WO2006025154A1

WO2006025154A1 - Modified oligonucleotides

Info

Publication number: WO2006025154A1
Application number: PCT/JP2005/012579
Authority: WO
Inventors: Yukio Kitade; Yoshihito Ueno; Yusuke Wataya
Original assignee: Gifu University; National University Corporation Okayama University
Priority date: 2004-08-30
Filing date: 2005-07-07
Publication date: 2006-03-09
Also published as: JPWO2006025154A1

Abstract

The invention aims at providing oligonucleotides modified at the 3' end which are excellent in penetration through cell membrane, resistance to nuclease, and the ability to form double strands and whose production was difficult in the prior art. The aim is attained by a modified oligonucleotide wherein the 5',3'-phosphoric diester linkage between the first and second nucleosides from the 3' end is replaced by a linkage represented by the general formula (I-1): -X1-C(=Y1)-Z1- (I) [wherein X1 is O, NH, or S; Y1 is O or S; and Z1 is O, NH, or S]. Such modified oligonucleotides can be produced through solid phase synthesis by using unit compounds for solid phase synthesis as represented by the general formula (X-1) as the starting raw material: (X-1) wherein R2, W11, W21, B1, B2, A, X2, X1, Y1, Z1 and M are each as defined in the description.

Description

Specification

Modified oligonucleotide

Technical field

[0001] The present invention relates to a modified oligonucleotide.

Background art

[0002] In recent years, a methodology for capturing genetic information itself as a treatment target has been spreading. One of the methodologies is an antisense method using oligonucleotides. The anti-sense method involves adding chemically synthesized oligonucleotides (for example, single-stranded DNA consisting of 15 to 20 base pairs) to cells to form ZmRNA double-stranded nucleic acids such as target messenger RNA (mRNA) and DNA. Thus, it is a method of inhibiting the translation process from mRNA to protein by specifically suppressing the expression of the target gene. In the antisense method, if the base sequence of the pathogenic virus or gene is known, it is possible to theoretically design an antisense molecule and synthesize it. It is expected as one of the effective treatment methods for diseases caused by various viruses and genetic diseases.

Recently, attention has been focused on a method using RNAi (RNA interference) as a gene expression suppression method using oligonucleotides. RNAi refers to a phenomenon in which RNA derived from a cell chromosome having the same base sequence is degraded and cleaved by introducing double-stranded RNA into a cell. The mechanism of RNAi is currently considered as follows. First, a long double-stranded RNA is called a double-stranded RNA (siRNA (short interfering RNA)) with a length of about 21 bases with a 3-UU-type dangling end structure by an enzyme (called Dicer). ). The siRNA forms an RNAZmRNA double-stranded nucleic acid with the target mRNA, and an intracellular protein that recognizes this double-stranded nucleic acid (called RNA-induced silencing complex (RISC)) binds to this double-stranded nucleic acid. The target mRNA is cleaved by this conjugate. In many cases, this RNAi-based method achieves the same effect using RNA at a concentration of about 1Z100 compared to the antisense method. Therefore, how to use RNAi However, expectations are rising as one of the effective treatment methods for diseases and gene diseases caused by various viruses that have been considered difficult to cure.

[0004] In a method using an oligonucleotide such as an antisense method or a method using RNAi, it is necessary to introduce the oligonucleotide into a cell. In addition, it is necessary to stably introduce the oligonucleotide introduced into the cell. Nucleic acid-hydrolyzing enzyme (nuclease) exists inside and outside the cell, and the introduced oligonucleotide, particularly the natural oligonucleotide. Had a problem with being easily disassembled.

[0005] Various modified oligonucleotides have been developed for the purpose of improving permeability to cell membranes and nuclease resistance. For example, a DNA oligonucleotide is known in which a modified TT dimer in which the 5 ′, 3′-phosphate diester bond between thymidine dimers is replaced with a force rubamate bond is introduced (for example, see Non-Patent Document 1). ). It has been confirmed that DNA oligonucleotides containing such modified TT dimers have slightly improved nuclease resistance. However, due to the nature of the production method, this modified TT dimer is capable of being introduced into sites other than the 3 'end of DNA oligonucleotides. .

[0006] In addition, nucleases are known to cleave oligonucleotides at their 3 'ends. For example, a DNA oligonucleotide containing a modified TT dimer as described above is considered to be normally degraded by a nuclease until the 3, end force of the DNA oligonucleotide is also modified TT dimer. In other words, although the nucleotide sequence of the DNA oligonucleotide containing the modified TT dimer is partially resolved, although it exhibits some nuclease resistance, the DNA oligonucleotide may not be conserved. There was a risk of decline. Considering the reaction characteristics of nucleases as described above, modification of the 5,3, monophosphate diester bond between the first and second nucleosides from the 3, end of the oligonucleotide is a modification that shows resistance to nucleases. Oligonucleotides can be expected. However, in oligonucleotide synthesis using solid phase synthesis technology, it is difficult to modify the site, and such a modified oligonucleotide has not been realized.

[0007] Furthermore, in the method using an oligonucleotide such as an antisense method or a method using RNAi, the ability of the oligonucleotide to form a double strand with a natural oligonucleotide and However, the stability of the formed double strand is required, but a non-natural oligonucleotide having a modified chemical structure usually has a problem that its ability to form a double strand is lowered. Non-patent literature 1: Adrian Waldner, Alain De Mesmaeker and Jacques Lebreton, “Synthesis of Oligodeoxyribonucleotides containing Dimers with Carbamate Moieties as Replacement of the Natural Phosphodiester linkagej, Bioorganic & Medicinal Chemistry Letters, 1994, IV, p.405

Disclosure of the invention

Problems to be solved by the invention

[0008] Therefore, an object of the present invention is to provide an oligonucleotide having a modified 3 'end that is excellent in three properties of permeability to a cell membrane, nuclease resistance, and duplex forming ability.

Means for solving the problem

[0009] The present invention relates to an oligonucleotide 3, 5, 3 between the first and second nucleosides from the end.

'Monophosphate diester bond strength A modified oligonucleotide substituted with a bond represented by the following formula (I 1).

^{^{-X 1 -C (= Y 1)}} -Z 1 - (1- 1)

In the formula (1-1), X ¹ is 0, NH or S, Y ¹ is O or S, Z ¹ is 0, NH or S

The invention's effect

[0010] The present invention has succeeded in producing an oligonucleotide in which the 5,3,3 monophosphate diester bond between the first and second nucleosides from the 3 'end has been modified, which has been difficult to produce. It is completed based on. In the present invention, as the modification, replacement with a bond represented by the above formula (1-1) is selected, and the modified oligonucleotide has improved nuclease resistance, improved permeability to cell membranes, and improved duplex formation ability. did. Furthermore, the improved nuclease resistance of the modified oligonucleotide of the present invention is far superior to that expected from the modification of the 3, termini.

Brief Description of Drawings FIG. 1 is a diagram showing an example of a modified double-stranded oligonucleotide.

FIG. 2 is a graph showing the RNaseL expression concentration of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide.

FIG. 3 is a graph showing the RNaseL expression concentration of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide.

FIG. 4 is a graph 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 graph showing intracellular RNaseL expression concentrations of an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide.

FIG. 6 is a graph showing the 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 a modified double-stranded oligonucleotide.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, “oligonucleotide” refers to, for example, a polymer of nucleoside subunits, and the number of subunits is not particularly limited. Among them, when the modified oligonucleotide of the present invention is DNA, the number of subunits is 4 to: L00 force S, preferably 4 to 30 is more preferable RNA. 4 to 50 is preferred 4 to 30 is more preferred. The “oligonucleotide” in the present invention is not particularly limited except that the 5,3, monophosphate diester bond between the first and second nucleosides from the end is modified. For example, for a nucleoside that is a subunit of an oligonucleotide, the sugar moiety (eg, 2′-substitution) and base may be modifications known to those skilled in the art.

In the modified oligonucleotide of the present invention, the bond represented by the formula (1-1) — ^ C ^ Y ¹ ) —Z 1 is conveniently attached to the 5′-position carbon atom of the sugar moiety of the nucleoside. The bond to be bonded is shown on the left, and the bond to be bonded to the 3 'carbon atom is shown on the right. For example, the 3 'end of the modified oligonucleotide is a clove, and the 5', 3 'phosphate diester bond between the TTs is represented by the formula (I 1). If it is replaced by a bond, it is expressed as follows.

[0014] [Chemical 36]

In the above formula, W ¹¹ is H or a group represented by the formula OR ¹¹ ;

W ²¹ is a group represented by H or the formula OR ²¹ ;

Here, R ¹¹ and R ²¹ are each independently a protecting group.

The bond represented by the formula (1-1) is, for example, —0—C (= 0) —NH 1, NH—C (

= 0) —O, 1 NH—C (= 0) —NH, 1 NH—C (= 0) —S, 1 S—C (= 0) 1 NH, 1 O—C (= S) —NH, One NH—C (═S) —O, one NH—C (═S) —NH, one NH—C (═S) —S, one S—C (═S) —NH, and the like. Among them, the bond represented by the formula (1-1) is preferably —0—C (= 0) —NH, one NH—C (= 0) —0 or NH—C (= 0) —NH. .

[0017] The modified oligonucleotide of the present invention preferably satisfies at least one of the following (i) and (ii). Similarly, for the bonds represented by the following formula (I 2) and the following formula (I 3), for convenience, the bond that binds to the 5′-position carbon atom of the sugar moiety of the nucleoside is shown on the left. The bond on the 'position carbon atom is shown on the right.

(i) 5,5,3, monophosphate diester bond strength between the 3rd and 2nd and 3rd nucleosides from the end of the oligonucleotide The bond is replaced by a bond represented by the following formula (1-2).

X ² -C (= Y ² ) Z (1- 2)

In the formula (1-2), X ² is 0, NH or S, Y ² is O or S, Z ² is 0, NH or S (ii) 5,3,3 phosphoric acid diester bond strength between the 3rd and 4th nucleosides from the 3rd end of the oligonucleotide and replaced by a bond represented by the following formula (1-3).

-X ³ -C (= Y ³ ) -Z ^3- (1-3)

In the formula (1-3), X ³ is 0, NH or S, Y ³ is O or S, Z ³ is 0, NH or S

[0018] The bond represented by the formula (I 2) and the bond represented by the formula (I 3) are independently of each other, for example, one OC (= 0) —NH—, —NH—C ( = 0) O, One NH— C (= 0) One NH, One NH— C (= 0) — S, One S— C (= 0) — NH, One OC (= S) — NH One, One NH —C (= S) O, 1 NH—C (= S) —NH, 1 NH—C (= S) —S—, —S—C (= S) —NH isotropic forces are also preferably selected. Among them, the bond represented by the formula (1-2) is more preferably O C (= 0) —NH—, —NH—C (= 0) 2 O or one NH—C (= 0) —NH 2. Among them, the bond represented by the formula (1-3) is —O—C (= 0) —NH—, —NH—C (= 0) —O or NH—C (= 0) —NH. Is more preferable.

[0019] 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. For example, 0−C (= 0 ) —NH and 0— C (= 0) NH—, — NH-C (= 0) O and one NH— C (= 0) O, one NH— C (= 0) — NH and NH— C (= 0) — NH, etc. The bond represented by the formula (I 1) and the bond represented by the formula (I 2) may be the same or different. Further, when satisfying the above (ii), the combination of the bond represented by the formula (I 1) and the bond represented by the formula (I 3) is not particularly limited. For example, OC (= 0) —NH And OC (= 0) — NH, 1 NH— C (= 0) O and 1 NH— C (= 0) O, 1 NH— C (= 0) — NH and NH— C (= O) NH etc. is there. The bond represented by formula (I 1) and the bond represented by formula (I 3) may be the same or different. Further, when both (i) and (ii) are satisfied, the combination of the bond represented by formula (I 1), the bond represented by formula (I 2), and the bond represented by formula (I 3) The combination is not particularly limited.For example, -0-C (= 0) —NH and NH—C (= 0) O and OC (= 0) —NH—, —NH-C (= 0) O OC (= 0) — NH And NH— C (= O) — O etc. The bond represented by formula (I 1), the bond represented by formula (I 2) and the bond represented by formula (1-3) may be the same or different.

[0020] 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 preferable for use as an antisense, siRNA or the like because of its excellent ability to form a double strand. Modified oligonucleotide force When double-stranded, 5, 3, 3, monophosphate between the first and second nucleosides from the 3, end of the single-stranded oligonucleotide of one or both of the double-stranded oligonucleotides It is preferable that the ester bond is replaced with the bond represented by the formula (I 1). The double-strand oligonucleotides have both the single-strand oligonucleotides 3, 5, 3, monophosphate diester binding strength between the first and second nucleosides from the end represented by the above formula (I 1) When it is replaced by a bond, the bond represented by both formulas (I 1) is the same and must be different.

[0021] When the modified oligonucleotide is double-stranded, it is an oligonucleotide that causes RNAi (RNA interference), and has the same base sequence as a part of mRNA encoding the modified oligonucleotide force endonuclease, etc. Modified oligonucleotides are preferred. Such modified oligonucleotides are also useful for RNAi research and the like. Examples of the endonuclease include RNaseL.

[0022] When the modified oligonucleotide is double-stranded, it is the modified oligonucleotide force siRNA (short interfering RNA), and a part of the mRNA is the first AA sequence upstream of 75 bases from the start codon. It is preferable that the partial strength of the mRNA is specific to the mRNA, and the modified oligonucleotide force is a combination of a sense strand and an antisense strand of the 19 base sequence. Such a sense chain can be obtained, for example, as follows. First, for example, the mRNA sequence of exonuclease can be obtained using known sites such as NCBI (National Center for Biotechnology Information) and EMBL—EBI (European Molecular Biology Laboratory-European Bioinformatics Institute). get. In the gene sequence, find the first AA sequence that is more than 75 bases downstream from the start codon. A 19-base sequence following the AA sequence, a total of 21 base sequences, is again obtained using a known gene database. Confirm that it is specific to the target gene. It is also confirmed that this 19 base sequence has a GC content of around 50%. Confirming these two, the resulting 19-base sequence is the sense strand as described above.

[0023] In the case of a modified oligonucleotide-force RNA as described above, it is preferable that one or both of the sense strand and the antisense strand have a nucleotide containing two more thymidines at its 3 'end. Having such a sequence at the 3 ′ end tends to cause RNAi. In addition, since this modified oligonucleotide is replaced by the bond represented by the formula (I 1), which is the first and second nucleosides from the 3 ′ end, the thymidine thymidine sequence ability is improved. It is preferable from the viewpoint of excellent siRNA function.

[0024] When the modified oligonucleotide is single-stranded, the modified oligonucleotide is an antisense DNA or an antisense RNA, and one of mRNAs encoding the modified oligonucleotide force endonuclease and the like. It is preferable to have a sequence complementary to part or all. Modified oligonucleotide force A force capable of forming a double strand with such mRNA and suppressing the expression of endonuclease and the like.

[0025] The modified oligonucleotide of the present invention is preferably nuclease resistant. The modified oligonucleotide of the present invention can be prevented from being degraded by a nuclease when incorporated into cells, and as a result, the modified oligonucleotide can maintain its activity in cells. is there.

[0026] The gene expression inhibitor of the present invention includes the modified oligonucleotide of the present invention. Such a gene expression inhibitor can modify the ability of modified oligonucleotide, for example, cleave the mRNA of the target gene or form a double strand with the mRNA of the target gene, thereby suppressing the gene expression.

[0027] The pharmaceutical composition of the present invention is for treating a disease associated with gene expression, and includes the gene expression inhibitor. When a disease associated with gene expression, for example, a disease is caused by the expression of a protein, this pharmaceutical composition can be used to suppress the gene expression and treat the disease associated with the gene expression. It is.

[0028] It is preferable that such a pharmaceutical composition further contains an excipient for cell introduction. Gene origin This is because it becomes possible to facilitate introduction into cells when treating a disease accompanying the present. Examples of the cell introduction excipient include a transfection reagent and the like. The transfection reagent includes, for example, a DNA molecule (the pharmaceutical composition) encapsulated in artificial lipid vesicles (ribosomes) composed of phospholipids, and the artificial lipid vesicles in a cell suspension. In addition, it is a reagent that forms artificial lipid vesicles that are used in the lipofection method to attach DNA molecules in artificial lipid vesicles by attaching them to the cell surface and fusing with cell membranes. Examples of the transfer reagent include, for example, Lipofectamine (Invitrogen), Lipofectamine 2000 (Invitrogen), Oligofectamin (Invitrogen), Transmessenger (Kigen (Kigen)) QIAGEN))), siRNA Transfection 'Kit.Jet SI (Ambion), Gene Slicer SiRNA Transfection Reagent (Gene Therapy Systems), etc. .

[0029] In addition to the inclusion of a cell introduction excipient as described above, the pharmaceutical composition of the present invention can also be introduced into cells using an electroporation method, a particle gun method, or the like. . The electopore position 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 pharmaceutical composition) are attached to gold fine particles, and using a particle gun (particle gun), the compressed fine helium gas is used to shoot the gold fine particles into the cell membrane. In this method, the DNA molecule (the pharmaceutical composition) is introduced into cells.

[0030] The RNAi kit of the present invention includes a modified oligonucleotide that is an siRNA. Other examples of such kits include a plate on which a well and a modified oligonucleotide are fixed, a fixed carrier such as a fiber and a nanochip, and the like. Such a kit may contain, in addition to the modified oligonucleotide, for example, a drug, a coloring reagent that reacts to develop color, a detection reagent that facilitates detection, and the like.

[0031] The reagent for RNAi research of the present invention includes a modified oligonucleotide that is siRNA. When researching RNA i, introduction of 30 bp or more dsRNA into cells activates cell-specific defense reactions In some cases, it may not be possible to determine whether RNAi is generated in the cell due to a reaction that randomly degrades mRNA. This random degradation of mRNA is considered to occur by the following mechanism. First, dsRNA activates 2-5 oligoadreic acid synthase (2-5AS) and activates 2-5A RNAaseL produced thereby. The RNAaseL degrades mRNA at random. Since the modified oligonucleotide, which is siRNA, can suppress the expression of RNAaseL, random degradation of mRNA can be suppressed. As a result, for example, it is easy to determine whether RNAi is occurring in the cell.

[0032] The gene expression suppression method of the present invention is a method for 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, thereby suppressing gene expression.

[0033] The method of causing RNAi of the present invention is a method of causing RNAi using a modified oligonucleotide that is siRNA. This method can cause RNAi since it is a modified oligonucleotide force RNA.

[0034] Next, the production method for producing the modified oligonucleotide of the present invention will be described by way of examples. Such a production method made it possible to produce the modified oligonucleotide of the present invention that could not be produced conventionally.

[0035] First, 3, 5, 5-phosphate diester bond strength between the first and second nucleosides from the 3, end of the oligonucleotide. Formula D (where X ¹ is 0, NH or S, A production example of a modified oligonucleotide in which Y ¹ is O or S, Z ¹ is 0, NH or S) will be described.

^{[0036] -X 1 -C (=} Y 1) -Z 1 - (1- 1)

For example, a solid-phase synthesis unit compound represented by the following formula (X-1) is used as a starting material, and R ² of the solid-phase synthesis unit compound represented by the formula (X-1) is removed. Then, the modified oligonucleotide can be obtained by extending a nucleotide to the 5 ′ end of the unit compound for solid phase synthesis represented by the formula (X-1), and then cutting out the solid phase carrier force.

[0037] [Chemical 37]

In the above 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 whose functional group is protected by a protecting group:

[0039] [Chemical 38]

[0040] A is a group represented by Formula 1 (CH) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

Here, R ¹¹ and R ²¹ are each independently a protecting group,

X ¹ and X ² are independently of each other 0, NH or S;

Y ¹ is O or S;

Z ¹ is 0, NH or S;

M is a solid support.

[0041] In addition, the 3, 5, 5-phosphate diester bond strength between the first and second nucleosides from the 3, end of the oligonucleotide is represented by the formula D (wherein X ¹ is 0, NH or S, Y ¹ is O or S, Z ¹ is 0, NH or S), and the 3rd, 2nd and 3rd positions of the oligonucleotide 5,3, Monophosphate diester bond strength between nucleosides of the following formula (1-2) (where X ² is 0, NH or S, Y ² is O or S, Z ² is 0, NH Or S) An example of production of a modified oligonucleotide substituted for the bond represented will be described.

[0042] -X ² -C (= Y ² ) -Z ^2- (1- 2)

For example, a solid-phase synthesis unit compound represented by the following formula (XI-1) is used as a starting material, and R ³ of the solid-phase synthesis unit compound represented by the formula (XI-1) is removed. Next, the modified oligonucleotide can be obtained by extending the nucleotide to the 5 ′ end of the solid-phase synthesis unity compound represented by the formula (XI-1) and then cutting out the solid phase carrier force. .

[0043] [Chemical 39]

[0044] In the above formula, is a protecting group,

B ² and B ³ are each independently a group selected from a group represented by the following formula and a group whose functional group is protected with a protecting group:

[0045] [Chemical 40]

[0046] A is a group represented by Formula 1 (CH) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

Wherein ¹ , R ²¹ and R ³¹ are each independently a protecting group;

X ¹ , X ² and X ³ are independently of each other 0, NH or S;

Y ¹ and Y ² are independently of each other O or S;

Z ¹ and Z ² are independently of each other 0, NH or S;

M is a solid support.

[0047] Further, the 3,, 5-phosphonate diester bond strength between the first and second nucleosides from the 3, terminal end of the oligonucleotide is represented by the formula D (wherein X ¹ is 0, NH or S, Y ¹ is O or S, Z ¹ is 0, NH or S), and 5, 3, 3, monophosphate diester bond strength between the 3rd and 4th nucleosides from the 3rd end of the oligonucleotide The following modified formula (1-3) (wherein X ³ is 0, NH or S, Y ³ is O or S, Z ³ is 0, NH or S) An example of production will be described.

[0048] -X ³ -C (= Y ³ ) -Z ^3- (1- 3)

For example, a solid-phase synthesis unit compound represented by the following formula (XII-1) is used as a starting material, and R ⁴ of the solid-phase synthesis unit compound represented by the formula (XII-1) is removed. Next, the modified oligonucleotide can be obtained by extending the nucleotide to the 5 ′ end of the solid-phase synthesis unity compound represented by the formula (XII-1), and then cutting out the solid phase carrier force. .

[0049] [Chemical 41]

[0050] In the above formula, R ⁴ and R are each independently a protecting group,

B ³ and B ⁴ are each independently a group selected from a group represented by the following formula and a group whose functional group is protected by a protective group:

[0051] [Chemical 42]

[0052] A is a group represented by Formula 1 (CH) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

W ⁴¹ is H or a group represented by the formula OR ⁴¹ , Wherein R ¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group, X ¹ , X ³ and X ⁴ are each independently 0, NH or S;

Y ¹ and Y ³ are independently of each other O or S;

Z ¹ and Z ³ are independently of each other 0, NH or S;

M is a solid support.

[0053] Further, the 3,, 5-phosphonate diester bond strength between the first and second nucleosides from the 3, terminal end of the oligonucleotide is represented by the formula D (wherein X ¹ is 0, NH or S, Y ¹ is O or S, Z ¹ is 0, NH or S), and 5 ′, 3 ′ phosphate diester bond strength between the 3rd and 3rd nucleosides from the 3rd end of the oligonucleotide A bond represented by formula (I 2) (wherein X ² is 0, NH or S, Y ² is O or S, Z ² is 0, NH or S), and 3 from the end of the oligonucleotide 3 5,3, monophosphate diester bond strength between the 4th and 4th nucleoside Formula (1-3) (where X ³ is 0, NH or S, Y ³ is O or S, Z ³ Illustrates an example of the preparation of a modified oligonucleotide substituted with a bond represented by 0, NH or S).

[0054] For example, the solid phase synthesis unity compound represented by the following formula (ΧΙΠ-1) is used as a starting material, and the solid phase synthesis unity compound R4 of the formula (XIII-1) is represented by R ⁴ Next, the nucleotide is extended to the 5 ′ end of the solid-phase synthesis unit compound represented by the formula (ΧΠΙ-1) in the following formula, and then the solid-phase carrier force is also cut out to obtain the modified oligonucleotide. be able to.

[0055] [Chemical 43]

[0056] In the above formula, R ⁴ independently of each other is a protecting group;

[0057] [Chemical 44]

[0058] A is a group represented by the formula — (CH 2) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

W ⁴¹ is H or a group represented by the formula OR ⁴¹ , Wherein ¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group, and X ¹ , X ² , X ³ and X ⁴ are each independently 0, NH or S Yes,

Υ ² and Υ ³ are, independently of each other, Ο or S;

Ζ ² and Ζ ³ are independently of each other 0, NH or S;

M is a solid support.

[0059] The solid phase carrier is not limited as long as it is a solid phase carrier suitable for synthesizing DNA, RNA, and the like on the solid phase carrier. For example, CPG (control pore glass), HCP (highly cross- lin ked polystyrene)

[0060] The protecting groups for R ² , R ³ and R ⁴ are selected from conventionally known protecting groups depending on the reactivity of X ² — H, — X ³ — H and — X ⁴ — H. . For example, when X ² , X ³ or X ⁴ is ο, a conventionally known primary alcohol protecting group, for example, 4,4′-dimethoxytrityl (DMTr), 4 known in the field of nucleoside chemistry Monomethoxytrityl (MMTr), (9-phenyl) xanthene mono 9-yl [pixyl] isotonic Can be used as its protecting group.

[0061] Further, R ^u, and the protecting group for R ^21, R ³¹ and R ^41, 2, depending on the reactivity of the position hydroxyl group, is selected from known protecting groups. For example, tert-butyldimethylsilyl (TBD MS), [(triisopropylpropylsilyl) oxy] methyl (Tom), tetrahydrobiral (THP) and the like can be used as its protecting group.

[0062] For R, the protecting group is selected from conventionally known protecting groups depending on the reactivity of phosphoric acid. For example, cyanoethyl (CE), allyl, trimethylsilylethyl (T MSE), p-trophenethyl (NPE) and the like can be used as its protecting group.

[0063] Also,

For B ³ and B ⁴ , the protecting group whose functional group is protected is selected from those known in nucleic acid chemistry. For example, benzoyl (Bz), isoptyryl (i Bu), phenoxycetyl (Pac), allyloxycarboxyl (AOC) and the like can be used as the protecting group.

[0064] In order to extend the nucleotide to the 5 'end of the solid-phase synthesis unity compound, a nucleoside is sequentially added according to the sequence of the modified oligonucleotide using a conventionally known technique in the field of oligonucleotide synthesis. It can be done by coupling. Nucleosides, coupling reagents, deprotection reagents, washing reagents, etc. are those usually used for nucleic acid solid phase synthesis. The modified oligonucleotide on the obtained solid phase carrier is subjected to deprotection of the oligonucleotide side chain if necessary, and then the solid phase carrier force is cut out 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 (the portion connecting the solid phase carrier and the modified oligonucleotide). The crude modified oligonucleotide may be purified by HPLC or the like, if necessary.

[0065] Next, 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 as 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. The resulting single-stranded modified oligonucleotide is then placed in an annealing buffer (eg, a buffer containing lOOmM KOAc aqueous solution, 2 mM MgOAc solution, and 30 mM HEPES-KOH (pH 7.4)). The solubilized product and the single-stranded natural oligonucleotide dissolved in the annealing buffer are mixed, treated at 95 ° C for 5 minutes, and then gradually cooled to 25 ° C. Thus, a double-stranded modified oligonucleotide can be obtained. This double-stranded modified oligonucleotide can be isolated and purified by further performing phenol Z chromatography, ethanol precipitation or the like, if necessary.

[0066] The unity compound for solid phase synthesis of formula (X-1), the unity compound for solid phase synthesis of formula (XI-1), and the solid phase synthesis of formula (XII-1) used in the above production method The unity compound for use and the unity compound for solid phase synthesis of the formula (ΧΠΙ1) can be produced, for example, by the following method.

[0067] First, the production of the unitary 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 (ΠΙ) are combined with a diimidazole derivative of the formula (V-1) and optionally a base (eg pyridine etc.), a catalyst (eg 4 Reaction is carried out in the presence of minopyridine (DMAP) etc. to obtain dimer (VI). Nucleosides of formula (IV-1) and formula (III) may be obtained commercially or may be made in-house using known literature. [0068] Next, the dimer (VI) thus obtained is mixed with an anhydride (VII), optionally with a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine (DMAP)). Etc.) to obtain dimer (VIII).

[0069] Next, the obtained dimer (VIII) and the solid phase carrier (IX) having an amino group are coupled with a coupling reagent (for example, WSC (1-ethyl-3- (3-dimethylamino) Propyl) -carbodiimide 'hydrochloride))) in the presence to give unit compound (X-1).

[0070] [Chemical 45]

^ Ν.

Scheme 1

[0071] In the above formula,

R ² is a protecting group,

[0072] [Chemical 46]

[0073] A is a group represented by Formula 1 (CH) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

Here, R ¹¹ and R ²¹ are each independently a protecting group.

X ¹ and X ² are independently of each other 0, NH or S;

Y ¹ is O or S;

Z ¹ is 0, NH or S;

M is a solid support.

Next, a production example of the unitary compound (XI-1) will be described with reference to Scheme 2. For example, R ² of the dimer of the formula (VI) is replaced with H by a method according to the property of R ² to obtain a dimer of the formula (VI-1) in which the 5 ′ position is free. Next, the dimer of the formula (VI-1) and the nucleoside derivative of the formula (I V-2) are converted into a diimidazole derivative of the formula (V-2) and optionally a base (for example, pyridine etc.), a catalyst Reaction in the presence of (eg, 4-dimethylaminopyridine (DMAP), etc.) gives the trimer (XIV-1). Nucleosides of the formula (IV-2) may be obtained through commercial sales! Or made in-house using known literature.

[0075] Next, the trimer (XIV-1) thus obtained is mixed with anhydride (VII), optionally with a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine ( React in the presence of DMAP) etc. to obtain the trimer (XV-1).

[0076] Next, the obtained trimer (XV-1) and solid phase carrier (IX) having an amino group are coupled with a coupling reagent (for example, WSC (1-ethyl-3- (3-dimethylamine)). Minopropyl) -carbodiimide 'hydrochloride))) in the presence to give unit compound (XI-1).

[0077] [Chemical 47]

In the above formula, R ² and R ³ are each independently a protecting group,

B ² and B ³ are each independently a group selected from a group represented by the following formula and a group whose functional group is protected with a protecting group: [0079] [Chemical 48]

[0080] A is a group represented by Formula 1 (CH) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

Wherein ¹ , R ²¹ and R ³¹ are each independently a protecting group.

X ¹ , X ² and X ³ are independently of each other 0, NH or S;

Y ¹ and Y ² are independently of each other O or S;

Z ¹ and Z ² are independently of each other 0, NH or S;

M is a solid support.

[0081] Next, a production example of the unitary compound (ΧΠ-1) will be described with reference to Scheme 3. For example, R ² of the dimer of formula (VI-2) is replaced with H by a method according to the properties of R ² to obtain a dimer of formula (VI-3) in which the 5 ′ position is free. . Next, the dimer of formula (VI-3) and the nucleoside derivative of formula (IV-3) are present in the presence of a phosphate binding reagent (for example, 2-cyanoethyl N, N-diisopropylchlorophosphoramidite, etc.) Subsequent condensation yields the trimer of formula (XIV-2). The nucleoside of the formula (IV-3) may be obtained commercially or made in-house using known literature.

[0082] Next, the resulting trimer (XIV-2) is added with anhydride (VII), optionally with a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine ( React in the presence of DMAP) etc. to obtain the trimer (XV-2).

Next, the obtained trimer (XV-2) and solid phase carrier (IX) having an amino group are coupled with a coupling reagent (for example, WSC (1-ethyl-3- (3-dimethyla Minopropyl) -carbodiimide 'hydrochloride))) in the presence to give unit compound (XII-1).

[0084] [Chemical 49]

In the above formula,

R ³ and R are, independently of one another, a protecting group;

B ¹ , B ² and B ³ are independently of each other a group represented by the following formula and its functional group is a protecting group: A group selected from the group protected by

[0086] [Chemical 50]

[0087] A is a group represented by Formula 1 (CH) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

Wherein ¹ , R ²¹ and R ³¹ are each independently a protecting group.

X ¹ and X ³ are independently of each other 0, NH or S;

Y ¹ is, independently of ^one another, O or S;

Z ¹ is independently of each other 0, NH or S;

M is a solid support.

[0088] Next, a production example of the unitary compound (V-1) will be described with reference to Scheme 4. For example, by a method corresponding to the formula (XIV 1) of the trimer of R ³ on the nature of R ^3, replaced with the H, 5 'position is a trimer of free expression (XIV 3) obtain. Next, a trimer of the formula (XIV-3) and a nucleoside derivative of the formula (IV-4) are optionally converted to a diimidazole derivative of the formula (V-3) and optionally a base (for example, pyridine), The reaction is carried out in the presence of a catalyst (for example, 4-dimethylaminopyridine (DMAP) etc.) to obtain a tetramer (XVI-1). The nucleoside of the formula (IV-4) may be obtained commercially or made in-house using known literature.

[0089] Next, to the resulting tetramer (XVI-1), an anhydride (VII) is optionally added with a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine ( Reaction in the presence of DMAP) etc. gives the tetramer (XVII-1).

[0090] Next, the resulting tetramer (XVII-1) and the solid phase carrier (IX) having an amino group are coupled with a coupling reagent (for example, WSC (1-ethyl-3- (3-dimethyl) Aminopropyl) -carbodiimi

[0092] [Chemical 52]

Scheme 4

[0093] In the above formula,

R ³ and R ⁴ are protecting groups,

[0094] [Chemical 53]

[0095] A is a group represented by Formula 1 (CH) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,

Wherein ¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group; X ¹ , X ² , X ³ and X ⁴ are independently of each other 0, NH or S;

Υ ² and Υ ³ are, independently of each other, Ο or S;

Ζ ² and Ζ ³ are independently of each other 0, NH or S;

M is a solid phase carrier.

[0096] In each step of Schemes 1 to 4, if necessary, a protecting group may be introduced into each functional group, the protecting group may be deprotected, or the protecting group may be changed. The selection of the protecting group according to the type of the functional group, the introduction of the protecting group, and the removal of the protecting group can be carried out according to a method known in the art. "Groups in Organic Synthesis, T. t ^ reene, John Wiley & ¾ons, Inc."

Example

[0097] In the description of the present specification, the following abbreviations are used.

Ar: Anolegon

APS: Ammonium peroxodisulfate CPG: Controlled pore glass

DIAD: diisopropylazocarboxylate

DMAP: 4-dimethylaminopyridine

DMTrCl: 4,4'—Dimethoxytritinochloride (4,4'—dimethoxytritylchloride)

DMF: dimethylformamide

EDTA: ethylenediamine-N, N, Ν ', Ν, —tetraacetic acid, disodium salt, dehydrate (ethylenediamine-Ν, Ν, Ν, Ν)

FAB: fast atom bombardment

HRMS: high-resolution mass spectrometry

Im CO: 1-1'-Carbonylbis- 1H-imidazole (1-1'-Carbonylbis-1H-imid

2

azole)

mRNA: messenger ribonucleic acid

NBA: 3--Trobenzylalchol (3-nitrobenzylalchol)

PAGE: polyacrylamide gel electrophoresis TBAF: Tributylammoniumfluoride TBE: Tris-boric acid-EDTA

TEAA: triethylamine-acetic acid

TBDMSCl: tert-butyldimethylsilylchloride TEMED: N, N, Ν ', N, monotetramethyl monoethylenediamine (Ν, Ν, Ν', Ν'- tetramet hyl-ethylenediamine)

THF: Tetrahydroforan

TRIS: Tris (hydroxymethyl) aminomethane WSC: 1-Ethyl 3- (3-Dimethylaminopropyl) -Carpositimide 'hydrochloride (1- Ethyl- 3-— (3-Dimethylaminopropyl) — carbodumide, hydrochloride j

The activity of the compound on CPG was calculated as follows.

[0098] Place 6 mg of dried CPG on a glass filter and add HCIO and ethanol

Four

The mixture (HCIO: EtOH = 3: 2) was poured. Absorbance of the obtained filtrate (wavelength 498nm

Four

(DMTr group absorption wavelength)) was measured, and the value was substituted into the following equation.

[0099] [Equation 1] 4b5. (498nm) x Fo /. (Solution) (mL) x l4.3 _ (,

Weight (support) (mg)

[0100] In the above formula, Abs. Is the absorbance of CPG at a wavelength of 498 nm,

Vol. Is the measured volume of the filtrate,

weight is the weight of the measured CPG.

[0101] 1. For production of modified single-stranded oligonucleotide or modified double-stranded oligonucleotide

, Monomer production

[0102] (1) Manufacture of 5'—O— (4,4'-dimethoxytrityl) thymidine (IV—11) (5'-0- (4,4 dimethoxytrityDthymidine) (S. Agrawal et al., Methods in Manufactured according to Molecular Biology vol.20, 360-366.)

A solution of thymidine (2.03 g, 8. 43 mmol) in pyridine (16 mL) at room temperature under Ar atmosphere Stir. DMTrCl (3.08 g, 9. l mmol, 1.1 eq) was added thereto and stirred at room temperature. After 12 hours, another equivalent of DMTrCl (0.28 g, 0.83 mmol) was added to the reaction mixture and the reaction mixture was further stirred. After confirming that no more thymidine was consumed by TLC analysis, methanol was added to the reaction mixture under ice cooling. After the solvent was distilled off from the reaction mixture under reduced pressure, the obtained residue was diluted with chloroform. The chloroform solution is mixed with saturated aqueous NaHCO solution (twice) and saturated aqueous NaCl solution.

Three

The solution was washed successively with the solution (once) and then dried over sodium sulfate. The solvent was distilled off from the chloroform solution under reduced pressure, and the resulting residue was azeotroped with toluene. The obtained residue was purified by silica gel column chromatography (MeOH: CHCl = 0-4%)

Three

The title compound was obtained as foamy crystals (3.55 g, 6.51 mmol, 77% yield).

[0103] ^J H NMR (400 MHz, CDC1) δ: 1.47 (3H, s, 5— CH), 2.32-2.41 (2H, m, 2′—

3 3

H), 3.35-3.49 (2H, m, 5'— H), 3.79 (6H, s, — OCH), 4.06 (1H, m, 4'— H),

Three

4.54 (1H, m, 3'— H), 6.44 (1H, t, J = 6.8 Hz, l'-H), 6.83—7.40 (13H, m, D

MTr), 7.59 (1H, s, 6- H), 8.66 (1H, s, 1-NH).

[0104] (2) Production of 3'—O-tert-butyldimethylsilylthymidine (III-12) (S. Agrawal et al.,

Manufactured according to Methods in Molecular Biology vol.20, 360-366. )

[0105] (i) 3, —O— tert-Butyldimethylsilyl-5, — O— (4, 4, —Dimethoxytrityl) thymidine (3'-0-tert-butyldimethylsibd—5'- 0- (4, 4'-dimethoxytrityl) thymidine)

5′—O— (4,4′-dimethoxytrityl) thymidine (1.04 g, 1.91 mmol) and imidazo monoole (0.32 g, 4.74 mmol, 2.5 eq) were added DMF ( 8. OmL) and the resulting solution was stirred under Ar atmosphere. TBDMSCKO. 36 g, 2. 37 mmol, 1. eq.) Was added to the solution and the reaction mixture was stirred under Ar atmosphere for 12 hours. After confirming by TLC analysis that no more 5′-O— (4,4′-dimethoxytrityl) thymidine was consumed, methanol (4 mL) was added to the reaction mixture under ice cooling. After the solvent was distilled off from the reaction mixture under reduced pressure, the resulting residue was diluted with ethyl acetate. The ethyl acetate solution was diluted with a saturated aqueous NaHCO solution (2 times) and a saturated aqueous NaCl solution (1 time).

Three

Washing sequentially, followed by drying over sodium sulfate. Solvent from the ethyl acetate solution The residue obtained by evaporation under reduced pressure was purified by silica gel column chromatography (MeOH: CHC1 = 0-5%) to give the title compound as white foam crystals (1.18 g, 1. 79mm

Three

ol, yield 94%).

[0106] ^J H NMR (400 MHz, CDC1) δ: 0.09 (6H, s, TBDMS— CH), 0.81 (9H, s, t—

3 3

Butinole), 1.47 (3H, s, 5— CH), 2.16—2.33 (2H, m, 2′— H), 3.23—3.46 (2H, m,

Three

5'-H), 3.77 (6H, s, OCH), 3.94—3.95 (1H, m, 4'—H), 4.49—4.50 (1H, m,

Three

3'-H), 6.32 (1H, t, J = 6.6 Hz, l'-H), 6.80—7.39 (13H, m, DMTr), 7.63 (1 H, s, 6-H), 8.15 (1H, s, NH).

[0107] (ii) 3, — Preparation of O—tert-butyldimethylsilylthymidine (III—12) (3′-0-tert-butyldime thylsilylthymidine)

80% acetic acid aqueous solution (26mL) was added to 3, 0-61 ^ -butyldimethylsilyl-5'-0- (4,4'-dimethoxytrityl) thymidine (1.18g, 1.79mmol) 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 then the solvent was removed from the reaction mixture under reduced pressure. Distilled off. The resulting residue was diluted with ethyl acetate. The ethyl acetate solution was washed successively with a saturated aqueous NaHCO solution (twice) and a saturated aqueous NaCl solution (once).

Three

And dried with sodium sulfate. The solvent was distilled off from the ethyl acetate solution under reduced pressure, and the resulting residue was purified by silica gel column chromatography (MeOH: CHC1 = 0 to 3%).

Three

To give the title compound as white crystals (0.60 g, 1.68 mmol, 94% yield).

[0108] ^J H NMR (400MHz, CDC1) δ: 0.03 (6H, s, TBDMS— CH), 0.81 (9H, s, t—

3 3

Butinole), 1.83 (3H, s, 5— CH), 2.10—2.41 (2H, m, 2′— H), 3.64—3.69 (1H, m,

Three

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 = 6.8 Hz, l'- 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. I, 1980, 306-310)

[0109] (i) 5, one azidothymidine - preparation of ⁽⁵ 'azidothymidine)

Thymidine (2.00 g, 8. 26 mmol), Ph P (2. 00 g, 8. 26 mmol, 1.0 equivalent) and N

Three

aN (2. 60 g, 41. 3 mmol, 5.0 eq) is dissolved in DMF (40 mU r (2.80 g, 8. 40 mmol, 1.0 equivalent) was added. The resulting reaction mixture was placed under Ar atmosphere

Four

The mixture was stirred at 85 ° C. After 25 hours, the disappearance of thymidine was confirmed by TLC analysis, and the reaction mixture was diluted with ethyl acetate, water (3 times), saturated aqueous NaHCO solution (1 time), saturated N

Three

The resultant was washed successively with an aCl aqueous solution (once) and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the ethyl acetate solution, and the resulting residue was purified by silica gel column chromatography (MeOH: CHCl = 1: 49-7: 93) to give the title compound as white crystals. Got

Three

Amount 2. Olg, 7.52 mmol, 91% yield).

[0110] ^J H NMR (400 MHz, DMSO— d) δ: 1.78 (3H, s, 5— CH), 2.06-2.28 (2H, m,

6 3

2'-H), 3.55 (2H, d, J = 4.8Hz, 5'— H), 3.83 (1H, m, 4'— H), 4.08 (1H, m, 3 and H), 3.40 (1H, d, J = 3.6Hz, 3-OH), 6.19 (1H, t, J = 6.8Hz, l'-H), 7.55 (1H, s, 6-H), 11.32 (1H, s, NH).

[0111] (ii) Production of 5, -aminothymidine (III-11) (5'-aminothymidine)

5, monoazidothymidine (0.23 g, 0.94 mmol) and 5% Pd catalyst (0.065 g) in a mixed solvent of ethanol and methylene chloride (EtOH: CH C1 = 1: 1) (3. lmL) , Hydrogen atmosphere

twenty two

Stir for 24 hours under. After confirming the disappearance of 5′-azidothymidine by TLC analysis, the reaction mixture was filtered through Celite. The solvent power of the obtained filtrate was also distilled off under reduced pressure to obtain the title compound as a white crystal (yield 0.22 g, 0.89 nmol, yield 95%).

[0112] ^J H NMR (400 MHz, DMSO— d) δ: 1.74 (3H, s, 5— CH), 2.00—2.16 (2H, m,

6 3

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 = 6.8 Hz, l'-H), 7.63 (1H, s, 6-H).

[0113] (4) Production of 3, monoamino-5, -O-tert-butyldimethylsilylthymidine (IV-12) (E. W. Harkins et al., Current Protocols in Nucleic Acid Chemistry, 4. 7. 15-4.

Manufactured according to 7.17. )

[0114] (i) Production of 5'-O-tert-butyldimethylsilylthymidine (5'-O-tert-butyldimethylsilylthymidine)

Thymidine (2.00 g, 8.26 mmol) was azeotroped with DMF and dissolved in DMF (16.5 mL). To the solution, Et N (l. 36 mL, 9. 91 mmol, 1.2 eq), DMAP (0. 05 g, 0.41 m mol, 0.05 equivalents) and TBDMSC1 (1.36 g, 9.02 mmol, 1.1 equivalents) were sequentially added, and the resulting reaction mixture was stirred at room temperature for 2 hours and a half in an Ar atmosphere. Further TBDMSC1 (0.25 g, 1.66 mmol, 0.2 eq) was added to the reaction mixture and stirred for another 30 minutes. After confirming disappearance of thymidine by TLC analysis, the reaction mixture was diluted with ethyl acetate. The obtained ethyl acetate solution was mixed with water (3 times), saturated aqueous NaHCO solution (1 time), saturated Na

Three

This was washed sequentially with an aqueous C1 solution (once) and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the ethyl acetate solution, and the resulting residue was subjected to silica gel column chromatography.

Purification by (MeOH: CHCl = 0: 1 to 1:20) gave the title compound as white crystals (yield 2

Three

44g, 6.84mmol, 83% yield).

[0115] ^J H NMR (400MHz, CDC1) δ: 0.12 (6H, m, TBDMS— CH), 0.92 (9H, s, t—

3 3

Butinole), 1.89 (3H, s, 5— CH), 2.08—2.39 (3H, m, 2′— H and 3′— OH), 3.82—3

Three

.92 (2H, m, 5'-H), 4.03 (1H, m, 4'—H), 4.47 (1H, m, 3'—H), 6.37 (1H, m, l'-H), 7.50 (1H, s, 6-H), 8.44 (1H, br, NH).

[0116] (ii) Preparation of 2, 3, 1-anhydro-5, -O-tert-butyldimethylsilylthymidine (2,3'-anhydro-o-O-tert-butyldimethylsuylthymidine)

5 '—O-tert-butyldimethylsilylthymidine (2. 19 g, 6. 14 mmol) and Ph P

Three

(2. 60 g, 9. 91 mmol, 1.6 eq) in DMF (7. OmL) solution, DIAD (3.2 mL, 9. OOmmol, 1.6 eq, 40% toluene solution) in DMF (3. lmL ) The solution was collected and the resulting reaction mixture was stirred at room temperature under Ar atmosphere. After 3 hours and a half, disappearance of 5′-O tert-butyldimethylsilylthymidine was confirmed by TLC analysis. The reaction mixture is diluted with ethyl acetate and washed with water (3 times), saturated aqueous NaHCO solution (1 time), saturated aqueous NaCl solution (1 time).

Three

Washing sequentially, followed by drying 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: Et OAc: MeOH = l: 1: 0 to 0: 10: 1). The title compound was obtained as white crystals (yield 1. 63 g, 4.82 mmol, yield 79%).

[0117] ^J H NMR (400MHz, CDC1) δ: 0.07 (6H, s, TBDMS— CH), 0.88 (9H, s, t—

3 3

Butinole), 1.95 (3H, s, 5— CH), 2.43-2.67 (2H, m, 2′— H), 3.73—3.83 (2H, m,

Three

5'-H), 4.28 (1H, t, J = 5.6Hz, 4'— H), 5.19 (1H, s, 3'— H), 5.47 (1H, s, 1 ' -H), 6.95 (1H, s, 6-H).

[0118] (iii) 3, monoazido 5, — O-tert-butyldimethylsilylthymidine (3'-azido

— 5'— 0— tert— butyldimethylsilylthymidine)

2,3'-Anhydro-5, -O-tert-butyldimethylsilylthymidine (0.48g, 1.41mmol) in DMF (4.2mL) solution with NaN (0.14g, 2.21mmol, 1. 5 equivalents)

Three

The reaction mixture was stirred at 120 ° C. under Ar atmosphere. After 48 hours, the disappearance of 2, 3, 1 anhydro-5, 1 O-tert-butyldimethylsilylthymidine was confirmed by TLC analysis. The reaction mixture was diluted with ethyl acetate, water (3 times), saturated aqueous NaHCO (1

3 times) and then with a saturated aqueous NaCl solution (1 time), and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the resulting ethyl acetate solution, and the resulting residue was purified by silica gel column chromatography (n-hexane: EtOAc = 0: 1) to give the title compound as an oil. (Yield 0.48 g, 1.25 mmol, yield 88%).

[0119] ^J H NMR (400 MHz, CDC1) δ: 0.01 (6H, m, TBDMS— CH), 0.80 (9H, m, t

3 3

—Butyl), 1.80 (3H, s, 5— CH), 2.07-2.34 (2H, m, 2'— H), 3.66—3.70 (1H,

Three

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).

[0120] (iv) 3,1-amino-5, —O—tert-butyldimethylsilylthymidine (IV—12) (3′-amin 0—5—O—tert-butyldimethylsilylthymidine)

3'-azido 5, -O-tert-butyldimethylsilylthymidine (1.15g, 3.02mmol) and 5% Pd catalyst (0.15g) in methanol (lOmL) at room temperature for 19 hours under Ar atmosphere Stir. 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 nmmol, yield 95%).

[0121] ^J H NMR (400MHz, DMSO— d) δ: 0.05 (6H, m, TBDMS— CH), 0.86 (9H, s,

6 3

t-butinole), 1.75 (3H, s, 5'-CH), 2.01—2.09 (2H, m, 2'—H), 3.30 (2H, s,

Three

NH), 3.41-3.46 (1H, m, 4'— H), 3.64-3.77 (2H, m, 5'— H), 3.80—3.84 (1H,

2

m, 3'-H), 6.11-6.16 (1H, t, 9.4 Hz, l'-H), 7.45 (1H, s, 6-H). [0122] 2. For production of modified single-stranded oligonucleotide or modified double-stranded oligonucleotide

, Manufacture of unity compound bound to solid support

[0123] (Example 1— 01)

(1) Production of unit compounds represented by (X—11)

[0124] [Chemical 54]

(IV-11) (III-11) (X-ll)

[0125] (i) 5, O— (4, 4, —Dimethoxytrityl) —3,-(5, —Aminothymidylyl) carbamoinoletin (5—〇— (4,4— dimethoxytrityl) — 3-( 0 -aminothymidilyl) carbamoylthymiai ne)

5, — O— (4, 4, — Dimethoxytrityl) thymidine (IV— 11) (0.76 g, 1.4 mmol) in a solution of pyridine (8 · OmL) with DMAP (small amount) and Im CO (0. 23g, 1 ・ 4mmol, 1 ・ 0

2

Eq.) 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. OmL) was added dropwise to the reaction mixture. After 48 hours, the reaction mixture was diluted with ethyl acetate. The obtained ethyl acetate solution was mixed with water (once), saturated aqueous NaHCO solution (once),

Three

The extract was washed successively with a saturated aqueous NaCl solution (once) and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the ethyl acetate solution, and the resulting residue was purified by silica gel column chromatography (MeOH: CHCl = 1: 20 to 1:10) to give the title compound as white crystals.

Three

(Yield 0.71 g, 0.87 mmol, 62% yield).

[0126] ^J H NMR (400 MHz, DMSO— d) δ: 1.38 (3H, s, 5— CH 5′T), 1.75 (3H, s, 5

6 3

— CH 3'T), 1.98-2.48 (4H, m, 2'— H), 3.14—3.33 (4H, m, 5'— H), 3.73 (6H,

Three

s, OCH), 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 and 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, CDC1) δ: 11.44, 12.34, 37.97, 38.90, 55.22, 63.94, 71

Three

.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 H N O (MH—), 810.29867; found, 810.29930.

42 45 2 12

[0127] Manufacture of unity compound (X—11)

5,-O- (4, 4, -dimethoxytrityl) -3, one (5,, aminothiomidylyl) strength rubamoyl thymidine (0.71 g, 0.87 mmol) in a solution of pyridine (8.7 mL) DMAP (small amount) and succinic anhydride (0.29 g, 2.87 mmol, 3.3 eq) were added and the reaction mixture was stirred at room temperature for 16 h under Ar atmosphere. After confirming the disappearance of 5'-0- (4,4'-dimethoxytrityl) -3 '(5',-aminominomidylyl) rubamoylthymidine by TLC analysis, the reaction mixture was treated with chloroform. Diluted with The black mouth form 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 portion of the resulting residue (0.80 g, 0.87 mmol, 4 eq) was dissolved in DMF (22 mL). CPG (136 / z molZg, 1.61 g, 0.22 mol) was added to the solution to swell CPG, and then WSC (0.17 g, 0.87 mmol, 4 equivalents) was added to the reaction mixture. . The reaction mixture was shaken at room temperature for 2 days. The reaction mixture was filtered under reduced pressure, and the resulting filtered product was washed with pyridine and then dried. To the above filtered product, a 0.1 M pyridine and acetic anhydride mixture solution (pyridine: acetic anhydride = 9: 1) (15 mL) of DMAP was added, and the mixture was shaken at room temperature for 15 hours. The reaction mixture was filtered under reduced pressure, and the resulting filtrate was washed successively with methanol and acetone, and then dried to give the title unity compound (CPG compound activity = 26.7 711010173) (yield) 1. 53 g) o

[0128] (Example 1 02)

(2) Production of unit compound represented by (X—12) [0129] [Chemical 55]

(IV-12) (III-12) (X-12)

[0130] (i) 5,0-0- 6-Butyldimethylsilyl-3, 1-amino- (3,-O-tert-butyldimethylsilylthymidylyl) force rubermoylthymidine (5'-0-tert-butyldimethylsilyl) -3 and a mino— (3 — O— tert— butyldimethylsilylthymidilyl) carbamoyltnymidine)

3, —O—tert-butyldimethylsilylthymidine (ΠΙ—12) (2. 27 g, 6. 37 mmol) in pyridine (35 mL) was added to DMAP (small amount) and Im CO (0. 76 g, 4. 70 mmol, 1 This

2

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 added. Stir for 1 day. The reaction mixture was diluted with ethyl acetate. The ethyl acetate solution was washed with water (once), saturated aqueous NaHCO solution (1

Three

) And saturated aqueous NaCl solution (1 time), and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the ethyl acetate solution, and the resulting residue was purified by silica gel column chromatography (MeOH: CHCl = 0: 1 to 1:10) to give the title compound of white crystals.

Three

The product was obtained (yield 2.46 g, 3.33 mmol, yield 70%).

[0131] ^J H NMR (400MHz, CDC1) δ: 0.08—0.13 (12H, m, TBDMS—CH 3), 0.89 an

3 3

d 0.93 (18H, s, t-butinole), 1.90 (3H, s, 5-CH 5'T), 1.93 (3H, s, 5-CH

3 3

3'T), 2.15-2.35 (4H, m, 2'- H), 3.85—3.94 (2H, m, 5 and H 5'T), 4.01—4.03 (2 H, 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, NH 3' T).

¹³ C— NMR (100 MHz, CDC1) δ: — 5.98, —4.91, 12.42, 18.30, 25.88, 40.46, 52

Three

.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 CHNO Si (MH "), 738.3566; found, 738.3579.

33 55 5 10 2

[0132] (ii) 3, Production of 3-amino-thymidylylcarbamoylthymidine

5 '0- 61 ^ -Butyldimethylsilyl-3'-amino- (3' '-O-tert-butyldimethylsilylthymidylyl) -powered rubamoylthymidine (0.90 g, 1. 44 mmol) in THF (14 mL) TBAF (2 mL, 2 mmol, 2 eq) was added and the reaction mixture was stirred for 1 day. The solvent was distilled off from the reaction mixture under reduced pressure, and the resulting residue was purified by silica gel column chromatography (MeOH: CHCl = 1: 20 to 1: 5) to give the title compound as white crystals.

Three

(Yield 0.72 g, 1. 43 mmol, 99% yield).

[0133] ^J H NMR (400MHz, DMSO— d) δ: 1.77 (6H, s, 5— CH), 2.00—2.24 (4H, m,

6 3

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, l'-H), 7.44-79 (2H, m, 6— H), 11.28 (1H, s, NH

5'T), 11.31 (1H, s, NH 3'T).

1 ³ C—NMR (100 MHz, DMSO— d) δ: 12.23, 12.29, 37.17, 50.80, 61.24, 64.53,

6

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 CHNO (MH ⁺ ), 510.18406; found, 510.18357.

21 27 5 10

[0134] (iii) 5, —O— (4,4, -Dimethoxytrityl) 3, -aminominomidylylcarbamoylthymine (5—0— (4,4-dimethoxytrityl) — — aminotnymidilyicarbamoylthymidine)

3, -Aminothymidylylcarbamoylthymidine (0.21 g, 0.41 mmol) in pyridine (1.4 mL) was added DMAP (small 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 with water (1 time), saturated aqueous NaHCO solution (1 time) and saturated NaCl.

Three

The solution was washed successively with an aqueous solution (once) and then dried over sodium sulfate. The solvent was distilled off from the obtained ethyl acetate solution under reduced pressure, and the resulting residue was purified by silica gel column chromatography (n hexane: AcOEt: MeOH = 9: 1: 0 to 0: 1: 20). White crystal title The compound was obtained (yield 0.19 g, 0.22 mmol, 54%).

[0135] ^J H NMR (400MHz, DMSO— d) δ: 1.46 (3H, s, 5— CH 5′T), 1.68 (3H, s,

6 3

5-CH 3'T), 1.90-2.36 (4H, m, 2'- H), 3.05-3.32 (4H, m, 5'- H), 3.71 (6H,

Three

s, OCH), 3.89-3.92 (2H, m, 4'— H), 4.02—4.31 (4H, m, 3′— H), 5.38 (1H,

Three

d, J = 4.4 Hz, OH), 6.15 (1H, t, J = 8.0Hz, 1 and H 5'T), 6.21 (1H, t, J = 8.0 Hz, l'-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) δ: 11.78, 12.14, 37.01, 38.60, 50.65, 54.98,

6

55.00, 63.11, 64.63, 70.68, 82.68, 83.56, 83.77, 84.06, 85.76, 109.50, 10 9.67, 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 CHNO (MH ⁺ ), 812.3143; found, 812.3137.

42 45 5 12

[0136] (iv) Manufacture of unitary compound (X-12)

5 '-0- (4,4'-dimethoxytrityl) -3'-aminominomidylylcarbamoylthymidine (0.50 g, 0.61 mmol) in pyridine (6. lmL) was added to DMAP (small amount) and anhydrous Citric acid (0.18 g, 1.83 mmol, 3.0 eq) was added and the reaction mixture was stirred for 2 days. The reaction mixture was diluted with black mouthform. The black mouth form solution was washed with water (3 times) and then dried over sodium sulfate. The chloroform solution solution solvent was distilled off under reduced pressure. A part of the obtained residue (0.55 g, 0.61 mmol, 4.0 equivalent) was dried in vacuo. The remaining DMF (15 mL) solution [CPG (1.65 g, 0.15 mmol, 90.4 μmol / g)] was added to swell the CPG. After 30 minutes, more WSC (0.12 g, 0.60 mmol, 1.0 equiv) was added to the reaction mixture and shaken for 3 days. The reaction mixture was filtered under reduced pressure, and the resulting filtered product was washed with pyridine and then dried. To the filtered product was added a DMAP solution of 0.1 M pyridine and acetic anhydride (pyridine: acetic anhydride = 9: 1) (20 mL), and the mixture was shaken at room temperature for 1 day. The reaction mixture was filtered under reduced pressure, and the resulting filtrate was washed successively with methanol and acetone and then dried to give the title unit. The compound was obtained (activity of the compound of CPG = 44.4 molZg) (yield 0.93 g).

[0137] (Example 1 03)

(3) Production of unit compound represented by (X—13)

[0138] [Chemical 56]

[0139] (i) 5, 0— 6-Butyldimethylsilyl-3, 1-amino- (5, -aminominomidylyl) ureten (5 -O-tert-butyldimetnyisilyl —3— amino— (o -aminothymidilyl) ureathy midine)

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 CO (0.12 g, 0.74 mmol, 1 eq) and the reaction mixture is stirred for 3.5 h.

2

Stir. A solution of 5, monoaminothymidine (III-11) (0.22 g, 0.89 mmol, 1.2 eq) in pyridine (2 mL) was added dropwise to the reaction mixture, and the reaction mixture was stirred for 12 hours. did. The reaction mixture was extracted with ethyl acetate, and the solvent was distilled off from the ethyl acetate solution under reduced pressure. The obtained residue was subjected to silica gel column chromatography (CHC1: CH 2 O

3 3

11 = 100: 3 to 10: 1) to give the title compound as white crystals (yield 0.26 g, 0.4 2 mmol, 57% yield).

[0140] ^J H NMR (400 MHz, DMSO— d) δ: 0.06 (6H, s, TBDMS— CH), 0.87 (9H, s,

6 3

t-butinole), 1.77 (3H, s, 5—CH 5'T), 1.79 (3H, s, 5—CH 3'T), 2.05—2.17

3 3

(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, l'-H 3'T), 7.48-7.50 (2 H, s, 6-H), 11.29- 11.32 (2H, s, NH). C-NMR (100MHz, DMSO- d) δ:-5.47, -5.43, 12.10, 12.26, 18.08, 25.81,

6

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 H N O Si (MH "), 623.28707; found, 623.28606

[0141] (ii) Preparation of 3, 1-amino- (5 "-aminothymi dily Dureathymidine)

To a THF (14 mL) solution of 5, 0- 61 ^ -butyldimethylsilyl-3, monoamino- (5 ,,-aminominomidyryl) urethymidine (0.90 g, 1.44 mmol) in TBAF (2 mL, 2 mmol , 1.4 eq) and the reaction mixture was stirred for 10 hours. The solvent was distilled off from the reaction mixture under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (CHC1: CH OH =

3 3

20: 1 to 5: 1) to give the title compound as white crystals (yield 0.72 g, 1. 43 mmo 1, yield 99%).

[0142] ^J H NMR (400MHz, DMSO— d) δ: 1.76 (3H, s, 5— CH 5'T), 1.78 (3H, s, 5

6 3

— CH 3'T), 1.94-2.20 (4H, m, 2'— H), 3.42—3.69 (4H, m, 5'— H), 4.10-4.24 (

Three

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) δ: 12.07, 12.25, 37.66, 38.35, 49.78, 61.23,

6

71.08, 83.33, 83.69, 85.43, 109.27, 109.74, 136.05, 150.39, 150.45, 157.6 3, 163.71, 163.73.

FAB- HRMS (NBA) calcd for CHNO (MH ⁺ ), 509.1996; found, 509.1996.

21 28 6 9

[Iii] 5, 1, O— (4, 4, 1 dimethoxytrityl) 3, 1 amino (5, 1 aminothymidylyl) urephine (— 0— (4,4— dimethoxytrityl) — — amino— (5 -aminothymidilyOureathym idine)

3, 1-amino- (5, 1-aminothymidyryl) ureathymidine (0.80 g, 1.57 mmol) pyridi To the solution (15.7 mL) was added DMAP (small amount) and DMTrCl (0.64 g, 1.90 mmol, 1.2 eq) and the reaction mixture was stirred for 1 day. The reaction mixture was diluted with ethyl acetate. The ethyl acetate solution was diluted with water (1 ×), saturated aqueous NaHCO 3 (1 ×) and saturated.

Three

The mixture was washed successively with aqueous NaCl solution (once) and then dried over sodium sulfate. The solvent was distilled off under reduced pressure from the ethyl acetate solution, and the resulting residue was purified by silica gel column chromatography (n hexane: AcOEt: MeOH = 9: 1: 0 to 0: 1: 20) to give a white The crystalline title compound was obtained (yield 0.30 g, 0.37 mmol, 24% yield).

[0144] ^J H NMR (400MHz, DMSO— d) δ: 1.43 (3H, s, 5— CH 5'T), 1.73 (3H, s,

6 3

5— CH 3'T), 1.98-2.32 (4H, m, 2'— H), 3.14—3.28 (4H, m, 5'— H), 3.72 (6H,

Three

s, OCH), 3.86-4.04 (2H, m, 4'— H), 4.07-4.36 (1H, m, 3'— H 5'T), 5.27 (

Three

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 (100MHz, DMSO- d) δ: 11.77, 12.08, 14.10, 20.78, 39.92, 40.13,

6

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. 149.63, 150.34, 150.48, 157.44, 158.13, 163.72.

FAB-MS (NBA) calcd for C H N O (MH ~), 809.31468; found, 809.31376.

42 46 6 11

[0145] (iv) Manufacture of unitary compound (X-13)

5, 1 O— (4, 4, 1-dimethoxytrityl) 3, 1-amino (5, 1-aminothymidyryl) urea thymidine (0.72 g, 0.88 mmol) in pyridine (8.8 mL) in DMAP (small amount) ) And succinic anhydride (0.29 g, 2. 87 mmol, 3.0 eq) were added and the reaction mixture was stirred for 2 days. The reaction mixture was diluted with chloroform. The black mouth form solution was washed with water (3 times) and then dried over sodium sulfate. The solvent was distilled off from the chloroform solution under reduced pressure. A part of the resulting residue (0.55 g, 0.61 mmol, 4.0 eq) was dried in vacuo. CPG (1.10g, 0.15mmol, 136 / z mol / g) to the residual DMF (15mL) Swelled and swollen. After 30 minutes, more WSC (0.12 g, 0.60 mmol, 1.0 equiv) was added to the reaction mixture and shaken for 3 days. The reaction mixture was filtered under reduced pressure, and the resulting filtered product was washed with pyridine and then dried. To the filtered product, a DMAP solution containing 0.1 M pyridine and acetic anhydride (pyridine: acetic anhydride = 9: 1) (20 mL) was added and shaken at room temperature for 1 day. The reaction mixture was filtered under reduced pressure, and the resulting filtrate was washed successively with methanol and acetone and then dried to give the title unity compound (activity of CPG compound = 38.4 / z molZg) (yield 1.09 g).

[0146] 3,5, -Phosphate diester bond between the first and second nucleoside from the end of single-stranded oligonucleotide is O—C (= 0) —NH—, —NH-C (= 0) — O or NH— C (= O) — Production of modified single-stranded oligonucleotide replaced by NH Target protein is homosapiens ribonuclease L (Homo sapiens ribonuclease L) (2 ', 5-oligoisoadenylate synthetase—dependent, RNase L) [gi: 30795246], and the target sequence was a nucleotide sequence consisting of SEQ ID NO: 1 from 92 to 114 in the start codon strength. As a modified single-stranded oligonucleotide, a modified RNA sense strand (SEQ ID NO: 2, 3 and 4) of the designed siRNA sequence and a modified RNA antisense strand (SEQ ID NO: 5) are compared with the base sequence consisting of SEQ ID NO: 1. 6) and 7). 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.

[0147] (Reference Example 1 Is)

The RNA sense strand consisting of SEQ ID NO: 8 was a commercially available dT resin (coupled to CPG resin) (manufactured by Darren Research) 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 the 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.

[0148] The oligonucleotide of SEQ ID NO: 8 bound to the CPG resin was synthesized by an automatic nucleic acid synthesizer with the DMTr group removed.

[0149] The oligonucleotides bound to the CPG resin were reacted at 55 ° C for 12 hours in ethanol and ammonia mixed (EtOH: NH = 1: 3) solution (2 mL), respectively. reaction The mixture was filtered under reduced pressure, and the resulting filtrate was transferred to an Eppendorf tube, and then concentrated under reduced pressure. To the obtained concentrate, 1M TBAF in THF (1 mL) was added, and the mixture was shaken for 12 hours. Each reaction solution was diluted to 30 mL with 0.1 M TEAA buffer. This diluted solution was added to a C-18 reverse phase column (Sep—Pak) (CH

Three

CN (lOmL) and 0.1 M TEAA buffer (lOmL) were sequentially passed in advance and equilibrated, respectively, and adsorbed onto the column. This column was washed with sterilized water to remove salts, and then eluted with a mixture of acetonitrile and water (50% CH CN in water (3 mL)).

Three

The obtained eluate was concentrated under reduced pressure. The resulting concentrates were each dissolved in packing solution (1% XTBE solution in 90% formamide) (100 L). The solutions were separated using 20% PAGE (20A, 6 hours) (1 XTBE buffer was used for the electrophoresis buffer), and the target oligonucleotide band was excised. Each cut band was filled with 0.1 M EDTA aqueous solution (20 mL) and allowed to stand. The liquid portion of this EDTA aqueous solution is purified by C-18 reverse phase column chromatography (Sep-Pak) (eluent: in water, 50% CH CN (3 mL)) to obtain the desired oligonucleotide.

Three

It was.

[0150] A method for preparing the following solution used in the above Reference Example will be described.

[0151] A 0.1 M TEAA buffer was prepared as follows. First, water was added to 1 L in a mixture of 2N acetic acid (114. 38 mL) and triethylamine (277. 6 mL). The solution was prepared by adding acetic acid to adjust the pH to 7.0, and then diluting the solution 20 times.

[0152] 20% PAGE was prepared as follows. First, 40% acrylamide (acrylamide: N, N, -methylenebisacrylamide = 19: 1) solution (45 mL), urea (37.8 g) and 10 XTBE buffer solution (9 mL) are mixed and dissolved, and then water is added. Was adjusted to 90 mL. APS (62 mg) was added to the solution and dissolved, and then 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.

[0153] A 0.1 M aqueous EDTA solution was prepared by dissolving EDTA'4Na (l. 80 g) in water (40 mL). [0154] A 40% acrylamide (acrylamide: N, N, monomethylene bisacrylamide = 19: 1) solution contains acrylamide (190 g) and N, N, monobisacrylamide (10 g).

Prepared by dissolving in water and rubbing 500 mU.

[0155] 10 XTBE buffer was prepared by dissolving 1 U of Tris (109 g), boric acid (55 g) and EDTA '2Na (7.43 g) in water.

[0156] The obtained oligonucleotide was dissolved in water (lmL) and diluted 100-fold with water to prepare a diluted solution. The absorbance (260 nm) of the diluted solution was measured, and the yield was calculated. The absorbance ε value, absorbance at 260 nm, and yield are shown in Table 1. The molecular weight of the obtained oligonucleotide was confirmed by MALDI-TOFZMS. The results are also shown in Table 1.

[0157] (Reference Example 1 la)

An RNA antisense strand consisting of SEQ ID NO: 9 was produced in the same manner as Reference Example 1 Is except that SEQ ID NO: 9 was used instead of SEQ ID NO: 8.

[0158] (Reference Example 1 2)

Each single-stranded oligonucleotide consisting of SEQ ID NO: 9 was prepared in the same manner as Reference Example 1-la, except that the 5 'end of the modified single-stranded oligonucleotide consisting of SEQ ID NO: 9 was labeled with a ³² P label. Manufactured.

[0159] (Reference Example 1 3a)

Similarly, the modified single-stranded oligonucleotide consisting of SEQ ID NO: 9 modified at the 5 ′ end with fluorescein is labeled with the fluorescein phosphoramidite HFluorescin Phophoramidite (Glen 'Research (Glen A single-stranded oligonucleotide consisting of SEQ ID NO: 9 was prepared in the same manner as in Reference Example 1—Is except that the 5 ′ end was modified with fluorescein, except that (Research) was added.

[0160] (Reference Example 1 4)

An RNA-stranded chain consisting of SEQ ID NO: 12 was produced in the same manner as Reference Example 1 Is except that SEQ ID NO: 12 was used instead of SEQ ID NO: 8.

[0161] (Example 1 Is)

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

[0162] (Example 1 la)

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

[0163] (Example 1 2s)

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

[0164] (Example 1-2a)

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

[0165] (Example 1 3s)

The modified single-stranded oligonucleotide having SEQ ID NO: 4 was the same as Reference Example 1 Is except that the unity compound represented by (X-13) was used instead of the dT resin. Thus, a modified single-stranded oligonucleotide having SEQ ID NO: 4 was also produced.

[Example 1 1 3a]

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

[Example 1 4a]

Similarly, a modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 modified at the 5 ′ end with fluorescein is labeled with a fluorescein phosphoramidite (HFluorescin Phophoramidite) (Glen's Research (Glen A modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 and having a 5 ′ end modified with fluorescein was prepared in the same manner as in Example 1-2a except that (Research) was added. [Example 1 5a]

The modified single-stranded oligonucleotide consisting of SEQ ID NO: 6 was modified with a ³² P label at the 5 'end consisting of SEQ ID NO: 6 in the same manner as Example 1-2a except that the 5' end was labeled with a ³² P label. Single-stranded oligonucleotides were produced.

[Example 1 1 6]

The modified single-stranded oligonucleotide consisting of SEQ ID NO: 10 was the same as Reference Example 1-Is except that the unity compound represented by (X-12) was used instead of the dT resin. Thus, a modified single-stranded oligonucleotide consisting of SEQ ID NO: 10 was produced.

[0170] (Example 1 7)

Further, the modified single-stranded oligonucleotide consisting of SEQ ID NO: 11 was the same as Reference Example 1-Is except that the unity compound represented by (X-13) was used instead of the dT resin. Accordingly, a modified single-stranded oligonucleotide consisting of SEQ ID NO: 11 was produced.

[0171] The oligonucleotides of SEQ ID NOs: 8 and 9 obtained in Reference Example 1 Is and 1 la are hereinafter referred to as (dT s) and (dTa), respectively. The oligonucleotide consisting of SEQ ID NO: 9 obtained in Reference Example 12 is hereinafter referred to as (PdT-a). The oligonucleotide obtained from Reference Example 1-3a and consisting of SEQ ID NO: 9 modified at the 5 ′ end with fluorescein 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).

Example 1—Modified single-stranded oligonucleotides consisting of SEQ ID NOs: 2 and 5 obtained in Is and 1-la 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 consisting of SEQ ID NO: 6 obtained in Example 1-4a and modified at the end with fluorescein 5 is 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 (P12a). The modified single-stranded oligonucleotides consisting of SEQ ID NOs: 10 and 11 obtained in Examples 1-6 and 1-7 are respectively represented by the following ( — Sa) and (17— sa). 1]

Example or Reference SEQ ID No. Sample Name ε Value (X Absorbance Yield Calculated MALDI-TOF / MS Example 1000) (OD260) 誦 1 MW observd Reference Example 1 1 1 s 8 (dT-s) 216.3 15.2 70.3 6717.10 6717.52 Reference Example 1 — 1 a 9 (dT- a) 202.3 10.4 51.4 6567.93 6568.58 Reference Example 1 1 4 1 2 (d-sa) 97.8 25.0 255.62 3117.94 ― Example 1 1 1 s 2 (1 1-s) 216.3 10.0 46.2 6680.15 6680.53 Implementation Example 1 1 1 a 5 (1 1-a) 202.3 13.9 68.7 6530.98 6532.32 Example 1 1 2 s 3 (1 2 s) 216.3 14.5 67.0 6680.15 6683.54 Example 1 1 2 a 6 (1-a) 202.3 19.8 97.9 6530.98 6531.28 Example 1 1 3 s 4 (1 3-s) 216.3 8.3 38.4 6679.16 6679.16 Example 1 1 3 a 7 (1 3-a) 202.3 10.1 49.9 6529.99 6538.39 Example 1 1 4 a 6 (F 1 2-a ) 216.3 14.5 67.4 7129.54 7127.63 Example 1 1 5 a 6 (P 1 2-a) 202.3 12.5 57.8 7223.71 7220.40 Example 1 1 6 1 0 (1 6-sa) 97.8 32.7 334.36 3080.99 3082.30 Example 1 1 7 1 1 (1 7-sa) 97.8 20.8 212.68 3080.00 3081.60

[0174] The absorbance and yield of the oligonucleotides were measured as follows.

[0175] The oligonucleotide was dissolved in water to prepare an aqueous solution, and the aqueous solution was diluted so that the absorbance (Abs) at a wavelength of 260 nm was within the effective range of the absorptiometer. Optical path length (

260

1) Abs was measured at room temperature using a lcm absorbance measurement quartz cell. Total OD value

260 260 The calculation was performed using the following formula (1).

OD (ε • M -mL " ¹ ) = Abs (ε-cm- M) XV" ¹ (mL) XI " ¹ (cm) (1)

260 260

In the formula (1), Abs is the absorbance of the oligonucleotide solution at a wavelength of 260 nm.

260

Degrees, V is the total volume of the solution, 1 is the optical path length, M is the molar concentration.

In the formula (1), the molar extinction coefficient ε 260 of the oligonucleotide was calculated using the following formula (2).

ε = 2 {ε (Ν Ν) + ε (Ν Ν) + · · + ε (Ν Ν)}-{ε (Ν) + ε (Ν) +… +

1ρ 2 2ρ 3 n-lp η 2 3

ε (Ν)} (2)

η-1

In the formula (2), ε (Ν) represents ε of a certain nucleic acid Ν, and ε (Ν)) represents a nucleic acid dimer.

η η 260 n-lp n

Shows ε of N N. (11 s), (11 a), (12—s), (12—a), (13—s), (n-lp n 260

For 13—a) ゝ (F12—s), (F12—a) ゝ (P—12—a) and (P13—a), ε is calculated using the modified 3 ′ end TT sequence as the UU sequence. did.

260

[0177] The concentration C (molZL) was calculated using the following equation (3).

C = Abs X ε "'Γ Γ ¹ (3)

260 260

In the formula (3), Abs, ε, and 1 are as described above.

260 260

Production of modified double-stranded oligonucleotides

[0179] (Example 2-1)

Example 1— (11—s) (35 nmol) prepared in Is and (11—a) (35 nmol) prepared in Example 1—la were added to an annealing buffer (10 mM Tris—HCl (pH 7.5) and 100 mM NaCl). The solution was incubated at 90 ° C. for 1 minute and then at 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.

[0180] (Example 2—2)

Except for using (12-s) and (12-a) instead of (11 s) and (11 a), In the same manner as in Example 2-1, a modified double-stranded oligonucleotide consisting of SEQ ID NO: 3 and SEQ ID NO: 6 as shown in FIG. 1 was obtained.

[0181] (Example 2-3)

SEQ ID NO: as shown in Fig. 1 in the same manner as in Example 2-1, except that (13-s) and (13-a) were used instead of (11 s) and (11 a). A modified double-stranded oligonucleotide consisting of 4 and SEQ ID NO: 7 was obtained.

[0182] (Example 2—4)

An arrangement as shown in FIG. 1 is performed in the same manner as in Example 2-1, except that (12-s) and (F12-a) are used instead of (11-s) and (11-a). A modified double-stranded oligonucleotide composed of No. 3 and a sequence further modified with fluorescein at the 5 ′ end in SEQ ID NO: 6 was obtained.

[0183] (Example 2-5)

(12—s) (80 pmmol) prepared in Example 1-2s and (P12-a) (80 pmmol) prepared in Example 1-5a were added to annealing buffer (10 mM Tris-HCl (pH 7.5) and lOOmM NaCl). The solution is heated at 95 ° C. for 3 minutes and then incubated at room temperature for 1 hour and consists of SEQ ID NO: 3 and SEQ ID NO: 6 with ³² P label at the 5 ′ end as shown in FIG. A modified double stranded oligonucleotide was obtained.

[0184] (Example 2—6)

7 except that (16-sa) and (16-sa) were used in place of (11 s) and (11 a), and SEQ ID NO: 10 as shown in FIG. And a modified double-stranded oligonucleotide consisting of SEQ ID NO: 10 was obtained.

[0185] (Example 2-7)

In the same manner as in Example 2-1, except that (17-sa) and (17-sa) were used instead of (11 s) and (11 a), SEQ ID NO: 11 as shown in FIG. And a modified double-stranded oligonucleotide consisting of SEQ ID NO: 11 was obtained.

[0186] (Reference Example 2-1)

As in Example 2-1, except that (dT-s) and (dT-a) were used instead of (11-s) and (11-a), Two consisting of SEQ ID NO: 8 and SEQ ID NO: 9 A double-stranded oligonucleotide was obtained.

[0187] (Reference Example 2-2)

In the same manner as in Example 2-1, except that (dT s) and (PdT a) were used in place of (11 s) and (11 a), SEQ ID NO: 8 and SEQ ID NO: A double-stranded oligonucleotide consisting of number 9 was obtained.

[0188] (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), SEQ ID NO: 12 and A double-stranded oligonucleotide consisting of SEQ ID NO: 12 was obtained.

[0189] (Evaluation of modified double-stranded oligonucleotide)

1. Evaluation of intracellular RNaseL expression

(1) Cell culture

Cell culture HT1080 cells were grown at 37 ° C in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units Zml) and streptomycin (0.1 mgZml). Cells were passaged regularly to maintain exponential growth. HT1080 cells were trypsinized 24 hours before transfer at approximately 25% confluency, diluted with medium without antibiotics, and transferred to 35 ml dishes (2 ml Z dishes) ).

[0190] (2) Transfection of modified double-stranded oligonucleotide into cells

The modified double-stranded oligonucleotide was transfected to the adherent cell line using Lipofuectamine 2000 reagent (Invitrogen) by the method described below. First, double-stranded oligonucleotide (50, ΙΟΟηΜ or 200 nM per 250 1 of the medium) (Example 2-2 obtained double-stranded oligonucleotide) was dissolved in RPMI1640 medium (without antibiotics and FBS). And incubated for 5 minutes at room temperature. To the solution, an equal volume of ribofactoramine 2000 reagent diluted 50-fold was added, and then incubated for 20 minutes at room temperature. Of the resulting solution, 5001 was collected and transferred to the 35 millidish prepared in (1) above for transfection. The cells in the 35 millidivision were incubated 24 hours and 48 hours after transfection. After that, in Example 2-3 or Reference Example 2-1 None of the resulting double-stranded oligonucleotides was confirmed to be toxic to the transfected cells.

[0191] (3) Monitoring RNaseL 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 (—). The resulting cell pellet was then added to 2 volumes of hypotonic buffer A (0.5% (vZv) Nonidet P-40, 20 mM Hepes (pH 7.5), 10 mM CH. CO K, 15mM (CH

3 2

CO) Mg, ImM dithiothreitol, ImM phenylmethylsulfonylfluora

3 2 2

Id, 10 gZml of Aprotune).

[0192] After incubating the resulting cell suspension on ice for 10 minutes, use a tight 'fitting-cuffs' Dough ~~ 's' homogenizer ~~ (Ίlght—fitting glass dounce homogenizer) 30 times on ice Stroke up and down to homogenize. The obtained homogenized solution was centrifuged at 4 ° C for 10 minutes using an ultracentrifuge (lOOOO X g). The centrifuged supernatant is then collected, and an equal volume of 2X sample buffer (0.14M Tris—HCl p H6.8, 0.2% (vZv) glycerol, 2 8% (vZv) SDS, 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 (Millipore).

[0193] The PVDF membrane was incubated with 5% bovine serum albumin at 25 ° C for 1 hour. Thereafter, the PVDF membrane was rinsed twice with TBST (200 mM Tris pH 7.6, 1.37 M sodium chloride sodium salt, 0.1% Tween-20). The PVDF membrane was incubated with 0.5 gZml of anti-RNaseL monoclonal antibody at 4 ° C for 16 hours. Thereafter, the PVDF membrane was washed with TBST once for 15 minutes and then 3 times for 5 minutes. The PVDF membrane was then incubated for 1 hour at 25 ° C. with anti-mouse IgG (500 μg ZmL diluted 100000 fold) labeled with Horse radish peroxidase. Thereafter, the PVDF membrane was washed with TBST once for 15 minutes and then 3 times for 5 minutes. [0194] The detection of the labeling of Candida peroxidase on the PVDF membrane was performed using an ECL plus 'Western' blotting detection kit (manufactured by Amersham). Quantification of C. peroxidase labeling on the PVDF membrane was performed using a densitometer (Kodak Digital Science DC290 Zoom Digital Camera). When the treatment with the double-stranded oligonucleotide was carried out, the relative ratio of the RNase L strength of the cells treated with the double-stranded oligonucleotide was determined by setting the RNase L strength in the cells to 1. The obtained values were standardized with the GAPDH intensity set to 1. The obtained results are shown in Fig. 2 (a).

[0195] (4) Instead of the double-stranded oligonucleotide obtained in Example 2-2, the double-stranded oligonucleotide obtained in Example 2-3 and Reference Example 2-1 was used, respectively. In the same manner as in (1) 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 Figures 2 (b) and (c), respectively.

[0196] As shown in Figs. 2 (a) to (c), the modified double-stranded oligonucleotide of the present invention has an effect of suppressing the expression of RNaseL as compared to the unmodified double-stranded oligonucleotide. It was confirmed that there was a marked improvement. This is presumably because the modified double-stranded oligonucleotide of the present invention was excellent in cell membrane permeability and improved in stability or retention in cells.

[0197] 2. Evaluation of cytotoxicity

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

[0198] (2) Instead of the double-stranded oligonucleotide obtained in Example 2-2, Examples 2-1 and 2

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

[0199] 3. Measurement of IC for RNaseL in cells

50

(1) 1. The concentration of the double-stranded oligonucleotide used in (2) is the same as that described in 1. (1) to (1) above except that 1ηΜ, 5nM or ΙΟηΜ is used instead of 50, ΙΟΟηΜ or 200nM per 250 1 of the medium. In the same manner as in 4), the relative ratio of the RNase L intensity of the cells treated with the double-stranded oligonucleotide was determined. The results are shown in Fig. 3.

[0200] (2) Further, instead of the double-stranded oligonucleotide obtained in Example 2-2, Example 2

3. Relative ratio of RNase L intensity of cells treated with double-stranded oligonucleotide was determined in the same manner as in (1) except that the double-stranded oligonucleotide obtained in 3 and Reference Example 2-1 was used. . The results are shown in Fig. 3.

[0201] (3) IC was calculated from the graph of FIG.

50

[0202] [Table 2]

[0203] As shown in Table 2 above, the modified double-stranded oligonucleotide of the present invention has a markedly improved effect of suppressing the expression of RNaseL compared to the unmodified double-stranded oligonucleotide. It could be confirmed. This remarkable improvement is more than expected because the modified double-stranded oligonucleotide of the present invention has excellent cell membrane permeability and improved nuclease resistance.

[0204] 4. Evaluation of introduction effect of modified double-stranded oligonucleotide into cells

(1) Observation of intracellular RNaseL by immunostaining

A cover glass coated with poly L-lysine (manufactured by Sigma) was placed in a 35 millidish 24 hours before the transfer. By adding 2 mL of the HT1080 cell solution onto the cover glass in the 35 millidish, and then culturing it, HT1080 cells were pre-fixed on a cover glass.

[0205] The double-stranded oligonucleotide obtained in Example 2-2 was diluted with RPMI1640 medium (GIBCO) without antibiotics and FBS !, and then incubated at room temperature for 5 minutes (dilution) Concentration = Double-stranded oligonucleotide 培地 ηΜ or 50 nM per medium 250). Separately, Lipofectamine 2000 reagent (Invitrogen) (5 / zg) was diluted with RPMI1640 medium (GIBCO) (2501) not containing antibiotics and FBS, and then incubated at room temperature for 5 minutes. After the incubation, 250 μ1 of the diluted double-stranded oligonucleotide solution was mixed with Lipofectamine 2000 reagent solution 2501 after the incubation, and the mixture was incubated at room temperature for 20 minutes. The mixture 5001 was added to a cover glass preliminarily fixed with the HT1080 cells prepared previously, and transfection was performed.

[0206] After incubation from the transfection for 24 hours, the T1080 cells on the cover glass were washed twice with PBS (-). Thereafter, the cells on the coverslips were incubated with 4% paraformaldehyde at 4 ° C for 15 minutes. After washing three times with PBS (-), the cells on the cover glass were incubated with 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 (1) and then incubated with 1 μg Zml of anti-RNase L monoclonal antibody for 60 minutes at room temperature. Cells on the cover glass were washed 3 times with PBS (—). 10 g of Zml of fluorescent (Alexa 488) labeled mouse IgG was added to the cells on the cover glass and incubated at room temperature for 30 minutes. Then, after washing 3 times with PBS (-), 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 except that the double-stranded oligonucleotide obtained in Reference Example 2-1 was used instead of the double-stranded oligonucleotide obtained in Example 2-2. Also shown.

[0207] As shown in Fig. 5 above, the modified double-stranded oligonucleotide of the present invention has a significant decrease in RNaseL fluorescence intensity, that is, strong and suppressed RNaseL expression compared to the unmodified double-stranded oligonucleotide. It was confirmed that the effect was demonstrated.

[0208] (2) Calculation method of intracellular introduction effect of modified double-stranded oligonucleotide by fluorescence microscope The concentration of the double-stranded oligonucleotide (10nM or 50nM) obtained in Example 2-2 was changed to 50nM or 25nM instead of ΙΟηΜ, and the concentration of the double-stranded oligonucleotide obtained in Example 2-2 Instead, the double-stranded oligonucleotide was transferred to HT1080 cells on the cover glass as described in 4 (1) except that the fluorescein-labeled double-stranded oligonucleotide obtained in Example 2-4 was used. Transfusion.

[0209] After incubation for 24 hours from the transfection, HT1080 cells on the cover glass were washed twice with PBS (-). Thereafter, the cells on the coverslips were incubated with 4% paraformaldehyde at 4 ° C for 15 minutes. After washing 3 times with PBS (-), the cells on the cover glass were immediately observed with a fluorescence microscope. The results are shown in Fig. 4. From the obtained results, the ratio of the number of cells positive for fluorescein-labeled double-stranded oligonucleotide per total number of cells was calculated. The results are shown in Table 3. Also, as a control, instead of the above mixture, only HT1080 cells prepared previously with 500 μl of lipofucamine amine reagent solution after incubation were placed on a previously fixed cover glass, and HT1080 subjected to transfection. The results obtained with the cells are also shown in FIG.

[0210] [Table 3]

[0211] As shown in FIG. 4 and Table 3, the modified double-stranded oligonucleotide of the present invention was confirmed to have a remarkable decrease in RNAaseL fluorescence intensity, ie, suppression of RNAaseL.

[0212] 5. modified single-stranded and double-stranded oligonucleotide of E exonuclease resistance Evaluation Example 1 consisting of SEQ ID NO: 6 obtained in 5a-modified single-stranded oligonucleotides (5 'end of the ³² P of Unmodified single-stranded oligonucleotide consisting of SEQ ID NO: 9 obtained in Reference Example 1-2 (5, end labeled with ³² P), modification obtained in Example 2-5 Obtained from double-stranded oligonucleotide (5, end labeled with ³² P) and Reference Example 2-2 The unmodified double-stranded oligonucleotide (5, labeled with ³² P at the end) was evaluated for exonuclease resistance. As the exonuclease, snake venom phosphorodiesterase (SVP) was used. SVP selectively cleaves phosphodiester bonds and cleaves oligonucleotides into 5, monomonophosphate nucleotides.

[0213] From the reaction solution having the following composition, dispensed to each Eppendorf tube after 1, 3, 5, 10, 30, 60 minutes, and into the filled solution (7M urea XC BPB) (5 1) The reaction was stopped by sampling the reaction solution (5 μ 1). The obtained samples at each time were separated by electrophoresis using 20% polyacrylamide gel (containing 7Μ urea) at 20 mA for 180 minutes, and the remaining percentage (%) of the complete strand of the modified oligonucleotide was displayed as a log. Things on the vertical axis

Ten

Figures 6 (a) and (b) were prepared with the reaction time as the horizontal axis. Then, from the graphs of FIGS. 6 (a) and (b), the time (t) at which the remaining rate of the complete chain was 50% was calculated. The results are shown in Table 4.

50

Show.

[0214] [Table 4]

[0215] Composition of reaction solution of modified single-stranded oligonucleotide (Example 1 5a)

20 / z M modified single-stranded oligonucleotide aqueous solution

Buffer solution (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 6 L

2

2u / mL SVP aqueous solution

Water 26 / z L

40 / z L in total

[0216] Composition of reaction solution of unmodified single-stranded oligonucleotide (Reference Example 1 2)

20 / z M Unmodified single-stranded oligonucleotide aqueous solution

Buffer solution (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 6 L 2u / mL SVP aqueous solution 4μL

Water 2δμL

40 μL total

[0217] Composition of reaction solution of modified double-stranded oligonucleotide (Example 2-5)

20 / zM modified double-stranded oligonucleotide aqueous solution 4μL

Buffer (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 6 μL ·

2

2u / mL SVP aqueous solution 4μL

Water 2δμL

40 μL total

[0218] Composition of reaction solution of unmodified double-stranded oligonucleotide (Reference Example 2-2)

20 / zM unmodified double-stranded oligonucleotide aqueous solution 4μL ·

Buffer (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 6 μL ·

2

2u / mL SVP aqueous solution 4μL

Water 2δμL

40 μL total

[0219] From FIG. 6 and Table 4, it was confirmed that the modified single-stranded oligonucleotide has improved exonuclease resistance compared to the unmodified single-stranded oligonucleotide. In addition, it was confirmed that the modified double-stranded oligonucleotide has significantly improved resistance to exonuclease compared to the unmodified double-stranded oligonucleotide. In particular, double-stranded modified oligonucleotides were confirmed to have significantly improved nuclease resistance even when compared to the results of single-stranded modified oligonucleotides, and this result exceeds the expected range.

[0220] 6. Evaluation of thermodynamic stability of modified double-stranded oligonucleotides

Modified double-stranded oligonucleotide consisting of SEQ ID NO: 10 obtained in Example 2-6, modified double-stranded oligonucleotide consisting of SEQ ID NO: 11 obtained in Example 2-7, and obtained in Reference Example 2-3 The resulting double-stranded oligonucleotide consisting of SEQ ID NO: 12 was evaluated for thermodynamic stability.

[0221] For the oligonucleotides, according to Marky and Bleslaur, van't Hoff transition enthalpy (Δ ピー °), entropy (AS °), free energy at 298K Lugi (AG °) was calculated. AG ° indicates the degree of inclination of the equilibrium state force thermodynamic energy of the reaction at a certain temperature when lmol single strand becomes lmol double strand in the standard state. The thermodynamic stability of the chain can be evaluated. The 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. ΔΗ ° represents the change in thermal energy balance when lmol single strand becomes lmol double strand in the standard state, and the one sign represents the exothermic reaction. Is stable in terms of thermal energy. AS ° represents the change in randomness when lmol single strand becomes lmol double strand in the standard state, and the sign indicates that it tends to a more disordered state. A smaller value indicates that it is more advantageous for duplex formation. The relationship between AG °, ΔΗ °, and AS ° is expressed by the following equation (4).

△ G. = ΔΗ °-TAS ° (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.

[0222] Tm was measured by dividing the concentration of oligonucleotides into several steps. This Tm value was measured with a Beck man Coulter DU 640 spectrophotometer. In the measurement, 10 mM NaHPO-NaHPO, 1 M NaCl (pH 7.0) was used as a buffer. Obtained Tm value

2 4 2 4

A graph of lZTm vs. In CtZ4 (Ct is the total oligonucleotide concentration) was drawn based on Equation (5), and ΔΗ ° and AS ° were calculated from the slope and intercept. Then, AG ° was found from equation (4). The results obtained are shown in Table 5.

l / Tm = (R / AH °) lnCt / 4 + (AS ° / ΔΗ °) '' (5)

[0223] [Table 5]

Tm (X) — A VJT _{2 9 8} -Δ Η

(kcal / moi) (kcai / mol)

Example 2—6 56.9 15.4 77.9 210 Example 2—7 54.5 14.8 74.1 199 Reference Example 2—3 55.7 13.6 62.7 165 [0224] As shown in Table 5 above, the modified double-stranded oligonucleotide of the present invention is more sure to increase the absolute value of -AG ° and the absolute value of ΔΗ than the unmodified oligonucleotide.

298

It was confirmed that the stability as a double strand was improved. In addition, it was confirmed that the modified double-stranded oligonucleotide of the present invention forms a more ordered double strand because the absolute value of AS is increased compared to the unmodified oligonucleotide. It was.

[0225] 7. Evaluation of endonuclease resistance of modified double-stranded oligonucleotides

Example 1—Modified single-stranded oligonucleotide consisting of SEQ ID NO: 5 obtained in 5a (5 ′ end labeled with ³² P), unmodified consisting of SEQ ID NO: 9 obtained in Reference Example 1-2 Single-stranded oligonucleotide (5, end labeled with ³² P), modified double-stranded oligonucleotide obtained in Example 2-5 (5, end labeled with ³² P) and Reference Example 2- Endonuclease resistance of the unmodified double-stranded oligonucleotide obtained in 2 (5, labeled with ³² P at the end) was evaluated. RNase A was used as the endonuclease.

[0226] From the reaction solution having the composition shown below, the reaction solution (10 1) was sampled in the filling solution (8M urea XC BPB) (10 1) dispensed to each Eppendorf tube 30 minutes later. To stop the reaction. The obtained samples at each time were separated by electrophoresis using 20% polyacrylamide gel (containing 7M urea) at 20mA for 180 minutes to obtain the complete strand of the modified or unmodified single-stranded oligonucleotide. The residual rate (%) was measured. The results are shown in Table 6.

[0227] [Table 6]

Composition of reaction solution of modified single-stranded oligonucleotide (Example 1 5a)

20 / z M modified single-stranded oligonucleotide aqueous solution 2 μL

Buffer (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 1.5 μL ·

2

10 ^ g / mL RNase aqueous solution 0.5 ^ L Water 6; zL

LO ^ L

[0229] Composition of reaction solution of unmodified single-stranded oligonucleotide (Reference Example 1 2)

20 / zM unmodified single-stranded oligonucleotide aqueous solution 2μL ·

Buffer (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 1.5 μL

2

10 ^ g / mL RNase aqueous solution 0.5 ^ L

Water 6; zL

LO ^ L

[0230] In addition, a charged solution (8M urea XC BPB) (10M) was dispensed into each Etbendorf tube after 5, 10, 20, 30, 60, 90 minutes from the reaction solution having the composition shown below. / zl), the reaction solution (10 μ1) was sampled to stop the reaction. The obtained samples at each time were separated by electrophoresis using 20% polyacrylamide gel (containing 7% urea) at 20 mA for 180 minutes to leave the complete strand of the modified or unmodified double-stranded oligonucleotide. Figure 8 was created with the percentage (%) on the vertical axis and the reaction time on the horizontal axis.

[0231] Composition of reaction solution of modified double-stranded oligonucleotide (Example 2-5)

20 / zM modified double-stranded oligonucleotide aqueous solution 2μL ·

Buffer (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 1.5 μL

2

10 ^ g / mL RNase aqueous solution 0.5 ^ L

Water 6; zL

LO ^ L

[0232] Composition of reaction solution of unmodified double-stranded oligonucleotide (Reference Example 2-2)

20 / zM unmodified double-stranded oligonucleotide aqueous solution 2μL ·

Buffer solution (250 mM Tris-HCU 50 mM MgCl (pH 7.0)) 1.5; zL

2

10 ^ g / mL RNase aqueous solution 0.5 ^ L

Water 6; zL

LO ^ L

[0233] From Table 6, it was confirmed that the modified double-stranded oligonucleotide had significantly improved endonuclease resistance compared to the unmodified double-stranded oligonucleotide. From Figure 8 The modified double-stranded oligonucleotide was confirmed to have significantly improved endonuclease resistance compared to the unmodified double-stranded oligonucleotide.

Industrial applicability

[0234] The modified oligonucleotide of the present invention is useful, for example, as a disease therapeutic agent.

Sequence listing free text

[0235] SEQ ID NO: 1 Target sequence of RNaseL

SEQ ID NO: 2 Modified sense strand of siRNA sequence against the target sequence of SEQ ID NO: 1

SEQ ID NO: 3 Modified sense strand of siRNA sequence against the target sequence of SEQ ID NO: 1

SEQ ID NO: 4 Modified sense strand of siRNA sequence against the target sequence of SEQ ID NO: 1

SEQ ID NO: 5 Modified antisense strand of the siRNA sequence against the target sequence of SEQ ID NO: 1 SEQ ID NO: 6 Modified antisense strand of the siRNA sequence against the target sequence of SEQ ID NO: 1 SEQ ID NO: 7 Modified antisense of the siRNA sequence against the target sequence of SEQ ID NO: 1 Strand SEQ ID NO: 8 sense strand of siRNA sequence against the target sequence of SEQ ID NO: 1

SEQ ID NO: 9 Antisense strand of siRNA sequence against target sequence of SEQ ID NO: 1 SEQ ID NO: 10 Modified strand of siRNA sequence against target sequence of SEQ ID NO: 12

SEQ ID NO: 11 Another modified strand of siRNA sequence against the target sequence of SEQ ID NO: 12 SEQ ID NO: 12 Target sequence

Claims

Claims [1] 3,5,3, monophosphate diester bond strength between the first and second nucleosides from the end of the oligonucleotide was replaced by a bond represented by the following formula (1-1) Modified oligonucleotide. -X1-C (= Y1) -Z1- (1-1) In the formula (1-1), X1 is 0, NH or S, Y1 is O or S, Z1 is 0, NH or S [2 ] The bond represented by the formula (1-1) is O—C (= 0) —NH, one NH—C (= 0) —O one, one NH—C (= 0) —NH, one NH — C (= 0) — S, One S— C (= 0) — NH—, O— C (= S) — NH, One NH— C (= S) — O, One NH— C (= S) The modified oligonucleotide according to claim 1, selected from the group consisting of: —NH, one N HC (═S) —S and one S—C (═S) —NH. [3] The bond represented by the formula (1-1) is composed of O—C (= 0) —NH, one NH—C (= 0) —O one and NH—C (═O) —NH. The modified oligonucleotide according to claim 2, which is selected from the group. [4] The modified oligonucleotide according to claim 1, which satisfies at least one of the following (i) and (ii): (i) 5,5,3, monophosphate diester bond strength between the 3rd and 2nd and 3rd nucleosides from the end of the oligonucleotide The bond is replaced by a bond represented by the following formula (1-2). X2-C (= Y2)-Z2-(1-2) In the formula (1-2), X2 is 0, NH or S, Y2 is O or S, Z2 is 0, NH or S (ii) 5,5,3, phosphodiester bond strength between the 3rd and 4th nucleosides from the 3rd end of the oligonucleotide is replaced by a bond represented by the following formula (1-3). -X3-C (= Y3) -Z3- (1-3) In the formula (1-3), X3 is 0, NH or S, Y3 is O or S, Z3 is 0, NH or S [5 ] The bond represented by the formula (I 2) and the bond represented by the formula (I 3) are independently of each other, one OC (= 0) —NH, one NH—C (= 0) —O , 1 NH— C (= 0) — NH—, 1 NH— C (= 0) — S, 1 S— C (= 0) — NH, 1 OC (= S) — NH, 1 NH— C (= 5. A group force of S) —0—, one NH—C (= S) —NH, one NH—C (= S) —S and —S—C (= S) —NH force is selected. Modified oligonucleotide. [6] The modified oligonucleotide according to [1], which is a double-stranded oligonucleotide. [7] 3 ′ of at least one single-stranded oligonucleotide of the double-stranded oligonucleotide, 5 ′, 3 ′ phosphate diester binding force between the first and second nucleosides from the end In the formula (I 1) The modified oligonucleotide according to claim 6, wherein the modified oligonucleotide is an oligonucleotide that causes RNAi (RNA interference), and mRN encoding the modified oligonucleotide force endonuclease The modified oligonucleotide according to claim 6, which has the same base sequence as a part of A. [9] The modified oligonucleotide is siRNA (short interfering RNA), and a part of the mRNA is a 19 base sequence following the first AA sequence at least 75 bases upstream from the start codon, The modified oligonucleotide according to claim 8, wherein the modified oligonucleotide is specific for the mRNA, and is a combination of the sense strand and the antisense strand of the 19-base sequence. 10. The modified oligonucleotide according to claim 9, wherein the modified oligonucleotide has nucleotides containing at least one of the sense strand and the antisense strand, and further three thymidines at the ends. [11] The modified oligonucleotide according to [1], which is a single-stranded oligonucleotide. [12] The modified oligonucleotide according to [11], wherein the modified oligonucleotide is antisense DNA or antisense RNA and has a sequence complementary to at least a part of mRNA encoding the modified oligonucleotide force endonuclease. [13] The modified oligonucleotide according to [1], wherein the oligonucleotide is nuclease resistant. [14] A gene expression inhibitor comprising an oligonucleotide, wherein the oligonucleotide is the modified oligonucleotide according to claim 1. [15] A pharmaceutical composition for treating a disease associated with gene expression, wherein the pharmaceutical composition comprises the gene expression inhibitor according to claim 14. 16. The pharmaceutical composition according to claim 15, further comprising an excipient for cell introduction. 17. The pharmaceutical composition according to claim 16, wherein the cell introduction excipient is a transfection reagent. [18] An RNAi kit, wherein the kit comprises the modified oligonucleotide according to claim 8. [19] A reagent for RNAi research, wherein the reagent comprises the modified oligonucleotide according to claim 8. [20] A method for suppressing gene expression using an oligonucleotide, wherein the oligonucleotide is the modified oligonucleotide according to claim 1. [21] A method for causing RNAi using an oligonucleotide, wherein the oligonucleotide is the modified oligonucleotide according to claim 8. [22] A method for producing the modified oligonucleotide according to claim 1, using a unity compound for solid-phase synthesis represented by the following formula (X-1) as a starting material, wherein the formula (X-1) R2 of the solid phase synthesis unity compound represented by the formula (X-1) is removed, and a nucleotide is extended to the 5 'end of the solid phase synthesis unity compound represented by the formula (X-1). A production method for obtaining the modified oligonucleotide by cutting out the carrier force.

[Chemical 1]

In the formula (X-1), R ² is a protecting group,

[Chemical 2]

A is a group represented by the formula — (CH 2) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

Here, R ¹¹ and R ²¹ are each independently a protecting group,

X ¹ and X ² are independently of each other 0, NH or S;

Y ¹ is O or S;

Z ¹ is 0, 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 unity compound for solid phase synthesis represented by the following formula (XI-1) as a starting material, XI—Removes R ³ from the solid phase synthesis unit compound represented by 1) A production method wherein the modified oligonucleotide is obtained by extending a nucleotide at the 5 ′ end of a unity compound for solid phase synthesis represented by the formula (XI-1) and then cutting out the solid phase carrier force.

[Chemical 3]

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 whose functional group is protected by a protecting group:

[Chemical 4]

A is a group represented by Formula 1 (CH 2) 1, wherein n is an integer of 1 to 6

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ; W ³¹ is a group represented by H or the formula OR ³¹ ;

Wherein ¹ , R ²¹ and R ³¹ are each independently a protecting group;

X ¹ , X ² and X ³ are independently of each other 0, NH or S;

Y ¹ and Y ² are independently of each other O or S;

Z ¹ and Z ² are independently of each other 0, NH or S;

M is a solid phase carrier.

[24] The method for producing the modified oligonucleotide according to claim 4, wherein (iii) is satisfied using a unit compound for solid phase synthesis represented by the following formula (XII-1) as a starting material: Removing R ⁴ of the unit compound for solid phase synthesis represented by the formula (XII-1),

A production method wherein the modified oligonucleotide is obtained by extending a nucleotide to the 5 ′ end of the unity compound for solid phase synthesis represented by the formula (XII-1) and then cutting out the solid phase carrier force.

[Chemical 5]

In the above formula, R ⁴ and R independently of each other are protecting groups; B ³ and B ⁴ are each independently a group selected from a group represented by the following formula and a group whose functional group is protected by a protective group:

[Chemical 6]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,

Wherein ¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group;

X ¹ , X ³ and X ⁴ are independently of each other 0, NH or S;

Y ¹ and Y ³ are independently of each other O or S;

Z ¹ and Z ³ are independently of each other 0, NH or S;

M is a solid phase carrier.

5. The method for producing the modified oligonucleotide according to claim 4, wherein a solid-phase synthesis unit compound represented by the following formula (ΧΙΠ-1) is used as a starting material and satisfies both (i) and (ii): Because

R ⁴ of the solid phase synthesis unit compound represented by the formula (ΧΙΠ—1) is removed,

A production method for obtaining the modified oligonucleotide by extending a nucleotide to the 5 ′ end of a unity compound for solid phase synthesis represented by the formula (ΧΙΠ-1), and then cutting out the solid phase carrier force.

[Chemical 7]

In the above formula, R ⁴ is a protecting group,

B ¹ , B ² , B ³ and B ⁴ are each independently a group selected from a group represented by the following formula and a group whose functional group is protected with a protective group:

[Chemical 8]

A is a group represented by Formula 1 (CH 3) 1, wherein n is an integer of 1 to 6,

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

Υ ² and Υ ³ are, independently of each other, Ο or S;

Ζ ² and Ζ ³ are independently of each other 0, NH or S;

M is a solid phase carrier.

[26] A unit compound for solid phase synthesis represented by the following formula (X-1) for producing the modified oligonucleotide according to claim 1.

[Chemical 9]

In the formula (X-1), R ² is a protecting group,

[Chemical 10]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ , W ²¹ is a group represented by H or the formula OR ²¹ ;

Here, R ¹¹ and R ²¹ are each independently a protecting group,

X ¹ and X ² are independently of each other 0, NH or S;

Y ¹ is O or S;

Z ¹ is 0, NH or S;

M is a solid phase carrier.

[27] A method for producing a unitary compound for solid phase synthesis represented by the 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 (ΠΙ) are condensed with a diimidazole derivative represented by the following formula (V-1), and the following formula: To obtain a dimer (VI) represented by (VI)

[Chemical 11]

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

[Chemical 12]

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

[Chemical 13]

j

R ² is a protecting group,

[Chemical 14]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

Here, R ¹¹ and R ²¹ are each independently a protecting group,

X ¹ and X ² are independently of each other 0, NH or S;

Y ¹ is O or S;

Z ¹ is 0, 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.

[Chemical 15]

In the above formula, R ³ is a protecting group,

[Chemical 16]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

Wherein ¹ , R ²¹ and R ³¹ are each independently a protecting group;

X ¹ , X ² and X ³ are independently of each other 0, NH or S;

Y ¹ and Y ² are independently of each other O or S;

Z ¹ and Z ² are independently of each other 0, 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,

Replacing R ² of the dimer represented by the formula (VI) with H to obtain a dimer represented by the formula (VI-1),

[Chemical 17]

The dimer represented by the formula (VI-1) and the 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,

[Chemical 18]

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),

[Chemical 19]

The trimer represented by the formula (XV-1) is condensed with a solid support having an amino group represented by the following formula (IX) to produce a solid phase synthesis represented by the formula (XI-1). A manufacturing method for obtaining a unity compound.

[Chemical 20]

In the above formula, IT and R ³ are independently of each other a protecting group, B ² and B ³ are each independently a group selected from a group represented by the following formula and a group whose functional group is protected with a protecting group:

[Chemical 21]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

Wherein ¹ , R ²¹ and R ³¹ are each independently a protecting group;

X ¹ , X ² and X ³ are independently of each other 0, NH or S;

Y ¹ and Y ² are independently of each other O or S;

Z ¹ and Z ² are independently of each other 0, 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.

[Chemical 22]

Wherein R ⁴ and R are protecting groups;

[Chemical 23]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,

Wherein ¹ , R ²¹ , R ³¹ and R ⁴¹ are each independently a protecting group; X ¹ , X ³ and X ⁴ are independently of each other 0, NH or S;

Y ¹ and Y ³ are independently of each other O or S;

Z ¹ and Z ³ are independently of each other 0, NH or S;

M is a solid phase carrier.

A method for producing a unitary compound for solid phase synthesis represented by the 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),

[Chemical 24]

(VI-2) (VI-3) A dimer represented by the formula (VI-3) and a nucleoside derivative represented by the following formula (IV-3) are condensed with a phosphate binding reagent, A trimer represented by the following formula (XIV-2) is obtained,

[Chemical 25]

(VI-3) rxiv-2) OH W " The trimer represented by the formula (XIV-2) and the anhydride represented by the following formula (VII) are condensed to obtain a trimer represented by the following formula (XV-2),

[Chemical 26]

The trimer represented by the formula (XV-2) and the solid support having an amino group represented by the following formula (IX) are condensed to form a solid phase represented by the following formula (ΧΠ-1). A production method for obtaining a unity compound for synthesis.

[Chemical 27]

In the above formula,

R ³ and R are, independently of one another, a protecting group;

[Chemical 28]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

Wherein ¹ , R ²¹ and R ³¹ are each independently a protecting group;

X ¹ and X ³ are independently of each other 0, NH or S;

Y ¹ is, independently of ^one another, O or S;

Z ¹ is independently of each other 0, NH or S;

M is a solid phase carrier.

[32] A unitary compound for solid-phase synthesis represented by the following formula (1-1) for producing the modified oligonucleotide according to claim 4.

[Chemical 29]

In the above formula, R ⁴ is a protecting group,

[Chemical 30]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,

X ¹ , X ² , X ³ and X ⁴ are independently of each other 0, NH or S;

Υ ² and Υ ³ are, independently of each other, Ο or S; Z ² and Z ³ are independently of each other 0, NH or S;

M is a solid phase carrier.

A method for producing a unitary compound for solid phase synthesis represented by the formula (ΧΙΠ-1) according to claim 32, comprising:

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),

[Chemical 31]

The trimer represented by the formula (XIV-3) and the nucleoside derivative represented by the following formula (IV-4) are condensed with a diimidazole derivative represented by the following formula (V-3). To obtain a tetramer represented by the following formula (XVI-1),

[Chemical 32]

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

[Chemical 33]

Solid phase synthesis represented by the formula (xm-1) by condensing the tetramer represented by the formula (XVII-1) and a solid phase carrier having an amino group represented by the following formula (IX) A manufacturing method for obtaining a unity compound.

[Chemical 34]

Eye ij self-style

R ³ and R ⁴ are protecting groups, B ³ and B ⁴ are each independently a group selected from a group represented by the following formula and a group whose functional group is protected by a protective group:

[Chemical 35]

2 n

W ¹¹ is a group represented by H or the formula OR ¹¹ ,

W ²¹ is a group represented by H or the formula OR ²¹ ;

W ³¹ is a group represented by H or the formula OR ³¹ ;

W ⁴¹ is H or a group represented by the formula OR ⁴¹ ,

X ¹ , X ² , X ³ and X ⁴ are independently of each other 0, NH or S;

Υ ² and Υ ³ are, independently of each other, Ο or S;

Ζ ² and Ζ ³ are independently of each other 0, NH or S;

M is a solid phase carrier.