ is O, NH or S,
M is a solid phase carrier. A unit compound for solid phase synthesis represented by the following formula (XI-1) for producing the modified oligonucleotide according to claim 4.

In the above formula, R ³ is a protecting group,
B ¹ , B ² and B ³ are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
Here, R ¹¹ , R ²¹ and R ³¹ are each independently a protecting group,
X ¹ , X ² and X ³ , independently of each other, are O, NH or S,
Y ¹ and Y ² , independently of each other, are O or S,
Z ¹ and Z ² , independently of each other, are O, NH or S,
M is a solid phase carrier. A method for producing a unit compound for solid phase synthesis represented by the formula (XI-1) according to claim 28,
R ² of the dimer represented by the formula (VI) is replaced with H to obtain a dimer represented by the formula (VI-1),

A dimer represented by the formula (VI-1) and a nucleoside derivative represented by the following formula (IV-2) are condensed with a diimidazole derivative represented by the following formula (V-2), A trimer represented by the formula (XIV-1) is obtained,

The trimer represented by the formula (XIV-1) and the anhydride represented by the following formula (VII) are condensed to obtain a trimer represented by the following formula (XV-1),

For solid phase synthesis represented by the formula (XI-1) by condensing the trimer represented by the formula (XV-1) and a solid phase carrier having an amino group represented by the following formula (IX). A method for producing a unit compound.

In the above formula, R ² and R ³ are each independently a protecting group,
B ¹ , B ² and B ³ are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
Here, R ¹¹ , R ²¹ and R ³¹ are each independently a protecting group,
X ¹ , X ² and X ³ , independently of each other, are O, NH or S,
Y ¹ and Y ² , independently of each other, are O or S,
Z ¹ and Z ² , independently of each other, are O, NH or S,
M is a solid phase carrier. A unit compound for solid phase synthesis represented by the following formula (XII-1) for producing the modified oligonucleotide according to claim 4.

In the above formula, R ⁴ and R are protecting groups,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ and Y ³ , independently of each other, are O or S,
Z ¹ and Z ³ , independently of each other, are O, NH or S,
M is a solid phase carrier. A method for producing a unit compound for solid phase synthesis represented by formula (XII-1) according to claim 30,
R ² of the dimer represented by the following formula (VI-2) is replaced with H to obtain a dimer represented by the following formula (VI-3),

The dimer represented by the above formula (VI-3) and the nucleoside derivative represented by the following formula (IV-3) are condensed with a phosphate binding reagent to form a dimer represented by the following formula (XIV-2). A trimer

The trimer represented by the formula (XIV-2) and the anhydride represented by the formula (VII) are condensed to obtain a trimer represented by the formula (XV-2).

Solid phase synthesis represented by the following formula (XII-1) by condensing the trimer represented by the formula (XV-2) and a solid support having an amino group represented by the following formula (IX). Manufacturing method for obtaining a unit compound for use.

In the above formula, R ² , R ³ and R independently of one another are protective groups,
B ¹ , B ² and B ³ are each independently a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
Here, R ¹¹ , R ²¹ and R ³¹ are each independently a protecting group,
X ¹ and X ³ , independently of each other, are O, NH or S,
Y ¹ is, independently of each other, O or S,
Z ¹ is, independently of each other, O, NH or S,
M is a solid phase carrier. A unit compound for solid phase synthesis represented by the following formula (XIII-1), for producing the modified oligonucleotide according to claim 4.

In the above formula, R ⁴ is a protecting group,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ² , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ , Y ² and Y ³ , independently of each other, are O or S,
Z ¹ , Z ² and Z ³ , independently of one another, are O, NH or S,
M is a solid phase carrier. A method for producing a unit compound for solid phase synthesis represented by formula (XIII-1) according to claim 32,
R ³ of the trimer represented by the following formula (XIV-1) is replaced with H to obtain a trimer represented by the formula (XIV-3),

A trimer represented by the above formula (XIV-3) and a nucleoside derivative represented by the following formula (IV-4) are condensed with a diimidazole derivative represented by the following formula (V-3), A tetramer represented by the following formula (XVI-1) is obtained,

The tetramer represented by the formula (XVI-1) and the anhydride represented by the formula (VII) are condensed to obtain a tetramer represented by the formula (XVII-1):

Solid-phase synthesis represented by the formula (XIII-1) by condensing the tetramer represented by the formula (XVII-1) and a solid support having an amino group represented by the following formula (IX). Manufacturing method for obtaining a unit compound for use.

In the above formula,
R ³ and R ⁴ are protecting groups,
B ¹ , B ² , B ³ and B ⁴ are, independently of each other, a group selected from a group represented by the following formula and a group having a functional group protected by a protecting group,

A has the formula - _{(CH 2) n} - with a group represented, n is an integer from 1 to 6 in the formula,
W ¹¹ is H or a group represented by the formula OR ¹¹ ,
W ²¹ is H or a group represented by the formula OR ²¹ ,
W ³¹ is H or a group represented by the formula OR ³¹ ,
W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,
Here, the R ¹¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group,
X ¹ , X ² , X ³ and X ⁴ , independently of one another, are O, NH or S,
Y ¹ , Y ² and Y ³ , independently of each other, are O or S,
Z ¹ , Z ² and Z ³ , independently of one another, are O, NH or S,
M is a solid phase carrier.

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